Extraction method of high-purity microalgae polysaccharide

Abstract

The invention discloses an extraction method of high-purity microalgae polysaccharide, which specifically comprises the following steps: step 1, enzymolysis wall breaking: preparing microalgae powder into an algae solution, adjusting the pH value, and adding a compound enzyme for enzymolysis wall breaking reaction to obtain a microalgae enzymolysis solution; the solid-liquid ratio of the algae liquid is (1: 10)-(1: 20) (g / mL); adjusting the pH (Potential of Hydrogen) to 6.0 to 7.5; the enzymolysis temperature is 45-55 DEG C, and the reaction time is 3-6 hours; the compound enzyme is composed of cellulase, pectinase and alkaline protease according to the mass ratio of 1: 2: 1; according to the method disclosed by the invention, multiple centrifugation and transfer operations between the extracting solution and the purifying solution are canceled, so that polysaccharide adsorption and residual loss caused by the operations are avoided, the accumulative loss rate of the polysaccharide in the purifying link is reduced, the total extraction rate of the final product is increased, meanwhile, the technological process is greatly shortened, and the total production time is shortened; the energy consumption and the labor cost are remarkably reduced, the purity and the quality of the product are remarkably improved, and efficient synchronization of deproteinization and decolorization is realized.

Description

Technical Field

[0001] This invention belongs to the field of bio-extraction technology, specifically relating to a method for extracting high-purity microalgal polysaccharides. Background Technology

[0002] Microalgal polysaccharides are natural macromolecules composed of different monosaccharide groups linked by glycosidic bonds. They are widely found both intracellularly and extracellularly in microalgae, encompassing types such as spirulina polysaccharides and laver polysaccharides. Their monosaccharide composition includes D-glucose and D-galactose. They possess various biological activities, including antiviral, antitumor, immune-enhancing, antioxidant, and lipid-lowering effects. In the pharmaceutical field, they can be developed into anticancer drugs and antiviral agents; in the food industry, they can be used as thickeners and emulsifiers; and they can also act as prebiotics to promote the growth of probiotics. Extracting microalgal polysaccharides can increase their added value, promote the comprehensive utilization of microalgal resources, and reduce bioenergy costs, showing broad application prospects.

[0003] In a high-purity microalgal polysaccharide and its extraction method disclosed in prior art publication CN116178577B, extraction and purification typically rely on a multi-step, intermittent physicochemical process. A typical method includes steps such as enzymatic hydrolysis to disrupt cell walls, solvent extraction, deproteinization, decolorization and impurity removal, membrane filtration, concentration, and drying. Although such processes can yield high-purity products on a laboratory scale, their inherent technical defects severely restrict the efficiency and effectiveness of industrial production. The core problem lies in the excessively long process flow and the involvement of multiple phase separation operations. After each purification step, the supernatant must be obtained through centrifugation or filtration. This process inevitably involves liquid residue and sample transfer, leading to continuous loss of the target polysaccharide during impurity removal. Even if the loss rate at each step is low (e.g., deproteinization and decolorization steps each cause 1%-3% polysaccharide loss), the cumulative effect of multiple steps will significantly reduce the final product yield. Furthermore, the lengthy process implies longer production cycles, higher energy consumption and labor costs, and increases the risks to process control and batch stability. Summary of the Invention

[0004] The purpose of this invention is to provide a method for extracting high-purity microalgal polysaccharides, in order to solve the problem mentioned in the background art that the existing multi-step purification process for microalgal polysaccharides results in significant cumulative loss of polysaccharides and low efficiency due to frequent material transfer and phase separation operations.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for extracting high-purity microalgal polysaccharides, specifically comprising the following steps: Step 1: Enzymatic hydrolysis and cell wall breaking: Prepare microalgae powder into algal solution, adjust the pH, add compound enzyme to carry out enzymatic hydrolysis and cell wall breaking reaction to obtain microalgae hydrolysate; The material-to-liquid ratio of the algal solution is 1:10 to 1:20 (g / mL); the pH is adjusted to 6.0 to 7.5; the enzymatic hydrolysis temperature is 45 to 55℃, and the reaction time is 3 to 6 hours; the compound enzyme consists of cellulase, pectinase, and alkaline protease in a mass ratio of 1:2:1, and the total addition amount is 0.5% of the mass of the algal solution. Under these conditions, the cell wall breaking rate of microalgae can reach more than 95%, which is conducive to the full release of intracellular polysaccharides. Step 2: In-situ extraction: Add a deep co-crystallization solvent directly to the microalgae enzymatic hydrolysate, mix and extract to obtain a microalgae polysaccharide extract mixture; Step 3: Integrated purification: The microalgal polysaccharide extract mixture is directly pumped into an integrated purification column device without any intermediate separation treatment for simultaneous protein removal and decolorization. The effluent is collected to obtain the purified microalgal polysaccharide solution. The operating flow rate is 2-5 BV / h. After this step, the protein removal rate in the extract is ≥96%, the pigment removal rate is ≥95%, and the instantaneous loss rate of polysaccharides is less than 2%, which is much lower than the cumulative loss of about 5-8% in the stepwise purification process. Step 4: Membrane filtration: The purified microalgal polysaccharide solution is subjected to membrane filtration, and the filtrate is collected. The membrane filtration uses an ultrafiltration membrane with a molecular weight cutoff of 50 kDa and an operating pressure of 0.2 MPa to remove large molecular impurities. Subsequently, a nanofiltration membrane with a molecular weight cutoff of 500 Da is used for concentration at an operating pressure of 1.0 MPa. This combination can effectively remove small molecular impurities and pre-concentrate the solution, maintaining the polysaccharide recovery rate above 98%. Step 5: Precipitation and concentration: Add a precipitating agent to the filtrate to precipitate the polysaccharide, and obtain the polysaccharide precipitate after separation; Step Six: Drying: The polysaccharide precipitate is dried to obtain a high-purity microalgae polysaccharide product. The drying is performed by vacuum freeze drying, with pre-freezing at -40℃ for 4 hours and then drying at -20℃ and 10 Pa for 24 hours. The final product is a white flocculent solid. The polysaccharide purity can reach more than 98.5% by phenol-sulfuric acid method, and the total extraction rate (based on microalgae powder) reaches more than 8.5%, which significantly improves production efficiency and product yield.

[0006] As a preferred technical solution of the present invention, the integrated purification column device in step three is filled with two functionally complementary and tandem chromatographic media. The upper layer is a medium with deproteinization function, and the lower layer is a medium with decolorization function. The integrated purification column device adopts a radial flow chromatography column or a dynamic axial compression chromatography column. The radial flow chromatography column has a diameter-to-height ratio of 5:1. This design can significantly reduce column pressure and allow for processing of extraction mixtures containing trace amounts of insoluble matter at higher flow rates, thereby improving purification throughput.

[0007] As a preferred technical solution of the present invention, the medium with deproteinization function is at least one of phenylboronic acid-based affinity chromatography medium, metal chelate chromatography medium or hydrophobic chromatography medium; the medium with decolorization function is at least one of macroporous adsorption resin, activated carbon fiber or chemically modified silica gel.

[0008] As a preferred technical solution of the present invention, in step two, the deep eutectic solvent is a eutectic mixture formed by choline chloride and lactic acid, glucose or glycerol. The most preferred formulation is choline chloride: lactic acid = 1:2 (molar ratio). This formulation has moderate viscosity and exhibits extremely high solubility and selectivity for microalgal polysaccharides.

[0009] As a preferred technical solution of the present invention, in step five, the precipitant is food-grade ethanol, isopropanol or acetone, and its added volume is 2 to 4 times the volume of the filtered liquid.

[0010] As a preferred technical solution of the present invention, in step four, the membrane filtration is performed sequentially as ultrafiltration membrane filtration and nanofiltration membrane filtration, wherein the molecular weight cutoff of the ultrafiltration membrane is 10-100kDa and the molecular weight cutoff of the nanofiltration membrane is 200-1000Da.

[0011] As a preferred technical solution of the present invention, in step one, the complex enzyme is a mixture of cellulase, pectinase and alkaline protease in a mass ratio of 1:(1-3):(0.5-2).

[0012] As a preferred embodiment of the present invention, the integrated purification column device is a vertically arranged column, with the following components arranged sequentially from top to bottom: A liquid distributor is used to receive the microalgae polysaccharide extract mixture and distribute it evenly. The protein removal medium layer is filled with phenylboronic acid affinity chromatography medium or metal chelate chromatography medium for the adsorption and removal of proteins. Transition sieve plate, used to separate the upper and lower media layers and ensure liquid flow; The decolorizing medium layer is filled with macroporous adsorption resin or chemically modified silica gel to adsorb and remove pigments and small molecule impurities. The system also includes a collector for collecting the purified microalgal polysaccharide solution after deproteinization and decolorization treatment. This ensures that the liquid to be purified first comes into contact with the deproteinization medium, effectively removing proteins and preventing irreversible protein adsorption on the decolorization medium, which would contaminate the medium and affect the decolorization effect. This maximizes the lifespan and efficiency of both functional media. The entire system is capable of in-line cleaning (CIP) and sterilization (SIP), meeting industrial production standards.

[0013] Compared with the prior art, the beneficial effects of the present invention are: In this invention, by eliminating multiple centrifugation and transfer operations between the extract and purification solutions, the resulting polysaccharide adsorption and residual loss are prevented. This method reduces the cumulative loss rate of polysaccharides during purification, thereby increasing the total extraction rate of the final product. Simultaneously, the significantly shortened process reduces total production time, energy consumption, and labor costs, while significantly improving product purity and quality. It achieves efficient simultaneous deproteinization and decolorization, avoiding potential cross-contamination in step-by-step processing. Combined with ultrafiltration-nanofiltration membrane technology, the final microalgal polysaccharide product has high purity and a more concentrated molecular weight distribution, better preserving its natural biological activity. This invention has broad prospects for industrial application. The method is simple, continuous, and easy to automate, solving the pain point of difficulty in scaling up existing technologies. The integrated purification column device has high throughput and strong anti-contamination capabilities, and can be cleaned and regenerated online, making it highly suitable for large-scale, high-standard industrial production. It provides a reliable raw material guarantee for the application of microalgal polysaccharides in pharmaceuticals, high-end foods, and other fields. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the steps of the present invention. Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1 Please see Figure 1 This invention provides a technical solution: a method for extracting high-purity microalgal polysaccharides, specifically including the following steps: Step 1: Enzymatic hydrolysis and cell wall breaking: Prepare microalgae powder into algal solution, adjust the pH, add compound enzyme to carry out enzymatic hydrolysis and cell wall breaking reaction to obtain microalgae hydrolysate; The material-to-liquid ratio of the algal solution was 1:10 (g / mL); the pH was adjusted to 6.0; the enzymatic hydrolysis temperature was 45℃ and the reaction time was 3 hours; the compound enzyme consisted of cellulase, pectinase and alkaline protease in a mass ratio of 1:2:1, and the total addition amount was 0.5% of the mass of the algal solution. Under these conditions, the cell wall breaking rate of microalgae could reach more than 95%, which was conducive to the full release of intracellular polysaccharides. Step 2: In-situ Extraction: A deep co-crystallization solvent is directly added to the microalgae enzymatic hydrolysate, mixed, and then extracted to obtain a microalgae polysaccharide extract mixture. The deep co-crystallization solvent is a mixture of choline chloride and lactic acid in a molar ratio of 1:2, stirred at 70°C until a clear liquid is formed; the amount added is 30% of the volume of the microalgae enzymatic hydrolysate. The extraction process is carried out at 55°C with continuous stirring for 2 hours. The extraction efficiency of this solvent system for polysaccharides has been measured to be over 96.5%, and centrifugation is not required; the polysaccharides can be directly transferred to the next step. Step 3: Integrated purification: The microalgal polysaccharide extract mixture is directly pumped into an integrated purification column device for simultaneous deproteinization and decolorization without any intermediate separation treatment. The effluent is collected to obtain the purified microalgal polysaccharide solution. The operating flow rate is 2-5 BV / h. After this step, the protein removal rate in the extract is ≥96%, the pigment removal rate is ≥95%, and the instantaneous loss rate of polysaccharides is less than 2%, which is much lower than the cumulative loss of about 5-8% in the stepwise purification process. Step 4: Membrane Filtration: The purified microalgae polysaccharide solution is subjected to membrane filtration, and the filtrate is collected. The membrane filtration uses an ultrafiltration membrane with a molecular weight cutoff of 50 kDa and an operating pressure of 0.2 MPa to remove large molecular impurities. Subsequently, a nanofiltration membrane with a molecular weight cutoff of 500 Da is used for concentration at an operating pressure of 1.0 MPa. This combination can effectively remove small molecular impurities and pre-concentrate the solution, maintaining the polysaccharide recovery rate above 98%. Step 5: Precipitation and concentration: Add a precipitating agent to the filtrate to precipitate the polysaccharide, and obtain the polysaccharide precipitate after separation; Step Six: Drying: The polysaccharide precipitate is dried to obtain a high-purity microalgae polysaccharide product. The drying is performed by vacuum freeze drying, with pre-freezing at -40℃ for 4 hours and then drying at -20℃ and 10 Pa for 24 hours. The final product is a white flocculent solid. The polysaccharide purity can reach more than 98.5% by phenol-sulfuric acid method, and the total extraction rate (based on microalgae powder) reaches more than 8.5%, which significantly improves production efficiency and product yield.

[0017] In this embodiment, the integrated purification column device in step three is filled with two functionally complementary and tandem chromatographic media. The upper layer is a medium with deproteinization function, and the lower layer is a medium with decolorization function. The integrated purification column device adopts a radial flow chromatography column or a dynamic axial compression chromatography column. The radial flow chromatography column has a diameter-to-height ratio of 5:1. This design can significantly reduce column pressure and allow for processing of extraction mixtures containing trace amounts of insoluble matter at higher flow rates, thereby increasing purification throughput.

[0018] In this embodiment, the medium with deproteinization function is at least one of phenylboronic acid-based affinity chromatography medium, metal chelate chromatography medium, or hydrophobic chromatography medium; the medium with decolorization function is at least one of macroporous adsorption resin, activated carbon fiber, or chemically modified silica gel; the upper deproteinization medium is phenylboronic acid-based affinity chromatography medium (its ligand structure is B(OH)2-), which can efficiently remove proteins by utilizing its specific reversible binding with serine and threonine residues in polysaccharide proteins; the lower decolorization medium is D101 type macroporous adsorption resin, which has a high adsorption capacity for algal pigments such as chlorophyll and carotenoids.

[0019] In this embodiment, in step two, the deep eutectic solvent is a eutectic mixture of choline chloride and lactic acid, glucose or glycerol. The most preferred formulation is choline chloride: lactic acid = 1:2 (molar ratio). This formulation has moderate viscosity and exhibits extremely high solubility and selectivity for microalgal polysaccharides.

[0020] In this embodiment, in step five, the precipitant is food-grade ethanol, isopropanol, or acetone, and its added volume is twice the volume of the filtered liquid.

[0021] In this embodiment, in step four, membrane filtration is performed sequentially as ultrafiltration and nanofiltration. The ultrafiltration membrane has a molecular weight cutoff of 10 kDa, and the nanofiltration membrane has a molecular weight cutoff of 200 Da.

[0022] In this embodiment, in step one, the complex enzyme is a mixture of cellulase, pectinase and alkaline protease in a mass ratio of 1:(1-3):(0.5-2).

[0023] In this embodiment, the integrated purification column device is a vertically arranged column, with the following components arranged sequentially from top to bottom: Liquid distributor is used to receive the microalgae polysaccharide extraction mixture and distribute it evenly; The protein removal medium layer is filled with phenylboronic acid affinity chromatography medium or metal chelate chromatography medium for the adsorption and removal of proteins. Transition sieve plate, used to separate the upper and lower media layers and ensure liquid flow; The decolorizing medium layer is filled with macroporous adsorption resin or chemically modified silica gel to adsorb and remove pigments and small molecule impurities. The system also includes a collector for collecting the purified microalgal polysaccharide solution after deproteinization and decolorization treatment. This ensures that the liquid to be purified first comes into contact with the deproteinization medium, effectively removing proteins and preventing irreversible protein adsorption on the decolorization medium, which would contaminate the medium and affect the decolorization effect. This maximizes the lifespan and efficiency of both functional media. The entire system is capable of in-line cleaning (CIP) and sterilization (SIP), meeting industrial production standards.

[0024] Example 2 The difference from that in Example 1 is that; The material-to-liquid ratio of the algal solution was 1:20 (g / mL); the pH was adjusted to 7.5; the enzymatic hydrolysis temperature was 55℃ and the reaction time was 6 hours; the compound enzyme consisted of cellulase, pectinase and alkaline protease in a mass ratio of 1:2:1, and the total addition amount was 0.5% of the mass of the algal solution. Under these conditions, the cell wall breaking rate of microalgae could reach more than 95%, which was conducive to the full release of intracellular polysaccharides. In this embodiment, in step five, the precipitant is food-grade ethanol, isopropanol, or acetone, and its added volume is four times the volume of the filtered liquid.

[0025] In this embodiment, in step four, membrane filtration is performed sequentially as ultrafiltration and nanofiltration. The ultrafiltration membrane has a molecular weight cutoff of 100 kDa, and the nanofiltration membrane has a molecular weight cutoff of 1000 Da.

[0026] Example 2 The difference from the above embodiments is that; In this embodiment, in step five, the precipitant is food-grade ethanol, isopropanol, or acetone, and its added volume is three times the volume of the filtered liquid.

[0027] In this embodiment, in step four, membrane filtration is performed sequentially as ultrafiltration and nanofiltration. The ultrafiltration membrane has a molecular weight cutoff of 50 kDa, and the nanofiltration membrane has a molecular weight cutoff of 500 Da.

[0028] Although embodiments of the invention have been shown and described (see the detailed description above), it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for extracting high-purity microalgal polysaccharides, characterized in that: Specifically, the steps include the following: Step 1: Enzymatic hydrolysis and cell wall breaking: Prepare microalgae powder into algal solution, adjust the pH, add compound enzyme to carry out enzymatic hydrolysis and cell wall breaking reaction to obtain microalgae hydrolysate; The material-to-liquid ratio of the algae solution is 1:10 to 1:20 (g / mL); the pH is adjusted to 6.0 to 7.5; the enzymatic hydrolysis temperature is 45 to 55℃, and the reaction time is 3 to 6 hours; the compound enzyme is composed of cellulase, pectinase, and alkaline protease in a mass ratio of 1:2:

1. Step 2: In-situ extraction: Add a deep co-crystallization solvent directly to the microalgae enzymatic hydrolysate, mix and extract to obtain a microalgae polysaccharide extract mixture; Step 3: Integrated purification: The microalgal polysaccharide extraction mixture is directly pumped into an integrated purification column device without any intermediate separation treatment for simultaneous deproteinization and decolorization. The effluent is collected to obtain the purified microalgal polysaccharide solution. The operating flow rate is 2-5 BV / h. Step 4: Membrane filtration: The purified microalgae polysaccharide solution is subjected to membrane filtration, and the filtrate is collected; Step 5: Precipitation and concentration: Add a precipitating agent to the filtrate to precipitate the polysaccharide, and obtain the polysaccharide precipitate after separation; Step 6: Drying: The polysaccharide precipitate is dried to obtain a high-purity microalgae polysaccharide product. The drying is performed by vacuum freeze drying, with the conditions being pre-freezing at -40°C for 4 hours and then drying at -20°C and 10 Pa for 24 hours.

2. The method for extracting high-purity microalgal polysaccharides according to claim 1, characterized in that: In step three, the integrated purification column device is filled with two complementary and tandem chromatographic media: the upper layer is a medium with deproteinization function, and the lower layer is a medium with decolorization function. The integrated purification column device adopts a radial flow chromatography column or a dynamic axial compression chromatography column.

3. The method for extracting high-purity microalgal polysaccharides according to claim 2, characterized in that: The media with deproteinization function is at least one of phenylboronic acid-based affinity chromatography media, metal chelate chromatography media, or hydrophobic chromatography media; the media with decolorization function is at least one of macroporous adsorption resin, activated carbon fiber, or chemically modified silica gel.

4. The method for extracting high-purity microalgal polysaccharides according to claim 1, characterized in that: In step two, the deep eutectic solvent is a eutectic mixture formed by choline chloride and lactic acid, glucose, or glycerol.

5. The method for extracting high-purity microalgal polysaccharides according to claim 1, characterized in that: In step five, the precipitant is food-grade ethanol, isopropanol, or acetone, and its added volume is 2 to 4 times the volume of the filtered liquid.

6. The method for extracting high-purity microalgal polysaccharides according to claim 1, characterized in that: In step four, membrane filtration consists of ultrafiltration and nanofiltration performed sequentially. The ultrafiltration membrane has a molecular weight cutoff of 10–100 kDa, and the nanofiltration membrane has a molecular weight cutoff of 200–1000 Da.

7. The method for extracting high-purity microalgal polysaccharides according to claim 1, characterized in that: In step one, the complex enzyme is a mixture of cellulase, pectinase and alkaline protease in a mass ratio of 1:(1-3):(0.5-2).

8. The method for extracting high-purity microalgal polysaccharides according to claim 2, characterized in that: The integrated purification column device is a vertically arranged column, with the following components arranged from top to bottom: A liquid distributor is used to receive the microalgae polysaccharide extract mixture and distribute it evenly. The protein removal medium layer is filled with phenylboronic acid affinity chromatography medium or metal chelate chromatography medium for the adsorption and removal of proteins. Transition sieve plate, used to separate the upper and lower media layers and ensure liquid flow; The decolorizing medium layer is filled with macroporous adsorption resin or chemically modified silica gel to adsorb and remove pigments and small molecule impurities. And a liquid collector for collecting purified microalgal polysaccharide solution after deproteinization and decolorization treatment.

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