CN117568176B - DHA extraction process based on microalgae extraction - Google Patents
DHA extraction process based on microalgae extraction Download PDFInfo
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- CN117568176B CN117568176B CN202311291926.6A CN202311291926A CN117568176B CN 117568176 B CN117568176 B CN 117568176B CN 202311291926 A CN202311291926 A CN 202311291926A CN 117568176 B CN117568176 B CN 117568176B
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- C07C51/44—Separation; Purification; Stabilisation; Use of additives by change of the physical state, e.g. crystallisation by distillation
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- C—CHEMISTRY; METALLURGY
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/50—Use of additives, e.g. for stabilisation
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- C—CHEMISTRY; METALLURGY
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
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Abstract
The invention provides a process for extracting DHA based on microalgae extraction, and belongs to the technical field of DHA extraction. Fermenting Isatis tinctoria such as Schizochytrium and Isatis indicum with enzyme, precipitating with ethanol to obtain algal polysaccharide, layering the filtrate to obtain DHA-rich crude oil, performing catalytic enzymolysis with immobilized lipase, and performing molecular distillation to obtain DHA algal oil; fermenting the protein powder to obtain antioxidant protein peptide; adding whey protein isolate, algal polysaccharide, antioxidant protein peptide and DHA algae oil into water, homogenizing, adding acacia, regulating pH value of the solution, cooling the reaction, and settling the microcapsule to obtain the microcapsule embedded with DHA. The DHA extracted by the method has high extraction rate, high purity and simple process, almost does not contain EPA, and the DHA embedded microcapsule is prepared, so that the oxidative decomposition of DHA is avoided, the edible taste is improved, and meanwhile, the DHA embedded rate and the nutritional value are high.
Description
Technical Field
The invention relates to the technical field of DHA extraction, in particular to a process for extracting DHA based on microalgae extraction.
Background
Docosahexaenoic acid, english name Docosahexaenoic Acid, DHA for short, molecular formula C 22H32O2, is a long-chain Omega-3 polyunsaturated fatty acid. DHA is the main element for the growth and function maintenance of cells in the nervous system, is an important constituent of the brain and retina, and researches show that the DHA content in the human brain cortex is about 20 percent and the DHA content in the retina is up to 50 percent. DHA has remarkable promoting effect on infant brain and vision development, and can induce a series of symptoms such as growth retardation, dysnoesia, etc. when DHA is lacking. However, since DHA is rarely synthesized by the human body itself and is required to be obtained from food, it is necessary to add DHA to food, especially infant food.
DHA is prepared from deep sea fish oil rich in DHA and EPA by molecular distillation, and exists in the form of DHA and EPA mixture. Wherein the chemical name of EPA is eicosapentaenoic acid, the English name is Eicosapentaenonic Acid, and EPA is short for short. A large number of researches show that EPA has the side effects of inhibiting the growth and development of children and teenagers, affecting the growth of fetuses, damaging blood platelets and causing difficult blood coagulation, and the like, so that most DHA products extracted from fish oil are not suitable for children, teenagers and women in pregnancy and lactation. Secondly, DHA extracted from fish oil needs to be saponified or esterified into an ethyl ester form for convenient extraction and preservation, and belongs to semi-chemical synthetic products. After the DHA product is eaten by human body, the DHA product needs to be decomposed into alcohol, the discomfort or adverse reaction can be generated by heart patients when the absorption amount is large, and the residual product after the decomposition of the DHA product can damage the kidney of the human body. In addition, due to the biological chain enrichment effect, heavy metal pollution exists in the fish oil DHA product, and the damage to the health of the eating crowd can be caused. The microalgae can be fermented to synthesize DHA, and the prior art also discloses a process for producing microalgae DHA by taking microalgae such as schizochytrium limacinum, ukenella, crypthecodinium cohnii and the like as raw materials through fermentation, separation, purification and other processes. Compared with fish oil DHA products, the microalgae DHA products have the advantages of high DHA content, low EPA content, strong oxidation resistance, no heavy metal pollution and the like, so that the microalgae DHA is more suitable for children, teenagers and women in pregnancy and lactation period in the growth and development period. The european union and the united states food and drug administration (Foodand Drug Administration, FDA) confirmed that microalgae DHA is GRAS grade (GENERALLY RECOGNIZED ASSAFE), a purely natural safety grade, and identified microalgae DHA as the only DHA product that can be added to infant food.
At present, methods applied in actual production and used for separating microalgae DHA mainly comprise a low-temperature fractionation method, a urea inclusion method, a solvent method, a supercritical gas extraction method and the like. The low-temperature classification method needs large-scale refrigeration equipment, and has high energy consumption and low extraction efficiency; the urea inclusion method is a simple and effective separation method, but has the problems of high solvent loss, water drainage, waste treatment caused by urea additives and the like when the urea inclusion method is applied to actual production; the supercritical gas extraction method can separate high-purity DHA, but has poor separation effect on fatty acids with the same carbon number and different double bond numbers, and has the advantages of large equipment investment, high technical requirement and larger energy consumption; the organic solvent extraction method is a microalgae grease extraction method widely used at home and abroad at present, but most of the solvents are inflammable, the solvent steam has certain toxicity, and in addition, the solvents are remained in the grease extracted by the solvent method, so the quality of the product is poor. Meanwhile, DHA prepared by extraction often has the problem of easy oxidation, so that inactivation can occur, certain toxic and side effects can be caused by adding common antioxidants, the food safety problem is caused, and some antioxidants are forbidden to be added in infant foods.
The prior art discloses a plurality of processes for producing microalgae DHA by fermentation, for example, chinese patent CN101168501B discloses a method for extracting and refining DHA-rich fatty acid from Crypthecodinium, firstly, flocculating Crypthecodinium fermentation liquor, then performing solid-liquid separation, mechanically breaking cells after breaking cell walls by alkali, extracting the broken thalli by adopting an organic solvent to obtain DHA crude oil, and degumming, alkali refining, decolorizing and deodorizing the DHA crude oil to obtain DHA essential oil. Chinese patent CN101307341B discloses a production method for docosahexaenoic acid grease by using a biological enzyme method, wherein enzyme is added into Crypthecodinium cohnii fermentation liquor to break the wall, then ethanol is added to carry out solid-liquid separation, then organic solvent is added to extract, and DHA refined oil is obtained after hydration, alkali refining, decoloration and deodorization. According to the two methods, the DHA essential oil is extracted by taking the organic solvent as the extractant, so that the extraction rate is low, a complex post-treatment process is required, the problem of extractant residue exists, and the obtained DHA product is polluted by the organic solvent.
Chinese patent CN101585759B discloses a method for extracting DHA from dinoflagellate fermentation liquor, after flocculation and biological enzyme wall breaking, using high-speed centrifugal machine to make three-phase separation until there is no oil phase basically, combining obtained oil phases, making five-stage continuous molecular distillation, directly making the heavy component of the previous stage be fed into the next stage to make distillation so as to obtain the unsaturated fatty acid whose DHA content is 40% -50%. According to the method, organic solvents are not required to be used for extraction, the obtained DHA product is not easy to be polluted by the organic solvents, but the method needs to firstly carry out biological enzyme wall breaking and then five-molecule molecular distillation, and the whole extraction process is long in route, complex in flow and high in energy consumption.
Disclosure of Invention
The invention aims to provide a process for extracting DHA based on microalgae extraction, which has the advantages of high extraction rate, high purity, simple process, almost no EPA, capability of avoiding oxidative decomposition of DHA, improvement of edible taste, high DHA embedding rate and high nutritive value.
The technical scheme of the invention is realized as follows:
The invention provides a process for extracting DHA based on microalgae extraction, which comprises the steps of carrying out enzyme-assisted fermentation on schizochytrium limacinum, chlorella globosa and the like, adding ethanol for precipitation to obtain algal polysaccharide, layering filtrate to obtain crude oil rich in DHA, carrying out catalytic enzymolysis by immobilized lipase, and carrying out molecular distillation to obtain DHA algal oil; fermenting the protein powder to obtain antioxidant protein peptide; adding whey protein isolate, algal polysaccharide, antioxidant protein peptide and DHA algae oil into water, homogenizing, adding acacia, regulating pH value of the solution, cooling the reaction, and settling the microcapsule to obtain the microcapsule embedded with DHA.
As a further improvement of the invention, the method comprises the following steps:
S1, enzyme-assisted fermentation of composite algae: drying and pulverizing Isatis Glaucescens and Isatis Glaucescens, adding the obtained dry powder into water, adding compound enzyme for enzymolysis, sterilizing, inoculating activated compound fermentation fungus seed solution, fermenting with enzyme, filtering to obtain fermentation product, and drying the residue to obtain feed;
S2, alcohol precipitation: adding ethanol into the fermentation product obtained in the step S1, standing for precipitation, and filtering to obtain algal polysaccharide; standing and layering the filtrate, collecting an organic phase to obtain crude oil rich in DHA, and freeze-drying an aqueous phase to obtain algae nutrient substances serving as nutritional food;
S3, preparing immobilized lipase: dissolving ferric chloride and ferrous chloride in water, adding ammonia water under the protection of inert gas, heating, stirring, reacting, centrifuging to obtain magnetic ferroferric oxide particles, adding the magnetic ferroferric oxide particles into the water, adding dopamine hydrochloride and a catalyst, heating, stirring, reacting, centrifuging, washing, drying to obtain modified magnetic ferroferric oxide particles, adding the modified magnetic ferroferric oxide particles into the water, adding composite lipase, stirring, reacting, volatilizing a solvent, and obtaining the immobilized lipase;
S4, lipase catalytic reaction: adding the immobilized lipase prepared in the step S3 into the DHA-rich crude oil prepared in the step S2, heating, stirring, performing enzymolysis, separating by a magnet, and recovering the immobilized lipase to obtain enzymolysis filtrate;
s5, molecular distillation and purification: adding the enzymolysis filtrate obtained in the step S4 into a molecular distillation device, heating and distilling, and collecting non-distilled substances to obtain DHA algae oil;
S6, preparing antioxidant protein peptide: adding the protein powder into water, sterilizing, inoculating activated zymophyte strain seed liquid, fermenting, separating, freeze-drying to obtain antioxidant protein peptide;
S7, embedding: dissolving whey protein isolate in water, adding algal polysaccharide prepared in the step S2, antioxidant protein peptide prepared in the step S6 and DHA algae oil prepared in the step S5, homogenizing, adding Arabic gum, uniformly mixing, regulating the pH value of the solution, stirring for reaction, cooling at low temperature, settling the microcapsule, centrifuging, washing and drying to obtain the microcapsule embedded with DHA.
As a further improvement of the invention, in the step S1, the mass ratio of the schizochytrium and the dinoflagellate is 10-12:5-7, the mass ratio of the dry powder to the water to the compound enzyme is 10-15:100-120:1-2, the enzymolysis temperature is 40-45 ℃ and the time is 1-3h, the compound enzyme is at least two of cellulase, hemicellulase, pectinase, alpha-amylase, beta-amylase, papain, ficin, alkaline protease, neutral protease, trypsin and bromelain, preferably, the cellulase, hemicellulase and pectinase, the mass ratio of the dry powder to the water to the compound enzyme is 3-5:2-3:2-3, the inoculation amount of the activated compound fermentation bacteria seed liquid comprises 2-3v/v% and 1-2v/v% of the activated bacillus licheniformis bacteria seed liquid, the bacteria content of the bacteria seed liquid is 10 8-109 cfu/mL, and the conditions of the enzyme assisted fermentation are 40-42 min-36 h.
As a further improvement of the invention, the addition amount of the ethanol in the step S2 is 70-80wt% of ethanol added to the system, the standing and precipitating time is 6-8h, and the standing and layering time is 1-3h; in the step S4, the mass ratio of the DHA-rich crude oil to the immobilized lipase is 100:5-7, the temperature of heating and stirring enzymolysis is 35-40 ℃, and the time is 2-4h.
As a further improvement of the invention, in the step S3, the mass ratio of the ferric chloride to the ferrous chloride is 16-16.4:12.5-12.7, the concentration of the ammonia water is 22-25wt%, the temperature of the heating and stirring reaction is 50-60 ℃ for 3-5 hours, and the mass ratio of the magnetic ferroferric oxide particles, the dopamine hydrochloride and the catalyst is 15-20:17-22:2-3, wherein the catalyst is Tris-HCl solution with pH value of 8.5-9; the mass ratio of the modified magnetic ferroferric oxide particles to the composite lipase is 10:3-5, the composite lipase is at least one selected from Mucor miehei lipase, candida rugosa lipase, pseudomonas cepacia lipase and candida antarctica lipase, preferably is a mixture of Mucor miehei lipase and candida rugosa lipase, the mass ratio is 5-7:10, and the stirring reaction is carried out for 30-40min.
As a further improvement of the invention, the molecular distillation condition in the step S5 is that the preheating temperature of the feed is 27-32 ℃, the condensed water is 20-30 ℃, the operating pressure of the system is 0.2-0.4Pa, the distillation temperature is 235-245 ℃, the feeding rate is 1.5-2.5mL/min, and the rotating speed of the film scraper is 100-200r/min.
As a further improvement of the invention, the protein powder in the step S6 is soy protein isolate and casein powder, and the mass ratio is 10-12:7-10, wherein the mass ratio of the protein powder to the water is 15-22:100, the activated fermentation bacteria seed liquid is activated lactobacillus bulgaricus and lactobacillus helveticus seed liquid, the inoculation amount is 3-4v/v% and 1-2v/v% respectively, the bacterial content of the seed liquid is 10 8-109 cfu/mL, the condition of enzyme-assisted fermentation is 36-39 ℃,50-70r/min, and the fermentation is carried out for 48-60h.
As a further improvement of the invention, in the step S7, the mass ratio of whey protein isolate, algal polysaccharide, antioxidant protein peptide, DHA algal oil and acacia gum is 25-30:3-5:5-7:17-22:17-25, the homogenization condition is 12000-13000r/min for homogenizing for 5-10min, the pH value of the solution is regulated to 3.7-3.8, the stirring reaction is carried out for 30-50min, the low temperature is 2-5 ℃, and the sedimentation time is 1-2h.
As a further improvement of the invention, the method specifically comprises the following steps:
S1, enzyme-assisted fermentation of composite algae: drying 10-12 parts by weight of schizochytrium limacinum, 5-7 parts by weight of dinoflagellate and the like, crushing to obtain dry powder, adding 10-15 parts by weight of dry powder into 100-120 parts by weight of water, adding 1-2 parts by weight of complex enzyme, carrying out enzymolysis for 1-3 hours at 40-45 ℃, sterilizing, inoculating activated bacillus subtilis strain seed liquid and activated bacillus licheniformis strain seed liquid, wherein the inoculum sizes are respectively 2-3v/v% and 1-2v/v%, the inoculum size is 10 8-109 cfu/mL, the temperature is 40-42 ℃, the speed is 50-70r/min, carrying out enzyme-assisted fermentation for 36-48 hours, filtering to obtain a fermentation product, and drying filter residues to be used as feed;
The compound enzyme is cellulase, hemicellulase and pectase, and the mass ratio is 3-5:2-3:2-3;
S2, alcohol precipitation: adding ethanol into the fermentation product obtained in the step S1 until the ethanol content of the system is 70-80wt%, standing and precipitating for 6-8h, and filtering to obtain algal polysaccharide; standing and layering the filtrate for 1-3h, collecting organic phase to obtain DHA-rich crude oil, and freeze-drying the water phase to obtain algae nutrient substances as nutritional food;
S3, preparing immobilized lipase: dissolving 16-16.4 parts by weight of ferric chloride and 12.5-12.7 parts by weight of ferrous chloride in 100 parts by weight of water, adding 10 parts by weight of 22-25wt% ammonia water under the protection of nitrogen, heating to 50-60 ℃, stirring and reacting for 3-5 hours, centrifuging to obtain magnetic ferroferric oxide particles, adding 15-20 parts by weight of magnetic ferroferric oxide particles into 100 parts by weight of water, adding 17-22 parts by weight of dopamine hydrochloride and 2-3 parts by weight of catalyst, heating to 40-45 ℃, stirring and reacting for 3-4 hours, centrifuging, washing, drying to obtain modified magnetic ferroferric oxide particles, adding 10 parts by weight of modified magnetic ferroferric oxide particles into 100 parts by weight of water, adding 3-5 parts by weight of composite lipase, stirring and reacting for 30-40 minutes, volatilizing a solvent to obtain immobilized lipase;
the catalyst is Tris-HCl solution with pH=8.5-9;
the composite lipase is a mixture of Mucor miehei lipase and candida rugosa lipase, and the mass ratio is 5-7:10;
S4, lipase catalytic reaction: adding 5-7 parts by weight of the immobilized lipase prepared in the step S3 into 100 parts by weight of the DHA-rich crude oil prepared in the step S2, heating to 35-40 ℃, stirring for enzymolysis for 2-4 hours, separating by a magnet, and recovering the immobilized lipase to obtain enzymolysis filtrate;
s5, molecular distillation and purification: adding the enzymolysis filtrate obtained in the step S4 into a molecular distillation device, heating and distilling, and collecting non-distilled substances to obtain DHA algae oil;
The molecular distillation conditions are that the preheating temperature of the feed is 27-32 ℃, the condensed water is 20-30 ℃, the system operation pressure is 0.2-0.4Pa, the distillation temperature is 235-245 ℃, the feeding rate is 1.5-2.5mL/min, and the rotating speed of the film scraping device is 100-200r/min;
S6, preparing antioxidant protein peptide: adding 15-22 parts by weight of protein powder into 100 parts by weight of water, sterilizing, inoculating activated lactobacillus bulgaricus and lactobacillus helveticus strain seed solutions, wherein the inoculum sizes are respectively 3-4v/v% and 1-2v/v%, the bacterial content of the strain seed solutions is 10 8-109 cfu/mL,36-39 ℃ and 50-70r/min, fermenting for 48-60h, separating, and freeze-drying to obtain antioxidant protein peptide;
The protein powder is soybean protein isolate and casein powder, and the mass ratio is 10-12:7-10;
S7, embedding: dissolving 25-30 parts by weight of whey protein isolate in 200 parts by weight of water, adding 3-5 parts by weight of algal polysaccharide prepared in the step S2, 5-7 parts by weight of antioxidant protein peptide prepared in the step S6 and 17-22 parts by weight of DHA algae oil prepared in the step S5, homogenizing for 5-10min with 12000-13000r/min, adding 17-25 parts by weight of acacia, uniformly mixing, regulating the pH value of the solution to 3.7-3.8, stirring and reacting for 30-50min, cooling at 2-5 ℃, settling the microcapsule for 1-2h, centrifuging, washing and drying to obtain the DHA embedded microcapsule.
The invention further provides the DHA-embedded microcapsule prepared by the process for extracting DHA based on microalgae extraction.
The invention has the following beneficial effects: the dinoflagellate such as schizochytrium and chlorella is rich in DHA, and the content of EPA is low, the content of DHA in algal fatty acid is more than 45%, so that the dinoflagellate and chlorella is a good DHA extraction raw material, the microalgae cell fatty acid composition is simple, the content of DHA is high, the content of other long-chain polyunsaturated fatty acids is not more than 1%, the dinoflagellate is basically free of EPA, the purification and separation of DHA are easy, the DHA extracted from the cells of the microalgae is basically free of fishy smell, the acceptance is high, and meanwhile, the microalgae has high content of proteins, trace elements, vitamins and antioxidant substances, and the extracted byproducts can be recycled and used as raw materials of feeds, nutritional foods and the like.
However, since microalgae are small, and most of microalgae cells contain polysaccharide, the culture solution is very viscous at high cell concentration, which brings great trouble to collection. In the invention, compound enzymes including cellulase, hemicellulase and pectase are adopted, wherein the cellulase and pectase can hydrolyze and destroy cell walls of microalgae cells, and the hemicellulase can hydrolyze polysaccharides (except cellulose and pectic substances) forming plant cell walls, so that the firm cell walls of the microalgae cells are destroyed and hydrolyzed, the dissolution of polysaccharide, protein, vitamin, DHA and other fatty acids in the cells is promoted, under the fermentation action of the microbial bacillus subtilis and the bacillus licheniformis, the microbial generates a large amount of enzymes and the microbial degradation action is used for carrying out enzymolysis on viscous high molecular weight polysaccharide, complex protein and the like, so that the dissolution of fatty acids coated or nearby the microbial polysaccharides is promoted, and the extraction rate of DHA is greatly improved;
After enzymatic fermentation, the product is filtered, the filter residue is mainly algae cell waste residue, the filter residue is a good feed raw material, the filter liquor is a fermentation product, ethanol is added for precipitation to obtain algal polysaccharide, the filter liquor is stood for layering, the upper layer is an algal oil layer, DHA is mainly distributed in the layer, and the water layer is a good nutrient raw material after freeze drying; the method fully utilizes the nutrition of the algae, recycles byproducts, reduces waste and improves the economic value of the algae.
The immobilized lipase is characterized in that the immobilized lipase is prepared by modifying the prepared magnetic particles with polydopamine, and the polyhydroxy, polyamino and polycarboxylic on the surface of the immobilized lipase can well form hydrogen bonds with amino, carboxyl and the like on the lipase, so that the immobilized lipase is stable.
After the prepared enzymolysis filtrate is distilled by a molecular distillation technology, most of other hydrolyzed fatty acids are distilled at a lower temperature, so that the purity of DHA is further improved, DHA algae oil is obtained, the high-temperature limitation of the traditional deodorization process is overcome, and the quality of the produced DHA product is higher.
The antioxidant active peptide has the characteristics of low toxicity, high efficiency and the like, is a safe and effective food antioxidant additive, has good in vitro free radical removal and reduction capacity, omits the steps of enzyme separation and purification by a fermentation method in soy protein isolate and casein powder, reduces production procedures and cost, and meanwhile, the terminal peptidase produced by microorganisms has a modification effect on the tail end of small peptide, so that the prepared peptide has no bitter taste, has natural fragrance of a certain fermented product, has good palatability, and further avoids the rejection of infants to the product and has high acceptance.
The algal polysaccharide is also a high-efficiency antioxidant, and in microalgae, the polysaccharide content is high, the nutrition is rich, the algal polysaccharide is a high-content byproduct in the DHA extraction process, and has good anti-inflammatory, antioxidant, antitumor and other activities, and meanwhile, the molecular weight of the polysaccharide prepared by fermentation is reduced, so that the infant has better tolerance to the polysaccharide, better absorbability and good health care and DHA oxidation prevention effects.
DHA taken from exogenous sources can treat mental diseases such as manic depression, major depressive disorder, post-traumatic stress disorder, psychosis and the like, maintain normal brain development and functions, promote infant brain and optic nerve development, improve organism inflammation through interaction with receptors, well prevent diseases related to cardiovascular and obesity through improving good regulation effects of blood fat, blood sugar and the like on in vivo metabolism, and simultaneously has important influence on peroxidation-antioxidation balance.
The DHA extracted by the method has high extraction rate, high purity and simple process, almost does not contain EPA, and the DHA embedded microcapsule is prepared, so that the oxidative decomposition of DHA is avoided, the edible taste is improved, and meanwhile, the DHA embedded rate and the nutritional value are high.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Whey protein isolate is WPI90, purchased from Dai Weilin international trade Shanghai limited; soy protein isolate, model ISOPRO552, protein content >90%, purchased from shandong jiawa biotechnology company limited; casein powder, protein content >95%, purchased from shanxi pannier biotechnology limited.
Mucor miehei lipase, 250 IUN/g (transesterification activity), available from Hangzhou department of catalysis, inc.; candida rugosa lipase, 700U/mg, purchased from Beijing Walker biotechnology Co. Cellulase, 5 ten thousand U/g, pectase, 2.5 ten thousand U/g, hemicellulase, 2.5 ten thousand U/g, purchased from Xia Cheng (Beijing) Biotech development Co., ltd.
Lactobacillus bulgaricus, 100 hundred million cfu/g, purchased from Wilkihai Sisi (Shandong) bioengineering Co., ltd; lactobacillus helveticus, 100 hundred million cfu/g, purchased from Hebei Patau bioengineering Co., ltd; bacillus subtilis, 1000 hundred million cfu/g, purchased from Shanxi Chenming Biotechnology Co., ltd; bacillus licheniformis, 1000 hundred million cfu/g, purchased from Weifang Yi Hao Biotechnology Co.
The schizochytrium limacinum is purchased from Shaanxi He bioengineering Co.Ltd; isatis like the ball was purchased from Shanghai optical and biological technology Co.
The bacillus subtilis or bacillus licheniformis is prepared by inoculating the strain into a Gao's culture medium, and performing activation culture at 42 ℃ for 24 hours at 70r/min to obtain bacillus subtilis strain seed liquid or bacillus licheniformis strain seed liquid with the bacterial content of 10 9 cfu/mL. The method for activating the Lactobacillus bulgaricus or Lactobacillus helveticus comprises inoculating the strain into a Gao's culture medium, and performing activation culture at 37deg.C and 70r/min for 24 hr to obtain seed solution of Bacillus subtilis strain or seed solution of Bacillus licheniformis strain with a bacterial content of 10 9 cfu/mL.
Example 1
The embodiment provides a process for extracting DHA based on microalgae extraction, which specifically comprises the following steps:
S1, enzyme-assisted fermentation of composite algae: drying 10 parts by weight of schizochytrium limacinum, 5 parts by weight of dinoflagellate and the like, crushing to obtain dry powder, adding 10 parts by weight of dry powder into 100 parts by weight of water, adding 1 part by weight of compound enzyme, carrying out enzymolysis for 1h at 40 ℃, sterilizing, inoculating activated bacillus subtilis strain seed liquid and activated bacillus licheniformis strain seed liquid, wherein the inoculation amount is respectively 2v/v% and 1v/v%, the bacterial seed liquid contains 10 9 cfu/mL, the temperature is 40 ℃ and 50r/min, carrying out enzyme-assisted fermentation for 36h, filtering to obtain a fermentation product, and drying filter residues to be used as feed;
the compound enzyme is cellulase, hemicellulase and pectase, and the mass ratio is 3:2:2;
S2, alcohol precipitation: adding ethanol into the fermentation product in the step S1 until the ethanol content of the system is 70wt%, standing and precipitating for 6 hours, and filtering to obtain algal polysaccharide; standing and layering the filtrate for 1h, collecting an organic phase to obtain DHA-rich crude oil, and freeze-drying an aqueous phase to obtain algae nutrient substances serving as nutritional food;
s3, preparing immobilized lipase: dissolving 16 parts by weight of ferric chloride and 12.5 parts by weight of ferrous chloride in 100 parts by weight of water, adding 10 parts by weight of 22wt% ammonia water under the protection of nitrogen, heating to 50 ℃, stirring and reacting for 3 hours, centrifuging to obtain magnetic ferroferric oxide particles, adding 15 parts by weight of magnetic ferroferric oxide particles into 100 parts by weight of water, adding 17 parts by weight of dopamine hydrochloride and 2 parts by weight of catalyst, heating to 40 ℃, stirring and reacting for 3 hours, centrifuging, washing and drying to obtain modified magnetic ferroferric oxide particles, adding 10 parts by weight of modified magnetic ferroferric oxide particles into 100 parts by weight of water, adding 3 parts by weight of composite lipase, stirring and reacting for 30 minutes, volatilizing solvent to obtain immobilized lipase;
the catalyst is Tris-HCl solution with pH=8.5;
the composite lipase is a mixture of Mucor miehei lipase and candida rugosa lipase, and the mass ratio is 5:10;
S4, lipase catalytic reaction: adding 5 parts by weight of immobilized lipase prepared in the step S3 into 100 parts by weight of DHA-rich crude oil prepared in the step S2, heating to 35 ℃, stirring for enzymolysis for 2 hours, separating by a magnet, and recovering the immobilized lipase to obtain enzymolysis filtrate;
s5, molecular distillation and purification: adding the enzymolysis filtrate obtained in the step S4 into a molecular distillation device, heating and distilling, and collecting non-distilled substances to obtain DHA algae oil;
The molecular distillation conditions are that the preheating temperature of the feed is 27 ℃, the condensate water is 20 ℃, the system operation pressure is 0.2Pa, the distillation temperature is 235 ℃, the feed rate is 1.5mL/min, and the rotating speed of the film scraping device is 100r/min;
S6, preparing antioxidant protein peptide: adding 15 parts by weight of protein powder into 100 parts by weight of water, sterilizing, inoculating activated lactobacillus bulgaricus and lactobacillus helveticus strain seed solutions, wherein the inoculum sizes are respectively 3v/v% and 1v/v%, the bacterial content of the strain seed solutions is 10 9 cfu/mL,36 ℃ and 50r/min, fermenting for 48 hours, separating, and freeze-drying to obtain antioxidant protein peptide;
the protein powder is soybean protein isolate and casein powder, and the mass ratio is 10:7, preparing a base material;
S7, embedding: dissolving 25 parts by weight of whey protein isolate in 200 parts by weight of water, adding 3 parts by weight of algal polysaccharide prepared in the step S2, 5 parts by weight of antioxidant protein peptide prepared in the step S6 and 17 parts by weight of DHA algae oil prepared in the step S5, homogenizing for 5min at 12000r/min, adding 17 parts by weight of acacia, uniformly mixing, adjusting the pH value of the solution to 3.7, stirring for reaction for 30min, cooling at 2 ℃, settling the microcapsule for 1h, centrifuging, washing and drying to obtain the DHA-embedded microcapsule.
Example 2
The embodiment provides a process for extracting DHA based on microalgae extraction, which specifically comprises the following steps:
S1, enzyme-assisted fermentation of composite algae: drying 12 parts by weight of schizochytrium limacinum, 7 parts by weight of dinoflagellate and the like, crushing to obtain dry powder, adding 15 parts by weight of dry powder into 120 parts by weight of water, adding 2 parts by weight of compound enzyme, carrying out enzymolysis for 3 hours at 45 ℃, sterilizing, inoculating activated bacillus subtilis strain seed liquid and activated bacillus licheniformis strain seed liquid, wherein the inoculation amount is 3v/v% and 2v/v% respectively, the bacterial content of the strain seed liquid is 10 9 cfu/mL, the temperature is 42 ℃, the fermentation is carried out for 48 hours in an enzyme-assisted manner, filtering to obtain a fermentation product, and drying filter residues to be used as feed;
the compound enzyme is cellulase, hemicellulase and pectase, and the mass ratio is 5:3:3;
S2, alcohol precipitation: adding ethanol into the fermentation product in the step S1 until the ethanol content of the system is 80wt%, standing and precipitating for 8 hours, and filtering to obtain algal polysaccharide; standing and layering the filtrate for 3 hours, collecting an organic phase to obtain crude oil rich in DHA, and freeze-drying an aqueous phase to obtain algae nutrient substances serving as nutritional food;
S3, preparing immobilized lipase: dissolving 16.4 parts by weight of ferric chloride and 12.7 parts by weight of ferrous chloride in 100 parts by weight of water, adding 10 parts by weight of 25wt% ammonia water under the protection of nitrogen, heating to 60 ℃, stirring and reacting for 5 hours, centrifuging to obtain magnetic ferroferric oxide particles, adding 20 parts by weight of magnetic ferroferric oxide particles into 100 parts by weight of water, adding 22 parts by weight of dopamine hydrochloride and 3 parts by weight of catalyst, heating to 45 ℃, stirring and reacting for 4 hours, centrifuging, washing, drying to obtain modified magnetic ferroferric oxide particles, adding 10 parts by weight of modified magnetic ferroferric oxide particles into 100 parts by weight of water, adding 5 parts by weight of composite lipase, stirring and reacting for 40 minutes, volatilizing solvent to obtain immobilized lipase;
The catalyst is Tris-HCl solution with pH=9;
the composite lipase is a mixture of Mucor miehei lipase and candida rugosa lipase, and the mass ratio is 7:10;
S4, lipase catalytic reaction: adding 7 parts by weight of immobilized lipase prepared in the step S3 into 100 parts by weight of DHA-rich crude oil prepared in the step S2, heating to 40 ℃, stirring for enzymolysis for 4 hours, separating by a magnet, and recovering the immobilized lipase to obtain enzymolysis filtrate;
s5, molecular distillation and purification: adding the enzymolysis filtrate obtained in the step S4 into a molecular distillation device, heating and distilling, and collecting non-distilled substances to obtain DHA algae oil;
The molecular distillation conditions are that the feed preheating temperature is 32 ℃, the condensate water is 30 ℃, the system operating pressure is 0.4Pa, the distillation temperature is 245 ℃, the feed rate is 2.5mL/min, and the rotating speed of a film scraping device is 200r/min;
S6, preparing antioxidant protein peptide: adding 22 parts by weight of protein powder into 100 parts by weight of water, sterilizing, inoculating activated lactobacillus bulgaricus and lactobacillus helveticus strain seed solutions, wherein the inoculum sizes are respectively 4v/v% and 2v/v%, the bacterial content of the strain seed solutions is 10 9 cfu/mL,39 ℃,70r/min, fermenting for 60 hours, separating, and freeze-drying to obtain antioxidant protein peptide;
the protein powder is soybean protein isolate and casein powder, and the mass ratio is 12:10;
S7, embedding: 30 parts by weight of whey protein isolate are dissolved in 200 parts by weight of water, 5 parts by weight of algal polysaccharide prepared in the step S2, 7 parts by weight of antioxidant protein peptide prepared in the step S6 and 22 parts by weight of DHA algae oil prepared in the step S5 are added, 13000r/min is homogenized for 10min, 25 parts by weight of acacia gum is added, after uniform mixing, the pH value of the solution is regulated to 3.8, stirring reaction is carried out for 50min, cooling is carried out at 5 ℃, microcapsule sedimentation is carried out for 2h, centrifugation, washing and drying are carried out, and the microcapsule embedded with DHA is prepared.
Example 3
The embodiment provides a process for extracting DHA based on microalgae extraction, which specifically comprises the following steps:
S1, enzyme-assisted fermentation of composite algae: drying 11 parts by weight of schizochytrium limacinum, 6 parts by weight of dinoflagellate and the like, crushing to obtain dry powder, adding 12 parts by weight of dry powder into 110 parts by weight of water, adding 1.5 parts by weight of compound enzyme, carrying out enzymolysis for 2 hours at 42 ℃, sterilizing, inoculating activated bacillus subtilis strain seed liquid and activated bacillus licheniformis strain seed liquid, wherein the inoculation amount is 2.5v/v% and 1.5v/v%, respectively, the bacterial content of the strain seed liquid is 10 9 cfu/mL,41 ℃ and 60r/min, carrying out enzyme-assisted fermentation for 42 hours, filtering to obtain a fermentation product, and drying filter residues to be used as feed;
the compound enzyme is cellulase, hemicellulase and pectase, and the mass ratio is 4:2.5:2.5;
S2, alcohol precipitation: adding ethanol into the fermentation product in the step S1 until the ethanol content of the system is 75wt%, standing and precipitating for 7h, and filtering to obtain algal polysaccharide; standing and layering the filtrate for 2 hours, collecting an organic phase to obtain crude oil rich in DHA, and freeze-drying an aqueous phase to obtain algae nutrient substances serving as nutritional food;
S3, preparing immobilized lipase: dissolving 16.2 parts by weight of ferric chloride and 12.6 parts by weight of ferrous chloride in 100 parts by weight of water, adding 10 parts by weight of 23.5wt% ammonia water under the protection of nitrogen, heating to 55 ℃, stirring and reacting for 4 hours, centrifuging to obtain magnetic ferroferric oxide particles, adding 17 parts by weight of magnetic ferroferric oxide particles into 100 parts by weight of water, adding 20 parts by weight of dopamine hydrochloride and 2.5 parts by weight of catalyst, heating to 42 ℃, stirring and reacting for 3.5 hours, centrifuging, washing, drying to obtain modified magnetic ferroferric oxide particles, adding 10 parts by weight of modified magnetic ferroferric oxide particles into 100 parts by weight of water, adding 4 parts by weight of composite lipase, stirring and reacting for 35 minutes, volatilizing solvent to obtain immobilized lipase;
the catalyst is Tris-HCl solution with pH=8.7;
the composite lipase is a mixture of Mucor miehei lipase and candida rugosa lipase, and the mass ratio is 6:10;
S4, lipase catalytic reaction: adding 6 parts by weight of immobilized lipase prepared in the step S3 into 100 parts by weight of DHA-rich crude oil prepared in the step S2, heating to 37 ℃, stirring for enzymolysis for 3 hours, separating by a magnet, and recovering the immobilized lipase to obtain enzymolysis filtrate;
s5, molecular distillation and purification: adding the enzymolysis filtrate obtained in the step S4 into a molecular distillation device, heating and distilling, and collecting non-distilled substances to obtain DHA algae oil;
The molecular distillation conditions are that the feed preheating temperature is 30 ℃, the condensed water is 25 ℃, the system operating pressure is 0.3Pa, the distillation temperature is 240 ℃, the feed rate is 2mL/min, and the rotating speed of a film scraping device is 150r/min;
S6, preparing antioxidant protein peptide: adding 20 parts by weight of protein powder into 100 parts by weight of water, sterilizing, inoculating activated lactobacillus bulgaricus and lactobacillus helveticus strain seed solutions, wherein the inoculum sizes are respectively 3.5v/v% and 1.5v/v%, the bacterial content of the strain seed solutions is 10 9 cfu/mL, the temperature is 37 ℃, the speed is 60r/min, fermenting for 56 hours, separating, and freeze-drying to obtain antioxidant protein peptide;
the protein powder is soybean protein isolate and casein powder, and the mass ratio is 11:8.5;
S7, embedding: dissolving 27 parts by weight of whey protein isolate in 200 parts by weight of water, adding 4 parts by weight of algal polysaccharide prepared in the step S2, 6 parts by weight of antioxidant protein peptide prepared in the step S6 and 20 parts by weight of DHA algae oil prepared in the step S5, homogenizing for 7min at 12500r/min, adding 22 parts by weight of acacia, uniformly mixing, adjusting the pH value of the solution to 3.75, stirring for reacting for 40min, cooling at 4 ℃, settling the microcapsule for 1.5h, centrifuging, washing and drying to obtain the DHA-embedded microcapsule.
Example 4
The difference compared with example 3 is that the complex enzyme is cellulase and pectase with the mass ratio of 6.5:2.5.
Example 5
The difference compared to example 3 is that the complex enzyme is a hemicellulase and a pectinase in a mass ratio of 6.5:2.5.
Example 6
The difference compared to example 3 is that the complex lipase is a single Mucor miehei fat.
Example 7
The difference compared to example 3 is that the complex lipase is a single candida rugosa lipase.
Comparative example 1
The difference compared to example 3 is that the complex enzyme in step S1 is pectase.
The method comprises the following steps:
S1, enzyme-assisted fermentation of composite algae: drying 11 parts by weight of schizochytrium limacinum, 6 parts by weight of dinoflagellate and the like, crushing to obtain dry powder, adding 12 parts by weight of dry powder into 110 parts by weight of water, adding 1.5 parts by weight of pectase, carrying out enzymolysis for 2 hours at 42 ℃, sterilizing, inoculating activated bacillus subtilis strain seed liquid and activated bacillus licheniformis strain seed liquid, wherein the inoculation amount is 2.5v/v% and 1.5v/v%, respectively, the bacterial content of the strain seed liquid is 10 9 cfu/mL,41 ℃ and 60r/min, carrying out enzyme-assisted fermentation for 42 hours, filtering to obtain a fermentation product, and drying filter residues to be used as feed.
Comparative example 2
In comparison with example 3, the difference is that no complex enzyme was added in step S1.
The method comprises the following steps:
S1, fermenting composite algae: drying 11 parts by weight of schizochytrium limacinum, 6 parts by weight of dinoflagellate and the like, crushing to obtain dry powder, adding 12 parts by weight of dry powder into 110 parts by weight of water, sterilizing, inoculating 2.5v/v% and 1.5v/v% of activated bacillus subtilis strain seed liquid, respectively, fermenting for 42h at the temperature of 41 ℃ at the temperature of 60r/min, filtering to obtain a fermentation product, and drying filter residues to be used as feed.
Comparative example 3
The difference compared with example 3 is that the activated seed solution of Bacillus subtilis strain was not inoculated in step S1.
The method comprises the following steps:
s1, enzyme-assisted fermentation of composite algae: drying 11 parts by weight of schizochytrium limacinum, 6 parts by weight of dinoflagellate such as ball and the like, crushing to obtain dry powder, adding 12 parts by weight of dry powder into 110 parts by weight of water, adding 1.5 parts by weight of compound enzyme, carrying out enzymolysis for 2 hours at 42 ℃, sterilizing, inoculating activated bacillus licheniformis strain seed liquid with the inoculation amount of 4v/v%, wherein the bacterial content of the strain seed liquid is 10 9 cfu/mL, the temperature is 41 ℃, the temperature is 60r/min, carrying out enzyme-assisted fermentation for 42 hours, filtering to obtain a fermentation product, and drying filter residues to obtain feed.
Comparative example 4
The difference compared to example 3 is that in step S1 the seed liquor of the activated Bacillus licheniformis bacteria is not inoculated.
The method comprises the following steps:
S1, enzyme-assisted fermentation of composite algae: drying and crushing 11 parts by weight of schizochytrium limacinum, 6 parts by weight of dinoflagellate and the like, obtaining dry powder, adding 12 parts by weight of dry powder into 110 parts by weight of water, adding 1.5 parts by weight of compound enzyme, carrying out enzymolysis for 2 hours at 42 ℃, sterilizing, inoculating activated bacillus subtilis strain seed liquid with the inoculation amount of 4v/v%, wherein the bacterial content of the strain seed liquid is 10 9 cfu/mL, the temperature is 41 ℃, the temperature is 60r/min, carrying out enzyme-assisted fermentation for 42 hours, filtering, obtaining a fermentation product, and drying filter residues to be used as feed.
Comparative example 5
The difference compared to example 3 is that in step S1, the activated seed solution of Bacillus subtilis strain and the activated seed solution of Bacillus licheniformis strain are not inoculated.
The method comprises the following steps:
S1, enzyme-assisted fermentation of composite algae: drying and crushing 11 parts by weight of schizochytrium limacinum, 6 parts by weight of dinoflagellate and the like, obtaining dry powder, adding 12 parts by weight of the dry powder into 110 parts by weight of water, adding 1.5 parts by weight of compound enzyme, carrying out enzymolysis at 42 ℃ for 2h, at 41 ℃ for 60r/min, stirring and reacting for 42h, filtering, obtaining a product, and drying filter residues to obtain feed.
Comparative example 6
The difference from example 3 is that polydopamine modification is not performed in step S3.
The method comprises the following steps:
S3, preparing immobilized lipase: dissolving 16.2 parts by weight of ferric chloride and 12.6 parts by weight of ferrous chloride in 100 parts by weight of water, adding 10 parts by weight of 23.5wt% ammonia water under the protection of nitrogen, heating to 55 ℃, stirring and reacting for 4 hours, centrifuging to obtain magnetic ferroferric oxide particles, adding 10 parts by weight of magnetic ferroferric oxide particles into 100 parts by weight of water, adding 4 parts by weight of composite lipase, stirring and reacting for 35 minutes, and volatilizing a solvent to obtain the immobilized lipase.
Comparative example 7
In comparison with example 3, the difference is that no complex lipase was added in step S3.
The method comprises the following steps:
S3, preparing modified magnetic ferroferric oxide particles: dissolving 16.2 parts by weight of ferric chloride and 12.6 parts by weight of ferrous chloride in 100 parts by weight of water, adding 10 parts by weight of 23.5wt% ammonia water under the protection of nitrogen, heating to 55 ℃, stirring and reacting for 4 hours, centrifuging to obtain magnetic ferroferric oxide particles, adding 17 parts by weight of magnetic ferroferric oxide particles into 100 parts by weight of water, adding 20 parts by weight of dopamine hydrochloride and 2.5 parts by weight of catalyst, heating to 42 ℃, stirring and reacting for 3.5 hours, centrifuging, washing, and drying to obtain modified magnetic ferroferric oxide particles.
Comparative example 8
In comparison with example 3, the difference is that steps S3 and S4 are not performed.
The method comprises the following steps:
S1, enzyme-assisted fermentation of composite algae: drying 11 parts by weight of schizochytrium limacinum, 6 parts by weight of dinoflagellate and the like, crushing to obtain dry powder, adding 12 parts by weight of dry powder into 110 parts by weight of water, adding 1.5 parts by weight of compound enzyme, carrying out enzymolysis for 2 hours at 42 ℃, sterilizing, inoculating activated bacillus subtilis strain seed liquid and activated bacillus licheniformis strain seed liquid, wherein the inoculation amount is 2.5v/v% and 1.5v/v%, respectively, the bacterial content of the strain seed liquid is 10 9 cfu/mL,41 ℃ and 60r/min, carrying out enzyme-assisted fermentation for 42 hours, filtering to obtain a fermentation product, and drying filter residues to be used as feed;
the compound enzyme is cellulase, hemicellulase and pectase, and the mass ratio is 4:2.5:2.5;
S2, alcohol precipitation: adding ethanol into the fermentation product in the step S1 until the ethanol content of the system is 75wt%, standing and precipitating for 7h, and filtering to obtain algal polysaccharide; standing and layering the filtrate for 2 hours, collecting an organic phase to obtain crude oil rich in DHA, and freeze-drying an aqueous phase to obtain algae nutrient substances serving as nutritional food;
S3, molecular distillation and purification: adding the crude oil rich in DHA obtained in the step S2 into a molecular distillation device, heating and distilling, and collecting non-distilled substances to obtain DHA algae oil;
The molecular distillation conditions are that the feed preheating temperature is 30 ℃, the condensed water is 25 ℃, the system operating pressure is 0.3Pa, the distillation temperature is 240 ℃, the feed rate is 2mL/min, and the rotating speed of a film scraping device is 150r/min;
s4, preparing antioxidant protein peptide: adding 20 parts by weight of protein powder into 100 parts by weight of water, sterilizing, inoculating activated lactobacillus bulgaricus and lactobacillus helveticus strain seed solutions, wherein the inoculum sizes are respectively 3.5v/v% and 1.5v/v%, the bacterial content of the strain seed solutions is 10 9 cfu/mL, the temperature is 37 ℃, the speed is 60r/min, fermenting for 56 hours, separating, and freeze-drying to obtain antioxidant protein peptide;
the protein powder is soybean protein isolate and casein powder, and the mass ratio is 11:8.5;
S5, embedding: dissolving 27 parts by weight of whey protein isolate in 200 parts by weight of water, adding 4 parts by weight of algal polysaccharide prepared in the step S2, 6 parts by weight of antioxidant protein peptide prepared in the step S4 and 20 parts by weight of DHA algae oil prepared in the step S3, homogenizing for 7min at 12500r/min, adding 22 parts by weight of acacia, uniformly mixing, adjusting the pH value of the solution to 3.75, stirring for reacting for 40min, cooling at 4 ℃, settling the microcapsule for 1.5h, centrifuging, washing, and drying to obtain the DHA-embedded microcapsule.
Comparative example 9
In comparison with example 3, the difference is that step S5 is not performed.
The method comprises the following steps:
S1, enzyme-assisted fermentation of composite algae: drying 11 parts by weight of schizochytrium limacinum, 6 parts by weight of dinoflagellate and the like, crushing to obtain dry powder, adding 12 parts by weight of dry powder into 110 parts by weight of water, adding 1.5 parts by weight of compound enzyme, carrying out enzymolysis for 2 hours at 42 ℃, sterilizing, inoculating activated bacillus subtilis strain seed liquid and activated bacillus licheniformis strain seed liquid, wherein the inoculation amount is 2.5v/v% and 1.5v/v%, respectively, the bacterial content of the strain seed liquid is 10 9 cfu/mL,41 ℃ and 60r/min, carrying out enzyme-assisted fermentation for 42 hours, filtering to obtain a fermentation product, and drying filter residues to be used as feed;
the compound enzyme is cellulase, hemicellulase and pectase, and the mass ratio is 4:2.5:2.5;
S2, alcohol precipitation: adding ethanol into the fermentation product in the step S1 until the ethanol content of the system is 75wt%, standing and precipitating for 7h, and filtering to obtain algal polysaccharide; standing and layering the filtrate for 2 hours, collecting an organic phase to obtain crude oil rich in DHA, and freeze-drying an aqueous phase to obtain algae nutrient substances serving as nutritional food;
S3, preparing immobilized lipase: dissolving 16.2 parts by weight of ferric chloride and 12.6 parts by weight of ferrous chloride in 100 parts by weight of water, adding 10 parts by weight of 23.5wt% ammonia water under the protection of nitrogen, heating to 55 ℃, stirring and reacting for 4 hours, centrifuging to obtain magnetic ferroferric oxide particles, adding 17 parts by weight of magnetic ferroferric oxide particles into 100 parts by weight of water, adding 20 parts by weight of dopamine hydrochloride and 2.5 parts by weight of catalyst, heating to 42 ℃, stirring and reacting for 3.5 hours, centrifuging, washing, drying to obtain modified magnetic ferroferric oxide particles, adding 10 parts by weight of modified magnetic ferroferric oxide particles into 100 parts by weight of water, adding 4 parts by weight of composite lipase, stirring and reacting for 35 minutes, volatilizing solvent to obtain immobilized lipase;
the catalyst is Tris-HCl solution with pH=8.7;
the composite lipase is a mixture of Mucor miehei lipase and candida rugosa lipase, and the mass ratio is 6:10;
S4, lipase catalytic reaction: adding 6 parts by weight of immobilized lipase prepared in the step S3 into 100 parts by weight of DHA-rich crude oil prepared in the step S2, heating to 37 ℃, stirring for enzymolysis for 3 hours, separating by a magnet, and recovering the immobilized lipase to obtain enzymolysis filtrate;
s5, preparing antioxidant protein peptide: adding 20 parts by weight of protein powder into 100 parts by weight of water, sterilizing, inoculating activated lactobacillus bulgaricus and lactobacillus helveticus strain seed solutions, wherein the inoculum sizes are respectively 3.5v/v% and 1.5v/v%, the bacterial content of the strain seed solutions is 10 9 cfu/mL, the temperature is 37 ℃, the speed is 60r/min, fermenting for 56 hours, separating, and freeze-drying to obtain antioxidant protein peptide;
the protein powder is soybean protein isolate and casein powder, and the mass ratio is 11:8.5;
S6, embedding: dissolving 27 parts by weight of whey protein isolate in 200 parts by weight of water, adding 4 parts by weight of algal polysaccharide prepared in the step S2, 6 parts by weight of antioxidant protein peptide prepared in the step S5 and 20 parts by weight of enzymolysis filtrate prepared in the step S4, homogenizing for 7min at 12500r/min, adding 22 parts by weight of acacia, uniformly mixing, adjusting the pH value of the solution to 3.75, stirring for reacting for 40min, cooling at 4 ℃, settling the microcapsule for 1.5h, centrifuging, washing and drying to obtain the DHA-embedded microcapsule.
Comparative example 10
In comparison with example 3, the difference is that no soy protein isolate was added in step S6.
The method comprises the following steps:
S6, preparing antioxidant protein peptide: adding 20 parts by weight of casein powder into 100 parts by weight of water, sterilizing, inoculating activated lactobacillus bulgaricus and lactobacillus helveticus strain seed solutions, wherein the inoculum sizes are respectively 3.5v/v% and 1.5v/v%, the bacterial content of the strain seed solutions is 10 9 cfu/mL, the temperature is 37 ℃, the speed is 60r/min, fermenting for 56 hours, separating, and freeze-drying to obtain the antioxidant protein peptide.
Comparative example 11
The difference from example 3 is that no casein powder was added in step S6.
The method comprises the following steps:
S6, preparing antioxidant protein peptide: adding 20 parts by weight of soybean protein isolate into 100 parts by weight of water, sterilizing, inoculating activated lactobacillus bulgaricus and lactobacillus helveticus strain seed solutions, wherein the inoculum sizes are respectively 3.5v/v% and 1.5v/v%, the bacterial content of the strain seed solutions is 10 9 cfu/mL, the temperature is 37 ℃, the speed is 60r/min, fermenting for 56 hours, separating, and freeze-drying to obtain the antioxidant protein peptide.
Comparative example 12
In comparison with example 3, the difference is that no fermentation is performed in step S6.
The method comprises the following steps:
s6, preparing protein powder: the soybean protein isolate and casein powder are mixed according to the mass ratio of 11:8.5, mixing to obtain the protein powder.
Comparative example 13
The difference from example 3 is that no antioxidant protein peptide was added in step S7.
The method comprises the following steps:
S7, embedding: dissolving 27 parts by weight of whey protein isolate in 200 parts by weight of water, adding 10 parts by weight of algal polysaccharide prepared in the step S2 and 20 parts by weight of DHA algal oil prepared in the step S5, homogenizing for 7min at 12500r/min, adding 22 parts by weight of acacia, uniformly mixing, adjusting the pH value of the solution to 3.75, stirring for reacting for 40min, cooling at 4 ℃, settling the microcapsule for 1.5h, centrifuging, washing and drying to obtain the DHA-embedded microcapsule.
Comparative example 14
In comparison with example 3, the difference is that algal polysaccharide is not added in step S7.
The method comprises the following steps:
S7, embedding: and (3) dissolving 27 parts by weight of whey protein isolate in 200 parts by weight of water, adding 10 parts by weight of the antioxidant protein peptide prepared in the step S6 and 20 parts by weight of DHA algae oil prepared in the step S5, homogenizing for 7min at 12500r/min, adding 22 parts by weight of acacia, uniformly mixing, regulating the pH value of the solution to 3.75, stirring for reacting for 40min, cooling at 4 ℃, settling the microcapsule for 1.5h, centrifuging, washing and drying to obtain the DHA-embedded microcapsule.
Comparative example 15
In comparison with example 3, the difference is that the antioxidant protein peptide and algal polysaccharide are not added in step S7.
The method comprises the following steps:
S7, embedding: dissolving 27 parts by weight of whey protein isolate in 200 parts by weight of water, adding 20 parts by weight of DHA algae oil prepared in the step S5, homogenizing for 7min at 12500r/min, adding 22 parts by weight of acacia, uniformly mixing, adjusting the pH value of the solution to 3.75, stirring for reacting for 40min, cooling at 4 ℃, settling the microcapsule for 1.5h, centrifuging, washing, and drying to obtain the DHA-embedded microcapsule.
Test example 1
The DHA-entrapped microcapsules prepared in examples 1-3 of the present invention were subjected to encapsulation efficiency test, and the results are shown in Table 1.
Determination of surface oil: 1g of the dried microcapsule sample was dispersed in 10mL of isohexane, followed by shaking at 200r/min for 5min, the suspension was filtered with a 5 μm filter membrane, the solid particles on the filter paper were washed 3 times with 10mL of isohexane each time, the filtrates were combined, the solvent residue was removed, and the mass M s of the surface oil was obtained by weighing.
Determination of total oil: the total oil content was determined using 4mol/LHCl. Dispersing 1g of microcapsule powder in 10mL of 4mol/L HCl, shaking for 15min at a rotating speed of 200r/min to dissolve wall materials, adding 15mL of isohexane into the mixture, shaking for 18h at room temperature to extract oil, centrifuging the mixture, collecting microalgae oil containing isohexane phase, removing residual solvent, and weighing to obtain the mass M t of total oil.
Encapsulation efficiency (%) = (M t-Ms）/Mt x 100%
TABLE 1
As can be seen from the above table, the encapsulation rate of DHA in the DHA-embedded microcapsules prepared in examples 1-3 of the present invention is between 95.2-96.7%.
Test example 2 test for peroxide value
The peroxide value of the oil is a main index for judging the rancidity degree of the oil and the products thereof. Weighing 5g of DHA-embedded microcapsules prepared in examples 1-7 and comparative examples 1-15, adding 50mL of chloroform-glacial acetic acid (volume ratio of 3:1) mixed solution into a 250mL iodometric bottle, shaking to dissolve a sample, adding saturated potassium iodide, stirring for 30s, standing in a dark place for 15min, adding 100mL of deionized water, immediately titrating with sodium thiosulfate, adding 1mL of starch indicator after light yellow color appears, and continuing to titrate until blue color disappears. And simultaneously performing a blank test. The peroxide value is measured by referring to a colorimetric method in an analysis method (GB/T5009.37-2003) of national standard-edible vegetable oil sanitation standard in China, and the calculation formula is as follows:
X1=（V1-V0×C×0.1269）/M×100
X2=X1×78.8
Wherein X 1 is the peroxide value in the sample, and the unit is meq/kg; x 2 -the peroxide value of DHA-embedded microcapsules, unit meq/kg; v 1 -volume of microencapsulated microalgae oil consumed sodium thiosulfate standard titration (mL); v 0 -microalgae oil blank consumption sodium thiosulfate standard titration volume (mL); c-sodium thiosulfate standard titration solution concentration (mol/L); m-sample mass (g); 0.1269-the mass of iodine (g) equivalent to 1.00mL of Na 2S2O3 standard titration solution; 78.8-conversion factor.
The results are shown in Table 2.
TABLE 2
As is clear from the above table, the microcapsule prepared in examples 1 to 3 of the present invention has a low peroxide value, and the DHA is not oxidized and decomposed, so that the quality is high.
Test example 3 storage stability test
The DHA-entrapped microcapsules prepared in examples 1-3 and comparative examples 10-15 were subjected to experiments.
1. Illumination of
The DHA-embedded microcapsule sample was sampled after 7 hours of ultraviolet irradiation, and the peroxide value was measured by the method of test example 2. The control 1 group is a commercial similar product, and the control 2 group is food antioxidant BHA with equal quality by replacing antioxidant protein peptide and algal polysaccharide.
2. Oxygen gas
The experimental temperature was 25℃and the light intensity was 0 LX. The DHA-embedded microcapsule sample was spread on a petri dish, left for 15 days under aerobic conditions, sampled, and the peroxide value was measured according to the method of test example 2. The control 1 group is a commercial similar product, and the control 2 group is food antioxidant BHA with equal quality by replacing antioxidant protein peptide and algal polysaccharide.
3. Temperature (temperature)
The microcapsule sample in which DHA was embedded was stored in an aluminum foil bag at 45℃and vacuum-sealed, and samples were taken every 15 days after storage, and the peroxide value was measured by the method of test example 2. The control 1 group is a commercial similar product, and the control 2 group is food antioxidant BHA with equal quality by replacing antioxidant protein peptide and algal polysaccharide.
The results are shown in Table 3.
TABLE 3 Table 3
As shown in the table above, DHA-embedded microcapsules prepared in examples 1-3 of the present invention have good stability to light, oxygen and high temperature.
Test example 4 sensory scoring
The DHA-entrapped microcapsules prepared in examples 1-7 and comparative examples 1-15, as well as the commercial products of the same type, were subjected to sensory evaluation in a well ventilated, odorless environment by 20 professional food researchers. The sensory evaluation total score was 100, specifically 40 for odor, 20 for color and 40 for tissue status. The scoring details are shown in Table 4.
TABLE 4 Table 4
The results are shown in Table 5.
TABLE 5
As can be seen from the above table, the DHA-entrapped microcapsules prepared in examples 1-3 of the present invention have a higher sensory score.
Test example 5
DHA algae oils prepared in the methods of examples 1-7 and comparative examples 1-15 were subjected to extraction rate and purity tests, and DHA purity was detected by using a high performance liquid chromatography and ultraviolet detection combined technique, and the results are shown in Table 6.
TABLE 6
As can be seen from the table, the DHA algae oil prepared by the method in the embodiment 1-3 has high extraction rate, high purity, simple process method and low cost, and is suitable for industrial application.
Examples 4 and 5 compare with example 3, the compound enzyme is cellulase and pectase with a mass ratio of 6.5:2.5 or the compound enzyme is hemicellulase and pectase with a mass ratio of 6.5:2.5. Comparative example 1 in comparison with example 3, the complex enzyme in step S1 was pectase. In comparative example 2, in contrast to example 3, no complex enzyme was added in step S1. The extraction rate is obviously reduced, and the sensory score is reduced. In the invention, compound enzymes including cellulase, hemicellulase and pectase are adopted, wherein the cellulase and pectase can hydrolyze and destroy cell walls of microalgae cells, and the hemicellulase can hydrolyze polysaccharides (except cellulose and pectic substances) forming plant cell walls, so that the microalgae cells can destroy and hydrolyze firmly, and the dissolution of polysaccharide, protein, vitamin, DHA and other fatty acids in the cells is promoted, so that the extraction rate of DHA is greatly improved.
Examples 6 and 7 compare with example 3, the complex lipase was a single Mucor miehei lipase or a Candida rugosa lipase. In comparative example 7, in contrast to example 3, no complex lipase was added in step S3. Comparative example 6 compared with example 3, polydopamine modification was not performed in step S3. Comparative example 8 in comparison with example 3, steps S3 and S4 were not performed. The purity and the extraction rate are obviously reduced, and the sensory components are reduced. The immobilized lipase is characterized in that the immobilized lipase is prepared by modifying the prepared magnetic particles with polydopamine, and the polyhydroxy, polyamino and polycarboxylic on the surface of the immobilized lipase can well form hydrogen bonds with amino, carboxyl and the like on the lipase, so that the immobilized lipase is stable.
Comparative examples 3 and 4 in comparison with example 3, the activated bacillus subtilis seed solution or the activated bacillus licheniformis seed solution was not inoculated in step S1. Comparative example 5 in comparison with example 3, the activated bacillus subtilis seed solution and the activated bacillus licheniformis seed solution were not inoculated in step S1. The extraction rate is obviously reduced, and the sensory score is reduced. Under the fermentation action of the microorganism bacillus subtilis and the bacillus licheniformis, the microorganism generates a large amount of enzymes and the microorganism degradation action is used for carrying out enzymolysis on viscous high molecular weight polysaccharide, complex proteins and the like, so that the dissolution of fatty acids coated or nearby the viscous high molecular weight polysaccharide is promoted, the extraction rate of DHA is greatly improved, and meanwhile, the fermentation technology also reduces the smell of DHA and improves the taste and color of DHA.
Comparative example 9 compared to example 3, step S5 was not performed. The purity is obviously reduced, and the sensory score is reduced. According to the invention, after the prepared enzymolysis filtrate is distilled by a molecular distillation technology, most of other hydrolyzed fatty acids are distilled at a lower temperature, so that the purity of DHA is further improved, DHA algae oil is obtained, the high-temperature limitation of the traditional deodorization process is overcome, and the quality of the produced DHA product is higher.
Comparative examples 10 and 11 in comparison with example 3, no soy protein isolate or casein powder was added in step S6. Comparative example 12 compared to example 3, no fermentation was performed in step S6. The sensory component is reduced, the peroxide value is increased, and the stability is reduced. The antioxidant active peptide has the characteristics of low toxicity, high efficiency and the like, is a safe and effective food antioxidant additive, has good in vitro free radical removal and reduction capacity, omits the steps of enzyme separation and purification by a fermentation method in soy protein isolate and casein powder, reduces production procedures and cost, and meanwhile, the terminal peptidase produced by microorganisms has a modification effect on the tail end of small peptide, so that the prepared peptide has no bitter taste, has natural fragrance of a certain fermented product, has good palatability, and further avoids the rejection of infants to the product and has high acceptance.
In comparative examples 13 and 14, no antioxidant protein peptide or algal polysaccharide was added in step S7, as compared with example 3. Comparative example 15 in comparison with example 3, no antioxidant protein peptide and algal polysaccharide were added in step S7. The sensory component is reduced, the peroxide value is increased, and the stability is reduced. The antioxidant active peptide has the characteristics of low toxicity, high efficiency and the like, is a safe and effective food antioxidant additive, has good in vitro free radical removal and reduction capacity, omits the steps of enzyme separation and purification by a fermentation method in soy protein isolate and casein powder, reduces production procedures and cost, and meanwhile, the terminal peptidase produced by microorganisms has a modification effect on the tail end of small peptide, so that the prepared peptide has no bitter taste, has natural fragrance of a certain fermented product, has good palatability, and further avoids the rejection of infants to the product and has high acceptance. The algal polysaccharide is also a high-efficiency antioxidant, and in microalgae, the polysaccharide content is high, the nutrition is rich, the algal polysaccharide is a high-content byproduct in the DHA extraction process, and has good anti-inflammatory, antioxidant, antitumor and other activities, and meanwhile, the molecular weight of the polysaccharide prepared by fermentation is reduced, so that the infant has better tolerance to the polysaccharide, better absorbability and good health care and DHA oxidation prevention effects. The antioxidant protein peptide and the algal polysaccharide have a synergistic effect, and can play a role in efficient antioxidant.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (2)
1. The process for extracting DHA based on microalgae extraction is characterized by comprising the following steps:
S1, enzyme-assisted fermentation of composite algae: drying 10-12 parts by weight of schizochytrium limacinum, 5-7 parts by weight of dinoflagellate and the like, crushing to obtain dry powder, adding 10-15 parts by weight of dry powder into 100-120 parts by weight of water, adding 1-2 parts by weight of complex enzyme, carrying out enzymolysis for 1-3 hours at 40-45 ℃, sterilizing, inoculating activated bacillus subtilis strain seed liquid and activated bacillus licheniformis strain seed liquid, wherein the inoculum sizes are respectively 2-3v/v% and 1-2v/v%, the inoculum size is 10 8-109 cfu/mL, the temperature is 40-42 ℃, the speed is 50-70r/min, carrying out enzyme-assisted fermentation for 36-48 hours, filtering to obtain a fermentation product, and drying filter residues to be used as feed;
The compound enzyme is cellulase, hemicellulase and pectase, and the mass ratio is 3-5:2-3:2-3;
S2, alcohol precipitation: adding ethanol into the fermentation product obtained in the step S1 until the ethanol content of the system is 70-80wt%, standing and precipitating for 6-8h, and filtering to obtain algal polysaccharide; standing and layering the filtrate for 1-3h, collecting organic phase to obtain DHA-rich crude oil, and freeze-drying the water phase to obtain algae nutrient substances as nutritional food;
S3, preparing immobilized lipase: dissolving 16-16.4 parts by weight of ferric chloride and 12.5-12.7 parts by weight of ferrous chloride in 100 parts by weight of water, adding 10 parts by weight of 22-25wt% ammonia water under the protection of nitrogen, heating to 50-60 ℃, stirring and reacting for 3-5 hours, centrifuging to obtain magnetic ferroferric oxide particles, adding 15-20 parts by weight of magnetic ferroferric oxide particles into 100 parts by weight of water, adding 17-22 parts by weight of dopamine hydrochloride and 2-3 parts by weight of catalyst, heating to 40-45 ℃, stirring and reacting for 3-4 hours, centrifuging, washing, drying to obtain modified magnetic ferroferric oxide particles, adding 10 parts by weight of modified magnetic ferroferric oxide particles into 100 parts by weight of water, adding 3-5 parts by weight of composite lipase, stirring and reacting for 30-40 minutes, volatilizing a solvent to obtain immobilized lipase;
the catalyst is Tris-HCl solution with pH=8.5-9;
the composite lipase is a mixture of Mucor miehei lipase and candida rugosa lipase, and the mass ratio is 5-7:10;
S4, lipase catalytic reaction: adding 5-7 parts by weight of the immobilized lipase prepared in the step S3 into 100 parts by weight of the DHA-rich crude oil prepared in the step S2, heating to 35-40 ℃, stirring for enzymolysis for 2-4 hours, separating by a magnet, and recovering the immobilized lipase to obtain enzymolysis filtrate;
s5, molecular distillation and purification: adding the enzymolysis filtrate obtained in the step S4 into a molecular distillation device, heating and distilling, and collecting non-distilled substances to obtain DHA algae oil;
The molecular distillation conditions are that the preheating temperature of the feed is 27-32 ℃, the condensed water is 20-30 ℃, the system operation pressure is 0.2-0.4Pa, the distillation temperature is 235-245 ℃, the feeding rate is 1.5-2.5mL/min, and the rotating speed of the film scraping device is 100-200r/min;
S6, preparing antioxidant protein peptide: adding 15-22 parts by weight of protein powder into 100 parts by weight of water, sterilizing, inoculating activated lactobacillus bulgaricus and lactobacillus helveticus strain seed solutions, wherein the inoculum sizes are respectively 3-4v/v% and 1-2v/v%, the bacterial content of the strain seed solutions is 10 8-109 cfu/mL,36-39 ℃ and 50-70r/min, fermenting for 48-60h, separating, and freeze-drying to obtain antioxidant protein peptide;
The protein powder is soybean protein isolate and casein powder, and the mass ratio is 10-12:7-10;
S7, embedding: dissolving 25-30 parts by weight of whey protein isolate in 200 parts by weight of water, adding 3-5 parts by weight of algal polysaccharide prepared in the step S2, 5-7 parts by weight of antioxidant protein peptide prepared in the step S6 and 17-22 parts by weight of DHA algae oil prepared in the step S5, homogenizing for 5-10min with 12000-13000r/min, adding 17-25 parts by weight of acacia, uniformly mixing, regulating the pH value of the solution to 3.7-3.8, stirring and reacting for 30-50min, cooling at 2-5 ℃, settling the microcapsule for 1-2h, centrifuging, washing and drying to obtain the DHA embedded microcapsule.
2. A DHA-embedded microcapsule prepared by the microalgae extraction-based process of claim 1.
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