A method for extracting spirulina polysaccharide

Abstract

The invention provides a method for extracting spirulina polysaccharide, belonging to the technical field of plant polysaccharide extraction; the extraction method comprises wall breaking treatment, enzymolysis, separation and purification; the enzymolysis step comprises adding a hydrochloric acid solution to a spirulina polysaccharide extract, adjusting the pH value, and then adding a functional enzyme for enzymolysis to obtain an enzymolysis solution; the preparation method of the functional enzyme comprises biochar pretreatment, cross-linking and loading; the cross-linking step comprises placing pretreated chitin and pretreated biochar in deionized water, stirring evenly, adding a glutaraldehyde solution, stirring at room temperature for 3.8-4.2 hours, and after the stirring is completed, standing and centrifuging and washing, and then freeze-drying to obtain a cross-linked carrier; by adopting the method of the invention, the yield of spirulina polysaccharide is high, the total sugar content is high, and the reusability of the functional enzyme is good.

Description

Technical Field

[0001] The invention belongs to the technical field of plant polysaccharide extraction, and particularly relates to a method for extracting spirulina polysaccharide. Background Art

[0002] Spirulina polysaccharide is a physiologically active polysaccharide extracted from Spirulina. It is a water-soluble polysaccharide and is recommended by the Food and Agriculture Organization of the United Nations as the "most ideal food in the 21st century". It is widely used in cosmetics, health care, medicine and other fields. Spirulina polysaccharide has anti-tumor, anti-radiation, DNA synthesis and immune enhancement effects. In recent years, Spirulina polysaccharide can also be used as an auxiliary treatment for hepatitis, diabetes, and gastric ulcers, which is beneficial to the body's anti-aging effect. It also has the effects of immune enhancement, improving hematopoietic function and promoting protein synthesis.

[0003] At present, the existing methods for extracting spirulina polysaccharides include alkaline leaching extraction, ultrasonic fragmentation extraction, microwave-assisted extraction, repeated freeze-thaw wall breaking, enzyme-assisted extraction or a combination of two of these methods; however, all of them have the disadvantage of low yield of spirulina polysaccharides;

[0004] In this regard, the prior art discloses the following:

[0005] CN108727510A discloses a spirulina polysaccharide extract, a preparation method thereof and an application thereof for enhancing immune function, specifically disclosing that spirulina dry powder is mixed with a sodium pyrophosphate solution for wall breaking, then protease is added for enzymolysis, then the polysaccharide is precipitated by an alcohol precipitation method, and finally washed and dried to obtain the spirulina polysaccharide;

[0006] The yield of Spirulina polysaccharide obtained by this method reached 10.64%, and the total content was 25.35%; however, the total sugar content of Spirulina polysaccharide extracted by this method was low. Summary of the invention

[0007] In order to solve the technical problems existing in the prior art, the present invention provides a method for extracting spirulina polysaccharide, which has a high yield and a high total sugar content.

[0008] In view of the above technical problems, the present invention adopts the following technical solutions:

[0009] A method for extracting spirulina polysaccharides, comprising wall breaking, enzymolysis, separation and purification steps, the specific operations are as follows:

[0010] 1. Wall breaking process

[0011] The spirulina powder is placed in deionized water, monoglycerol fatty acid ester is added, stirred for 0.8-1.2 hours, and then homogenized for 4-6 minutes, the homogenization pressure is 6.2-6.7 MPa, and the homogenization times are 3 times. After the homogenization, 10-14wt% sodium hydroxide solution is added, the pH value is controlled to be 9.8-10.2, and the mixture is water bathed at 78-82°C for 5.8-6.2 hours to obtain a spirulina polysaccharide extract;

[0012] The mass ratio of the spirulina powder, deionized water and monoglycerol fatty acid ester is 8-12:290-310:0.10-0.14;

[0013] 2. Enzymatic hydrolysis

[0014] Add 8-12wt% hydrochloric acid solution to the spirulina polysaccharide extract to adjust the pH to 6.1-6.4, then add functional enzyme for ultrasonic treatment, the ultrasonic time is 23-27min, the ultrasonic power is 83-88W, the ultrasonic frequency is 20-25kHz, after the ultrasonic treatment, heat to 50-54°C at a rate of 1.7-2.2°C / min for enzymolysis, the enzymolysis time is 9-12h, after the enzymolysis, obtain enzymolysis solution;

[0015] The mass ratio of the spirulina polysaccharide extract to the functional enzyme is 4.0-4.2 kg:3.5-3.7 g;

[0016] The method for preparing the functional enzyme comprises the following steps:

[0017] (1) Biochar pretreatment

[0018] The biochar is placed in a sealed container, sprayed with a pre-dispersed liquid, and the temperature is controlled to be 43-47°C, the stirring speed is 210-230rpm, and the spraying rate is controlled to be 3.7-4.2g / min. After the spraying is completed, it is dried at 78-82°C for 5.8-6.2h to obtain pre-dispersed biochar; the pre-dispersed biochar is placed in a pre-treatment liquid, stirred and reacted at room temperature, the reaction time is 9.8-10.3h, the stirring speed is 195-206rpm, and after the stirring is completed, it is filtered, washed, and dried to obtain pre-treated biochar;

[0019] The particle size of the biochar is 145-155 nm;

[0020] The mass ratio of the biochar to the pre-dispersed liquid is 10.3-10.5:82-86;

[0021] The pre-dispersion liquid is a mixture of 30-34wt% ethanol solution, rhamnolipid and sucrose fatty acid ester, and the mass ratio of the 30-34wt% ethanol solution, rhamnolipid and sucrose fatty acid ester is 78-83:1.1-1.5:1.5-1.8;

[0022] The mass volume ratio of the pre-dispersed biochar to the pre-treatment liquid is 9.6-10.3 g:490-508 mL;

[0023] The pretreatment liquid is prepared by uniformly mixing deionized water, tris(hydroxymethyl)aminomethane hydrochloride) and dopamine hydrochloride, wherein the mass ratio of the deionized water, tris(hydroxymethyl)aminomethane hydrochloride) and dopamine hydrochloride is 495-505:0.63-0.67:2.1-2.3;

[0024] (2) Cross-linking

[0025] The chitosan was placed in an acid solution, the temperature was raised to 50-54°C, and ultrasonic treatment was performed. The ultrasonic time was 18-22 minutes, the ultrasonic power was 226-235W, and the ultrasonic frequency was 15-20kHz. After the ultrasonic treatment, the temperature was raised to 80-84°C at a rate of 1.8-2.2°C / min, and ball milling was performed in a nitrogen atmosphere. The ball milling time was 1.4-1.6 hours, the ball milling speed was 116-124rpm, and the ball-to-material ratio was 3-5:1. After the ball milling was completed, the pretreated chitosan was filtered, washed, and dried to obtain pretreated chitosan. The pretreated chitosan and pretreated biochar were placed in deionized water, stirred evenly, and then glutaraldehyde solution was added. The mixture was stirred at room temperature for 3.8-4.2 hours. After the stirring was completed, the mixture was allowed to stand and centrifuged for washing, and then freeze-dried. The drying temperature was -34 to -30°C and the drying time was 10-13 hours. After the drying was completed, the mixture was naturally restored to room temperature to obtain a cross-linked carrier.

[0026] The chitin has a length of 190-210 nm and a width of 35-45 nm;

[0027] The mass ratio of chitosan to acid solution is 10.0-10.5:96-105;

[0028] The acid solution is a mixture of hydrochloric acid, salicylic acid and deionized water, and the mass ratio of the hydrochloric acid, salicylic acid and deionized water is 5.0-5.2:1.8-2.2:50-55;

[0029] The mass volume ratio of the pretreated chitin, pretreated biochar, deionized water and glutaraldehyde solution is 3.4-3.8 g: 2.7-3.3 g: 115-124 g: 58-63 mL;

[0030] The mass concentration of the glutaraldehyde solution is 3.4-3.7%;

[0031] (3) Load

[0032] The cross-linked carrier is placed in deionized water, the temperature is raised to 58-62°C, papain is added, and the mixture is stirred at a stirring speed of 163-175 rpm for 2.0-2.5 hours. After the stirring is completed, the mixture is centrifuged and dried at 58-63°C for 7.8-8.4 hours to obtain a functional enzyme.

[0033] The mass ratio of the cross-linked carrier, deionized water, and papain is 1.1-1.5:17.1-17.8:0.6-0.8;

[0034] 3. Separation

[0035] The enzymatic hydrolysate is centrifuged at a speed of 2680-2730 rpm for 12-17 min. After the centrifugation, the supernatant is concentrated to 1 / 4 of the original volume of the supernatant. The concentration temperature is 72-75°C and the vacuum degree is 0.080-0.084 MPa. After the concentration is completed, it is cooled to room temperature to obtain a concentrated solution.

[0036] 4. Purification

[0037] The concentrate was mixed with 6-8 times the volume of 93-97wt% ethanol solution, allowed to stand for 11-13 hours, and centrifuged to obtain a primary supernatant and a primary precipitate, which was washed with 2.5-3.5 times the mass of anhydrous ethanol and filtered to obtain a wet solid one; the primary supernatant was mixed with 4.8-5.2 times the volume of 93-97wt% ethanol solution, allowed to stand for 10-13 hours, and centrifuged to obtain a secondary supernatant and a secondary precipitate, which was washed with 2.5-3.5 times the mass of anhydrous ethanol and filtered to obtain a wet solid two; the wet solid one and the wet solid two were mixed and vacuum dried at a drying temperature of 58-62°C, a vacuum degree of 0.088-0.093MPa, and a drying time of 8.0-8.4h. After drying, Spirulina polysaccharide was obtained.

[0038] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0039] 1. The present invention adopts an enzymatic hydrolysis method to extract spirulina polysaccharide, and adopts a specific carrier to solidify the enzyme, wherein the carrier is biochar and chitin as the carrier, the biochar is firstly dispersed by a surfactant to avoid aggregation between particles, and then a pretreatment liquid is used to introduce more amino groups on its surface, and in the cross-linking step, the pretreated biochar and chitin are cross-linked under the action of glutaraldehyde, so that the pretreated biochar and chitin achieve a stable cross-linking structure, on the one hand, the adsorption performance of the cross-linking carrier is improved, more enzyme active centers are evenly distributed on the cross-linking carrier, the catalytic activity of the enzyme is fully exerted, and the immobilization performance of the enzyme is improved, so that the enzymatic hydrolysis step is more sufficient, and the yield and total sugar content of spirulina polysaccharide are increased; on the other hand, the stable structure of the cross-linking carrier enhances the stability of the functional enzyme, so that the functional enzyme can still ensure stable high activity and high catalytic performance after repeated use, and the reusability is good;

[0040] 2. The method for extracting spirulina polysaccharides according to the present invention has a yield of 9.28-9.33% and a total sugar content of 67.6-68.1%;

[0041] 3. The functional enzyme prepared by the present invention is recovered. Specifically, in the separation step, after the enzymatic hydrolyzate is centrifuged, the precipitate is washed with purified water, dried at a temperature of 58° C. and a vacuum degree of 0.08 MPa for 8.0 h, and then reused 20 times. After each use, the functional enzyme is recovered. The yield of Spirulina polysaccharide extracted for the 20th time is measured to be 9.10-9.24%, and the total sugar content is 66.0-67.2%. DETAILED DESCRIPTION

[0042] In order to more clearly understand the technical features, purposes and effects of the present invention, specific embodiments of the present invention are now described.

[0043] Example 1

[0044] 1. Wall breaking process

[0045] 10 g of spirulina powder was placed in 300 g of deionized water, 0.12 g of monoglycerol fatty acid ester was added, and the mixture was stirred for 1.0 h, and then homogenized for 5 min at a homogenization pressure of 6.5 MPa and 3 times. After the homogenization, 12 wt % sodium hydroxide solution was added, the pH value was controlled to be 10, and the mixture was in a water bath at 80° C. for 6.0 h to obtain a spirulina polysaccharide extract.

[0046] 2. Enzymatic hydrolysis

[0047] Add 10 wt % hydrochloric acid solution to 4.1 kg of spirulina polysaccharide extract to adjust the pH to 6.3, then add 3.6 g of functional enzyme for ultrasonic treatment, the ultrasonic time is 25 min, the ultrasonic power is 85 W, the ultrasonic frequency is 23 kHz, after the ultrasonic treatment, heat to 52 ° C at a rate of 2.0 ° C / min for enzymolysis, the enzymolysis time is 10 h, after the enzymolysis, obtain an enzymolysis solution;

[0048] The method for preparing the functional enzyme comprises the following steps:

[0049] (1) Biochar pretreatment

[0050] 10.5 g of biochar was placed in a sealed container, and 84 g of pre-dispersed liquid was sprayed. The temperature was controlled to be 45 ° C, the stirring speed was 220 rpm, and the spraying rate was controlled to be 4.0 g / min. After the spraying, it was dried at 80 ° C for 6.0 h to obtain pre-dispersed biochar; 10.0 g of pre-dispersed biochar was placed in 500 mL of pre-treatment liquid, and stirred at room temperature for reaction. The reaction time was 10 h and the stirring speed was 200 rpm. After the stirring was completed, it was filtered, washed and dried to obtain pre-treated biochar;

[0051] The particle size of the biochar is 150 nm;

[0052] The pre-dispersion liquid is a mixture of 32wt% ethanol solution, rhamnolipid and sucrose fatty acid ester, and the mass ratio of the 32wt% ethanol solution, rhamnolipid and sucrose fatty acid ester is 80:1.3:1.7;

[0053] The pretreatment solution is prepared by uniformly mixing deionized water, tris(hydroxymethyl)aminomethane hydrochloride) and dopamine hydrochloride, wherein the mass ratio of the deionized water, tris(hydroxymethyl)aminomethane hydrochloride) and dopamine hydrochloride is 500:0.65:2.2;

[0054] (2) Cross-linking

[0055] 10.2g chitosan was placed in 100g acid solution, the temperature was raised to 52°C, and ultrasonic treatment was performed. The ultrasonic time was 20min, the ultrasonic power was 230W, and the ultrasonic frequency was 17kHz. After the ultrasonic treatment, the temperature was raised to 82°C at a rate of 2.0°C / min, and ball milling was performed in a nitrogen atmosphere. The ball milling time was 1.5h, the ball milling speed was 120rpm, and the ball-to-material ratio was 4:1. After the ball milling was completed, it was filtered, washed, and dried to obtain pretreated chitosan; 3.6g pretreated chitosan and 3.0g pretreated biochar were placed in 120g deionized water, stirred evenly, and then 60mL glutaraldehyde solution was added. The mixture was stirred at room temperature for 4.0h. After the stirring was completed, it was centrifuged and washed, and then freeze-dried. The drying temperature was -32°C and the drying time was 12h. After the drying was completed, it was naturally restored to room temperature to obtain a cross-linked carrier;

[0056] The chitosan has a length of 200 nm and a width of 40 nm;

[0057] The acid solution is a mixture of hydrochloric acid, salicylic acid and deionized water, and the mass ratio of the hydrochloric acid, salicylic acid and deionized water is 5.1:2.0:52;

[0058] The mass concentration of the glutaraldehyde solution is 3.5%;

[0059] (3) Load

[0060] 1.3 g of the cross-linked carrier was placed in 17.5 g of deionized water, the temperature was raised to 60°C, 0.7 g of papain was added, and the mixture was stirred at a speed of 170 rpm for 2.3 h. After the stirring was completed, the mixture was centrifuged and dried at 60°C for 8.0 h to obtain a functional enzyme.

[0061] 3. Separation

[0062] The enzymatic hydrolysate was centrifuged at a speed of 2700 rpm for 14 min. After the centrifugation, the supernatant was concentrated to 1 / 4 of the original volume of the supernatant. The concentration temperature was 73° C. and the vacuum degree was 0.082 MPa. After the concentration was completed, it was cooled to room temperature to obtain a concentrated solution.

[0063] 4. Purification

[0064] The concentrate was mixed with 7 times the volume of 95wt% ethanol solution, allowed to stand for 12 hours, and centrifuged to obtain a primary supernatant and a primary precipitate. The primary precipitate was washed with 3 times the mass of anhydrous ethanol and filtered to obtain wet solid one; the primary supernatant was mixed with 5 times the volume of 95wt% ethanol solution, allowed to stand for 12 hours, and centrifuged to obtain a secondary supernatant and a secondary precipitate. The secondary precipitate was washed with 3 times the mass of anhydrous ethanol and filtered to obtain wet solid two; wet solid one and wet solid two were mixed and vacuum dried at a drying temperature of 60°C, a vacuum degree of 0.090MPa, and a drying time of 8.2 hours. After drying, Spirulina polysaccharide was obtained.

[0065] The method for extracting Spirulina polysaccharide in Example 1 was used, and the yield was 9.33%, and the total sugar content was 68.1%, wherein the yield = (Spirulina polysaccharide weight g / Spirulina dry powder weight g) × 100%;

[0066] The functional enzyme of Example 1 was recovered. Specifically, in the separation step, after the enzymatic hydrolysate was centrifuged, the precipitate was washed with purified water, dried at a temperature of 58° C. and a vacuum degree of 0.08 MPa for 8.0 h, and then reused 20 times using the method of Example 1. After each use, the functional enzyme was recovered. The yield of Spirulina polysaccharides extracted for the 20th time was measured to be 9.24%, and the total sugar content was 67.2%.

[0067] Example 2

[0068] 1. Wall breaking process

[0069] 8 g of spirulina powder was placed in 290 g of deionized water, 0.10 g of monoglycerol fatty acid ester was added, and the mixture was stirred for 0.8 h, and then homogenized for 4 min at a homogenization pressure of 6.2 MPa for 3 times. After the homogenization, 10 wt % sodium hydroxide solution was added, the pH value was controlled to be 9.8, and the mixture was in a water bath at 78 ° C for 5.8 h to obtain a spirulina polysaccharide extract.

[0070] 2. Enzymatic hydrolysis

[0071] 8 wt % hydrochloric acid solution was added to 4.0 kg of spirulina polysaccharide extract to adjust the pH to 6.1, and then 3.5 g of functional enzyme was added for ultrasonic treatment. The ultrasonic time was 23 min, the ultrasonic power was 83 W, and the ultrasonic frequency was 20 kHz. After the ultrasonic treatment, the temperature was raised to 50 ° C at a rate of 1.7 ° C / min for enzymolysis. The enzymolysis time was 9 h. After the enzymolysis was completed, an enzymolysis solution was obtained;

[0072] The method for preparing the functional enzyme comprises the following steps:

[0073] (1) Biochar pretreatment

[0074] 10.3 g of biochar was placed in a sealed container, and 82 g of pre-dispersed liquid was sprayed. The temperature was controlled at 43 ° C, the stirring speed was 210 rpm, and the spraying rate was controlled at 3.7 g / min. After spraying, it was dried at 78 ° C for 5.8 h to obtain pre-dispersed biochar; 9.6 g of pre-dispersed biochar was placed in 490 mL of pre-treatment liquid, and stirred at room temperature for reaction. The reaction time was 9.8 h and the stirring speed was 195 rpm. After stirring, it was filtered, washed, and dried to obtain pre-treated biochar;

[0075] The particle size of the biochar is 145 nm;

[0076] The pre-dispersion liquid is a mixture of 30wt% ethanol solution, rhamnolipid and sucrose fatty acid ester, and the mass ratio of the 30wt% ethanol solution, rhamnolipid and sucrose fatty acid ester is 78:1.1:1.5;

[0077] The pretreatment solution is prepared by uniformly mixing deionized water, tris(hydroxymethyl)aminomethane hydrochloride) and dopamine hydrochloride, wherein the mass ratio of the deionized water, tris(hydroxymethyl)aminomethane hydrochloride) and dopamine hydrochloride is 495:0.63:2.1;

[0078] (2) Cross-linking

[0079] 10.0g chitosan was placed in 96g acid solution, the temperature was raised to 50°C, and ultrasonic treatment was performed. The ultrasonic time was 18min, the ultrasonic power was 226W, and the ultrasonic frequency was 15kHz. After the ultrasonic treatment, the temperature was raised to 80°C at a rate of 1.8°C / min, and ball milling was performed in a nitrogen atmosphere. The ball milling time was 1.4h, the ball milling speed was 116rpm, and the ball-to-material ratio was 3:1. After the ball milling was completed, it was filtered, washed, and dried to obtain pretreated chitosan; 3.4g pretreated chitosan and 2.7g pretreated biochar were placed in 115g deionized water, stirred evenly, and then 58mL glutaraldehyde solution was added. The mixture was stirred at room temperature for 3.8h. After the stirring was completed, it was centrifuged and washed, and then freeze-dried. The drying temperature was -34°C and the drying time was 10h. After the drying was completed, it was naturally restored to room temperature to obtain a cross-linked carrier;

[0080] The chitin has a length of 190 nm and a width of 35 nm;

[0081] The acid solution is a mixture of hydrochloric acid, salicylic acid and deionized water, and the mass ratio of the hydrochloric acid, salicylic acid and deionized water is 5.0:1.8:50;

[0082] The mass concentration of the glutaraldehyde solution is 3.4%;

[0083] (3) Load

[0084] 1.1 g of the cross-linked carrier was placed in 17.1 g of deionized water, the temperature was raised to 58°C, 0.6 g of papain was added, and the mixture was stirred at a speed of 163 rpm for 2.0 h. After the stirring, the mixture was centrifuged and dried at 58°C for 8.4 h to obtain a functional enzyme.

[0085] 3. Separation

[0086] The enzymatic hydrolysate was centrifuged at a speed of 2680 rpm for 17 min. After the centrifugation, the supernatant was concentrated to 1 / 4 of the original volume of the supernatant. The concentration temperature was 72° C. and the vacuum degree was 0.080 MPa. After the concentration was completed, it was cooled to room temperature to obtain a concentrated solution.

[0087] 4. Purification

[0088] The concentrate was mixed with 6 times the volume of 93wt% ethanol solution, allowed to stand for 11 hours, and centrifuged to obtain a primary supernatant and a primary precipitate. The primary precipitate was washed with 2.5 times the mass of anhydrous ethanol and filtered to obtain wet solid one; the primary supernatant was mixed with 4.8 times the volume of 93wt% ethanol solution, allowed to stand for 10 hours, and centrifuged to obtain a secondary supernatant and a secondary precipitate. The secondary precipitate was washed with 2.5 times the mass of anhydrous ethanol and filtered to obtain wet solid two; wet solid one and wet solid two were mixed and vacuum dried at a drying temperature of 58°C, a vacuum degree of 0.088MPa, and a drying time of 8.4h. After drying, Spirulina polysaccharide was obtained.

[0089] The method for extracting Spirulina polysaccharide in Example 2 was used, and the yield was 9.28%, and the total sugar content was 67.6%, wherein the yield = (Spirulina polysaccharide weight g / Spirulina dry powder weight g) × 100%;

[0090] The functional enzyme of Example 2 was recovered. Specifically, in the separation step, after the enzymatic hydrolyzate was centrifuged, the precipitate was washed with purified water, dried at a temperature of 58° C. and a vacuum degree of 0.08 MPa for 8.0 h, and then reused 20 times using the method of Example 2. After each use, the functional enzyme was recovered. The yield of Spirulina polysaccharides extracted for the 20th time was measured to be 9.10%, and the total sugar content was 66.0%.

[0091] Example 3

[0092] 1. Wall breaking process

[0093] 12 g of spirulina powder was placed in 310 g of deionized water, 0.14 g of monoglycerol fatty acid ester was added, and the mixture was stirred for 1.2 h, and then homogenized for 6 min, at a homogenization pressure of 6.7 MPa, and for 3 times. After the homogenization, 14 wt % sodium hydroxide solution was added, the pH value was controlled to be 10.2, and the mixture was in a water bath at 82° C. for 6.2 h to obtain a spirulina polysaccharide extract.

[0094] 2. Enzymatic hydrolysis

[0095] Add 12 wt % hydrochloric acid solution to 4.2 kg of spirulina polysaccharide extract to adjust the pH to 6.4, then add 3.7 g of functional enzyme for ultrasonic treatment, the ultrasonic time is 27 min, the ultrasonic power is 88 W, the ultrasonic frequency is 25 kHz, after the ultrasonic treatment, heat to 54 ° C at a rate of 2.2 ° C / min for enzymolysis, the enzymolysis time is 12 h, after the enzymolysis, obtain an enzymolysis solution;

[0096] The method for preparing the functional enzyme comprises the following steps:

[0097] (1) Biochar pretreatment

[0098] 10.7 g of biochar was placed in a sealed container, and 86 g of pre-dispersed liquid was sprayed. The temperature was controlled to be 47 ° C, the stirring speed was 230 rpm, and the spraying rate was controlled to be 4.2 g / min. After the spraying, it was dried at 82 ° C for 6.2 h to obtain pre-dispersed biochar; 10.3 g of pre-dispersed biochar was placed in 508 mL of pre-treatment liquid, and stirred at room temperature for reaction. The reaction time was 10.3 h and the stirring speed was 206 rpm. After the stirring was completed, it was filtered, washed, and dried to obtain pre-treated biochar;

[0099] The particle size of the biochar is 155 nm;

[0100] The pre-dispersion liquid is a mixture of 34wt% ethanol solution, rhamnolipid and sucrose fatty acid ester, and the mass ratio of the 34wt% ethanol solution, rhamnolipid and sucrose fatty acid ester is 83:1.5:1.8;

[0101] The pretreatment solution is prepared by uniformly mixing deionized water, tris(hydroxymethyl)aminomethane hydrochloride) and dopamine hydrochloride, wherein the mass ratio of the deionized water, tris(hydroxymethyl)aminomethane hydrochloride) and dopamine hydrochloride is 505:0.67:2.3;

[0102] (2) Cross-linking

[0103] 10.5g chitosan was placed in 105g acid solution, the temperature was raised to 54°C, and ultrasonic treatment was performed. The ultrasonic time was 22min, the ultrasonic power was 235W, and the ultrasonic frequency was 20kHz. After the ultrasonic treatment, the temperature was raised to 84°C at a rate of 2.2°C / min, and ball milling was performed in a nitrogen atmosphere. The ball milling time was 1.6h, the ball milling speed was 124rpm, and the ball-to-material ratio was 5:1. After the ball milling was completed, it was filtered, washed, and dried to obtain pretreated chitosan; 3.8g pretreated chitosan and 3.3g pretreated biochar were placed in 124g deionized water, stirred evenly, and then 63mL glutaraldehyde solution was added. The mixture was stirred at room temperature for 4.2h. After the stirring was completed, it was centrifuged and washed, and then freeze-dried. The drying temperature was -30°C and the drying time was 13h. After the drying was completed, it was naturally restored to room temperature to obtain a cross-linked carrier;

[0104] The chitin has a length of 210 nm and a width of 45 nm;

[0105] The acid solution is a mixture of hydrochloric acid, salicylic acid and deionized water, and the mass ratio of the hydrochloric acid, salicylic acid and deionized water is 5.2:2.2:55;

[0106] The mass concentration of the glutaraldehyde solution is 3.7%;

[0107] (3) Load

[0108] 1.5 g of the cross-linked carrier was placed in 17.8 g of deionized water, the temperature was raised to 62°C, 0.8 g of papain was added, and the mixture was stirred at a speed of 175 rpm for 2.5 h. After the stirring was completed, the mixture was centrifuged and dried at 63°C for 7.8 h to obtain a functional enzyme.

[0109] 3. Separation

[0110] The enzymatic hydrolysate was centrifuged at a speed of 2730 rpm for 12 min. After the centrifugation, the supernatant was concentrated to 1 / 4 of the original volume of the supernatant. The concentration temperature was 75°C and the vacuum degree was 0.084 MPa. After the concentration was completed, it was cooled to room temperature to obtain a concentrated solution.

[0111] 4. Purification

[0112] The concentrate was mixed with 8 times the volume of 97wt% ethanol solution, allowed to stand for 13 hours, and centrifuged to obtain a primary supernatant and a primary precipitate. The primary precipitate was washed with 3.5 times the mass of anhydrous ethanol and filtered to obtain wet solid one; the primary supernatant was mixed with 5.2 times the volume of 97wt% ethanol solution, allowed to stand for 13 hours, and centrifuged to obtain a secondary supernatant and a secondary precipitate. The secondary precipitate was washed with 3.5 times the mass of anhydrous ethanol and filtered to obtain wet solid two; wet solid one and wet solid two were mixed and vacuum dried at a drying temperature of 62°C, a vacuum degree of 0.093MPa, and a drying time of 8.0 hours. After drying, Spirulina polysaccharide was obtained.

[0113] The method of extracting Spirulina polysaccharide in Example 3 was used, and the yield was 9.30%, and the total sugar content was 67.9%, wherein the yield = (Spirulina polysaccharide weight g / Spirulina dry powder weight g) × 100%;

[0114] The functional enzyme of Example 3 was recovered. Specifically, in the separation step, after the enzymatic hydrolysate was centrifuged, the precipitate was washed with purified water, dried at a temperature of 58° C. and a vacuum degree of 0.08 MPa for 8.0 h, and then reused 20 times using the method of Example 3. After each use, the functional enzyme was recovered. The yield of Spirulina polysaccharides extracted for the 20th time was measured to be 9.16%, and the total sugar content was 66.5%.

[0115] Comparative Example 1

[0116] Based on Example 1, the changes are as follows:

[0117] In the method for preparing functional enzymes in the enzymatic hydrolysis process, the biochar pretreatment step is omitted;

[0118] In the cross-linking step, the pre-treated biochar is replaced by an equal amount of biochar without any treatment, and the particle size of the biochar is 150 nm;

[0119] The rest of the operations are the same.

[0120] The method of extracting Spirulina polysaccharide in Comparative Example 1 was used, and the yield was 8.11%, and the total sugar content was 56.6%, wherein the yield = (Spirulina polysaccharide weight g / Spirulina dry powder weight g) × 100%;

[0121] The functional enzyme of Comparative Example 1 was recovered. Specifically, in the separation step, the enzymatic hydrolyzate was centrifuged, the precipitate was washed with purified water, and dried at a temperature of 58° C. and a vacuum degree of 0.08 MPa for 8.0 h. Then, the method of Comparative Example 1 was used repeatedly for 20 times, and the enzyme was recovered after each use. The yield of Spirulina polysaccharides extracted for the 20th time was 7.04%, and the total sugar content was 47.7%.

[0122] Comparative Example 2

[0123] Based on Example 1, the changes are as follows: in the method for preparing the functional enzyme in the enzymatic hydrolysis process,

[0124] In the cross-linking step, the pretreated chitosan component was omitted and an equal amount of the pretreated chitosan was replaced with the pretreated biochar component;

[0125] The rest of the operations are the same.

[0126] The method of extracting Spirulina polysaccharide in Comparative Example 2 was used, and the yield was 8.54%, and the total sugar content was 60.3%, wherein the yield = (Spirulina polysaccharide weight g / Spirulina dry powder weight g) × 100%;

[0127] The functional enzyme of Comparative Example 2 was recovered. Specifically, in the separation step, after the enzymatic hydrolysate was centrifuged, the precipitate was washed with purified water, dried at a temperature of 58°C and a vacuum degree of 0.08 MPa for 8.0 h, and then reused 20 times using the method of Comparative Example 2, and recovered after each use. The yield of Spirulina polysaccharides extracted for the 20th time was measured to be 7.55%, and the total sugar content was 52.8%.

[0128] The invention adopts an enzymatic hydrolysis method to extract spirulina polysaccharide, adopts a specific carrier to solidify the enzyme, wherein the carrier is biochar and chitin as the carrier, the biochar is firstly dispersed by adopting a surfactant to avoid aggregation between particles, and then a pretreatment liquid is adopted to introduce more amino groups on its surface, and in the cross-linking step, the pretreated biochar and chitin are cross-linked under the action of glutaraldehyde, so that the pretreated biochar and chitin achieve a stable cross-linking structure, on the one hand, the adsorption performance of the cross-linking carrier is improved, more enzyme active centers are evenly distributed on the cross-linking carrier, the catalytic activity of the enzyme is fully exerted, and the immobilization performance of the enzyme is improved, so that the enzymatic hydrolysis step is more sufficient, and the yield and total sugar content of the spirulina polysaccharide are improved; on the other hand, the stable structure of the cross-linking carrier enhances the stability of the functional enzyme, so that the functional enzyme can still ensure stable high activity and high catalytic performance after repeated use, and the repeated use performance is good.

[0129] Comparative Example 1 omitted the pretreatment step of biochar, and directly used untreated biochar to crosslink with chitin in the crosslinking step. The biochar had poor dispersion performance and serious aggregation, and no amino groups were introduced on its surface, so it could not be effectively crosslinked with chitin, which in turn affected the stability and dispersion performance of the crosslinked carrier, reduced the loading performance for the enzyme active center, and the enzyme was unevenly distributed on the crosslinked carrier, which affected the catalytic activity of the enzyme, thereby affecting the enzymatic hydrolysis step, and ultimately reducing the product yield and total sugar content, and the reusability was poor;

[0130] In Comparative Example 2, chitin was omitted in the cross-linking step, and only biochar and glutaraldehyde were used for cross-linking, which reduced the loading efficiency and weakened the loading performance for the enzyme. The resulting carrier structure was not stable, resulting in poor recycling performance of the functional enzyme, which ultimately affected the product yield.

[0131] Unless otherwise specified, all percentages used in the present invention are by mass.

[0132] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for extracting spirulina polysaccharides, characterized in that it comprises the steps of cell wall breaking, enzymolysis, separation and purification; The cell wall breaking step comprises placing spirulina dry powder in deionized water, adding monoglycerol fatty acid ester, stirring for 0.8-1.2 hours, and then homogenizing for 4-6 minutes, at a homogenizing pressure of 6.2-6.7 MPa, and for 3 times. After the homogenization, adding 10-14wt% sodium hydroxide solution, controlling the pH value to 9.8-10.2, and water bathing at 78-82°C for 5.8-6.2 hours to obtain a spirulina polysaccharide extract; The mass ratio of the spirulina powder, deionized water and monoglycerol fatty acid ester is 8-12:290-310:0.10-0.14; The enzymatic hydrolysis step comprises adding 8-12 wt % hydrochloric acid solution to the spirulina polysaccharide extract, adjusting the pH to 6.1-6.4, then adding a functional enzyme for ultrasonic treatment, wherein the ultrasonic time is 23-27 min, the ultrasonic power is 83-88 W, and the ultrasonic frequency is 20-25 kHz. After the ultrasonic treatment, the temperature is raised to 50-54° C. at a rate of 1.7-2.2° C. / min for enzymatic hydrolysis, and the enzymatic hydrolysis time is 9-12 h. After the enzymatic hydrolysis is completed, the water bath is treated at 72-76° C. for 3.1-3.5 h to obtain an enzymatic hydrolyzate. The mass ratio of the spirulina polysaccharide extract to the functional enzyme is 4.0-4.2 kg:3.5-3.7 g; The method for preparing the functional enzyme includes biochar pretreatment, cross-linking, and loading steps; The biochar pretreatment step comprises placing the biochar in a sealed container, spraying the pre-dispersed liquid, controlling the temperature to 43-47° C., stirring the speed to 210-230 rpm, and controlling the spraying rate to 3.7-4.2 g / min while spraying, and drying at 78-82° C. for 5.8-6.2 h after spraying to obtain pre-dispersed biochar; placing the pre-dispersed biochar in the pretreatment liquid, stirring and reacting at room temperature, the reaction time is 9.8-10.3 h, the stirring speed is 195-206 rpm, and after stirring, filtering, washing, and drying to obtain pre-treated biochar; The pre-dispersion liquid is a mixture of 30-34wt% ethanol solution, rhamnolipid and sucrose fatty acid ester, wherein the mass ratio of the 30-34wt% ethanol solution, rhamnolipid and sucrose fatty acid ester is 78-83: 1.1-1.5:1.5-1.8； The pretreatment liquid is prepared by uniformly mixing deionized water, tris(hydroxymethyl)aminomethane hydrochloride) and dopamine hydrochloride, wherein the mass ratio of the deionized water, tris(hydroxymethyl)aminomethane hydrochloride) and dopamine hydrochloride is 495-505:0.63-0.67:2.1-2.3; The cross-linking step comprises placing the pretreated chitin and the pretreated biochar in deionized water, stirring them evenly, adding glutaraldehyde solution, stirring them at room temperature for 3.8-4.2 hours, standing and centrifuging them for washing, and then freeze-drying them at a drying temperature of -34 to -30°C for 10-13 hours. After drying, the temperature is naturally restored to room temperature to obtain a cross-linked carrier. The mass volume ratio of the pretreated chitin, pretreated biochar, deionized water and glutaraldehyde solution is 3.4-3.8 g: 2.7-3.3 g: 115-124 g: 58-63 mL; The mass concentration of the glutaraldehyde solution is 3.4-3.7%; The preparation method of the pretreated chitosan comprises placing the chitosan in an acid solution, raising the temperature to 50-54° C., performing ultrasonic treatment, wherein the ultrasonic time is 18-22 min, the ultrasonic power is 226-235 W, and the ultrasonic frequency is 15-20 kHz. After the ultrasonic treatment, the temperature is raised to 80-84° C. at a rate of 1.8-2.2° C. / min, and ball milling is performed in a nitrogen atmosphere, wherein the ball milling time is 1.4-1.6 h, the ball milling speed is 116-124 rpm, and the ball-to-material ratio is 3-5:

1. After the ball milling is completed, the pretreated chitosan is filtered, washed, and dried to obtain the pretreated chitosan. The chitin has a length of 190-210 nm and a width of 35-45 nm; The mass ratio of chitosan to acid solution is 10.0-10.5:96-105; The acid solution is a mixture of hydrochloric acid, salicylic acid and deionized water, and the mass ratio of the hydrochloric acid, salicylic acid and deionized water is 5.0-5.2:1.8-2.2:50-55; The loading step is to place the cross-linked carrier in deionized water, raise the temperature to 58-62° C., add papain, and stir at a stirring speed of 163-175 rpm for 2.0-2.5 hours. After the stirring is completed, centrifuge and dry at 58-63° C. for 7.8-8.4 hours to obtain a functional enzyme; The mass ratio of the cross-linked carrier, deionized water and papain is 1.1-1.5:17.1-17.8:0.6-0.

8.

2. The method for extracting Spirulina polysaccharides according to claim 1, characterized in that: In the biochar pretreatment step, the particle size of the biochar is 145-155 nm; The mass ratio of the biochar to the pre-dispersed liquid is 10.3-10.5:82-86.

3. The method for extracting Spirulina polysaccharides according to claim 1, characterized in that: In the biochar pretreatment step, the mass volume ratio of the pre-dispersed biochar to the pretreatment liquid is 9.6-10.3 g:490-508 mL.

4. The method for extracting Spirulina polysaccharide according to claim 1, characterized in that: The separation step comprises the following steps: centrifuging the enzymatic hydrolysate at a centrifugal speed of 2680-2730 rpm and a centrifugal time of 12-17 min; concentrating the supernatant after the centrifugation to 1 / 4 of the original volume of the supernatant; the concentration temperature is 72-75° C. and the vacuum degree is 0.080-0.084 MPa; after the concentration is completed, cooling to room temperature to obtain a concentrated solution.

5. The method for extracting Spirulina polysaccharide according to claim 1, characterized in that: The purification step comprises: mixing the concentrated solution with 6-8 times the volume of 93-97wt% ethanol solution, standing for 11-13h, and centrifuging to obtain a primary supernatant and a primary precipitate, washing the primary precipitate with 2.5-3.5 times the mass of anhydrous ethanol, filtering and drying to obtain a wet solid one; mixing the primary supernatant with 4.8-5.2 times the volume of 93-97wt% ethanol solution, standing for 10-13h, and centrifuging to obtain a secondary supernatant and a secondary precipitate, washing the secondary precipitate with 2.5-3.5 times the mass of anhydrous ethanol, and filtering and drying to obtain a wet solid two; mixing the wet solid one and the wet solid two, and then vacuum drying, the drying temperature is 58-62°C, the vacuum degree is 0.088-0.093MPa, and the drying time is 8.0-8.4h. After drying, Spirulina polysaccharide is obtained.