A method for extracting and refining DHA from marine microalgae fermentation liquid

Abstract

The invention discloses a method for extracting and refining DHA from marine microalgae fermentation liquid, belonging to the technical field of DHA algae oil refining. The method comprises the following steps: enzymolysis, extraction, degumming, deacidification, decolorization and deodorization; in the enzymolysis, after mixing schizochytrium fermentation liquid, alkaline protease and enzymolysis auxiliary agent, the pH value is adjusted to 8-9, and the mixture is stirred at 50-60 DEG C for 2.5-3h to obtain enzymolysis wall-breaking liquid; in the decolorization, the deacidified DHA crude oil and the decolorizer are mixed, stirred at room temperature for 30-40min, centrifuged, and the supernatant is taken to obtain the decolorized DHA crude oil; the preparation method of the decolorizer comprises the following steps: primary coating and secondary coating; the invention can improve the wall-breaking rate and the extraction rate, shorten the enzymolysis time, and fully remove the pigment in DHA.

Description

Technical Field

[0001] The invention relates to the technical field of DHA algae oil refining, and in particular to a method for extracting and refining DHA from marine microalgae fermentation liquid. Background Art

[0002] DHA (docosahexaenoic acid), commonly known as brain gold, is a polyunsaturated fatty acid that is very important to the human body and an important nutrient for the growth and maintenance of nervous system cells.

[0003] The production sources of DHA mainly include marine fish oil and marine microalgae. Among them, marine fish oil as a production source has the disadvantages of unstable yield, low extraction rate, and strong fishy smell. Marine fish oil contains cholesterol, fat-soluble vitamins, and ω-6 fatty acids that are difficult to remove. Excessive intake has great side effects on the human body. In addition, marine fish oil also contains EPA (eicosapentaenoic acid), so the refining process is complicated and the cost is high. Marine microalgae as a production source has the advantages of stable yield, high extraction rate, and no strong fishy smell. The fatty acid composition of marine microalgae is simple, with DHA accounting for 30-50% of the total fatty acids, and the content of other long-chain polyunsaturated fatty acids does not exceed 1%. It does not contain EPA and is easy to refine DHA, making it the most ideal source of DHA.

[0004] When marine microalgae is used as the production source, the main production method is to extract the marine microalgae fermentation liquid to obtain a crude oil containing a large amount of impurities such as phospholipids, proteins, colloid substances, pigments, free fatty acids, etc., and then refine it, specifically degumming, deacidification, decolorization, and deodorization to obtain DHA. At present, the commonly used extraction method is to use alcohol to extract after enzymatic wall breaking of the marine microalgae fermentation liquid, and remove the residue by centrifugation; the commonly used degumming method is acid degumming, specifically by adding acidic substances such as phosphoric acid and citric acid to remove the colloid substances in the crude oil; the commonly used deacidification method is to add alkali and free fatty acids to the degummed crude oil for saponification, and then remove the saponified products, thereby reducing the acid value of the degummed crude oil; the commonly used decolorization method is to add activated carbon to the deacidified crude oil for physical adsorption, after the pigment is adsorbed, the physical adsorbent after adsorption is removed by centrifugation or filtration; the commonly used deodorization method is to pass steam into the deacidified crude oil, and remove the odor by using the volatility difference between DHA and odorous substances.

[0005] However, the above method has the following problems: First, the enzymatic cell wall breaking has the problem of product inhibition of enzyme activity, resulting in low cell wall breaking rate and extraction rate, and long enzymatic hydrolysis time; second, the decolorization ability of activated carbon used in decolorization is limited, resulting in a dark color of the obtained DHA. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for extracting and refining DHA from marine microalgae fermentation broth, which can improve the wall breaking rate and extraction rate, shorten the enzymatic hydrolysis time, and fully remove the pigment in DHA.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for extracting and refining DHA from marine microalgae fermentation liquid, comprising the following steps: enzymatic hydrolysis, extraction, degumming, deacidification, decolorization, and deodorization;

[0009] The enzymatic hydrolysis comprises mixing the Schizochytrium fermentation broth, alkaline protease and an enzymatic hydrolysis aid, adjusting the pH to 8-9, stirring at 50-60° C. and at a stirring speed of 150-200 rpm for 2.5-3 hours to obtain an enzymatic hydrolysis cell wall-breaking liquid;

[0010] In the enzymolysis, the mass ratio of Schizochytrium fermentation broth to the enzymolysis aid is 1000:14-15;

[0011] The mass ratio of alkaline protease to dry matter of Schizochytrium fermentation broth is 0.9-1:100;

[0012] The enzymatic activity of the alkaline protease is 500,000 U / g;

[0013] The preparation method of the enzymolysis aid comprises the following steps: preparing carboxylated activated carbon, grafting, and cross-linking;

[0014] The preparation of carboxylated activated carbon comprises mixing activated carbon and nitric acid, stirring at room temperature at a stirring speed of 300-400 rpm for 20-30 minutes, stirring and refluxing at a stirring speed of 300-400 rpm for 4-5 hours at 85-95° C., centrifuging at a centrifugal speed of 4000-5000 rpm for 8-10 minutes, washing the precipitate with distilled water for 3-4 times, and drying at 120-130° C. for 9-10 hours to obtain carboxylated activated carbon;

[0015] In the preparation of carboxylated activated carbon, the mass ratio of activated carbon to nitric acid is 150-160:2800-3000;

[0016] The molar concentration of the nitric acid is 7 mol / L;

[0017] The average particle size of the activated carbon is 200 μm and the average pore size is 2 nm;

[0018] The grafting comprises mixing β-cyclodextrin, carboxylated activated carbon and N,N-dimethylformamide, stirring and refluxing at 80-85°C at a stirring speed of 400-500 rpm for 17-19 hours, standing at room temperature for 10-12 hours, centrifuging at a centrifugal speed of 4000-5000 rpm for 8-10 minutes, washing the precipitate with acetone for 3-4 times, and drying at 80-90°C for 18-20 hours to obtain grafted activated carbon;

[0019] In the grafting, the mass ratio of β-cyclodextrin, carboxylated activated carbon, and N,N-dimethylformamide is 50-54:12-13:1400-1600;

[0020] The cross-linking comprises mixing the grafted activated carbon, β-cyclodextrin and sodium hydroxide aqueous solution, stirring at a stirring speed of 400-500 rpm for 30-40 min at room temperature, adding glutaraldehyde aqueous solution dropwise, stirring at a stirring speed of 400-500 rpm for 30-40 min at room temperature after the addition is completed, stirring at a stirring speed of 400-500 rpm for 2.5-3 h at 55-60° C., centrifuging at a centrifugal speed of 4000-5000 rpm for 8-10 min, washing the precipitate with distilled water for 3-4 times, and drying at 120-130° C. for 12-13 h to obtain an enzymatic hydrolysis aid;

[0021] In the cross-linking, the mass ratio of the grafted activated carbon, β-cyclodextrin, sodium hydroxide aqueous solution, and glutaraldehyde aqueous solution is 50-52:30-35:1000-1100:30-35;

[0022] The molar concentration of sodium hydroxide aqueous solution is 0.2 mol / L;

[0023] The mass concentration of the glutaraldehyde aqueous solution is 25%;

[0024] The dropping speed of the glutaraldehyde aqueous solution is 1-2 g / min;

[0025] The extraction comprises mixing the enzymatic cell wall breaking liquid with n-hexane, stirring the mixture at 65-70° C. at a stirring speed of 400-500 rpm for 2-2.5 hours, standing the mixture at 65-70° C. for 2-3 hours, taking the oil phase, centrifuging the mixture at a centrifugal speed of 10000-11000 rpm for 9-10 minutes, taking the supernatant, and evaporating the mixture under reduced pressure to remove the n-hexane to obtain DHA crude oil;

[0026] In the extraction, the mass ratio of the Schizochytrium fermentation broth in the enzymatic hydrolysis to the n-hexane in the extraction is 1000:1900-2000;

[0027] The degumming comprises mixing citric acid and fumaric acid to obtain a mixed acid, adding distilled water to the mixed acid to prepare a mixed acid aqueous solution, adding DHA crude oil, stirring at 75-85° C. at a stirring speed of 50-100 rpm for 50-60 min, adding a sodium hydroxide aqueous solution, stirring at room temperature for 20-30 min, heating to 80-90° C., centrifuging at a centrifugal speed of 10000-11000 rpm for 9-10 min, taking the supernatant, and drying to remove water to obtain the degummed DHA crude oil;

[0028] In the degumming, the mass ratio of citric acid to fumaric acid is 5:1-2;

[0029] The mass ratio of DHA crude oil to mixed acid is 100:0.2-0.3.

[0030] The mass ratio of DHA crude oil to sodium hydroxide aqueous solution is 100:2.5-3;

[0031] The mass concentration of the mixed acid aqueous solution is 14-15%;

[0032] The mass concentration of the sodium hydroxide aqueous solution is 5%;

[0033] The deacidification comprises mixing the degummed DHA crude oil with a sodium hydroxide aqueous solution, stirring at 35-40° C. at a stirring speed of 50-100 rpm for 50-60 min, heating to 80-90° C., centrifuging at a centrifugal speed of 10000-11000 rpm for 9-10 min, taking the supernatant, adding distilled water at a temperature of 80-90° C., stirring at room temperature for 20-30 min, centrifuging at a centrifugal speed of 10000-11000 rpm for 9-10 min, taking the supernatant, and drying to remove water to obtain the deacidified DHA crude oil;

[0034] In the deacidification, the mass ratio of the degummed DHA crude oil to the sodium hydroxide aqueous solution is 100:2.5-3;

[0035] The mass ratio of the degummed DHA crude oil to distilled water at a temperature of 80-90°C is 100:7-8;

[0036] The mass concentration of the sodium hydroxide aqueous solution is 5%;

[0037] The decolorization comprises mixing the deacidified crude DHA oil and the decolorizing agent, stirring at a stirring speed of 50-100 rpm for 30-40 min at room temperature, centrifuging at a centrifugal speed of 10000-11000 rpm for 9-10 min, and taking the supernatant to obtain the decolorized crude DHA oil;

[0038] In the decolorization, the mass ratio of the deacidified DHA crude oil to the decolorizing agent is 100:5-6;

[0039] The preparation method of the decolorizing agent comprises the following steps: primary coating, secondary coating;

[0040] The primary coating comprises mixing the acidic silica sol with distilled water, stirring the mixture at a stirring speed of 200-300 rpm for 20-30 min at room temperature, adding activated carbon, stirring the mixture at a stirring speed of 200-300 rpm for 3.5-4 h at room temperature, standing the mixture at room temperature for 17-20 h, centrifuging the mixture at a centrifugal speed of 4000-5000 rpm for 8-10 min, washing the mixture with distilled water for 3-4 times, and drying the mixture at 120-130° C. for 10-12 h to obtain the primary coated activated carbon;

[0041] In the primary coating, the mass volume ratio of acidic silica sol, distilled water and activated carbon is 95-100:370-400:40-42;

[0042] The mass content of silicon dioxide in the acidic silica sol is 30%, the pH is 3, and the average particle size is 10 nm;

[0043] The average particle size of the activated carbon is 200 μm and the average pore size is 2 nm;

[0044] The secondary coating comprises mixing alkaline silica sol with distilled water, stirring at a stirring speed of 200-300 rpm for 15-25 min at room temperature, adding primary coated activated carbon, stirring at a stirring speed of 200-300 rpm for 2.5-3 h at room temperature, standing at room temperature for 12-13 h, centrifuging at a centrifugal speed of 3000-4000 rpm for 8-10 min, washing with distilled water for 3-4 times, and drying at 120-130° C. for 14-15 h to obtain a decolorizing agent;

[0045] In the secondary coating, the mass ratio of alkaline silica sol, distilled water, and primary coated activated carbon is 85-90:370-400:54-57;

[0046] The mass content of silicon dioxide in the alkaline silica sol is 20%, the pH is 9, and the average particle size is 40 nm;

[0047] In the deodorization, steam at a temperature of 90-100° C. is introduced into the bleached DHA crude oil at a temperature of 140-150° C. and a vacuum degree of 200-400 Pa for 30-40 minutes to obtain DHA algae oil.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) The method for extracting and refining DHA from marine microalgae fermentation broth of the present invention is aimed at the prior art, and the improvement is that an enzymatic hydrolysis aid is added in the enzymatic hydrolysis step, and a decolorizing agent that has been coated in two stages is added in the decolorization step. Among them, the preparation method of the enzymatic hydrolysis aid includes preparing carboxylated activated carbon, grafting, and cross-linking. The preparation of carboxylated activated carbon is to use nitric acid to treat the surface of activated carbon to introduce carboxyl groups; the grafting is to mix β-cyclodextrin with carboxylated activated carbon, stir and reflux at high temperature, and the hydroxyl groups in the β-cyclodextrin and the carboxyl groups in the carboxylated activated carbon undergo esterification reaction to introduce β-cyclodextrin, thereby fixing the β-cyclodextrin on the surface of the carboxylated activated carbon to obtain grafted activated carbon; the cross-linking is to add the grafted activated carbon into an alkaline solution containing β-cyclodextrin, disperse it evenly, and then drop glutaraldehyde aqueous solution. Under the action of glutaraldehyde, β-cyclodextrin cross-links with β-cyclodextrin on the surface of the grafted activated carbon to form a β-cyclodextrin cross-linked network on the surface of the grafted activated carbon to obtain an enzymatic hydrolysis aid with a core-shell structure, wherein the core of the enzymatic hydrolysis aid is activated carbon with adsorption function, and the shell is a β-cyclodextrin cross-linked network. In the enzymatic hydrolysis step, alkaline protease is uniformly adsorbed on the surface of the β-cyclodextrin cross-linked network, which can increase the contact area with Schizochytrium during stirring, thereby promoting the enzymatic hydrolysis of alkaline protease. The amino acids obtained after enzymatic hydrolysis can be adsorbed on the surface of activated carbon after passing through the β-cyclodextrin cross-linked network due to their small molecular weight. Water-insoluble amino acids and partially water-insoluble fatty acids can also be included in the cavity of β-cyclodextrin, thereby reducing the concentration of amino acids in the Schizochytrium fermentation broth, reducing the product inhibition, further shortening the enzymatic hydrolysis time, and improving the wall breaking rate; in the extraction step, n-hexane is added and stirred at high temperature, thereby promoting the release of fatty acids included in β-cyclodextrin and avoiding the influence on the total ester extraction rate.The preparation method of the decolorizer includes primary coating and secondary coating. The primary coating is to use acidic silica sol with an average particle size of 10nm to coat the activated carbon. Since the average particle size of the acidic silica sol is larger than the pore size of the activated carbon, the pores of the activated carbon will not be blocked during the primary coating, but a layer of porous silica will be formed on the surface of the activated carbon. The secondary coating is to use alkaline silica sol with an average particle size of 40nm to coat the primary coated activated carbon. The alkaline silica sol is adsorbed on the surface of the primary coated activated carbon by utilizing the different charged properties of the alkaline silica sol and the acidic silica sol. Moreover, since the average particle size of the alkaline silica sol is larger than the pore size of the porous silica formed in the primary coating, Thus, a layer of porous silica is formed on the surface of the primary coated activated carbon to obtain a decolorizer. The decolorizer has three layers of porous structures with different pore sizes. When adsorbing pigments, it can fix small molecule pigments and large molecule pigments respectively, thereby improving the ability to fix the pigments. Since silica and activated carbon are made of different materials, the adsorption capacity for different pigments is also different. After the silica and activated carbon are compounded, the adsorption range of the pigment can be increased, thereby improving the adsorption capacity of the pigment, improving the appearance of the DHA algae oil, and further adsorbing more pigments, resulting in a decrease in the weight of the obtained DHA algae oil, but it will not affect the quality of DHA in the DHA algae oil.

[0050] (2) The method of extracting and refining DHA from marine microalgae fermentation broth of the present invention can improve the cell wall breaking rate and shorten the enzymatic hydrolysis time. The enzymatic hydrolysis time required in the enzymatic hydrolysis step is 2.5-3h, and the cell wall breaking rate of the obtained enzymatic cell wall breaking liquid can reach 98.7-100%;

[0051] (3) The method of extracting and refining DHA from marine microalgae fermentation broth of the present invention can improve the extraction rate. The total ester extraction rate of the obtained DHA algae oil is 86.6-88.9%, wherein the mass content of DHA is 46.4-47.1%;

[0052] (4) The method of extracting and refining DHA from marine microalgae fermentation broth of the present invention can fully remove the pigment in DHA to obtain golden DHA algae oil. DETAILED DESCRIPTION

[0053] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific implementation methods of the present invention are now described.

[0054] Example 1

[0055] A method for extracting and refining DHA from marine microalgae fermentation broth, specifically comprising:

[0056] 1. Enzymatic hydrolysis: 1000 g of Schizochytrium fermentation broth, 0.9% of the dry matter mass of the Schizochytrium fermentation broth alkaline protease, and 14 g of an enzymatic hydrolysis aid were mixed, the pH was adjusted to 8, and the mixture was stirred at 50°C and 150 rpm for 2.5 h to obtain an enzymatic hydrolysis cell wall-breaking liquid;

[0057] The mass content of total esters in the Schizochytrium fermentation broth is 10.1%;

[0058] The enzymatic activity of the alkaline protease is 500,000 U / g;

[0059] The preparation method of the enzymolysis aid is:

[0060] (1) Preparation of carboxylated activated carbon: 150 g of activated carbon and 2800 g of nitric acid with a molar concentration of 7 mol / L were mixed, stirred at room temperature at a stirring speed of 300 rpm for 20 min, stirred and refluxed at a stirring speed of 300 rpm for 4 h at 85° C., centrifuged at a centrifugal speed of 4000 rpm for 8 min, washed the precipitate with distilled water 3 times, and dried at 120° C. for 9 h to obtain carboxylated activated carbon;

[0061] The average particle size of the activated carbon is 200 μm and the average pore size is 2 nm;

[0062] (2) Grafting: 50 g of β-cyclodextrin, 12 g of carboxylated activated carbon, and 1400 g of N,N-dimethylformamide were mixed, stirred and refluxed at 80 °C at a stirring speed of 400 rpm for 17 h, allowed to stand at room temperature for 10 h, centrifuged at a centrifugal speed of 4000 rpm for 8 min, washed the precipitate with acetone three times, and dried at 80 °C for 18 h to obtain grafted activated carbon;

[0063] (3) Cross-linking: 50 g of grafted activated carbon, 30 g of β-cyclodextrin, and 1000 g of a sodium hydroxide aqueous solution with a molar concentration of 0.2 mol / L were mixed, and the mixture was stirred at a stirring speed of 400 rpm for 30 min at room temperature. 30 g of a 25% glutaraldehyde aqueous solution was added dropwise at a dropping speed of 1 g / min. After the addition was completed, the mixture was stirred at a stirring speed of 400 rpm for 30 min at room temperature, stirred at a stirring speed of 400 rpm for 2.5 h at 55 ° C, and centrifuged at a centrifugal speed of 4000 rpm for 8 min. The precipitate was washed 3 times with distilled water and dried at 120 ° C for 12 h to obtain an enzymatic hydrolysis aid.

[0064] The cell wall breaking rate of the enzymatic cell wall breaking solution is 98.7%;

[0065] 2. Extraction: After the enzymatic cell wall-breaking liquid obtained in step 1 is mixed with 1900 g of n-hexane, the mixture is stirred at 65°C at a stirring speed of 400 rpm for 2 h, and then allowed to stand at 65°C for 2 h. The oil phase is taken and centrifuged at a centrifugal speed of 10,000 rpm for 9 min. The supernatant is taken and the n-hexane is removed by evaporation under reduced pressure to obtain crude DHA oil.

[0066] 3. Degumming: citric acid and fumaric acid were mixed in a mass ratio of 5:1 to obtain a mixed acid, distilled water was added to the mixed acid to prepare a mixed acid aqueous solution with a mass concentration of 14%, the DHA crude oil obtained in step 2 was added, the mass ratio of the DHA crude oil to the mixed acid was controlled to be 100:0.2, and then stirred at 75°C at a stirring speed of 50 rpm for 50 min, a 5% sodium hydroxide aqueous solution was added, the mass ratio of the DHA crude oil to the sodium hydroxide aqueous solution was controlled to be 100:2.5, stirred at room temperature for 20 min, heated to 80°C, centrifuged at a centrifugal speed of 10000 rpm for 9 min, the supernatant was taken, and dried to remove water to obtain the degummed DHA crude oil;

[0067] 4. Deacidification: The degummed DHA crude oil obtained in step 3 is mixed with a sodium hydroxide aqueous solution having a mass concentration of 5%, and the mass ratio of the degummed DHA crude oil to the sodium hydroxide aqueous solution is controlled to be 100:2.5, stirred at 35°C at a stirring speed of 50 rpm for 50 min, heated to 80°C, centrifuged at a centrifugal speed of 10,000 rpm for 9 min, the supernatant is taken, distilled water at a temperature of 80°C is added, the mass ratio of the degummed DHA crude oil to the distilled water at a temperature of 80°C is controlled to be 100:7, stirred at room temperature for 20 min, centrifuged at a centrifugal speed of 10,000 rpm for 9 min, the supernatant is taken, and dried to remove water to obtain deacidified DHA crude oil;

[0068] 5. Decolorization: Mix the deacidified DHA crude oil obtained in step 4 with a decolorizing agent, control the mass ratio of the deacidified DHA crude oil to the decolorizing agent to be 100:5, stir at room temperature at a stirring speed of 50 rpm for 30 minutes, centrifuge at a centrifugal speed of 10,000 rpm for 9 minutes, take the supernatant, and obtain the decolorized DHA crude oil;

[0069] The preparation method of the decolorizing agent is:

[0070] (1) Primary coating: 95 g of acidic silica sol was mixed with 370 g of distilled water, stirred at room temperature at a stirring speed of 200 rpm for 20 min, 40 g of activated carbon was added, stirred at room temperature at a stirring speed of 200 rpm for 3.5 h, allowed to stand at room temperature for 17 h, centrifuged at a centrifugal speed of 4000 rpm for 8 min, washed with distilled water 3 times, and dried at 120°C for 10 h to obtain primary coated activated carbon;

[0071] The mass content of silicon dioxide in the acidic silica sol is 30%, the pH is 3, and the average particle size is 10 nm;

[0072] The average particle size of the activated carbon is 200 μm and the average pore size is 2 nm;

[0073] (2) Secondary coating: 85 g of alkaline silica sol was mixed with 370 g of distilled water, and stirred at room temperature at a stirring speed of 200 rpm for 15 min, 54 g of primary coated activated carbon was added, and stirred at room temperature at a stirring speed of 200 rpm for 2.5 h, and allowed to stand at room temperature for 12 h, and centrifuged at a centrifugal speed of 3000 rpm for 8 min, and washed with distilled water for 3 times, and dried at 120°C for 14 h to obtain a decolorizing agent;

[0074] The mass content of silicon dioxide in the alkaline silica sol is 20%, the pH is 9, and the average particle size is 40 nm;

[0075] 6. Deodorization: At a temperature of 140°C and a vacuum degree of 200 Pa, steam at a temperature of 90°C was introduced into the decolorized DHA crude oil obtained in step 5 for 30 minutes to obtain 87.5 g of golden DHA algae oil with a total ester extraction rate of 86.6%, of which the mass content of DHA was 46.4%.

[0076] Example 2

[0077] A method for extracting and refining DHA from marine microalgae fermentation broth, specifically comprising:

[0078] 1. Enzymatic hydrolysis: 1000 g of Schizochytrium fermentation broth, 0.9% of the dry matter mass of the Schizochytrium fermentation broth alkaline protease, and 14.5 g of an enzymatic hydrolysis aid were mixed, the pH was adjusted to 8.5, and the mixture was stirred at 55°C at a stirring speed of 200 rpm for 3 h to obtain an enzymatic hydrolysis cell wall-breaking liquid;

[0079] The mass content of total esters in the Schizochytrium fermentation broth is 10.1%;

[0080] The enzymatic activity of the alkaline protease is 500,000 U / g;

[0081] The preparation method of the enzymolysis aid is:

[0082] (1) Preparation of carboxylated activated carbon: 155 g of activated carbon and 2900 g of nitric acid with a molar concentration of 7 mol / L were mixed, stirred at room temperature at a stirring speed of 350 rpm for 25 min, stirred and refluxed at a stirring speed of 350 rpm at 90° C. for 4.5 h, centrifuged at a centrifugal speed of 4500 rpm for 9 min, washed the precipitate with distilled water 3 times, and dried at 125° C. for 9.5 h to obtain carboxylated activated carbon;

[0083] The average particle size of the activated carbon is 200 μm and the average pore size is 2 nm;

[0084] (2) Grafting: 52 g of β-cyclodextrin, 12.5 g of carboxylated activated carbon, and 1500 g of N,N-dimethylformamide were mixed, stirred and refluxed at 82 °C at a stirring speed of 450 rpm for 18 h, allowed to stand at room temperature for 11 h, centrifuged at a centrifugal speed of 4500 rpm for 9 min, the precipitate was washed 3 times with acetone, and dried at 85 °C for 19 h to obtain grafted activated carbon;

[0085] (3) Cross-linking: 51 g of grafted activated carbon, 32 g of β-cyclodextrin, and 1050 g of a sodium hydroxide aqueous solution with a molar concentration of 0.2 mol / L were mixed, and stirred at a stirring speed of 450 rpm for 35 min at room temperature. 32 g of a 25% glutaraldehyde aqueous solution was added dropwise at a dropping speed of 1 g / min. After the addition was completed, the mixture was stirred at a stirring speed of 450 rpm for 35 min at room temperature, stirred at a stirring speed of 450 rpm for 2.5 h at 57 ° C, and centrifuged at a centrifugal speed of 4500 rpm for 9 min. The precipitate was washed 3 times with distilled water and dried at 125 ° C for 12.5 h to obtain an enzymatic hydrolysis aid.

[0086] The cell wall breaking rate of the enzymatic cell wall breaking solution is 100%;

[0087] 2. Extraction: After mixing the enzymatic cell wall-breaking liquid obtained in step 1 with 1950 g of n-hexane, stir at 67°C at a stirring speed of 450 rpm for 2 h, let stand at 67°C for 2.5 h, take the oil phase, centrifuge at a centrifugal speed of 11000 rpm for 9.5 min, take the supernatant, evaporate under reduced pressure to remove n-hexane, and obtain DHA crude oil;

[0088] 3. Degumming: citric acid and fumaric acid were mixed in a mass ratio of 5:1.5 to obtain a mixed acid, distilled water was added to the mixed acid to prepare a mixed acid aqueous solution with a mass concentration of 15%, the DHA crude oil obtained in step 2 was added, the mass ratio of the DHA crude oil to the mixed acid was controlled to be 100:0.2, and then stirred at 80°C at a stirring speed of 60 rpm for 55 minutes, a sodium hydroxide aqueous solution with a mass concentration of 5% was added, the mass ratio of the DHA crude oil to the sodium hydroxide aqueous solution was controlled to be 100:2.7, stirred at room temperature for 25 minutes, heated to 85°C, centrifuged at a centrifugal speed of 11000 rpm for 10 minutes, the supernatant was taken, and dried to remove water to obtain the degummed DHA crude oil;

[0089] 4. Deacidification: The degummed DHA crude oil obtained in step 3 is mixed with a sodium hydroxide aqueous solution having a mass concentration of 5%, and the mass ratio of the degummed DHA crude oil to the sodium hydroxide aqueous solution is controlled to be 100:2.8, stirred at 37°C at a stirring speed of 60 rpm for 55 min, heated to 85°C, centrifuged at a centrifugal speed of 11000 rpm for 10 min, the supernatant is taken, distilled water at a temperature of 85°C is added, the mass ratio of the degummed DHA crude oil to the distilled water at a temperature of 85°C is controlled to be 100:7.5, stirred at room temperature for 25 min, centrifuged at a centrifugal speed of 11000 rpm for 10 min, the supernatant is taken, and dried to remove water to obtain deacidified DHA crude oil;

[0090] 5. Decolorization: Mix the deacidified DHA crude oil obtained in step 4 with a decolorizing agent, control the mass ratio of the deacidified DHA crude oil to the decolorizing agent to be 100:5.5, stir at room temperature at a stirring speed of 60 rpm for 35 minutes, centrifuge at a centrifugal speed of 11000 rpm for 10 minutes, take the supernatant, and obtain the decolorized DHA crude oil;

[0091] The preparation method of the decolorizing agent is:

[0092] (1) Primary coating: 97 g of acidic silica sol was mixed with 380 g of distilled water, stirred at room temperature at a stirring speed of 250 rpm for 25 min, 41 g of activated carbon was added, stirred at room temperature at a stirring speed of 250 rpm for 4 h, allowed to stand at room temperature for 18 h, centrifuged at a centrifugal speed of 4500 rpm for 9 min, washed with distilled water for 3 times, and dried at 125°C for 11 h to obtain primary coated activated carbon;

[0093] The mass content of silicon dioxide in the acidic silica sol is 30%, the pH is 3, and the average particle size is 10 nm;

[0094] The average particle size of the activated carbon is 200 μm and the average pore size is 2 nm;

[0095] (2) Secondary coating: 87 g of alkaline silica sol was mixed with 380 g of distilled water, and the mixture was stirred at 250 rpm for 20 min at room temperature. 55 g of primary coated activated carbon was added, and the mixture was stirred at 250 rpm for 3 h at room temperature. The mixture was allowed to stand at room temperature for 12.5 h, and the mixture was centrifuged at 3500 rpm for 9 min. The mixture was washed with distilled water for 3 times, and dried at 125° C. for 14.5 h to obtain a decolorizing agent.

[0096] The mass content of silicon dioxide in the alkaline silica sol is 20%, the pH is 9, and the average particle size is 40 nm;

[0097] 6. Deodorization: At a temperature of 145°C and a vacuum degree of 300 Pa, steam at a temperature of 95°C was introduced into the decolorized DHA crude oil obtained in step 5 for 35 minutes to obtain 89.8 g of golden DHA algae oil with a total ester extraction rate of 88.9%, of which the mass content of DHA was 46.9%.

[0098] Example 3

[0099] A method for extracting and refining DHA from marine microalgae fermentation broth, specifically comprising:

[0100] 1. Enzymatic hydrolysis: 1000 g of Schizochytrium fermentation broth, 1% of the dry matter mass of the Schizochytrium fermentation broth, and 15 g of an enzymatic hydrolysis aid were mixed, the pH was adjusted to 9, and the mixture was stirred at 60°C and 200 rpm for 3 h to obtain an enzymatic hydrolysis cell wall-breaking liquid;

[0101] The mass content of total esters in the Schizochytrium fermentation broth is 10.1%;

[0102] The enzymatic activity of the alkaline protease is 500,000 U / g;

[0103] The preparation method of the enzymolysis aid is:

[0104] (1) Preparation of carboxylated activated carbon: 160 g of activated carbon and 3000 g of nitric acid with a molar concentration of 7 mol / L were mixed, stirred at room temperature at a stirring speed of 400 rpm for 30 min, stirred and refluxed at a stirring speed of 400 rpm at 95° C. for 5 h, centrifuged at a centrifugal speed of 5000 rpm for 10 min, washed the precipitate with distilled water 4 times, and dried at 130° C. for 10 h to obtain carboxylated activated carbon;

[0105] The average particle size of the activated carbon is 200 μm and the average pore size is 2 nm;

[0106] (2) Grafting: 54 g of β-cyclodextrin, 13 g of carboxylated activated carbon, and 1600 g of N,N-dimethylformamide were mixed, stirred and refluxed at 85 °C at a stirring speed of 500 rpm for 19 h, allowed to stand at room temperature for 12 h, centrifuged at a centrifugal speed of 5000 rpm for 10 min, the precipitate was washed 4 times with acetone, and dried at 90 °C for 20 h to obtain grafted activated carbon;

[0107] (3) Cross-linking: 52 g of grafted activated carbon, 35 g of β-cyclodextrin, and 1100 g of a sodium hydroxide aqueous solution with a molar concentration of 0.2 mol / L were mixed, and the mixture was stirred at a stirring speed of 500 rpm for 40 min at room temperature. 35 g of a 25% glutaraldehyde aqueous solution was added dropwise at a dropping speed of 2 g / min. After the addition was completed, the mixture was stirred at a stirring speed of 500 rpm for 40 min at room temperature, stirred at a stirring speed of 500 rpm for 3 h at 60°C, and centrifuged at a centrifugal speed of 5000 rpm for 10 min. The precipitate was washed 4 times with distilled water, and dried at 130°C for 13 h to obtain an enzymatic hydrolysis aid.

[0108] The cell wall breaking rate of the enzymatic cell wall breaking solution is 100%;

[0109] 2. Extraction: After mixing the enzymatic cell wall-breaking liquid obtained in step 1 with 2000 g of n-hexane, stir at 70°C at a stirring speed of 500 rpm for 2.5 hours, let stand at 70°C for 3 hours, take the oil phase, centrifuge at a centrifugal speed of 11000 rpm for 10 minutes, take the supernatant, evaporate under reduced pressure to remove n-hexane, and obtain DHA crude oil;

[0110] 3. Degumming: citric acid and fumaric acid are mixed in a mass ratio of 5:2 to obtain a mixed acid, distilled water is added to the mixed acid to prepare a mixed acid aqueous solution with a mass concentration of 15%, the DHA crude oil obtained in step 2 is added, and the mass ratio of the DHA crude oil to the mixed acid is controlled to be 100:0.3, and then stirred at 85°C at a stirring speed of 100 rpm for 60 minutes, a sodium hydroxide aqueous solution with a mass concentration of 5% is added, and the mass ratio of the DHA crude oil to the sodium hydroxide aqueous solution is controlled to be 100:3, and stirred at room temperature for 30 minutes, heated to 90°C, and centrifuged at a centrifugal speed of 11000 rpm for 10 minutes, the supernatant is taken, and dried to remove water to obtain the degummed DHA crude oil;

[0111] 4. Deacidification: The degummed DHA crude oil obtained in step 3 is mixed with a sodium hydroxide aqueous solution having a mass concentration of 5%, and the mass ratio of the degummed DHA crude oil to the sodium hydroxide aqueous solution is controlled to be 100:3. The mixture is stirred at a stirring speed of 100 rpm for 60 min at 40°C, heated to 90°C, and centrifuged at a centrifugal speed of 11000 rpm for 10 min. The supernatant is taken, and distilled water at a temperature of 90°C is added to control the mass ratio of the degummed DHA crude oil to the distilled water at a temperature of 90°C to be 100:8. The mixture is stirred at room temperature for 30 min, and centrifuged at a centrifugal speed of 11000 rpm for 10 min. The supernatant is taken, and dried to remove water to obtain deacidified DHA crude oil.

[0112] 5. Decolorization: Mix the deacidified DHA crude oil obtained in step 4 with a decolorizing agent, control the mass ratio of the deacidified DHA crude oil to the decolorizing agent to be 100:6, stir at room temperature at a stirring speed of 100 rpm for 40 minutes, centrifuge at a centrifugal speed of 11000 rpm for 10 minutes, take the supernatant, and obtain the decolorized DHA crude oil;

[0113] The preparation method of the decolorizing agent is:

[0114] (1) Primary coating: 100 g of acidic silica sol was mixed with 400 g of distilled water, stirred at 300 rpm for 30 min at room temperature, 42 g of activated carbon was added, stirred at 300 rpm for 4 h at room temperature, allowed to stand at room temperature for 20 h, centrifuged at 5000 rpm for 10 min, washed with distilled water for 4 times, and dried at 130 °C for 12 h to obtain primary coated activated carbon;

[0115] The mass content of silicon dioxide in the acidic silica sol is 30%, the pH is 3, and the average particle size is 10 nm;

[0116] The average particle size of the activated carbon is 200 μm and the average pore size is 2 nm;

[0117] (2) Secondary coating: 90 g of alkaline silica sol was mixed with 400 g of distilled water, and stirred at 300 rpm for 25 min at room temperature, 57 g of primary coated activated carbon was added, and stirred at 300 rpm for 3 h at room temperature, and allowed to stand at room temperature for 13 h, and centrifuged at 4000 rpm for 10 min, and washed with distilled water for 4 times, and dried at 130°C for 15 h to obtain a decolorizing agent;

[0118] The mass content of silicon dioxide in the alkaline silica sol is 20%, the pH is 9, and the average particle size is 40 nm;

[0119] 6. Deodorization: At a temperature of 150°C and a vacuum degree of 400 Pa, steam at a temperature of 100°C was introduced into the decolorized DHA crude oil obtained in step 5 for 40 minutes to obtain 87.7 g of golden DHA algae oil with a total ester extraction rate of 86.8%, of which the mass content of DHA was 47.1%.

[0120] Comparative Example 1

[0121] Based on Example 2, the addition of the enzymatic hydrolysis aid in the first enzymatic hydrolysis step is omitted;

[0122] The cell wall breaking rate of the enzymatic hydrolysis liquid obtained in the first enzymatic hydrolysis step was 84.2%;

[0123] In the sixth step of deodorization, 75.0 g of golden yellow DHA algae oil was obtained, and the total ester extraction rate was 74.3%, of which the mass content of DHA was 45.6%.

[0124] Comparative Example 2

[0125] On the basis of Comparative Example 1, in the first enzymatic hydrolysis step, the enzymatic hydrolysis time was increased from 3 h to 4.5 h;

[0126] The cell wall breaking rate of the enzymatic hydrolysis liquid obtained in the first enzymatic hydrolysis step is 100%;

[0127] In the sixth step of deodorization, 89.4 g of golden yellow DHA algae oil was obtained, and the total ester extraction rate was 88.5%, of which the mass content of DHA was 46.8%.

[0128] Comparative Example 3

[0129] On the basis of Example 2, in the fifth decolorization step, an activated carbon having an average particle size of 200 μm is used in an equal mass to replace the addition of the decolorizing agent;

[0130] In the sixth step of deodorization, 91.3 g of brown-yellow DHA algae oil was obtained, and the total ester extraction rate was 90.4%, of which the mass content of DHA was 44.5%.

[0131] Comparative Example 4

[0132] On the basis of Example 2, in the fifth decolorization step, in the preparation of the decolorant, the second-stage coating step (2) is omitted, that is, the first-stage coated activated carbon obtained in the first-stage coating step (1) is directly used as the decolorant;

[0133] In the sixth step of deodorization, 89.4 g of light brown-yellow DHA algae oil was obtained, and the total ester extraction rate was 88.5%, of which the mass content of DHA was 45.8%.

[0134] It can be seen from the results of Examples 1-3 and Comparative Examples 1-4 that by adding an enzymatic hydrolysis aid in the first enzymatic hydrolysis step, the enzymatic hydrolysis time can be shortened, the wall breaking rate can be increased, and the weight of the obtained DHA algae oil and the total ester extraction rate can be further increased; by adding a decolorizing agent in the decolorization step, although the weight of the obtained DHA algae oil is reduced, the mass content of DHA in the DHA algae oil can be increased, and the adsorption capacity of pigments can also be increased, thereby improving the appearance of the DHA algae oil.

[0135] Unless otherwise specified, all percentages used in the present invention are by mass.

[0136] Finally, it should be noted that the above 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 and refining DHA from marine microalgae fermentation broth, characterized in that: It consists of the following steps: enzymatic hydrolysis, extraction, degumming, deacidification, decolorization, and deodorization; The enzymatic hydrolysis comprises mixing the Schizochytrium fermentation liquid, alkaline protease and an enzymatic hydrolysis aid, adjusting the pH to 8-9, and stirring at 50-60° C. for 2.5-3 hours to obtain an enzymatic hydrolysis cell wall-breaking liquid; The preparation method of the enzymolysis aid comprises the following steps: preparing carboxylated activated carbon, grafting, and cross-linking; The preparation of carboxylated activated carbon comprises mixing activated carbon and nitric acid, stirring at room temperature for 20-30 minutes, stirring and refluxing at 85-95° C. for 4-5 hours, centrifuging, washing and drying the precipitate to obtain carboxylated activated carbon; The grafting comprises mixing β-cyclodextrin, carboxylated activated carbon and N,N-dimethylformamide, stirring and refluxing at 80-85° C. for 17-19 hours, standing at room temperature for 10-12 hours, centrifuging, washing and drying the precipitate to obtain grafted activated carbon; The cross-linking comprises mixing the grafted activated carbon, β-cyclodextrin and sodium hydroxide aqueous solution, stirring at room temperature for 30-40 minutes, adding glutaraldehyde aqueous solution dropwise, stirring at room temperature for 30-40 minutes after the addition is completed, stirring at 55-60° C. for 2.5-3 hours, centrifuging, washing and drying the precipitate to obtain an enzymatic hydrolysis aid; The decolorization comprises mixing the deacidified DHA crude oil and the decolorizing agent, stirring for 30-40 minutes at room temperature, centrifuging, and taking the supernatant to obtain the decolorized DHA crude oil; The preparation method of the decolorizing agent comprises the following steps: primary coating, secondary coating; The primary coating comprises mixing the acidic silica sol with distilled water, stirring at room temperature for 20-30 minutes, adding activated carbon, stirring at room temperature for 3.5-4 hours, standing at room temperature for 17-20 hours, centrifuging, washing and drying the precipitate to obtain the primary coated activated carbon; For the secondary coating, alkaline silica sol is mixed with distilled water, stirred at room temperature for 15-25 minutes, primary coated activated carbon is added, stirred at room temperature for 2.5-3 hours, allowed to stand at room temperature for 12-13 hours, centrifuged, and the precipitate is washed and dried to obtain a decolorizing agent.

2. The method for extracting and refining DHA from marine microalgae fermentation broth according to claim 1, characterized in that: In the enzymolysis, the mass ratio of Schizochytrium fermentation broth to the enzymolysis aid is 1000:14-15; The mass ratio of alkaline protease to dry matter of Schizochytrium fermentation broth is 0.9-1:100; The enzymatic activity of the alkaline protease is 500,000 U / g.

3. The method for extracting and refining DHA from marine microalgae fermentation broth according to claim 1, characterized in that: In the preparation of carboxylated activated carbon, the mass ratio of activated carbon to nitric acid is 150-160:2800-3000; The molar concentration of the nitric acid is 7 mol / L; The average particle size of the activated carbon is 200 μm, and the average pore size is 2 nm.

4. The method for extracting and refining DHA from marine microalgae fermentation broth according to claim 1, characterized in that: In the grafting, the mass ratio of β-cyclodextrin, carboxylated activated carbon and N,N-dimethylformamide is 50-54:12-13:1400-1600.

5. The method for extracting and refining DHA from marine microalgae fermentation broth according to claim 1, characterized in that: In the cross-linking, the mass ratio of the grafted activated carbon, β-cyclodextrin, sodium hydroxide aqueous solution, and glutaraldehyde aqueous solution is 50-52:30-35:1000-1100:30-35; The molar concentration of sodium hydroxide aqueous solution is 0.2 mol / L; The mass concentration of the glutaraldehyde aqueous solution is 25%; The dropping speed of the glutaraldehyde aqueous solution is 1-2 g / min.

6. The method for extracting and refining DHA from marine microalgae fermentation broth according to claim 1, characterized in that: The extraction comprises mixing the enzymatic cell-breaking liquid with n-hexane, stirring at 65-70° C. for 2-2.5 hours, standing at 65-70° C. for 2-3 hours, taking the oil phase, centrifuging, taking the supernatant, and evaporating under reduced pressure to remove the n-hexane to obtain DHA crude oil; In the extraction, the mass ratio of the Schizochytrium fermentation broth in the enzymatic hydrolysis to the n-hexane in the extraction is 1000:1900-2000.

7. The method for extracting and refining DHA from marine microalgae fermentation broth according to claim 1, characterized in that: The degumming comprises mixing citric acid and fumaric acid to obtain a mixed acid, adding distilled water to the mixed acid to prepare a mixed acid aqueous solution, adding DHA crude oil, stirring at 75-85° C. for 50-60 minutes, adding a sodium hydroxide aqueous solution, stirring at room temperature for 20-30 minutes, heating to 80-90° C., centrifuging, taking a supernatant, and drying to remove water to obtain degummed DHA crude oil; In the degumming, the mass ratio of citric acid to fumaric acid is 5:1-2; The mass ratio of DHA crude oil to mixed acid is 100:0.2-0.

3. The mass ratio of DHA crude oil to sodium hydroxide aqueous solution is 100:2.5-3; The mass concentration of the mixed acid aqueous solution is 14-15%; The mass concentration of the sodium hydroxide aqueous solution is 5%.

8. The method for extracting and refining DHA from marine microalgae fermentation broth according to claim 1, characterized in that: The deacidification comprises mixing the degummed DHA crude oil with a sodium hydroxide aqueous solution, stirring at 35-40° C. for 50-60 min, heating to 80-90° C., centrifuging, taking the supernatant, adding distilled water at a temperature of 80-90° C., stirring at room temperature for 20-30 min, centrifuging, taking the supernatant, and drying to remove water to obtain the deacidified DHA crude oil; In the deacidification, the mass ratio of the degummed DHA crude oil to the sodium hydroxide aqueous solution is 100:2.5-3; The mass ratio of the degummed DHA crude oil to distilled water at a temperature of 80-90°C is 100:7-8; The mass concentration of the sodium hydroxide aqueous solution is 5%.

9. The method for extracting and refining DHA from marine microalgae fermentation broth according to claim 1, characterized in that: In the decolorization, the mass ratio of the deacidified DHA crude oil to the decolorizing agent is 100:5-6; In the primary coating, the mass volume ratio of acidic silica sol, distilled water and activated carbon is 95-100:370-400:40-42; The mass content of silicon dioxide in the acidic silica sol is 30%, the pH is 3, and the average particle size is 10 nm; The average particle size of the activated carbon is 200 μm and the average pore size is 2 nm; In the secondary coating, the mass ratio of alkaline silica sol, distilled water, and primary coated activated carbon is 85-90:370-400:54-57; The mass content of silicon dioxide in the alkaline silica sol is 20%, the pH value is 9, and the average particle size is 40 nm.

10. The method for extracting and refining DHA from marine microalgae fermentation broth according to claim 1, characterized in that: In the deodorization, steam at a temperature of 90-100° C. is introduced into the bleached DHA crude oil at a temperature of 140-150° C. and a vacuum degree of 200-400 Pa for 30-40 minutes to obtain DHA algae oil.