阅读说明：本技术 一种从墨角藻中综合提取岩藻黄质和海藻多糖的方法 (Method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus ) 是由 田军 刘青 卢伊娜 于 2020-12-30 设计创作，主要内容包括：本发明属于海洋天然产物提取技术领域,具体是指一种可以充分提取墨角藻中的海藻多糖和岩藻黄质的工艺,以便使墨角藻的经济价值最大化。亦可适用于其他种类的褐藻综合提取岩藻黄质和岩藻聚糖硫酸酯、生物糖胶等海藻多糖。本发明所公开的一种从墨角藻中综合提取岩藻黄质和海藻多糖的方法包括以下步骤(1)破壁；(2)提取岩藻黄质；(3)提取海藻多糖；(4)海藻多糖粗产品提纯。本发明具有操作简单,岩藻黄质和岩藻聚糖硫酸酯的得率及纯度较高等优点。(The invention belongs to the technical field of marine natural product extraction, and particularly relates to a process capable of fully extracting algal polysaccharides and fucoxanthin in Fucus vesiculosus, so as to maximize the economic value of the Fucus vesiculosus. It can also be used for extracting fucoxanthin, fucoidan, biological carbohydrate gum, etc. from other brown algae. The invention discloses a method for comprehensively extracting fucoxanthin and algal polysaccharide from Fucus vesiculosus, which comprises the following steps of (1) breaking the wall; (2) extracting fucoxanthin; (3) extracting algal polysaccharide; (4) and (5) purifying the crude seaweed polysaccharide product. The method has the advantages of simple operation, higher yield and purity of fucoxanthin and fucoidan sulfate, and the like.)

1. A method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus is characterized by comprising the following steps:

(1) taking all fronds of fresh Fucus vesiculosus containing sporophyll, drying, pulverizing, sieving, soaking and washing with calcium bicarbonate water solution, taking out Fucus vesiculosus, spreading in a container, freezing and hardening at a temperature below 0 deg.C, and thawing by microwave; then, the container is placed at the temperature below 0 ℃ again, the container is unfrozen by a microwave oven after freezing and hardening, so that the cell wall of the fucus is broken, and then the water is drained for later use;

(2) extracting fucoxanthin: placing the fucus processed in the step (1) in a container, adding a large amount of absolute ethyl alcohol, placing in a constant temperature environment to keep the temperature, uniformly mixing and fully stirring, carrying out suction filtration on the mixture, adding absolute ethyl alcohol again into the solid phase object after suction filtration under the same condition to carry out suction filtration extraction once, mixing the liquid phases obtained by two extractions together, placing in a centrifugal machine for centrifugation, removing the solid phase object, and carrying out rotary evaporation on the liquid phase obtained after centrifugation to obtain a fucoxanthin liquid concentrated solution; then further purifying the fucoxanthin liquid concentrated solution to obtain fucoxanthin;

(3) extracting fucosan sulfate: mixing the solid phase substances obtained by suction filtration and centrifugation in the step (2), adding a solution containing calcium ions, fully stirring, heating for a certain time, cooling to room temperature, centrifuging, retaining liquid phase supernatant, and discarding the solid phase substances. Evaporating and concentrating the supernatant, adding absolute ethyl alcohol until the solution generates flocculent precipitate, and removing the liquid phase to obtain precipitate which is the crude product of algal polysaccharide;

(4) and (3) purifying the crude algal polysaccharide, namely adding a small amount of deionized water into the crude algal polysaccharide obtained in the step (3) to completely dissolve the crude algal polysaccharide, and then purifying to obtain the purified fucosan sulfate.

2. The method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus of claim 1, wherein the step (2) of further purifying the fucoxanthin concentrate comprises the following steps: heating silica gel, pouring petroleum ether, standing for more than 24 hours, and adding the treated silica gel into a chromatographic column; pouring the fucoxanthin concentrated solution after rotary evaporation into the fucoxanthin collecting tank, eluting with petroleum ether-ethyl acetate mixed liquid, collecting fucoxanthin eluent, finally, performing rotary evaporation and freeze drying on the fucoxanthin eluent, and volatilizing a solvent to obtain fucoxanthin.

3. The method of claim 1, wherein the further purification of the fucoxanthin concentrate comprises the following steps: adding deionized water into the rotary evaporated fucoxanthin liquid concentrated solution, diluting the concentration of ethanol to 40-50%, standing at 4-8 ℃, centrifuging, and removing a liquid phase to obtain a precipitate which is a fucoxanthin crude product; dissolving the fucoxanthin crude product by using high-concentration ethanol, then adding nonpolar macroporous resin into the liquid phase, and stirring and adsorbing; concentrating the liquid phase after resin adsorption and purification, standing at 4-8 deg.C, centrifuging, and removing liquid phase to obtain fucoxanthin as solid phase.

4. The method of claim 3, wherein the further purification of the fucoxanthin concentrate comprises the following steps: adding the nonpolar macroporous resin into the liquid phase in a weight ratio of 3-5%; and concentrating the liquid phase after the resin adsorption and purification to 45-60% of the original volume.

5. The method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus according to claim 1, wherein the solution containing calcium ions in the step (3) is a calcium chloride aqueous solution with a mass concentration of 2-5%.

6. The method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus according to claim 1, wherein the solution containing calcium ions is added in the step (3) in such a ratio that the mass ratio of the solid phase obtained by suction filtration and centrifugation to the solution containing calcium ions is 1: 10 to 15.

7. The method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus of claim 1, wherein the step (4) of purifying crude algal polysaccharides comprises the following steps: adding absolute ethyl alcohol into the dissolved crude seaweed polysaccharide product solution until a little insoluble substances are generated in the solution, centrifuging, taking supernatant, adding a proper amount of ethyl alcohol into the supernatant to generate white precipitate in the solution, centrifuging, removing the supernatant, washing the precipitate with diethyl ether, and freeze-drying to obtain white powder, namely the purified fucosan sulfate.

8. The method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus of claim 1, wherein the step (4) of purifying crude algal polysaccharides comprises the following steps: adding an adsorbent into the dissolved crude algal polysaccharide product solution, and stirring and adsorbing to remove protein; carrying out ultrafiltration on the liquid phase adsorbed by the adsorbent; adding anhydrous ethanol into the ultrafiltered liquid phase, washing the precipitate with diethyl ether, and lyophilizing to obtain white powder as purified fucoidan sulfate.

9. The method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus of claim 8, wherein the step (4) of purifying crude algal polysaccharides comprises the following steps: the adsorbent added into the dissolved crude algal polysaccharide product solution is polyamide, and the ultrafiltration membrane with the molecular weight cut-off of 3k is selected as the ultrafiltration membrane.

10. The method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus according to claim 1, wherein the crushed particle size of Fucus vesiculosus in step (1) is 50-70 mesh, the centrifugation condition is 5000r/min, 10-15min, before being sent into a microwave oven, the Fucus vesiculosus is spread on a tray with the thickness of the spread layer not exceeding 3 cm, and the microwave thawing temperature is less than 25 ℃.

11. The method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus according to claim 1, wherein the concentration of said calcium bicarbonate aqueous solution in said step (1) is 5-10%.

12. The method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus according to claim 1, wherein the mass ratio of the absolute ethyl alcohol to the Fucus vesiculosus powder in the step (2) is 20: 1-30: 1, the rotation speed during centrifugation is 8000r/min, the centrifugation is 10min, and the water bath temperature is 35-45 ℃.

13. The method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus of claim 2, wherein the step (2) of further purifying the fucoxanthin concentrate comprises the following steps: concentrating the volume of the fucoxanthin liquid concentrate to below 1/4 of the centrifugate, heating the silica gel for 1h at 120 ℃, wherein the volume ratio of the petroleum ether-ethyl acetate mixed liquid is 2: 3.

Technical Field

The invention belongs to the technical field of marine natural product extraction, and particularly relates to a method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus.

Background

Fucus vesiculosus (Latin's name Fucus vesiculosus) is a brown algae growing on rocks attached to the shore. Its algal body is black brown, bifurcate branch, and is a flat angular leaf. Most are distributed in tropical and subtropical oceans, and some are distributed in cold zones. Is distributed in coastal areas such as Liaoning, Shandong, etc. The compound leaves of Fucus vesiculosus are covered with a layer of mucus, which can prevent the seaweed from dying when the sea tide subsides. The algae is perennial, has two branches and no central main axis. Seminal vesicles and oocysts grow in the fossa genitalis (or sporophyll) on the enlarged pessary at the top of the algae. The base part is provided with a disk-shaped fixer and a flat belt-shaped blade, a middle rib which is obviously and longitudinally arranged on the blade belt from the base part to the top end, and a pair of air bags are usually arranged on the blade belt at two sides of the middle rib so as to be beneficial to floating.

Fucus vesiculosus contains fucoidan (fucoidan, also known as fucoidan and fucoidan), fucoxanthin (fucoxanthin), and other components beneficial to the human body. The sodium alginate contained in the fucus extract has strong binding capacity with strontium and cadmium with carcinogenic effect, and the sodium alginate and the strontium and the cadmium are discharged out of the body, so that the fucus extract has a certain cancer prevention effect, and particularly has a huge application prospect in improving the health level of people in cadmium-polluted areas.

Research shows that the fucosan sulfate has obvious tumor inhibiting effect, multiple biological activities of anticoagulation, blood fat reduction, virus resistance, organism immunity enhancement and the like, is a hot spot of current research on marine functional foods and medicines, and fucosan sulfate powder and drink containing the fucosan sulfate are sold in markets at home and abroad. Fucosan sulfate, as a water-soluble natural polymer compound, can be used as a thickening agent, a dispersing agent, an emulsifying agent, a stabilizing agent, a binding agent, a film-forming agent, a gelling agent and an ion exchanger, and is widely applied to various aspects of industrial and agricultural production.

The common fucosan sulfate extraction method comprises the following steps: water extraction, enzymolysis, acid extraction and ultrasonic extraction, which have the following advantages and disadvantages: the water extraction method can avoid the degradation of fucoidan sulfate, is a common extraction method, but has low yield; the enzymolysis method has mild conditions, has little influence on the structure of the fucoidan sulfate, can also improve the yield, but has higher cost and limits the application of the fucoidan sulfate; the acid extraction method can improve the yield of the fucoidan, but can obviously degrade fucoidan and influence the biological activity of the fucoidan; the ultrasonic extraction method can improve the yield of the fucoidan sulfate, but the degradation of the fucoidan sulfate can be caused by the ultrasonic extraction method for a long time.

The research finds that fucoidan sulfate and other substances are mainly present in the mucus matrix of undaria pinnatifida sporophyll. If firstSeparating spore leaf mucus, and extracting bioactive substances such as fucoidan sulfate from the mucus, so as to reduce enzyme preparation dosage and reduce extraction cost of active substances such as fucoidan sulfate. The prior art discloses the use of NaOH and Na₂CO₃The composite liquid is used for treating the brown algae sporophyll to achieve the effect of separating mucus. However, fucoidan sulfate is less stable in alkaline environment, and the use of alkaline extract results in the loss of fucoidan sulfate contained in the raw material.

Fucoxanthin, another beneficial ingredient contained in the fucus is fat-soluble pigment which is easily soluble in a non-polar organic solvent, and has poor heat resistance and poor stability in an acidic environment. Currently, non-polar solution extraction is usually employed. Although reports have shown that supercritical fluid extraction with carbon dioxide can be used. QuitaINAT and the like extract fucoxanthin from undaria pinnatifida by supercritical extraction for 180min at 40 ℃ and 40MPa, and the recovery rate is 80 percent. However, the method takes long time and has strict process requirements, which can cause the production cost to rise greatly. At the current market price level of fucoxanthin, the technology has no industrial application value.

Fucoidan sulfate is not alkali-resistant, fucoxanthin is not acid-resistant; and the two are not temperature-resistant, the temperature resistance of fucoxanthin is inferior to that of fucoidan sulfate, and the fucoxanthin is easy to decompose under the illumination condition. The prior art usually only extracts fucosan sulfuric acid or fucoxanthin from Fucus vesiculosus, and the prior art lacks an extraction process which can extract fucosan sulfuric acid ester and fucoxanthin contained in Fucus vesiculosus. In addition, the prior art usually only extracts the effective components in the fucus vesiculosus spore leaves, and the effective components contained in other parts of the fucus vesiculosus are difficult to extract.

Disclosure of Invention

In order to solve the problems, the invention provides a process for fully extracting fucoxanthin and fucoxanthin from Fucus vesiculosus, so as to maximize the economic value of Fucus vesiculosus. The method can also be used for extracting fucoxanthin and algal polysaccharides from other brown algae.

In order to realize the functions, the technical scheme adopted by the invention is as follows: a method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus comprises the following steps:

(1) wall breaking: taking all fronds of fresh Fucus vesiculosus containing sporophyll, drying, pulverizing, sieving, soaking and washing with calcium bicarbonate water solution, taking out Fucus vesiculosus, spreading in a container, freezing and hardening at a temperature below 0 deg.C, and thawing by microwave; and then, the container is placed at the temperature below 0 ℃ again, the container is unfrozen by a microwave oven after freezing hardening, the cell wall of the fucus is broken, and then the water is drained for standby.

(2) Extracting fucoxanthin: placing the fucus processed in the step (1) in a container, adding a large amount of absolute ethyl alcohol, placing in a constant temperature environment to keep the temperature, uniformly mixing and fully stirring, carrying out suction filtration on the mixture, adding absolute ethyl alcohol again into the solid phase object after suction filtration under the same condition to carry out suction filtration extraction once, mixing the liquid phases obtained by two times of extraction together, placing in a centrifugal machine for centrifugation, removing the solid phase object, and carrying out rotary evaporation on the liquid phase obtained after centrifugation to obtain the fucoxanthin liquid concentrated solution.

(2-a) purification of fucoxanthin method a. Heating silica gel, pouring petroleum ether, standing for more than 24 hours, and adding the treated silica gel into a chromatographic column. Pouring the concentrated solution of fucoxanthin after rotary evaporation into the solution, eluting with petroleum ether-ethyl acetate mixed solution, collecting fucoxanthin eluent, finally, carrying out rotary evaporation and freeze drying on the fucoxanthin eluent, and volatilizing a solvent to obtain fucoxanthin.

(2-b) purification of fucoxanthin method b. Adding deionized water into the rotary evaporated fucoxanthin liquid concentrate, diluting the concentration of ethanol to 40-50%, standing at 4-8 deg.C for a period of time, centrifuging, and removing large polar impurities along with the liquid phase to obtain a precipitate which is fucoxanthin crude product. Dissolving fucoxanthin crude product with high concentration ethanol, adding 3-5% (by weight) nonpolar macroporous resin into the liquid phase, and stirring to adsorb small polar impurities (such as esters). Concentrating the liquid phase after resin adsorption and purification to 45-60% of the original volume, standing at 4-8 deg.C for a period of time, centrifuging, removing the liquid phase, and obtaining fucoxanthin as the solid phase.

(3) Extracting fucosan sulfate: mixing the solid phase substances obtained by suction filtration and centrifugation in the step (2), adding a solution containing calcium ions, fully stirring, heating for a certain time, cooling to room temperature, centrifuging, retaining the liquid phase supernatant, and discarding the solid phase substances. Evaporating and concentrating the supernatant, adding absolute ethyl alcohol until the solution generates flocculent precipitate, and removing the liquid phase to obtain precipitate which is the crude product of algal polysaccharide;

(4) purifying the crude seaweed polysaccharide product: and (4) adding a small amount of deionized water into the crude algal polysaccharide product obtained in the step (3) to completely dissolve the crude algal polysaccharide product.

(4-a) adding absolute ethyl alcohol into the dissolved crude algal polysaccharide product solution until a little insoluble substances are generated in the solution, centrifuging, taking supernatant, adding a proper amount of ethyl alcohol into the supernatant, generating white precipitate in the solution, centrifuging, removing the supernatant, washing the precipitate with diethyl ether, and freeze-drying to obtain white powder, namely the purified fucosan sulfate.

(4-b) adding polyamide into the dissolved crude algal polysaccharide product solution, and stirring and adsorbing to remove protein; and (3) carrying out ultrafiltration on the liquid phase after the polyamide adsorption to remove residual salt and small molecular impurities. Adding anhydrous ethanol into the ultrafiltered retentate, washing the precipitate with diethyl ether, and lyophilizing to obtain white powder as purified fucoidan sulfate.

(5) Determination of fucoxanthin purity: adopting high performance liquid chromatography, preparing solutions with different concentrations by using fucoxanthin standard solution, measuring at a wavelength of 450nm, and drawing a standard curve; and (3) taking the fucoxanthin sample obtained in the step (2), adding an organic solvent to dissolve the fucoxanthin sample, measuring at the wavelength of 450nm, and calculating the content of the fucoxanthin in the sample.

(6) And (3) measuring the purity of the algal polysaccharide: the content of polysaccharide is determined by a phenol-sulfuric acid method, and the ultraviolet detection wavelength is 490 nm. Taking 10mg of L-fucose as a standard substance, placing the standard substance in a 25ml volumetric flask to prepare a trehalose standard solution, preparing solutions with different concentrations by using the L-fucose standard solution, and drawing an L-fucose absorbance standard curve at the UV 490nm wavelength. And (4) taking the purified fucoidan sulfate sample obtained in the step (4), adding a certain amount of water for dissolving, and calculating the content of algal polysaccharide in the sample after measuring the absorbance of the solution under the UV 490nm wavelength.

Further, the size of the pre-crushed Fucus vesiculosus in the step (1) is 50-70 meshes.

Further, in the step (1), calcium bicarbonate water solution with the concentration of 5-10% is adopted for soaking and washing.

Further, the microwave thawing temperature in step (1) is <40 ℃. The container is a flat-bottomed petri dish or a plastic tray.

Further, the water drained in the step (1) adopts a centrifugal method, the centrifugal condition is 3000-,

further, the weight ratio of the absolute ethyl alcohol to the fucus in the step (2) is 20: 1-60: 1, the rotation speed during centrifugation is 8000r/min, the centrifugation is 10min, and the water bath temperature is 30-50 ℃.

Further, in the step (2-a), the volume of the fucoxanthin concentrate in the step (2-a) is 1/5 of the centrifugate, the silica gel is heated for 1h at the temperature of 120 ℃, and the volume ratio of the petroleum ether-ethyl acetate mixed liquor is 2: 3.

Further, the process of the step (2-a) of rotary steaming requires shading.

Further, the mass ratio of the filter residue to the calcium-containing ion solution in the step (3) is 1:15, and the calcium-containing ion solution is an aqueous solution containing more than 2% of calcium chloride.

Further, the water bath temperature in the step (3) is 98 ℃, the extraction time is 3h, and the centrifugation condition is 5000r/min and 15 min; adding absolute ethyl alcohol twice, wherein the absolute ethyl alcohol is added for the first time until the mass fraction of the ethyl alcohol in the solution is 20%, and the absolute ethyl alcohol is added for the second time until the mass fraction of the ethyl alcohol in the solution is more than 60%.

Further, the mass ratio of the crude seaweed polysaccharide product to the deionized water in the step (4) is 1: 5.

Further, the step (4-a) is to add absolute ethyl alcohol until the weight content of the ethyl alcohol in the solution is 30-70%.

Further, the weight of the polyamide added in the step (4-b) is more than 2 percent of the total weight of the liquid.

Further, the ultrafiltration cut-off of the step (4-b) was 3 k.

Compared with the prior art, the invention has the advantages and beneficial effects that: 1. the raw materials are fully utilized. The invention can take all the fronds of the Fucus vesiculosus as the extraction raw material, thereby avoiding the condition that the prior art only adopts the sporophyll but not all the fronds, which causes the waste of a large amount of raw materials; two beneficial components in the fucus can be extracted together, wherein the yield of fucoxanthin is over 74 percent, and the yield of fucoidan sulfate is over 85 percent; 2. compared with a simple method for extracting fucoxanthin, the method can economically and rapidly extract fucoxanthin, avoids the damage to fucosan sulfate in the extraction process, can continuously and fully extract fucosan sulfate, and improves the comprehensive utilization rate and economic benefit of raw materials; 3. the fucoxanthin and algal polysaccharide extracts obtained by the invention have higher purity: the silica gel activation and pretreatment method of the invention ensures that the purity of the purified fucoxanthin is not less than 90 percent (HPLC 450nm wavelength detection); the fucoidin is purified by combining ethanol precipitation and polyamide adsorption with ultrafiltration, the content of the fucoidin is not lower than 95% (detected by UV 490nm wavelength), and compared with the prior extraction technology, the method is obviously improved; 4. the method has the advantages of high extraction speed, high speed, low toxicity of the used extraction solvent, economy and environmental protection, avoids environmental pollution caused by adopting a large amount of resin in the prior art, effectively reduces the production cost and increases the enterprise profit, and simultaneously obtains the high-quality fucoxanthin and fucoidan, thereby meeting different market demands and having stronger advancement.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The first embodiment is as follows:

the embodiment of the invention provides a method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus, which comprises the following steps:

(1) wall breaking: taking all alga bodies of fresh fucus including sporophyll, drying, crushing into granules, sieving by a 50-mesh sieve, soaking fine powder passing through the sieve pores in 5-10% calcium bicarbonate water solution for 1 hour to wash and remove impurities on the surface, centrifuging (5000r/min, 10min) to remove water on the surface of the fucus, taking out the fucus, flatly paving the fucus in a plastic tray, wherein the thickness of a layer is not more than 3 cm, and putting the plastic tray in a refrigerator below-20 ℃ for more than 30 minutes to completely freeze and freeze the alga bodies; the plastic trays were then introduced into a microwave oven using low power continuous radiation for about 3 minutes to completely thaw and to control the thawing temperature to not exceed 25 ℃. Repeating the freezing and microwave thawing steps for 2 times to break cell wall and obtain Fucus vesiculosus powder.

(2) Extracting fucoxanthin: weighing the fucus vesiculosus powder obtained in the step (1) into an extraction tank, adding aqueous ethanol, wherein the mass ratio of the anhydrous ethanol to the fucus vesiculosus powder is 20: 1; placing the mixture in a constant-temperature water bath kettle, heating at the constant temperature of 40 +/-5 ℃, stirring for 60 minutes by using an electric stirrer, and performing suction filtration on the mixture; adding absolute ethyl alcohol (solid-liquid mass: 1: 20) into the residual filter residue after suction filtration under the same condition, stirring in a water bath at 40 +/-5 ℃ for 60 minutes, extracting, and carrying out suction filtration once; mixing the liquid phases obtained by the two suction filtration and extraction, and centrifuging in a centrifuge (the rotation speed is 8000r/min, and the centrifugation time is 10 min); and (4) carrying out rotary evaporation on the supernatant obtained by centrifugation until the volume of the centrifugate is reduced to below the original volume of 1/5, thus obtaining the fucoxanthin liquid concentrate.

(3) And (3) fucoxanthin purification: heating silica gel at the temperature of 120 ℃ for 1 hour, and then mixing the silica gel with the water according to the mass ratio of 1:1, adding petroleum ether, standing for 24 hours, and naturally cooling; adding the treated silica gel into a chromatographic column, draining petroleum ether, pouring the fucoxanthin concentrate obtained by rotary evaporation and concentration into the chromatographic column, eluting with a petroleum ether-ethyl acetate mixed solution (the volume ratio of petroleum ether to ethyl acetate is 2: 3), and collecting fucoxanthin eluent. And finally, carrying out rotary evaporation on the fucoxanthin eluent in a shading environment, fully volatilizing the solvent, and freeze-drying to obtain the purified fucoxanthin.

(4) Extracting algal polysaccharide: adding 2% calcium chloride aqueous solution into the filter residue obtained after twice suction filtration in the step (2), wherein the mass ratio of the filter residue to the calcium chloride aqueous solution is 1: heating in a water bath at the temperature of 10-15 ℃ and stirring for 180 minutes, standing and cooling to room temperature, centrifuging (5000r/min, 15min) for separation, taking supernatant, adding absolute ethyl alcohol into the supernatant until the content of the ethanol is more than 40%, wherein the solution generates flocculent precipitates, centrifuging again (5000r/min, 15min), and removing a liquid phase to obtain precipitates, namely crude seaweed polysaccharide products;

(5) purifying the crude seaweed polysaccharide product: adding deionized water into the crude algal polysaccharide product for dissolving, wherein the mass ratio of the crude algal polysaccharide product to the deionized water is 1:5, adding absolute ethyl alcohol until the content of the ethyl alcohol reaches 30 percent, and generating a little insoluble substances in the solution; centrifuging (5000r/min, 15min) to remove precipitate, collecting supernatant, adding anhydrous ethanol until ethanol content is no more than 70%, generating white precipitate in the solution, centrifuging, removing supernatant, washing precipitate with anhydrous diethyl ether, and freeze drying to obtain white powder as algal polysaccharide;

(6) determination of fucoxanthin purity: and (2) putting 10mg of fucoxanthin standard substance into a 25ml volumetric flask to prepare a fucoxanthin standard solution, using 90% methanol (volume ratio, methanol: water is 90: 10) as a solvent, preparing solutions with different concentrations by using the fucoxanthin standard solution, performing HPLC analysis at the wavelength of 450nm, drawing a fucoxanthin standard curve, taking the sample obtained in the step (3), adding a fixed amount of solvent for dissolving, measuring at the wavelength of 450nm, and calculating the purity of the fucoxanthin in the sample to be 90.6%.

(7) And (3) measuring the purity of the algal polysaccharide: the polysaccharide content is determined by a phenol-sulfuric acid method, the phenol concentration is 5%, the polysaccharide is prepared before the test, and the sulfuric acid concentration is more than 95%. Taking 10mg of L-fucose as a standard substance, placing the standard substance in a 25ml volumetric flask to prepare a trehalose standard solution, preparing solutions with different concentrations by using the L-fucose standard solution, and drawing an L-fucose absorbance standard curve at the UV 490nm wavelength. And (4) taking the sample obtained in the step (5), adding a certain amount of water for dissolving, and calculating the purity of the algal polysaccharide in the sample to be 71.5% after measuring the absorbance of the solution at the wavelength of UV 490 nm.

Example two:

the embodiment of the invention provides a method for comprehensively extracting fucoxanthin and algal polysaccharides from Fucus vesiculosus, which comprises the following steps:

(1) wall breaking: wall breaking: taking all alga bodies of fresh fucus including sporophyll, drying, crushing, sieving by a 70-mesh sieve, soaking fine powder passing through the sieve mesh in 5-10% calcium bicarbonate water solution for 30 minutes, washing to remove impurities on the surface, centrifuging (5000r/min, 15min), draining water on the surface of the fucus, taking out the fucus, flatly paving the fucus in a glass surface dish, wherein the thickness of a paving layer is not more than 2 cm, and placing in an environment below zero degree to freeze the alga bodies until the alga bodies are completely frozen; and then, the glass watch glass is sent into a microwave oven to be radiated in a pulse mode with the power of 1000 watts, the microwave radiation time is 5 seconds every time, the interval is 10 seconds, and the unfreezing is completed after 2 to 3 minutes. Repeating the freezing and microwave thawing steps for 2 times to break cell wall and obtain Fucus vesiculosus powder.

(2) Extracting fucoxanthin: weighing the fucus vesiculosus powder obtained in the step (1) into an extraction tank, adding absolute ethyl alcohol, wherein the mass ratio of the absolute ethyl alcohol to the fucus vesiculosus powder is 30:1, placing the mixture in a constant-temperature water bath kettle, heating at the constant temperature of 40 +/-5 ℃, stirring by using an electric stirrer, carrying out suction filtration on the mixture, removing an extracting solution for later use, adding absolute ethyl alcohol into residual filter residues after suction filtration under the same condition, carrying out suction filtration extraction once, mixing the extracting solutions obtained in two times of extraction, placing the mixture in a centrifugal machine for centrifugation (8000r/min, 10min) to remove precipitates, and carrying out rotary evaporation on the centrifugate until the original volume of the centrifugate is below 1/4 to obtain the fucoxanthin liquid concentrated solution.

(3) Purifying fucoxanthin. Adding deionized water into the rotary evaporated fucoxanthin liquid concentrate, diluting the concentration of ethanol to 40-50%, standing at 4-8 deg.C for 20 min, centrifuging, and removing large polar impurities with liquid phase to obtain fucoxanthin crude product as precipitate; dissolving fucoxanthin crude product with high-concentration ethanol in a shading environment, adding 3-5% (by weight) of styrene-divinylbenzene polymer resin into the liquid phase, and stirring to remove small polar impurities such as esters by adsorption; concentrating the liquid phase after resin adsorption and purification to 50% of the original volume, standing at 4-8 deg.C for 30 min, centrifuging (8000r/min, 15min), and removing the liquid phase to obtain solid phase of fucoxanthin.

(4) Extracting algal polysaccharide: adding 5% calcium chloride aqueous solution into the filter residue subjected to twice suction filtration in the step (2), wherein the mass ratio of the filter residue to the calcium chloride aqueous solution is 1: heating in a water bath at the temperature of 10-15 ℃, stirring for 120 minutes, standing and cooling to room temperature, centrifuging (5000r/min, 15 minutes) for separation, taking supernate, adding absolute ethyl alcohol twice, adding absolute ethyl alcohol for the first time until the mass fraction of the ethyl alcohol in the solution is 20%, standing for 5 minutes, and adding absolute ethyl alcohol again until the mass fraction of the ethyl alcohol in the solution is more than 60%; centrifuging (5000r/min, 15min), and separating to remove liquid phase to obtain precipitate as crude product of algal polysaccharide.

(5) Purifying the crude seaweed polysaccharide product: and (3) adding deionized water into the crude algal polysaccharide obtained in the step (4) for dissolving, wherein the mass ratio of the crude algal polysaccharide to the deionized water is 1: adding not less than 2 percent (weight ratio) of polyamide-6 into the dissolved crude algal polysaccharide solution, stirring for 30 minutes to adsorb and remove proteins, and then removing residual salt and small molecular impurities from the liquid phase by using an ultrafiltration membrane with the molecular weight cutoff of 3 k; adding anhydrous ethanol into the ultrafiltered liquid phase until the ethanol content is 70%, allowing a large amount of precipitate to appear, centrifuging (5000r/min, 15min), separating, removing the liquid phase, and vacuum filtering and cleaning the obtained precipitate with diethyl ether for 3 times, wherein the mass ratio of diethyl ether to precipitate is 6: and 1, freeze-drying the white precipitate obtained after cleaning to remove the solvent, thereby obtaining the purified fucosan sulfate.

(6) Measurement of fucoxanthin content: taking 90% methanol (volume ratio, methanol: water is 90: 10) as a solvent, placing 10mg fucoxanthin standard substance into a 25ml volumetric flask to prepare a fucoxanthin standard solution, using the fucoxanthin standard solution to prepare solutions with different concentrations, measuring by using HPLC at a wavelength of 450nm, drawing a fucoxanthin standard curve, taking the sample in the step (2), adding a certain amount of methanol for dissolution, and measuring at a wavelength of 450nm, wherein the purity of the fucoxanthin in the sample is 85.4%;

(7) and (3) measuring the content of seaweed polysaccharide: the polysaccharide content is determined by a phenol-sulfuric acid method, the phenol concentration is 5%, the polysaccharide is prepared before the test, and the sulfuric acid concentration is more than 95%. Taking 10mg of L-fucose as a standard substance, placing the standard substance in a 25ml volumetric flask to prepare a trehalose standard solution, preparing solutions with different concentrations by using the L-fucose standard solution, and drawing an L-fucose absorbance standard curve at the UV 490nm wavelength. And (4) adding a certain amount of water into the sample obtained in the step (4) for dissolving, and calculating the purity of the algal polysaccharide in the sample to be 96.5% after measuring the absorbance of the solution under the UV 490nm wavelength.

Comparative example one:

the present comparative example provides a method for extracting fucoxanthin from fucus. The method comprises the following steps:

(1) wall breaking: operation same as in step (1) of example one

(2) Extracting fucoxanthin: operation same as in step (2) of example one

(3) And (3) fucoxanthin purification: the difference from step (3) of the example is that the silica gel is not subjected to high temperature activation and petroleum ether pretreatment. Pouring the concentrated fucoxanthin solution into a silica gel column without any treatment, eluting with a petroleum ether-ethyl acetate mixed solution (the volume ratio of petroleum ether to ethyl acetate is 2: 3), and collecting fucoxanthin eluent. And finally, carrying out rotary evaporation on the fucoxanthin eluent in a shading environment, fully volatilizing the solvent, and freeze-drying to obtain the purified fucoxanthin.

(4) Determination of fucoxanthin purity: the procedure of step (6) of the example was performed, and the purity of fucoxanthin was 79.6%.

Examples of the experiments. And (5) measuring the extraction rate of fucoxanthin.

The fucus powder sample obtained in the step (1) of example 1 or example 2 is dried, 10 g of the dried fucus powder sample is taken, and 50 ml of absolute ethyl alcohol is added.

And (3) extracting the sample for more than 2 hours in a dark environment at the extraction temperature of not more than 45 ℃ by adopting a solid phase extractor. The clear extract after removal of the solids was weighed. Determining fucoxanthin content in the sample extract by high performance liquid chromatography, calculating to obtain theoretical fucoxanthin content according to formula I, and calculating to obtain fucoxanthin yield according to formula II (the yield is actual yield/theoretical yield, and the yield is actual yield/raw material quality).

Fucoxanthin theoretical content (100%; fucoxanthin content in extract).; extract mass)/sample mass (formula I)

Yield of 100% (. fucoxanthin content in product. product weight/raw material mass)/theoretical content of fucoxanthin (formula II)

TABLE 1 fucoxanthin yield calculation

	Example 1	Example 2
			Fucoxanthin content (%)	0.06	0.06
Extract quality (gram)	35.2	34.5
			Fucoxanthin content (%)	90.6	85.4
Product quality (gram)	3.0	1.8
			Raw material mass (kilogram)	1.5	1.0
Actual yield (%)	88.80	74.26

The results of the first example and the first comparative example show that the silica gel activation and pretreatment method can obviously improve the purity of fucoxanthin; the comparison of the first and second examples shows that the fucoidan purified by ethanol precipitation and polyamide adsorption combined with ultrafiltration has higher polysaccharide content.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.