Method for extracting fucoxanthine and polyunsaturated fatty acid from microalgae and extracted product

Abstract

The invention relates to the field of microalgae active component extraction, and discloses a method for extracting fucoxanthine and polyunsaturated fatty acid from microalgae and an extracted product thereof, the method comprises the following steps: S1, carrying out first extraction on the microalgae by using a low-concentration alcohol solution, and carrying out solid-liquid separation to obtain a first algae cake and a first extracting solution; and S2, carrying out second extraction on the first algae cake obtained in the step S1 by using a high-concentration alcohol solution, and carrying out solid-liquid separation to obtain a second algae cake and a second extracting solution. According to the method, the low-concentration alcoholic solution and the high-concentration alcoholic solution are matched for extraction, the method is simple, efficient, low in cost and suitable for large-scale production, the problem of residues of toxic solvents such as n-hexane is avoided, the alcoholic solution can be recycled, the proportion of the low-concentration alcoholic solution and the high-concentration alcoholic solution in a final finished product is close to that in natural microalgae cells, and the method is suitable for large-scale production. And the composition does not need to be compounded again.

Description

Technical Field

[0001] The present invention relates to the field of extraction of active components of microalgae, and in particular to a method for extracting fucoxanthin and polyunsaturated fatty acids from microalgae and the extracted products. Background Art

[0002] Fucoxanthin is a fat-soluble carotenoid that is insoluble in highly polar solvents such as water under neutral conditions, but readily soluble in non-polar organic solvents. Fucoxanthin is sensitive to light and heat and easily degrades. Currently, the most commonly used extraction methods are organic solvent extraction and supercritical extraction. Organic solvent extraction generally uses a single organic solvent, such as methanol, ethanol, acetone, or ethyl acetate, or a mixture of several solvents. While simple and easy to perform, it suffers from poor selectivity, high impurities in the extract, and the tendency for precipitation. This leads to poor product performance and prevents direct application in food, cosmetics, and other fields. Furthermore, residual organic solvents in some cases limit fucoxanthin's applications. Supercritical extraction uses carbon dioxide as the separation fluid, varying pressure and temperature to precipitate the target product, achieving separation and purification. However, this method requires high equipment requirements and is expensive, increasing production costs.

[0003] Polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA), are ω-3 fatty acids that are essential nutrients for the human body. Although the human body can convert EPA from linolenic acid, the conversion rate is very slow and the amount converted is very small, which cannot meet the human body's EPA needs and needs to be supplemented from food. Traditionally, fatty acids such as EPA or DHA can be obtained using polar and non-polar solvents or prepared using pressure-based methods. Polar solvents such as ethanol, ethyl acetate, and acetone can also be used to extract lipophilic components (such as fatty acids).

[0004] Research has shown that fucoxanthin has antioxidant, anti-obesity, metabolic regulation, anti-tumor, anti-inflammatory, immune-modulating, liver-protective, and neuroprotective effects. Eicosapentaenoic acid (EPA) plays an important role in cardiovascular health, anti-inflammatory and immune support, and brain and cognitive health. Both are important components that benefit human health.

[0005] Microalgae, especially diatoms, are rich in fucoxanthin and polyunsaturated fatty acids. A green, efficient, low-cost and industrializable extraction process is needed to fully utilize the nutrients in microalgae.

[0006] Existing research has found that fucoxanthin and fatty acids have significantly different polarities. Literature reports that the selectivity of fucoxanthin extracted using polar solvents is poor, and obtaining high-purity fucoxanthin requires complex purification processes. Kim et al. (2012) reported using n-hexane to remove lipids / fatty acids from ethanol extracts and found that fucoxanthin is virtually insoluble in n-hexane. Furthermore, most processes rely solely on extracting fucoxanthin and EPA separately using different solvents, resulting in complex process routes, making large-scale production on the same production line difficult and costly, making it unsuitable for industrialization.

[0007] For example, CN111286402A discloses a fucoxanthin-containing algae oil, an extraction method thereof, and a health food. In this method, fucoxanthin and EPA are extracted from fucoxanthin-containing algae using a weakly polar organic solvent (such as n-hexane) and a highly polar, fat-soluble organic solvent (such as ethyl acetate) in two steps. The solvents used in the two steps are different, and the second organic solvent cannot be ethanol. The process is complicated, pollutes the environment, and is costly. CN112876431A discloses a method for extracting fucoxanthin, fucoxanthol, and eicosapentaenoic acid from microalgae. In this method, algae mud is first incubated with inorganic salts and then extracted with liquid alcohol. An inorganic salt (such as ammonium sulfate)-liquid alcohol (such as tert-butanol) system is established to directly extract EPA, fucoxanthin, and fucoxanthin from the algae mud. The extraction rate of fucoxanthin is low, inorganic salts are used in the extraction process, and the process is complicated, which is not conducive to industrialization. Summary of the Invention

[0008] The purpose of the present invention is to overcome the problems of complex process, high cost, environmental pollution and low extraction rate in the prior art, and to provide a method for extracting fucoxanthin and polyunsaturated fatty acids from microalgae and the extracted products thereof. The method has the advantages of simple process, low extraction cost, avoidance of environmental pollution and improved extraction rate.

[0009] In order to achieve the above objectives, the present invention provides a method for extracting fucoxanthin and polyunsaturated fatty acids from microalgae in a first aspect, the method comprising:

[0010] S1. performing a first extraction on the microalgae using a low-concentration alcohol solution, and performing solid-liquid separation to obtain a first algae cake and a first extract;

[0011] S2, performing a second extraction on the first algae cake obtained in S1 using a high-concentration alcohol solution, and performing solid-liquid separation to obtain a second algae cake and a second extract;

[0012] The alcohol content in the first extraction system of step S1 is at least 20 vol% lower than the alcohol content in the second extraction system of step S2, and the alcohol in the low-concentration alcohol solution is the same as or different from the alcohol in the high-concentration alcohol solution.

[0013] A second aspect of the present invention provides an algae oil containing fucoxanthin and polyunsaturated fatty acids, wherein the fucoxanthin content of the algae oil is greater than 5 wt%, and the eicosapentaenoic acid content is greater than 15 wt%.

[0014] Preferably, the mass ratio of the prepared fucoxanthin to polyunsaturated fatty acids is 1:1-5, more preferably 1:2-4;

[0015] Preferably, the algae oil is prepared by the method described in the first aspect.

[0016] Through the above technical solution, the present invention can at least achieve the following beneficial effects:

[0017] (1) The method of the present invention uses a low-concentration alcohol solution and a high-concentration alcohol solution to extract fucoxanthin and polyunsaturated fatty acids from microalgae. The method is simple, efficient, low-cost, and suitable for large-scale production.

[0018] (2) The extraction solvents of the present invention are alcohol solutions of different concentrations, which avoids the problem of residual toxic solvents such as n-hexane.

[0019] (3) The method of the present invention is used to extract fucoxanthin and polyunsaturated fatty acids. The ratio of fucoxanthin and polyunsaturated fatty acids in the final product is close to that in natural microalgae cells, which is closer to nature. There is no need to re-mix them when using them, and there is little loss during the extraction process.

[0020] (4) The extraction solvent of the present invention is an alcohol solution of different concentrations, which can be recycled and reused, effectively avoiding environmental pollution during the extraction process. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 1 is a comparison diagram of the states of the algae oil prepared in Example 1 (right) and Comparative Example 1 (left) after treatment. DETAILED DESCRIPTION

[0022] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0023] A first aspect of the present invention provides a method for extracting fucoxanthin and polyunsaturated fatty acids from microalgae, the method comprising:

[0024] S1. performing a first extraction on the microalgae using a low-concentration alcohol solution, and performing solid-liquid separation to obtain a first algae cake and a first extract;

[0025] S2, performing a second extraction on the first algae cake obtained in S1 using a high-concentration alcohol solution, and performing solid-liquid separation to obtain a second algae cake and a second extract;

[0026] The alcohol content in the first extraction system of step S1 is at least 20 vol% lower than the alcohol content in the second extraction system of step S2, and the alcohol in the low-concentration alcohol solution is the same as or different from the alcohol in the high-concentration alcohol solution.

[0027] During their research, the inventors discovered that the present method, which utilizes a combination of low- and high-concentration alcohol solutions to extract fucoxanthin and polyunsaturated fatty acids from microalgae, is simple, efficient, low-cost, and suitable for large-scale production, avoiding the problem of residual toxic solvents such as n-hexane. Furthermore, by controlling the relative alcohol content in the low- and high-concentration alcohol solutions, the present invention achieves a specific ratio of fucoxanthin to polyunsaturated fatty acids in the final product, minimizing losses during the extraction process and enabling solvent recycling, effectively preventing environmental pollution during the extraction process.

[0028] It is understood that, in the present invention, the "first extraction system" and the "second extraction system" refer to systems composed of all liquid components, including but not limited to alcohol and water, etc. For example, the first and second extraction systems refer to liquid alcohol and water.

[0029] In the present invention, preferably, the alcohol content in the first extraction system of step S1 can be 15-50 vol% (for example, it can be a range formed by any two values of 15 vol%, 20 vol%, 25 vol%, 28 vol%, 30 vol%, 32 vol%, 35 vol%, 37 vol%, 40 vol%, 42 vol%, 45 vol%, 48 vol%, 50 vol%, and the value within the range), more preferably 20-40 vol%.

[0030] In the present invention, preferably, the amount of the low-concentration alcohol solution used can be 5-20 mL (for example, a range formed by any two values of 5 mL, 8 mL, 10 mL, 12 mL, 15 mL, 18 mL, and 20 mL, and a value within the range), relative to 1 g of microalgae on a dry weight basis, and more preferably 10-18 mL.

[0031] In the present invention, preferably, the concentration and amount of the low-concentration alcohol solution are not particularly limited, as long as the alcohol content in the first extraction system of step S1 can be met. For example, the concentration of the low-concentration alcohol solution can be 25-60 vol% (for example, it can be a range formed by any two values of 25 vol%, 28 vol%, 30 vol%, 32 vol%, 35 vol%, 37 vol%, 40 vol%, 42 vol%, 45 vol%, 48 vol%, 50 vol%, 53 vol%, 55 vol%, 58 vol%, and 60 vol%, and a value within the range), more preferably 30-50 vol%.

[0032] In the present invention, preferably, the alcohol in the low-concentration alcohol solution can be a C1-C4 low-carbon alcohol (for example, it can be at least one of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol and tert-butanol), more preferably methanol and / or ethanol.

[0033] In the present invention, preferably, the conditions for the first extraction may include: a temperature of 15-35°C (for example, it can be a range formed by any two values of 15°C, 18°C, 20°C, 22°C, 25°C, 28°C, 30°C, 32°C, and 35°C, and a value within the range), more preferably 20-30°C; a time of 10-80min (for example, it can be a range formed by any two values of 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, and 60min, and a value within the range), more preferably 30-60min.

[0034] In the present invention, preferably, the second extraction is performed in the presence of an antioxidant.

[0035] In the present invention, preferably, the antioxidant is selected from at least one of vitamin E, rosemary extract, vitamin C, vitamin C palmitate, grape seed extract, curcumin, caffeic acid and tea polyphenol palmitate.

[0036] In the present invention, preferably, the mass ratio of microalgae to antioxidant based on dry weight is 100:0.1-0.3.

[0037] In the present invention, preferably, the alcohol in the high-concentration alcohol solution can be a C1-C4 low-carbon alcohol (for example, it can be at least one of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol and tert-butanol), more preferably methanol and / or ethanol.

[0038] In the present invention, preferably, the alcohol content in the first extraction system of step S1 is 20-83 vol% lower than the alcohol content in the second extraction system of step S2 (for example, it can be a range formed by any two values of 20 vol%, 25 vol%, 29 vol%, 30 vol%, 34 vol%, 35 vol%, 37 vol%, 40 vol%, 43 vol%, 45 vol%, 48 vol%, 50 vol%, 55 vol%, 60 vol%, 65 vol%, 70 vol%, 75 vol%, 80 vol%, and 83 vol%, and the values within the range), more preferably 29-80 vol%, and further preferably 34-75 vol%.

[0039] In the present invention, preferably, the alcohol content in the second extraction system of step S2 can be 70-98 vol% (for example, it can be a range formed by any two values of 70 vol%, 72 vol%, 74 vol%, 76 vol%, 80 vol%, 82 vol%, 84 vol%, 86 vol%, 88 vol%, 90 vol%, 92 vol%, 95 vol%, 96 vol%, 98 vol%, and the value within the range), more preferably 74-95 vol%.

[0040] In the present invention, preferably, the amount of the high-concentration alcohol solution used can be 5-25 mL (for example, it can be a range formed by any two values of 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, and 20 mL, and a value within the range), with respect to 1 g of microalgae on a dry weight basis, more preferably 10-20 mL.

[0041] In the present invention, preferably, the concentration and amount of the high-concentration alcohol solution are not particularly limited, as long as the alcohol content in the second extraction system of step S2 can be met. For example, the concentration of the high-concentration alcohol solution can be 85-99 vol% (for example, it can be a range formed by any two values of 85 vol%, 86 vol%, 87 vol%, 88 vol%, 89 vol%, 90 vol%, 91 vol%, 92 vol%, 93 vol%, 94 vol%, 95 vol%, and 99 vol%, and a value within the range), more preferably 88-95 vol%.

[0042] In a preferred embodiment of the present invention, in step S2, the second extraction can be repeated by adding a high-concentration alcohol solution, preferably 2-4 times. For example, in the case of two second extractions, the alcohol content in the extraction system can be 70-85 vol%, preferably 74-82 vol%, for one second extraction; and 80-98 vol%, preferably 82-90 vol%, for a second second extraction. It is understood that repeating the second extraction can effectively increase the extraction yield of fucoxanthin and EPA. However, when the alcohol content in the extraction system exceeds 90 vol%, the ability to increase the extraction yield of fucoxanthin and EPA is limited. Furthermore, in industrial production, further increasing the alcohol content from 90 vol% significantly increases production costs.

[0043] In the present invention, preferably, the alcohol content in the low-concentration alcohol solution is at least 25 vol% lower than the alcohol content in the high-concentration alcohol solution, and more preferably, the alcohol content in the low-concentration alcohol solution is 38-62 vol% lower than the alcohol content in the high-concentration alcohol solution (for example, it can be a range formed by any two values of 38 vol%, 40 vol%, 42 vol%, 44 vol%, 46 vol%, 48 vol%, 50 vol%, 52 vol%, 54 vol%, 56 vol%, 58 vol%, 60 vol%, and 62 vol%, and the values within the range).

[0044] In the present invention, it can be understood that the "low concentration alcohol solution" and "high concentration alcohol solution" do not represent the "low" or "high" of the raw material alcohol solution used, but refer to the "low" or "high" alcohol concentration in the reaction system after the alcohol solution is added to the reaction system.

[0045] In the present invention, it is understood that the higher the concentration of the high-concentration alcohol solution, the better the effect of extracting fucoxanthin and polyunsaturated fatty acids from microalgae. However, economic benefits need to be considered in the industrial production process. Therefore, in combination with the economic benefits in the actual production process, the concentration of the high-concentration alcohol solution of the present invention is preferably 88-95 vol%.

[0046] In the present invention, preferably, the conditions for the second extraction may include: a temperature of 40-55°C (for example, it can be a range formed by any two values of 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, and 55°C, and a value within the range), more preferably 45-50°C; a time of 0.1-3h (for example, it can be a range formed by any two values of 0.1h, 0.5h, 1h, 1.5h, 2h, 2.5h, and 3h, and a value within the range), more preferably 0.5-1h.

[0047] In the present invention, preferably, the temperature of the first extraction is 5-40°C lower than the temperature of the second extraction (for example, it can be a range formed by any two values of 5°C, 7°C, 10°C, 12°C, 14°C, 15°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, and 40°C, and values within the range), more preferably 15-30°C lower.

[0048] In the present invention, by controlling the temperature difference between the first extraction temperature and the second extraction temperature, the loss rate of fucoxanthin and eicosapentaenoic acid during the extraction process can be reduced, and the extraction rate of fucoxanthin and eicosapentaenoic acid from microalgae can be effectively improved.

[0049] In the present invention, preferably, the microalgae is selected from at least one of Bacillariophyceae, Chrysophyceae and Haplophyceae, more preferably at least one of Dunaliella salina, Tetraselmis, Subcordiformes, Chlorella ellipsoidea, Chlorella, Nannochloropsis eye-spotted, Nannochloropsis, Pavlova viridis, Isochrysis, Pseudochloropsis triangularis and Rhodophyta, further preferably at least one of Chlorella, Isochrysis and Pseudochloropsis triangularis.

[0050] In the present invention, preferably, based on dry weight, the content of fucoxanthin in the microalgae may be 0.1-6wt%, and the content of polyunsaturated fatty acids may be 0.5-9wt%; more preferably, based on dry weight, the content of salinoaxanthin in the microalgae may be 0.5-1.5wt%, and the content of polyunsaturated fatty acids may be 3-4wt%.

[0051] In the present invention, preferably, the method may further include:

[0052] S3, mixing the second extract with a decolorizing agent for decolorization and then performing solid-liquid separation to obtain a third extract;

[0053] S4. The third extract is adsorbed by an adsorbent, and then solid-liquid separation is performed, and the solid phase is taken for analysis.

[0054] In the present invention, preferably, the decolorizing agent may be activated carbon, for example, at least one of wood activated carbon, fruit shell activated carbon, bamboo charcoal, coal charcoal and coconut shell activated carbon.

[0055] In a preferred embodiment of the present invention, the activated carbon has a size of >100 mesh and a specific surface area of ≥1150 m 2 / g, methylene blue adsorption value ≥200mg / g, total pore volume ≥1cm 2 / g, mesopore volume ≥ 0.2cm 2 / g; More preferably, the specification of the activated carbon can be 200-400 mesh, and the specific surface area can be 1160-1200m 2 / g (for example, it can be 1160m 2 / g、1165m 2 / g、1170m 2 / g、1175m 2 / g、1180m 2 / g、1185m 2 / g、1190m 2 / g、1195m 2 / g、1200m 2 / g, and values within the range formed by any two values thereof), the methylene blue adsorption value may be 220-260 mg / g (for example, it may be a range formed by any two values of 220 mg / g, 225 mg / g, 230 mg / g, 235 mg / g, 240 mg / g, 245 mg / g, 250 mg / g, 255 mg / g, and 260 mg / g, and values within the range), and the total pore volume may be 1.02-1.1 cm 2 / g (for example, it can be 1.02cm 2 / g, 1.03cm 2 / g, 1.04cm 2 / g, 1.05cm 2 / g, 1.06cm 2 / g, 1.07cm 2 / g, 1.08cm 2 / g, 1.09cm 2 / g, 1.1cm 2 / g) and the values within the range formed by any two values in the range, the mesopore volume can be 0.22-0.3 cm 2 / g (for example, it can be 0.22cm 2 / g, 0.23cm 2 / g, 0.24cm 2 / g, 0.25cm 2 / g, 0.26cm 2 / g, 0.27cm 2 / g, 0.28cm 2 / g, 0.29cm 2 / g, 0.3cm 2 / g forms a range of any two values and the value within the range).

[0056] In the present invention, the principle for selecting the decolorizing agent is that the decolorizing agent can decolorize low-polarity components with high selectivity.

[0057] In the present invention, the mass ratio of microalgae to decolorant, based on dry weight, is preferably 100:2-10 (for example, it can be a range formed by any two ratios of 100:2, 100:3, 100:4, 100:5, 100:6, 100:7, 100:8, 100:9, or 100:10, and ratios within that range), and more preferably 100:3-7. When the mass ratio of microalgae to decolorant, based on dry weight, exceeds 100:7, it can cause loss of fucoxanthin and EPA in the microalgae, thereby reducing the extraction yield of fucoxanthin and EPA. Furthermore, in industrial production processes, it can increase production costs and cause waste.

[0058] In the present invention, preferably, the decolorization conditions may include: a temperature of 20-30°C (for example, it can be a range formed by any two values of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, and 30°C, and a value within the range), and a time of 30-60min (for example, it can be a range formed by any two values of 30min, 35min, 40min, 45min, 50min, 55min, and 60min, and a value within the range).

[0059] In the present invention, preferably, the adsorbent is at least one of clay, silica gel, zeolite molecular sieve and diatomaceous earth.

[0060] In the present invention, the principle for selecting the adsorbent is that the adsorbent can adsorb moderately polar components with high selectivity.

[0061] In the present invention, preferably, the mass ratio of microalgae to adsorbent based on dry weight can be 100:10-100 (for example, it can be a range formed by any two ratios of 100:10, 100:20, 100:30, 100:40, 100:50, 100:55, 100:60, 100:70, 100:80, 100:90, 100:100, and ratios within the range), more preferably 100:30-55.

[0062] In the present invention, preferably, the analysis method is: the solid phase obtained in step S4 is mixed with an alcohol solution with a concentration of 85-95 vol% for analysis.

[0063] In the present invention, preferably, during the analysis process, the ratio of the added amount of the alcohol solution having a concentration of 85-95 vol% to the volume mass (mL / g) of the microalgae based on dry weight is 10-30:1.

[0064] In the present invention, preferably, the analysis conditions include: temperature of 15-50° C. and time of 30-120 min.

[0065] In a preferred embodiment of the present invention, step S4 may further include: concentrating the third extract while adsorbing it with an adsorbent, subjecting the concentrate to solid-liquid separation, and analyzing the solid phase after solid-liquid separation. Concentration can be carried out in the presence of an adsorbent, or concentration can be performed first and then adsorption; if concentration is performed first and then adsorption is performed, the adsorption conditions are not particularly limited, and can be common adsorption conditions in the art, for example, the adsorption temperature can be 35-45°C (for example, it can be a range formed by any two values of 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, and 45°C, and a value within the range), and the adsorption time can be 30-60 min (for example, it can be a range formed by any two values of 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, and 60 min, and a value within the range).

[0066] In the present invention, preferably, the concentration conditions are such that the ethanol concentration of the concentrate can be 0-20 vol% (for example, it can be a range formed by any two values of 0 vol%, 2 vol%, 4 vol%, 6 vol%, 8 vol%, 10 vol%, 12 vol%, 14 vol%, 16 vol%, 18 vol%, 20 vol%, and a value within the range). The concentration conditions can specifically include: a pressure of 0.09-0.1 MPa, a temperature of 35-55°C, and a time of 30-120 min.

[0067] In a preferred embodiment of the present invention, step S4 may further include: before desorption, washing the solid phase with a 10-20 vol% alcohol solution at a mass-to-volume (g / mL) ratio of 1:1-4 to remove impurities. The washing temperature may be 25-30°C, and the washing time may be 30-60 minutes.

[0068] In a preferred embodiment of the present invention, the method further comprises: after the parsing in step S4 is completed, the parsed filtrate can be subjected to a further decolorization. The further decolorization can be performed using activated carbon (the same as or different from that in step S3). The conditions for the further decolorization can be conventional decolorization conditions, for example, the same as or different from those in step S3, and will not be further described herein. The filtrate after further decolorization is concentrated and evaporated to dryness, mixed with an alcohol solution with a concentration of 80-95 vol%, and then allowed to stand. After solid-liquid separation, the filtrate is dried to obtain algae oil; wherein, the volume mass ratio (mL / g) of the alcohol solution with a concentration of 80-95 vol% to the microalgae based on dry weight is 0.5-1:1; the conditions for concentrating and evaporating the decolorized filtrate include: a pressure of 50-100 Pa, a temperature of 45-55° C., and a time of 30-120 min; the standing conditions may include: a standing temperature of 4-8° C. and a standing time of 10-14 h; the drying conditions may include: a pressure of 50-100 Pa, a drying temperature of 45-55° C., and a drying time of 30-120 min.

[0069] According to a preferred embodiment of the present invention, the method comprises:

[0070] S1: First extraction: Take wet algae of Phaeodactylum tricornutum, add ethanol aqueous solution so that the ethanol content in the extraction system is 20-40 vol%, stir at 20-30°C for 0.5-1h, and separate the solid and liquid to obtain an algae cake;

[0071] S2: Second extraction: The algae cake is added with ethanol aqueous solution for a first extraction, so that the ethanol content in the first extraction system is 74-82 vol%, vitamin E is added (the mass ratio of microalgae to vitamin E based on dry weight is 100:0.1-0.3), stirred at 45-50°C for 0.5-2h, and solid-liquid separation is performed; after solid-liquid separation, the algae cake is again added with ethanol aqueous solution for a second extraction, so that the ethanol content in the second extraction system is 82-95%, vitamin E is added (the mass ratio of microalgae to vitamin E based on dry weight is 100:0.1-0.3), stirred at 45-50°C for 0.5-2h, and finally top washed with 85-99 vol% ethanol, and all the extracts are collected;

[0072] S3: Add activated carbon and stir, the mass ratio of activated carbon to microalgae raw material (based on dry weight) is 3-6:100, the temperature is 20-30°C, the time is 0.5-1h, and solid-liquid separation is performed;

[0073] S4: adding clay to the extraction filtrate after solid-liquid separation in step S3 and concentrating under reduced pressure, wherein the mass ratio of the clay to the microalgae raw material (on a dry weight basis) is 30-50:100, and the ethanol concentration of the concentrate after reduced pressure concentration is 0-20 vol% (the conditions for reduced pressure concentration include: pressure of 0.09-0.1 MPa, temperature of 35-55° C., and time of 0.5-2 h), filtering, and washing the filter cake with a 5-10 vol% ethanol aqueous solution until the washing liquid is colorless or light yellow;

[0074] S5: Take out the filter cake, add 10-20 vol% ethanol aqueous solution and stir to remove impurities, the mass volume ratio (g / mL) of the filter cake and the 10-20 vol% ethanol aqueous solution is 1:1-4, stir at 25-30°C for 0.5-1h, and separate the solid and liquid to obtain a filter cake;

[0075] S6: adding 85-95 vol% ethanol aqueous solution to the filter cake, with the ratio of 85-95 vol% ethanol aqueous solution to the volume mass (mL / g) of the microalgae raw material (on a dry weight basis) being 15-30:1, stirring thoroughly to dissolve, and separating the solid and liquid to obtain a filtrate;

[0076] S7: Add the activated carbon to the filtrate and stir. The mass ratio of activated carbon to algae powder (based on dry weight) is 3-6:100. Stir at 20-30° C. for 0.5-1 h, and separate the solid and liquid to obtain a filtrate.

[0077] S8: The filtrate is concentrated and evaporated to dryness (pressure of 50-100 Pa, temperature of 45-55 ° C, and decompression time of 30-120 min), and 80-95 vol% ethanol aqueous solution is added, and the ratio of the amount of 80-95 vol% ethanol aqueous solution to the volume mass (mL / g) of the microalgae raw material (on a dry weight basis) is 0.5-1:1. The solution is placed at 4-8 ° C for 10-14 h, solid-liquid separation is performed, and the filtrate is dried (drying pressure of 50-100 Pa, temperature of 45-55 ° C, time of 30-120 min), ethanol is removed, and algal oil is harvested.

[0078] A second aspect of the present invention provides an algae oil containing fucoxanthin and polyunsaturated fatty acids, wherein the fucoxanthin content of the algae oil is ≥5wt%, and the eicosapentaenoic acid content is ≥15wt%;

[0079] Preferably, the mass ratio of the prepared fucoxanthin to polyunsaturated fatty acids is 1:1-5, more preferably 1:2-4;

[0080] Preferably, the algae oil is prepared by the method described in the first aspect.

[0081] In a preferred embodiment of the present invention, the content of fucoxanthin in the algae oil containing fucoxanthin and polyunsaturated fatty acids is 5-7 wt %, and the content of eicosapentaenoic acid is 15-21 wt %.

[0082] In a preferred embodiment of the present invention, the ratio of fucoxanthin to polyunsaturated fatty acids in the algae oil can be 1:2.95-3.38. This ratio is close to that found in natural microalgae cells, making it more natural. Therefore, during use, there is no need to reconstitute the fucoxanthin and polyunsaturated fatty acids to a ratio close to that found in natural microalgae, simplifying the process.

[0083] The present invention will be described in detail below through examples. In the following examples, the ethanol content is tested by first detecting the alcohol content and temperature using an alcohol meter and a thermometer, and then reading the alcohol temperature comparison table. Activated carbon was purchased from Jiangsu Zhuxi Activated Carbon Co., Ltd., and is a commercially available product with model ZX-313. Its specification is 300 mesh, specific surface area is 1350 m2 / g, methylene blue adsorption value is 240 mg / g, total pore volume is 1.05 cm3 / g, and mesopore volume is 0.27 cm3 / g. 3 / g.

[0084] 1. The detection method for fucoxanthin in raw materials (microalgae) and products adopts the fucoxanthin high performance liquid chromatography content detection method. The following is the specific detection method for fucoxanthin in raw materials (microalgae) (the detection method for fucoxanthin in products is the same and will not be repeated here):

[0085] (1) Instruments

[0086] Liquid chromatograph Agilent 1200 analytical balance 1

[0087] (2) Reagents

[0088] Acetonitrile Chromatographically pure Ethanol (95%) analytically pure Deionized water

[0089] (3)Liquid phase conditions

[0090] Chromatographic column: YMC ODS-A 250*4.6mm, 5μm;

[0091] Mobile phase: acetonitrile / deionized water = 85 / 15;

[0092] Detection wavelength: 450nm;

[0093] Column temperature: 40°C; flow rate: 1 mL / min.

[0094] (4) Preparation of working reference solution

[0095] Weigh about 100 mg (accurate to 0.01 mg) of the working reference substance into a 25 mL brown volumetric flask, add about 5 mL of deionized water and ultrasonicate to completely disperse it, then add an appropriate amount of 95% ethanol, ultrasonicate for 10 minutes, cool to room temperature, make up to volume, shake well, and filter with a 0.45 μm microporous membrane to obtain the product.

[0096] (5) Preparation of sample solution

[0097] When detecting fucoxanthin in the product, the method of step (5-1) is adopted; when detecting fucoxanthin in the raw material (microalgae), the method of step (5-2) is adopted.

[0098] (5-1) Oily sample (product sample solution)

[0099] Mix the product sample, weigh about 100 mg (accurate to 0.01 mg) of the oily sample into a 25 mL brown volumetric flask, dissolve it in 95% ethanol and make up to volume, shake well, and obtain the product;

[0100] (5-2) Microalgae sample (raw material sample solution)

[0101] Mix the raw material samples, weigh about 100 mg (accurate to 0.01 mg) of microalgae sample into a broken tube, add 5 mL of 95% ethanol, break the tube for 10 minutes, transfer the broken solution to a 50 mL volumetric flask, dissolve it with 95% ethanol and make up to volume, shake well, and filter with a 0.45 μm microporous membrane to obtain the product.

[0102] (6) Calculation formula

[0103]

[0104] Where:

[0105] A1: (Product / Raw Material) Area of Fucoxanthin in Sample Solution, mg;

[0106] A2: area of fucoxanthin in the working standard solution, mg;

[0107] C1: concentration of (product / raw material) sample solution, mg / mL;

[0108] C2: Concentration of fucoxanthin in the working standard solution, mg / mL.

[0109] 2. The detection method of eicosapentaenoic acid in raw materials (microalgae) and products is as follows (the detection method of eicosapentaenoic acid in products is the same and will not be repeated here). It specifically includes:

[0110] (1) Instruments: analytical balance, accurate to 0.01 mg, vortex mixer, constant temperature water bath, gas chromatography-flame ionization detector (GC-FID, Agilent 7890A), gas chromatography column: DB-FFAP, 30 m × 0.32 mm ID × 0.23 μm.

[0111] (2) Reagents: isooctane (analytical grade), methanol (analytical grade), chloroform (analytical grade), concentrated hydrochloric acid (analytical grade), chloroform:methanol (2 / 1, v / v), 1.5N hydrochloric acid-methanol solution: Accurately pipette 6.25 mL of concentrated hydrochloric acid (12 M) into a 50 mL volumetric flask, dilute to the mark with methanol, and shake well (prepare fresh before use), isooctane containing BHT: Add 50 mg of BHT to 1 L of isooctane, sonicate and dissolve, then set aside. Hexacosane was purchased from Aladdin, and a standard mixture of 37 fatty acids was purchased from NU-CHEK-PREP, INC.

[0112] (3) Chromatographic conditions:

[0113] Detector: FID;

[0114] Chromatographic column: DB-FFAP (30 mm × 0.32 mm × 0.23 μm);

[0115] Inlet temperature: 220°C;

[0116] Detector temperature: 250°C;

[0117] Nitrogen flow rate: 2.5 mL / min;

[0118] Air: 400 mL / min;

[0119] Hydrogen: 30 min;

[0120] Column temperature: 90°C for 1 min, increase to 160°C at 6°C / min and hold for 5 min; increase to 215°C at 5°C / min and hold for 8 min; increase to 245°C at 10°C / min and hold for 3 min (total process approximately 42.6 min);

[0121] Split ratio: 1 / 10;

[0122] Injection volume: 2ul.

[0123] (4) Preparation of internal standard solution: C26 internal standard solution: Accurately weigh 100 mg of hexacosane standard into a 100 mL volumetric flask, dissolve it in isooctane (containing BHT) and dilute to 100 mL, shake well, and set aside.

[0124] (5) Preparation of standard solution: Accurately weigh 50 mg of the 37 fatty acid mixture into a 25 mL volumetric flask, add 2 mL of C26 internal standard solution, then add an appropriate amount of isooctane (containing BHT) to completely dissolve it, and make up to 25 mL. Shake well to obtain the standard solution.

[0125] (6) Sample solution preparation:

[0126] When detecting eicosapentaenoic acid in the raw material (microalgae), the method of step (6.1) is adopted; when detecting eicosapentaenoic acid in the product, the method of step (6.2) is adopted.

[0127] (6.1) Microalgae raw material sample (raw material sample): Accurately weigh approximately 50 mg of freeze-dried microalgae sample and place them in two centrifuge tubes;

[0128] (6.2) Finished microalgae product (product sample): Accurately weigh approximately 25 mg of product sample and place in two centrifuge tubes;

[0129] (6.3) Add 1 mL of chloroform:methanol (2:1, v / v) to each centrifuge tube to dissolve the sample, then add 1.5 mL of 1.5 N hydrochloric acid-methanol solution.

[0130] (6.4) Cover all bottles, vortex for 30 seconds, place in a constant temperature water bath at 85°C for 1 hour, remove from the bottle, and cool to room temperature (approximately 20 minutes);

[0131] (6.5) Add 2 mL of C26 internal standard solution and 3 mL of isooctane (containing BHT) to each centrifuge tube. Cap the tube, vortex for 20 seconds, shake repeatedly for at least 10 times, and centrifuge at 3000 rpm for 10 minutes.

[0132] (6.6) Transfer 0.5 mL of the supernatant to another centrifuge tube, add 2 mL of isooctane (containing BHT), shake well, transfer to a sample injection bottle, and perform GC analysis (gas chromatography).

[0133] (7) Calculation of content:

[0134]

[0135] Where:

[0136] X i : The content of each fatty acid, in %;

[0137] A Xi : (Product / raw material) Peak area of each fatty acid in the sample solution;

[0138] C' s : (Product / raw material) internal standard concentration in sample solution, unit: mg / mL;

[0139] A S : Peak area of internal standard substance (C26) in standard solution;

[0140] C Ri : The content of each fatty acid in the standard solution, unit: mg / mL;

[0141] A'S : Peak area of internal standard in (product / raw material) sample solution;

[0142] C X : (Product / raw material) concentration of sample solution, unit: mg / mL;

[0143] A Ri : Peak area of each fatty acid in the standard solution;

[0144] C s : The concentration of the internal standard substance (C26) in the standard solution, unit: mg / mL.

[0145] Example 1:

[0146] S1: First extraction: 100 g of wet Phaeodactylum tricornutum (the fucoxanthin content of the raw material of Phaeodactylum tricornutum, based on dry weight, is 1.07 wt% and the EPA content is 3.53 wt%), with a water content of about 80 vol%, is added with ethanol aqueous solution so that the ethanol content in the extraction system is 35 vol% (the mass volume ratio (g / mL) of Phaeodactylum tricornutum based on dry algae to the extraction system is 1:14), stirred at 25°C for 0.5 h, and solid-liquid separation is performed to obtain an algae cake;

[0147] S2: Second extraction: The algae cake was added with ethanol aqueous solution for a primary extraction, so that the ethanol content in the primary extraction system was 74 vol% (the mass volume ratio (g / mL) of the triangular finger algae to the primary extraction system was 1:18), 0.2 wt% of vitamin E was added, and the mixture was stirred at 40°C for 2 h, and solid-liquid separation was performed; after solid-liquid separation, the algae cake was again added with ethanol aqueous solution for a secondary extraction, so that the ethanol content in the secondary extraction system was 86% (the mass volume ratio (g / mL) of the triangular finger algae to the secondary extraction system was 1:12), 0.2 wt% of vitamin E was added, and the mixture was stirred at 40°C for 0.5 h, and finally, 92 vol% of ethanol was used for top washing, and all the extracts were collected;

[0148] S3: Add activated carbon and stir, the mass ratio of triangular phytoplankton (based on dry weight) to activated carbon is 20:1, the temperature is 20℃, the time is 0.5h, and solid-liquid separation is carried out;

[0149] S4: adding clay to the extraction filtrate after solid-liquid separation in step S3 for vacuum concentration, wherein the mass ratio of clay to Phaeodactylus tricornutum (by dry weight) is 0.5:1, the pressure of vacuum concentration is 0.1 MPa, the temperature is 40° C., the vacuum compression time is 1 hour, and the ethanol concentration of the concentrate after vacuum concentration is 5 vol%, filtering, and washing the filter cake with a 5 vol% ethanol aqueous solution until the washing liquid is colorless or light yellow;

[0150] S5: The filter cake was taken out and added with 15 vol% ethanol aqueous solution to remove impurities by stirring. The mass volume ratio (g / mL) of the filter cake and the 15 vol% ethanol aqueous solution was 1:2. The mixture was stirred at 25°C for 0.5 h and solid-liquid separation was performed to obtain a filter cake.

[0151] S6: Add 90 vol% ethanol aqueous solution to the filter cake, with the volume mass ratio (mL / g) of 90 vol% ethanol aqueous solution to microalgae raw material (dry weight) being 15:1, stir thoroughly to dissolve, and separate the solid and liquid to obtain a filtrate;

[0152] S7: Add the activated carbon to the filtrate and stir. The mass ratio of activated carbon to microalgae raw material (based on dry weight) is 3:100. Stir at 25° C. for 0.5 h, and separate the solid and liquid to obtain a filtrate.

[0153] S8: The filtrate was concentrated and evaporated to dryness (pressure of 100 Pa, temperature of 50° C., time of 60 min), 95 vol% ethanol aqueous solution was added, the amount of 95 vol% ethanol aqueous solution added was 0.5:1 to the volume mass (mL / g) of the microalgae raw material (on a dry weight basis), and the mixture was placed at 6° C. for 12 h for solid-liquid separation. The filtrate was dried (drying conditions included: pressure of 100 Pa, temperature of 50° C., time of 60 min), ethanol and water were removed, and 2.94 g of algae oil was harvested (the obtained algae oil was mixed with medium-chain triglyceride oil in a mass ratio of 1:2, and the product state was as follows: Figure 1 (As shown in the right), the algae oil was detected by the above method, and the content of fucoxanthin in the algae oil was 6.16wt%, and the content of EPA was 19.37wt%.

[0154] Examples 2-7

[0155] The method of Example 1 was followed, except that the ethanol content in the extraction system in step S1 was changed, and the mass-to-volume ratio (g / mL) of Phaeodactylum tricornutum (dried algae) to the extraction system was controlled to remain consistent with that of Example 1 by varying the concentration of the ethanol solution. Specific information on the ethanol content, the mass of the harvested algae oil, the fucoxanthin content in the algae oil, and the EPA content in the algae oil are shown in Table 1.

[0156] Table 1

[0157]

[0158] Examples 8-10

[0159] The method of Example 1 was followed, except that the ethanol content in the primary extraction system in step S2 was varied. The mass-to-volume ratio (g / mL) of Phaeodactylum tricornutum (dried algae) to the primary extraction system in step S2 was controlled to remain consistent with that in Example 1 by varying the concentration of the ethanol solution. Specific information on the ethanol content, fucoxanthin content in the algae oil, and EPA content in the algae oil are shown in Table 2.

[0160] Table 2

[0161]

[0162] Examples 11-13

[0163] The method of Example 1 was followed, except that the ethanol content in the secondary extraction system in step S2 was varied, and the mass volume (g / mL) ratio of Phaeodactylum tricornutum (dried algae) to the secondary extraction system in step S2 was controlled to remain consistent with that in Example 1 by varying the concentration of the ethanol solution. Specific information on the ethanol content, fucoxanthin content in the algae oil, and EPA content in the algae oil are shown in Table 3.

[0164] Table 3

[0165]

[0166] Examples 14-16

[0167] The method of Example 1 was followed, except that in step S3, the mass ratio of the microalgae feedstock (by dry weight) to the decolorizing agent was varied. The specific mass ratio of the microalgae feedstock (by dry weight) to the decolorizing agent, the fucoxanthin content in the algae oil, and the EPA content in the algae oil are shown in Table 4.

[0168] Table 4

[0169]

[0170] Example 17

[0171] The method of Example 1 is followed, except that in step S3, activated carbon is replaced by aluminum oxide.

[0172] The mass of the algae oil was 2.16 g. After testing, the fucoxanthin content in the algae oil was 8.37 wt %, and the EPA content was 0.59 wt %.

[0173] Examples 18-20

[0174] The method of Example 1 was followed, except that in step S4, the mass ratio of the adsorbent to the microalgae feedstock (on a dry weight basis) was varied. The specific mass ratio of the adsorbent to the microalgae feedstock (on a dry weight basis), the fucoxanthin content in the algae oil, and the EPA content in the algae oil are shown in Table 5.

[0175] Table 5

[0176]

[0177] Example 21

[0178] The method of Example 1 is followed, except that in step S4, the white clay is replaced with diatomaceous earth.

[0179] The mass of the algae oil is 3.12 g. After testing, the fucoxanthin content in the algae oil is 5.81 wt %, and the EPA content is 18.3 wt %.

[0180] Example 22

[0181] The method of Example 1 was followed, except that the activated carbon and clay in steps S3 and S4 were simultaneously added to the extract obtained in step S2 for simultaneous decolorization and adsorption (specifically, the method for simultaneous decolorization and adsorption was as follows: coconut shell activated carbon and clay were added to the extract obtained in step S2 for 1 hour at a temperature of 40°C; the mass ratio of activated carbon to microalgae raw material (by dry weight) was 5:100, and the mass ratio of clay to microalgae raw material (by dry weight) was 0.5:1). After decolorization and adsorption, the extract was concentrated under reduced pressure (the conditions for reduced pressure concentration were the same as those in step S4) and filtered. The filter cake was then washed with a 5 vol% aqueous ethanol solution until the washed solution was colorless or light yellow. The filter cake was then subjected to the methods of steps S5-S8. The mass of the algae oil was 2.8 g. Testing revealed that the fucoxanthin content in the algae oil was 5.57 wt% and the EPA content was 16.83 wt%.

[0182] Example 23

[0183] The method of Example 1 is followed, except that only one extraction is performed in step S2. Specifically: Step S2: Second extraction: An ethanol-water solution is added to the algae cake for extraction so that the ethanol content in the extraction system is 74 vol%, 0.2 wt% of vitamin E is added, stirred at 40°C for 2 h, top washed with 92 vol% ethanol, and all the extracts are collected.

[0184] The final mass of the algae oil obtained was 2.38 g. After testing, the fucoxanthin content in the algae oil was 6.52 wt %, and the EPA content was 19.87 wt %.

[0185] Comparative Example 1

[0186] The method of Example 1 was followed, except that step S1 was omitted and ethanol aqueous solution was directly added to the microalgae for the remaining steps. The mass of algae oil was 3.15 g. After testing, the fucoxanthin content in the algae oil was 5.74 wt % and the EPA content was 18.10 wt %. The product state after the algae oil was mixed with medium-chain triglyceride oil in a ratio of 1:2 was as follows: Figure 1 (Left) shown.

[0187] By comparing the states obtained after mixing the algae oil prepared in Example 1 and Comparative Example 1 with medium-chain triglyceride oil, it can be seen that the product obtained by the method of the present invention has better fluidity and state, while the product obtained without low-concentration alcohol extraction has a large amount of granular impurities adhering to the bottle wall and poor fluidity. As can be seen from the results of the Examples and Comparative Examples and Tables 1-5, the products obtained by the present invention using low-alcohol and high-alcohol extraction avoid the problem of toxic solvent residues, also have a high recovery rate for EPA and fucoxanthin, and are more excellent in improving product fluidity and other aspects.

[0188] Test Case

[0189] The fat, protein, and carbohydrate contents of the algae oils obtained in Example 1 and Comparative Example 1 were respectively measured, and the results are shown in Table 6. The fat content was measured according to GB 5009.6-2016, the protein content was measured according to GB 5009.5-2016, and the carbohydrate content (g / 100g) was calculated as 100 - fat content (g / 100g) - protein content (g / 100g) - water content (g / 100g).

[0190] Table 6

[0191]

[0192] According to the data in Table 6, compared with Comparative Example 1, Example 1 showed a decrease in fat content by [(3.15 × 93.48%) - (2.94 × 93.79%)] / (3.15 × 93.48%) = 6.36%; a decrease in protein content by 20.15% (calculated using the same method as above); a decrease in carbohydrate content by 7.66% (calculated using the same method as above); a decrease in fucoxanthin by -0.16% (calculated using the same method as above); and a decrease in EPA by 0.12% (calculated using the same method as above). This indicates that the present invention employs a low-concentration alcohol solution for the first extraction of microalgae, resulting in virtually no loss of fucoxanthin and EPA in the product. The product primarily removes proteins, carbohydrates, and highly polar fats, thereby reducing substances that could affect the product's solubility and preventing such substances from precipitating in the mixture after the product is mixed with medium-chain triglyceride oil. This further improves the fluidity and state of the mixture, avoids any impact on the product's appearance, and thus on its sales, thus offering better commercial prospects.

[0193] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A method for extracting fucoxanthin and polyunsaturated fatty acids from microalgae, characterized in that: The method includes: S1. performing a first extraction on the microalgae using a low-concentration alcohol solution, and performing solid-liquid separation to obtain a first algae cake and a first extract; S2, performing a second extraction on the first algae cake obtained in S1 using a high-concentration alcohol solution, and performing solid-liquid separation to obtain a second algae cake and a second extract; The alcohol content in the first extraction system of step S1 is at least 20 vol% lower than the alcohol content in the second extraction system of step S2, and the alcohol in the low-concentration alcohol solution is the same as or different from the alcohol in the high-concentration alcohol solution.

2. The method according to claim 1, wherein The alcohol content in the first extraction system in step S1 is 15-50 vol%, preferably 20-40 vol%; And / or, the alcohol in the low-concentration alcohol solution is a C1-C4 low-carbon alcohol, preferably methanol and / or ethanol; And / or, the conditions for the first extraction include: a temperature of 15-35°C, preferably 20-30°C; and a time of 10-80 min, preferably 30-60 min.

3. The method according to claim 1, wherein The second extraction is performed in the presence of an antioxidant; Preferably, the antioxidant is selected from at least one of vitamin E, rosemary extract, vitamin C, vitamin C palmitate, grape seed extract, curcumin, caffeic acid and tea polyphenol palmitate; Preferably, the mass ratio of microalgae to antioxidant based on dry weight is 100:0.1-1.

4. The method according to claim 1 or 3, wherein The alcohol in the high-concentration alcohol solution is a C1-C4 low-carbon alcohol, preferably methanol and / or ethanol; and / or, the alcohol content in the first extraction system of step S1 is 20-83 vol%, preferably 34-75 vol% lower than the alcohol content in the second extraction system of step S2; And / or, the alcohol content in the second extraction system in step S2 is 70-98 vol%, preferably 74-95 vol%.

5. The method according to claim 1 or 3, wherein: The conditions of the second extraction include: temperature of 40-55°C, preferably 45-50°C; time of 0.1-3h, preferably 0.5-2h; And / or, the temperature of the first extraction is 5-40°C lower than the temperature of the second extraction, preferably 15-30°C lower.

6. The method according to any one of claims 1 to 5, wherein: The microalgae is selected from at least one of the classes Bacillariophyceae, Chrysophyceae and Haplophyceae, preferably at least one of Dunaliella salina, Tetraselmis, Platymonas subcordiformis, Chlorella ellipsoidea, Chlorella, Nannochloropsis eye-spotted, Nannochloropsis, Pavlova viridis, Isochrysis, Pseudomonas tricornutum and Porphyridium red algae, more preferably at least one of Chlorella, Isochrysis and Pseudomonas tricornutum.

7. The method according to claim 1, wherein The method further comprises: S3, mixing the second extract with a decolorizing agent for decolorization and then performing solid-liquid separation to obtain a third extract; S4. The third extract is adsorbed by an adsorbent, and then solid-liquid separation is performed, and the solid phase is taken for analysis.

8. The method according to claim 7, wherein: The decolorizing agent is activated carbon; And / or, the mass ratio of microalgae to decolorizing agent based on dry weight is 100:2-10, preferably 100:3-7.

9. The method according to claim 7, wherein: The adsorbent is at least one of clay, silica gel, zeolite molecular sieve and diatomaceous earth; and / or, the mass ratio of microalgae to adsorbent, based on dry weight, is 100:10-100, preferably 100:30-55; And / or, the analysis method is: mixing the solid phase obtained in step S4 with an alcohol solution with a concentration of 85-95 vol% for analysis.

10. An algae oil containing fucoxanthin and polyunsaturated fatty acids, characterized in that: The algae oil has a fucoxanthin content of ≥5wt% and an eicosapentaenoic acid content of ≥15wt%; Preferably, the mass ratio of the prepared fucoxanthin to polyunsaturated fatty acids is 1:1-5, more preferably 1:2-4; Preferably, the algae oil is prepared by the method according to any one of claims 1 to 9.

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