Preparation method of chlamydomonas reinhardtii polysaccharide granules

Abstract

The invention discloses a preparation method of chlamydomonas reinhardtii polysaccharide granules, which comprises the following steps: extracting chlamydomonas reinhardtii polysaccharide to prepare polysaccharide dry powder, and mixing the polysaccharide dry powder with starch and magnesium stearate to prepare the chlamydomonas reinhardtii polysaccharide granules; the chlamydomonas reinhardtii polysaccharide is extracted by adopting a compound enzyme; the compound enzyme is prepared from cellulase, pectinase and superoxide dismutase according to the mass ratio of (1.8 to 2.2) to 1 to (0.8 to 1.2). According to the preparation method of the chlamydomonas reinhardtii polysaccharide granules, chlamydomonas reinhardtii polysaccharide is extracted to prepare polysaccharide dry powder, and the polysaccharide dry powder is mixed with starch and magnesium stearate to prepare the chlamydomonas reinhardtii polysaccharide granules; the compound enzyme is adopted for enzymolysis, so that the cell wall structure of the chlamydomonas reinhardtii can be destroyed to promote the polysaccharide to be easily dissolved out, the problems of low extraction efficiency and low purity in the chlamydomonas reinhardtii polysaccharide extraction process are solved, the loss of the polysaccharide is also avoided to the greatest extent, and the obtained polysaccharide particles are high in antioxidant activity and good in water solubility.

Description

[0001] This application is a divisional application of the invention patent application with application date of June 11, 2024, application number 2024107423049, and invention name “A kind of Chlamydomonas reinhardtii polysaccharide particles and preparation method thereof”. Technical Field

[0002] The invention relates to the technical field of Chlamydomonas reinhardtii, and in particular to a method for preparing Chlamydomonas reinhardtii polysaccharide particles. Background Art

[0003] Chlamydomonas reinhardtii is a green microalgae with a valuable nutritional composition, including ≥30.0% protein, ≥10.0% polysaccharide, 8 essential amino acids, high cellulose and chlorophyll content, and a variety of trace elements required by the human body. Studies have shown that Chlamydomonas reinhardtii polysaccharide, as the main ingredient, has multiple functions such as scavenging free radicals, lowering blood lipids and blood sugar, resisting fatigue, enhancing human immunity, and regulating gastrointestinal discomfort.

[0004] Although Chlamydomonas reinhardtii polysaccharides have good pharmacological effects, there is currently no technical development for extracting and preparing Chlamydomonas reinhardtii polysaccharides into granules. Therefore, studying and optimizing the extraction process of Chlamydomonas reinhardtii polysaccharides and preparing them into granules is an important way to deeply utilize Chlamydomonas reinhardtii and increase its added value.

[0005] The prior art, such as the Chinese invention patent with publication number CN109021096A, discloses a method for separation, extraction and purification of Spirulina polysaccharides and phycocyanin. The method includes extraction, wall breaking, centrifugal filtration, salt washing and protein precipitation, and critical extraction of polysaccharides. Although the method is simple to operate and can obtain both Spirulina polysaccharides and phycocyanin at the same time, the method requires the use of high-pressure equipment for critical extraction when extracting polysaccharides, which is costly and has great safety risks.

[0006] Another example is the Chinese invention patent with publication number CN116178577A, which discloses a high-purity microalgae polysaccharide and its extraction method. The polysaccharide is extracted by compound enzyme enzymatic cell wall breaking; a new deep eutectic solvent is prepared and screened; a new protein treatment agent is prepared to remove protein efficiently; a new decolorization and impurity removal agent is prepared to effectively remove pigments and other impurities; membrane filtration; reduced pressure concentration; spray drying to obtain the finished product. Although this method is simple to operate and can obtain a microalgae polysaccharide product with high purity and few impurities, the enzyme used has poor specificity for the antioxidant activity of polysaccharides and cannot protect the antioxidant activity of polysaccharides.

[0007] The disclosure of the above background technology content is only used to assist in understanding the inventive concept and technical solution of the present invention. It does not necessarily belong to the prior art of this patent application. In the absence of clear evidence that the above content has been disclosed before the filing date of this patent application, the above background technology should not be used to evaluate the novelty and creativity of the present application. Summary of the invention

[0008] In view of this, in order to overcome the defects of the prior art, the object of the present invention is to provide a method for preparing Chlamydomonas reinhardtii polysaccharide particles.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A method for preparing Chlamydomonas reinhardtii polysaccharide particles comprises the following steps:

[0011] Extraction of Chlamydomonas reinhardtii polysaccharide:

[0012] Take Chlamydomonas reinhardtii, add water and mix to obtain a mixed solution;

[0013] Adding complex enzyme to the mixed solution, adding acetic acid to adjust the pH, keeping the mixture warm, and heating for extraction to obtain an extract;

[0014] The extract was filtered and concentrated into a clear paste of 1.05-1.15 g / mL;

[0015] adding anhydrous ethanol to the clear paste, centrifuging and collecting the precipitate, which is Chlamydomonas reinhardtii polysaccharide;

[0016] Preparation of Chlamydomonas reinhardtii polysaccharide particles:

[0017] Add water to dissolve the precipitate, add cyclodextrin and mix, and then spray dry to obtain polysaccharide powder;

[0018] The polysaccharide powder is mixed with starch and magnesium stearate, granulated, and sieved to obtain Chlamydomonas reinhardtii polysaccharide granules;

[0019] The complex enzyme comprises cellulase, pectinase and superoxide dismutase.

[0020] According to some preferred embodiments of the present invention, the mass volume ratio of Chlamydomonas reinhardtii to water in the mixed solution is 1:8 to 1:12.

[0021] According to some preferred implementation aspects of the present invention, the dosage of the complex enzyme is 0.2-0.5% of the mass of the mixed solution.

[0022] According to some preferred implementation aspects of the present invention, the complex enzyme is composed of cellulase, pectinase, and superoxide dismutase in a mass ratio of 1.8-2.2:1:0.8-1.2. Preferably, the complex enzyme is composed of cellulase, pectinase, and superoxide dismutase (SOD) in a mass ratio of 2:1:1. Cellulase destroys the dense structure of the cell wall and increases the conversion of insoluble cellulose into glucose; pectinase acts on the glycosidic bonds between D-galacturonic acid residues in pectin, breaks pectin molecules, degrades the polysaccharide chains of pectin, generates oligogalactose and a small amount of galacturonic acid small molecules, thereby reducing viscosity, increasing filtration speed, and improving polysaccharide extraction rate; superoxide dismutase (SOD) can play an antioxidant protective role on Chlamydomonas reinhardtii polysaccharides in the process of extracting Chlamydomonas reinhardtii polysaccharides, and enhances the ability of Chlamydomonas reinhardtii polysaccharides to scavenge superoxide anion free radicals. The combination of the three can efficiently extract Chlamydomonas reinhardtii polysaccharides and effectively protect the antioxidant activity of polysaccharides.

[0023] According to some preferred embodiments of the present invention, when preparing the extract, acetic acid is added to adjust the pH value to 4.0-6.0, and the mixed solution is heated to react at a temperature of 40-55°C for 8-24 hours; boiling and decocting, filtering to obtain a filtrate and a filter residue, and the filtrate is reserved; then water of several times the weight of the raw material Chlamydomonas reinhardtii is added to the filter residue, boiling and decocting, filtering to obtain a filtrate; and combining the two filtrates to obtain the extract.

[0024] According to some preferred implementation aspects of the present invention, the concentration is performed using a double-effect concentrator, the steam pressure of the concentrator is 0.09 to 0.1 MPa, the vacuum degree is -0.10 to -0.02 MPa, and the concentration temperature is 70 to 90°C.

[0025] According to some preferred implementation aspects of the present invention, the volume ratio of the clear paste to anhydrous ethanol is 1:4-6, and the centrifuge speed during centrifugal separation is 5500-6500r / min.

[0026] According to some preferred embodiments of the present invention, the mass ratio of the precipitate to water is 1:1.8-2.5, the amount of cyclodextrin added is 8-12% of the mass of the precipitate, the inlet air temperature of the spray drying is 180-200°C, and the outlet air temperature is 80-100°C.

[0027] According to some preferred embodiments of the present invention, the amount of starch used is 18-22% of the mass of the polysaccharide dry powder, and the amount of magnesium stearate used is 0.4-0.6% of the mass of the polysaccharide dry powder.

[0028] According to some preferred embodiments of the present invention, the granulation is dry granulation, the feeding speed is 22-28 rpm, the pressing wheel spacing is 0.08-0.12 mm, and the pressing wheel speed is 0.8-1.2 rpm.

[0029] The present invention also provides Chlamydomonas reinhardtii polysaccharide particles prepared by the above preparation method.

[0030] Due to the adoption of the above technical scheme, compared with the prior art, the advantages of the present invention are as follows: the preparation method of the Chlamydomonas reinhardtii polysaccharide particles of the present invention comprises extracting the Chlamydomonas reinhardtii polysaccharide to prepare the polysaccharide dry powder, and mixing the polysaccharide with starch and magnesium stearate to prepare the Chlamydomonas reinhardtii polysaccharide particles; using a composite enzyme for enzymolysis, which can destroy the cell wall structure of Chlamydomonas reinhardtii and facilitate the dissolution of the polysaccharide, solves the problems of low extraction efficiency and low purity in the extraction process of Chlamydomonas reinhardtii polysaccharide, and also avoids the loss of polysaccharides to the greatest extent, and the obtained polysaccharide particles have high antioxidant activity and good water solubility. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0032] The invention relates to Chlamydomonas reinhardtii polysaccharide and a preparation method thereof, and application of Chlamydomonas reinhardtii polysaccharide in preparing Chlamydomonas reinhardtii polysaccharide particles. One purpose is to provide a preparation method of Chlamydomonas reinhardtii polysaccharide, which can effectively extract and purify polysaccharides in Chlamydomonas reinhardtii, and the obtained Chlamydomonas reinhardtii polysaccharide has high purity, high antioxidant activity and good pharmacological effect; the second purpose is to provide a preparation method of particles based on the extracted Chlamydomonas reinhardtii polysaccharide, and the particles are convenient to carry, can be taken directly without decocting, are simple to take and have a rapid effect.

[0033] Specifically, the method for preparing Chlamydomonas reinhardtii polysaccharide particles of the present invention comprises the following steps:

[0034] (1) Take Chlamydomonas reinhardtii, add purified water and mix to obtain a mixed solution.

[0035] The mass volume ratio of Chlamydomonas reinhardtii to purified water is 1:8 to 1:12.

[0036] (2) Adding the complex enzyme to the mixed solution prepared in step (1), adjusting the pH by adding acetic acid, keeping the mixture warm overnight, heating and extracting, and combining the extracts.

[0037] Specifically, the dosage of the complex enzyme is 0.2-0.5% of the mass of the mixed solution. The complex enzyme is composed of cellulase, pectinase, and superoxide dismutase in a mass ratio of 1.8-2.2:1:0.8-1.2. Preferably, the mass ratio of cellulase, pectinase, and superoxide dismutase (SOD) is 2:1:1.

[0038] Add acetic acid to adjust the pH value to 4.0-6.0, heat the mixture to make the temperature of the mixture react at 40-55°C for 8-24 hours; boil and decoct for 2 hours, filter to obtain a filtrate and a filter residue, and reserve the filtrate; then add water 8 times the weight of the raw material Chlamydomonas reinhardtii to the filter residue, boil and decoct for 2 hours, filter to obtain a filtrate; combine the two filtrates to obtain the extract.

[0039] (3) Filter the extract obtained in step (2) and concentrate it into a clear paste of 1.05-1.15 g / mL for later use.

[0040] The concentration adopts a double-effect concentrator with a steam pressure of 0.09 to 0.1Mpa, a vacuum degree of -0.10 to -0.02Mpa, and a concentration temperature of 70 to 90°C.

[0041] (4) adding anhydrous ethanol to the clear paste obtained in step (3), centrifuging and collecting the precipitate, which is Chlamydomonas reinhardtii polysaccharide.

[0042] The volume ratio of the clear paste to anhydrous ethanol is 1:4-6, and the centrifuge speed during centrifugal separation is 5500-6500r / min. Preferably, the volume ratio of the clear paste to anhydrous ethanol is 1:5, and the centrifuge speed is 6000r / min.

[0043] (5) The precipitate obtained in step (4) is dissolved in purified water, mixed with cyclodextrin, and then spray-dried to obtain a polysaccharide dry powder.

[0044] The mass ratio of the precipitate to the purified water is 1:1.8-2.5, and the amount of cyclodextrin added is 8-12% of the mass of the precipitate. Preferably, the mass ratio of the precipitate to the purified water is 1:2, and the amount of cyclodextrin added is 10% of the mass of the precipitate. The inlet air temperature of the spray drying is 180-200°C, and the outlet air temperature is 80-100°C.

[0045] (6) The polysaccharide powder obtained in step (5) is mixed with starch and magnesium stearate, dry granulated, and sieved.

[0046] The amount of starch is 18-22% of the mass of the polysaccharide dry powder, and the amount of magnesium stearate is 0.4-0.6% of the mass of the polysaccharide dry powder; the feeding speed of dry granulation is 22-28rpm, the pressure wheel spacing is 0.08-0.12mm, and the pressure wheel speed is 0.8-1.2rpm. Preferably, the amount of starch is 20% of the mass of the polysaccharide dry powder, the amount of magnesium stearate is 0.5% of the mass of the polysaccharide dry powder; the dry powder is granulated by dry method, the feeding speed is 25rpm, the pressure wheel spacing is 0.1mm, and the pressure wheel speed is 1rpm.

[0047] The preparation of Chlamydomonas reinhardtii polysaccharide particles in this embodiment mainly includes two steps: extraction of Chlamydomonas reinhardtii polysaccharide (steps 1-4) and preparation of Chlamydomonas reinhardtii polysaccharide into polysaccharide particles (steps 5-6).

[0048] Example 1

[0049] The method for preparing Chlamydomonas reinhardtii polysaccharide particles in this embodiment comprises the following steps:

[0050] Take Chlamydomonas reinhardtii, add purified water at a mass volume ratio of Chlamydomonas reinhardtii to purified water of 1:8, and mix to obtain a mixed solution.

[0051] Add a complex enzyme composed of cellulase, pectinase and superoxide dismutase (SOD) in a mass ratio of 2:1:1 to the mixed solution, the amount of the complex enzyme is 0.2% of the mass of the mixed solution, and then add acetic acid to adjust the pH to 4.0. After keeping warm overnight, heat the mixed solution to make the temperature of the mixed solution react at 40°C for 24 hours for extraction; boil and decoct for 2 hours, filter to obtain a filtrate and a filter residue, and use the filtrate for later use; then add water 8 times the weight of the raw material Chlamydomonas reinhardtii to the filter residue, boil and decoct for 2 hours, filter to obtain a filtrate; combine the two filtrates to obtain an extract.

[0052] The extract was filtered and a double-effect concentrator was used. The steam pressure of the concentrator was set to 0.09 MPa, the vacuum degree was -0.10 MPa, and the concentration temperature was 70°C. The extract was concentrated into a clear paste of 1.05 g / mL for later use.

[0053] Take the clear paste, add anhydrous ethanol in a volume ratio of 1:5 to the clear paste and mix, and separate and collect the precipitate using a centrifuge at 6000 r / min.

[0054] Purified water twice the weight of the precipitate was added to dissolve it, and then cyclodextrin 10% of the weight of the precipitate was added to mix, and then dried in a spray dryer with an inlet air temperature of 180°C and an outlet air temperature of 80°C to obtain polysaccharide dry powder.

[0055] The polysaccharide powder was mixed with starch and magnesium stearate, wherein the amount of starch was 20% of the mass of the polysaccharide powder and the amount of magnesium stearate was 0.5% of the mass of the polysaccharide powder. The dry granulation was carried out with a feeding speed of 25 rpm, a pressing wheel spacing of 0.1 mm, a pressing wheel speed of 1 rpm, and sieving.

[0056] In this example, the yield of Chlamydomonas reinhardtii polysaccharide particles was 10.3%, and the purity of Chlamydomonas reinhardtii polysaccharide particles was 66.8%.

[0057] Example 2

[0058] The method for preparing Chlamydomonas reinhardtii polysaccharide particles in this embodiment comprises the following steps:

[0059] Take Chlamydomonas reinhardtii, add purified water at a mass volume ratio of Chlamydomonas reinhardtii to purified water of 1:12, and mix to obtain a mixed solution.

[0060] Add a complex enzyme composed of cellulase, pectinase and superoxide dismutase (SOD) in a mass ratio of 2:1:1 to the mixed solution, the amount of the complex enzyme is 0.5% of the mass of the mixed solution, and then add acetic acid to adjust the pH to 6.0. After keeping warm overnight, heat the mixed solution to make the temperature of the mixed solution react at 55°C for 8 hours for extraction; boil and decoct for 2 hours, filter to obtain a filtrate and a filter residue, and use the filtrate for later use; then add water 8 times the weight of the raw material Chlamydomonas reinhardtii to the filter residue, boil and decoct for 2 hours, filter to obtain a filtrate; combine the two filtrates to obtain an extract.

[0061] The extract was filtered and a double-effect concentrator was used. The steam pressure of the concentrator was set to 0.1 MPa, the vacuum degree was -0.02 MPa, and the concentration temperature was 90°C. The extract was concentrated into a clear paste of 1.15 g / mL for later use.

[0062] Take the clear paste, add anhydrous ethanol in a volume ratio of 1:5 to the clear paste and mix, and separate and collect the precipitate using a centrifuge at 6000 r / min.

[0063] Purified water twice the weight of the precipitate was added to dissolve it, and then cyclodextrin 10% of the weight of the precipitate was added to mix, and then dried in a spray dryer with an inlet air temperature of 200°C and an outlet air temperature of 100°C to obtain polysaccharide dry powder.

[0064] The polysaccharide powder was mixed with starch and magnesium stearate, wherein the amount of starch was 20% of the mass of the polysaccharide powder and the amount of magnesium stearate was 0.5% of the mass of the polysaccharide powder. The dry granulation was carried out with a feeding speed of 25 rpm, a pressing wheel spacing of 0.1 mm, a pressing wheel speed of 1 rpm, and sieving.

[0065] In this example, the yield of Chlamydomonas reinhardtii polysaccharide particles was 10.8%, and the purity of Chlamydomonas reinhardtii polysaccharide particles was 68.3%.

[0066] Example 3

[0067] The method for preparing Chlamydomonas reinhardtii polysaccharide particles in this embodiment comprises the following steps:

[0068] Take Chlamydomonas reinhardtii, add purified water at a mass volume ratio of Chlamydomonas reinhardtii to purified water of 1:8, and mix to obtain a mixed solution.

[0069] Add a complex enzyme composed of cellulase, pectinase and superoxide dismutase (SOD) in a mass ratio of 1.8:1:1.2 to the mixed solution, the amount of the complex enzyme is 0.2% of the mass of the mixed solution, and then add acetic acid to adjust the pH to 4.0. After keeping warm overnight, heat the mixed solution to make the temperature of the mixed solution react at 40°C for 24 hours for extraction; boil and decoct for 2 hours, filter to obtain a filtrate and a filter residue, and use the filtrate for later use; then add water 8 times the weight of the raw material Chlamydomonas reinhardtii to the filter residue, boil and decoct for 2 hours, filter to obtain a filtrate; combine the two filtrates to obtain an extract.

[0070] The extract was filtered and a double-effect concentrator was used. The steam pressure of the concentrator was set to 0.09 MPa, the vacuum degree was -0.10 MPa, and the concentration temperature was 70°C. The extract was concentrated into a clear paste of 1.05 g / mL for later use.

[0071] Take the clear paste, add anhydrous ethanol in a volume ratio of 1:4 to the clear paste and mix them, and separate and collect the precipitate using a centrifuge at 5500 r / min.

[0072] Purified water 1.8 times of the precipitate's mass was added to dissolve it, and then cyclodextrin 8% of the precipitate's mass was added to mix, and then dried in a spray dryer with an inlet air temperature of 180°C and an outlet air temperature of 80°C to obtain a polysaccharide dry powder.

[0073] The polysaccharide powder was mixed with starch and magnesium stearate, wherein the amount of starch was 18% of the mass of the polysaccharide powder and the amount of magnesium stearate was 0.4% of the mass of the polysaccharide powder. Dry granulation was adopted with a feeding speed of 22 rpm, a pressing wheel spacing of 0.08 mm, a pressing wheel speed of 0.8 rpm, and sieving.

[0074] In this example, the yield of Chlamydomonas reinhardtii polysaccharide particles was 11.5%, and the purity of Chlamydomonas reinhardtii polysaccharide particles was 65.7%.

[0075] Example 4

[0076] The method for preparing Chlamydomonas reinhardtii polysaccharide particles in this embodiment comprises the following steps:

[0077] Take Chlamydomonas reinhardtii, add purified water at a mass volume ratio of Chlamydomonas reinhardtii to purified water of 1:12, and mix to obtain a mixed solution.

[0078] Add a complex enzyme composed of cellulase, pectinase and superoxide dismutase (SOD) in a mass ratio of 2.2:1:0.8 to the mixed solution, the amount of the complex enzyme is 0.5% of the mass of the mixed solution, and then add acetic acid to adjust the pH to 6.0. After keeping warm overnight, heat the mixed solution to make the temperature of the mixed solution react at 55°C for 8 hours for extraction; boil and decoct for 2 hours, filter to obtain a filtrate and a filter residue, and use the filtrate for later use; then add water 8 times the weight of the raw material Chlamydomonas reinhardtii to the filter residue, boil and decoct for 2 hours, filter to obtain a filtrate; combine the two filtrates to obtain an extract.

[0079] The extract was filtered and a double-effect concentrator was used. The steam pressure of the concentrator was set to 0.1 MPa, the vacuum degree was -0.02 MPa, and the concentration temperature was 90°C. The extract was concentrated into a clear paste of 1.15 g / mL for later use.

[0080] Take the clear paste, add anhydrous ethanol in a volume ratio of 1:6 to the clear paste and mix them, and separate and collect the precipitate using a centrifuge at 6500 r / min.

[0081] Purified water at 2.2 times of the precipitate's own weight was added to dissolve it, and then cyclodextrin at 12% of the precipitate's own weight was added to mix, and then dried in a spray dryer with an inlet air temperature of 200°C and an outlet air temperature of 100°C to obtain a polysaccharide dry powder.

[0082] The polysaccharide powder was mixed with starch and magnesium stearate, wherein the amount of starch was 22% of the mass of the polysaccharide powder and the amount of magnesium stearate was 0.6% of the mass of the polysaccharide powder. Dry granulation was performed with a feeding speed of 28 rpm, a pressing wheel spacing of 0.12 mm, a pressing wheel speed of 1.2 rpm, and sieving.

[0083] In this example, the yield of Chlamydomonas reinhardtii polysaccharide particles was 10.9%, and the purity of Chlamydomonas reinhardtii polysaccharide particles was 67.8%.

[0084] Comparative Example 1

[0085] The difference between this comparative example and Example 2 is that the complex enzyme of this comparative example does not add superoxide dismutase (SOD) enzyme. Other steps and parameters are basically the same as those of Example 2.

[0086] In this embodiment, the yield of Chlamydomonas reinhardtii polysaccharide particles is 8.5%, and the purity of Chlamydomonas reinhardtii polysaccharide particles is 60.3%; compared with Example 2, the lack of the use of superoxide dismutase (SOD) enzyme cannot play an antioxidant protection role in the extraction of Chlamydomonas reinhardtii polysaccharide, resulting in low antioxidant activity of Chlamydomonas reinhardtii polysaccharide.

[0087] Comparative Example 2

[0088] The difference between this comparative example and Example 2 is that amylase is used to replace pectinase in this comparative example. The complex enzyme in this comparative example is composed of cellulase, amylase, and superoxide dismutase (SOD) in a mass ratio of 2:1:1. Other steps and parameters are basically the same as those in Example 2.

[0089] In this example, the yield of Chlamydomonas reinhardtii polysaccharide particles was 6.8%, and the purity of Chlamydomonas reinhardtii polysaccharide particles was 52.7%. Compared with Example 2, the use of amylase could not effectively hydrolyze, resulting in the yield and purity of Chlamydomonas reinhardtii polysaccharide particles being affected.

[0090] Comparative Example 3

[0091] The difference between this comparative example and Example 2 is that this comparative example does not use spray drying, but uses belt drying to prepare polysaccharide dry powder. Other steps and parameters are basically the same as those in Example 2.

[0092] In this example, the yield of Chlamydomonas reinhardtii polysaccharide particles was 10.2%, and the purity of Chlamydomonas reinhardtii polysaccharide particles was 50.3%. Compared with Example 2, belt drying would destroy the polysaccharide structure, resulting in the purity of Chlamydomonas reinhardtii polysaccharide particles being affected.

[0093] Tests and Results

[0094] The antioxidant activity (DPPH free radical scavenging ability) of the polysaccharides extracted from Examples 1-2 and Comparative Examples 1-3 was measured to evaluate the in vitro antioxidant activity of the prepared Chlamydomonas reinhardtii polysaccharides. The specific method is as follows:

[0095] Preparation of DPPH working solution: Accurately weigh 10 mg of DPPH powder, add anhydrous ethanol to dissolve it, and make up to 0.1 mg / mL DPPH working solution in a 100 mL volumetric flask.

[0096] Preparation of sample solution: Take the Chlamydomonas reinhardtii polysaccharide particles in each case, grind them into powder, take about 0.1g, weigh them accurately, put them in a conical flask, add 50mL of water, extract them by ultrasonic for 30 minutes, filter them at normal pressure, and then draw 0.5ml, 1mL, 2mL, 5mL, and 10mL from them to make up to the volume in a 10mL volumetric flask to prepare the sample solution for standby use.

[0097] Determination: Take 0.5mL sample solution and 1.5mL DPPH working solution and mix them in a test tube, keep it away from light for 30 minutes, and measure its absorbance at a wavelength of 517nm, which is recorded as A1. Replace the DPPH working solution with 1.5mL water and record the absorbance as A2; replace the sample solution in the blank control with water, which is recorded as A0.

[0098] The DPPH free radical scavenging activity (antioxidant rate) was calculated by the following formula: DPPH free radical scavenging rate = 1-(A1-A2) / A0. The test results are shown in Table 1 below.

[0099] Table 1 Antioxidant rate test results

[0100]

[0101] The test results in Table 1 show that the antioxidant rates of the corresponding samples in Example 1 and Example 2 are significantly better than those of the corresponding samples in Comparative Example 1 and Comparative Example 3. The yield and purity of the Chlamydomonas reinhardtii polysaccharide particles in the examples are significantly better than those in the comparative examples.

[0102] The present invention relates to a method for preparing Chlamydomonas reinhardtii polysaccharide particles, comprising the following steps: taking Chlamydomonas reinhardtii, adding purified water to form a mixed solution; adding a composite enzyme to the mixed solution for enzymolysis, heating and extracting, combining the extracts to concentrate them into a clear paste; adding anhydrous ethanol to the prepared clear paste, centrifugally collecting precipitation; dissolving the precipitate in purified water, adding cyclodextrin and mixing, and spray drying to form a dry powder; adding starch and magnesium stearate to the dry powder for mixed granulation, thereby preparing Chlamydomonas reinhardtii polysaccharide particles. The present invention adopts a composite enzyme for enzymolysis, which can destroy the cell wall structure of Chlamydomonas reinhardtii and promote the easy dissolution of polysaccharides. At the same time, the clear paste is precipitated with alcohol, redissolved, and spray-dried, thereby solving the problems of low extraction efficiency and low purity in the extraction process of Chlamydomonas reinhardtii polysaccharides, and also avoiding the loss of polysaccharides to the greatest extent. The obtained polysaccharide particles have high antioxidant activity and good water solubility. The invention has the following beneficial effects: 1. The invention adopts a composite enzyme composed of cellulase, pectinase and superoxide dismutase (SOD) to enzymolyze Chlamydomonas reinhardtii, so as to accelerate the decomposition, soften the cellulose, destroy the cell wall, increase the dissolution amount of polysaccharides in the plant cells, ensure that the system environment maintains a low viscosity during high-temperature extraction, promote the full dissolution of polysaccharides, improve the extraction rate of Chlamydomonas reinhardtii polysaccharides, and protect the antioxidant activity of polysaccharides; 2. The invention improves the purity and yield of Chlamydomonas reinhardtii polysaccharides by extracting, concentrating, purifying and spray-drying Chlamydomonas reinhardtii, and the process is simple, easy to operate and highly safe; 3. The invention adopts dry granulation, has a good forming rate, and the particles are uniform and stable, and have the advantages of being easy to carry, simple to take and having good water solubility.

[0103] The above embodiments prepared by the method of the present invention are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing Chlamydomonas reinhardtii polysaccharide particles, characterized in that: The steps include: After extracting Chlamydomonas reinhardtii polysaccharide, the polysaccharide dry powder is prepared, and the polysaccharide dry powder is mixed with starch and magnesium stearate to prepare Chlamydomonas reinhardtii polysaccharide particles; When extracting Chlamydomonas reinhardtii polysaccharide, a composite enzyme is used for extraction; the composite enzyme is composed of cellulase, pectinase and superoxide dismutase in a mass ratio of 1.8-2.2:1:0.8-1.

2.

2. The preparation method according to claim 1, characterized in that: The extraction of Chlamydomonas reinhardtii polysaccharide comprises the following steps: Take Chlamydomonas reinhardtii, add water and mix to obtain a mixed solution; Adding complex enzyme to the mixed solution, adding acetic acid to adjust the pH, keeping the mixture warm, and heating for extraction to obtain an extract; The extract is filtered and concentrated into a clear paste; Ethanol is added to the clear paste, and the mixture is centrifuged and the precipitate is collected. The precipitate is Chlamydomonas reinhardtii polysaccharide.

3. The preparation method according to claim 2, characterized in that: The mass volume ratio of Chlamydomonas reinhardtii to water in the mixed solution is 1:8 to 1:

12.

4. The preparation method according to claim 2, characterized in that: The mass of the complex enzyme is 0.2-0.5% of the mass of the mixed solution.

5. The preparation method according to claim 2, characterized in that: The complex enzyme consists of cellulase, pectinase and superoxide dismutase in a mass ratio of 2:1:

1.

6. The preparation method according to claim 2, characterized in that: When preparing the extract, acetic acid is added to adjust the pH value to 4.0-6.0, and the mixed solution is heated to react at a temperature of 40-55° C. for 8-24 hours; boiling and decocting, filtering to obtain a filtrate and a filter residue, and the filtrate is reserved; water is then added to the filter residue, boiling and decocting, filtering to obtain a filtrate; and the two filtrates are combined to obtain the extract.

7. The preparation method according to claim 2, characterized in that: The concentration is carried out by using a double-effect concentrator, the steam pressure of the concentrator is 0.09-0.1Mpa, the vacuum degree is -0.10--0.02Mpa, and the concentration temperature is 70-90°C.

8. The preparation method according to claim 2, characterized in that: The concentration of the clear paste is 1.05-1.15 g / mL; the volume ratio of the clear paste to ethanol is 1:4-6, and the centrifuge speed during centrifugal separation is 5500-6500 r / min.

9. The preparation method according to claim 1, characterized in that: The preparation of the polysaccharide dry powder comprises the following steps: adding water to the precipitate to dissolve it, adding cyclodextrin to mix it, and then drying it to form the polysaccharide dry powder.

10. The preparation method according to claim 9, characterized in that: When the precipitate is dissolved in water, the mass ratio of the precipitate to water is 1:1.8-2.5, and the added amount of cyclodextrin is 8-12% of the mass of the precipitate.

11. The preparation method according to claim 9, characterized in that: The drying is spray drying; the air inlet temperature of the spray drying is 180-200°C, and the air outlet temperature is 80-100°C.

12. The preparation method according to claim 1, characterized in that: The amount of starch used is 18-22% of the mass of the polysaccharide dry powder, and the amount of magnesium stearate used is 0.4-0.6% of the mass of the polysaccharide dry powder.

13. The preparation method according to claim 1, characterized in that: The granulation method for preparing polysaccharide granules is dry granulation, the feeding speed is 22-28 rpm, the pressing wheel spacing is 0.08-0.12 mm, and the pressing wheel speed is 0.8-1.2 rpm.

14. The preparation method according to claim 1, characterized in that: The steps include the step of measuring the antioxidant activity of Chlamydomonas reinhardtii polysaccharide particles: Prepare 0.1 mg / mL DPPH working solution; Prepare sample solution: Take Chlamydomonas reinhardtii polysaccharide particles, grind them into powder, take 0.1g, weigh them, put them in Erlenmeyer flasks, add 50mL water, filter after ultrasonication, and then take 0.5ml, 1mL, 2mL, 5mL, and 10mL of them to 10mL to prepare sample solution for standby use; Determination: Take 0.5mL sample solution and 1.5mL DPPH working solution and mix them evenly. Protect from light for 30 minutes and measure its absorbance at 517nm wavelength, record it as A1; replace DPPH working solution with 1.5mL water to measure the absorbance and record it as A2; replace the sample solution in the blank control with water, record it as A0; The DPPH free radical scavenging activity was calculated by the following formula: DPPH free radical scavenging rate = 1-(A1-A2) / A0.

15. The preparation method according to any one of claims 1 to 14, characterized in that: The superoxide dismutase is used in the process of extracting Chlamydomonas reinhardtii polysaccharide to play an antioxidant protection role on Chlamydomonas reinhardtii polysaccharide and enhance the ability of Chlamydomonas reinhardtii polysaccharide to remove superoxide anion free radicals.

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