1. A preparation method of crude algae oil is characterized by comprising the following steps:

a) adding sodium hydroxide into the algae culture solution, filtering to obtain algae mud, and drying the algae mud to obtain dry algae powder;

b) mixing the dry algae powder with ethanol, filtering and removing the ethanol to obtain algae oil;

c) adding lower alcohol into the algae oil to carry out ester exchange reaction; and

d) adding hexane and water to the system after the transesterification reaction to obtain a hexane phase, collecting the hexane phase and washing with water to obtain crude algal oil.

2. The preparation method according to claim 1, wherein in step a), the algal mud is obtained by filtration using Miracloth with a pore size of 25 μm.

3. The preparation method according to claim 1, wherein in the step b), the dried algae powder is mixed with 95% ethanol.

4. The preparation method according to claim 3, wherein, in the step b), the mixture of the dry algal powder and ethanol is stirred at 78 ℃.

5. The preparation method according to claim 1, wherein, in step b), filtration is performed using filter paper having a pore size of 50 μm.

6. The method of claim 1, wherein in step b), the ethanol is removed by evaporation.

7. The preparation method according to claim 1, wherein, in step c), the lower alcohol is selected from methanol, ethanol, propanol or butanol.

8. The method according to claim 7, wherein in step c), sulfuric acid is added as a catalyst, and the transesterification is performed at 70 ℃.

9. The method according to claim 1, wherein the hexane to water is added in step d) in a volume ratio of 1: 1.

10. The process according to claim 1, wherein in step d) the hexane phase is collected and washed with water until the pH is neutral.

Background

Polyunsaturated fatty acids (PUFA), such as omega-3 fatty acids, are of great importance for daily life. The benefits of polyunsaturated fatty acids such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) among others include lowering blood triglyceride levels, reducing the risk of cardiovascular disease. Meanwhile, other benefits of these polyunsaturated fatty acids include prevention or treatment of inflammation, neurodegenerative diseases, improvement of cognitive ability, etc. (Sugano, Michihiro, Balanced intake of polyunsaturated fatty acids for health bones. J. Oleo Sci.2001,50(5): 305-.

Omega-3 fatty acids are typically derived from marine fish. However, with the decrease of fish resources, the enrichment of fish body pollutants caused by the environment and the like, and more people devote vegetarian diet, the demand for omega-3 fatty acids from plants is increasing. Algal polyunsaturated fatty acids show great potential in plant resources, whether as dietary supplements or as pharmaceuticals. The algae has the advantages that the area occupied by algae cultivation is smaller than that of common land crops, and seawater or fresh water resources can be widely applied.

The lipid mixture extracted from algae without any purification treatment is called algal oil, which contains a large amount of pigments, mainly chlorophyll. The presence of chlorophyll gives algal oil a brownish black or dark green color. Specifically, the concentration of chlorophyll in algal oil is approximately 5,320 to 39,998ppm, which is approximately 117 to 3,076 times higher than that of typical canola oil. Chlorophyll is lipophilic and is carried along with lipids when the lipids are extracted using polar solvents such as hexane, with serious adverse effects on subsequent processing and oil quality. For example, the presence of chlorophyll complicates silica gel column chromatography, reduces the storage stability of the oil, causes oxidative deterioration of the oil under light, and causes an unpleasant odor. Therefore, when algae are used to produce unsaturated fatty acids, it is necessary to remove chlorophyll to the maximum extent.

There are many methods for removing chlorophyll, for example, enzymatic decolorization (CN200580027938.1), physical adsorption using silica (US5,053,169) or clay, extraction using an organic solution or a supercritical fluid, oxidation treatment, and chromatography. As described above, algae oil contains a large amount of chlorophyll, and the removal of pigments requires consideration of both time and material costs.

Disclosure of Invention

The invention relates to a method for decoloring crude algae oil, which comprises the following steps:

a) contacting the crude algae oil with activated carbon in ethanol at 60-75 deg.C;

b) suction filtering the mixture obtained in the step a) through filter paper;

c) contacting the filtrate obtained in step b) with activated carbon at 60-75 ℃, and

d) the mixture from step c) was filtered with suction through a filter paper and the ethanol was removed.

In step a), the contacting is carried out for about 1 hour, the temperature may be about 68 ℃, the ethanol is absolute ethanol, the mesh number of the activated carbon is 200 mesh, and the weight ratio of the crude algal oil to the activated carbon and ethanol is about 0.5:1:10 to 2:1:10, in particular 1:0.52:10 or 2:1: 20. The crude algal oil in step a) comprises fatty acid esters, especially esters of fatty acids with lower alcohols selected from the group consisting of methanol, ethanol, propanol and butanol. In one embodiment, the crude algae oil comprises fatty acid ethyl esters. The crude algal oil containing fatty acid esters is obtained by subjecting algal oil directly extracted from algae to transesterification with a lower alcohol selected from methanol, ethanol, propanol and butanol, and the transesterification may be carried out at 70 ℃ for 2 hours under the catalysis of sulfuric acid.

In step b), the filter paper may be a filter paper having a pore size of 50 μm or less.

In step c), the weight of activated carbon added is the same as in step a), and the weight ratio of filtrate to activated carbon is about 19.3:1 and the contacting is for about 1 hour, the temperature may be about 68 ℃.

In step d), the filter paper may be a filter paper with a pore size of 50 μm, and the ethanol is removed by evaporation.

The crude algae oil decoloring method is simple and convenient. By this method, all or almost all of the pigments including chlorophyll in the crude algal oil containing fatty acid esters can be removed. The crude algae oil is decolorized from dark brown to light yellow, the concentration of the contained fatty acid ester is improved, and the recovery rate of the fatty acid ester reaches 90 percent.

Drawings

FIG. 1 shows crude algal oil (or crude fatty acid ethyl ester) before and after decolorization with activated carbon.

Detailed Description

There are a large number of pigments, especially chlorophyll, in algal oil. The presence of chlorophyll can deteriorate the appearance quality of the algae oil and, more so, can cause oxidative deterioration of the algae oil. Thus, decolorizing algal oil is an essential procedure for using algal unsaturated fatty acids as dietary supplements or pharmaceuticals.

In the present invention, pigments, particularly chlorophyll, in the crude algal oil are adsorbed mainly by activated carbon. The activated carbon adsorption decoloration is simple and convenient to operate, has low cost and is suitable for large-scale application.

Specifically, the invention relates to a method for decoloring crude algae oil, which comprises the following steps:

a) contacting the crude algae oil with activated carbon in ethanol at 60-75 deg.C;

b) suction filtering the mixture obtained in the step a) through filter paper;

c) contacting the filtrate obtained in step b) with activated carbon at 60-75 ℃, and

d) the mixture from step c) was filtered with suction through a filter paper and the ethanol was removed.

The crude algal oil of the present invention comprises a fatty acid ester, which is obtained by transesterification of algal oil directly extracted from algae with a lower alcohol selected from the group consisting of methanol, ethanol, propanol and butanol. The algae oil directly extracted from algae is a mixture of triglyceride of fatty acid, glycolipid and phospholipid. Due to its sticky texture, subsequent decolorization, purification/concentration, etc. operations are difficult to perform. Therefore, in order to increase the efficiency of the treatment, three forms of fatty acids are generally converted to lower alcohol esters by transesterification with lower alcohols. The transesterification reaction may be carried out in the presence of an enzyme, an acid/base catalyst, and reaction conditions known to those skilled in the art. In one embodiment, algal oil extracted from dry algal powder by ethanol is subjected to transesterification reaction with ethanol to obtain fatty acid ethyl ester. In one embodiment, the transesterification reaction is carried out at 70 ℃ for 2 hours under the catalysis of sulfuric acid.

In step a), the contact time of the crude algal oil with the activated carbon can be adjusted by those skilled in the art according to actual needs. In one embodiment, the contact time is 1 hour. In step a), the contacting of the crude algal oil with the activated carbon may be carried out at a certain temperature, for example, 60-75 ℃, which may be adjusted by those skilled in the art according to actual needs. In one embodiment, the temperature of step a) is set at 68 ℃. To ensure that the crude algal oil and the activated carbon are in sufficient contact, continuous stirring may be performed using a magnetic stirrer.

In step a), the ethanol may be anhydrous ethanol or ethanol containing a small amount of moisture (e.g., less than 0.05 wt.%, less than 1 wt.%, or less than 5 wt.%). The mesh number of the activated carbon used in step a) may be 200 mesh. The weight ratio of crude algal oil to activated carbon and ethanol is about 0.5:1:10 to 2:1: 10. In one embodiment, the weight ratio of crude algal oil to activated carbon and ethanol is about 1:0.52:10 or 2:1: 20.

In step b), the mixture in step a) is filtered by suction through a filter paper, wherein the pore diameter of the filter paper can be less than 50 microns as long as the effect of filtering the activated carbon can be achieved.

In step c), activated carbon is added to the filtrate, the weight of the added activated carbon may be the same as or different from that in step a), and can be adjusted by the skilled person according to the actual situation, such as decolorization. In one embodiment, the weight ratio of filtrate to activated carbon is 19.3: 1. Likewise, the time and temperature of the contact of the filtrate with activated carbon in step c) can be adjusted by the person skilled in the art. In one embodiment, in step c), the filtrate is contacted with activated carbon for about 1 hour at a temperature of 60-75 ℃, in particular 68 ℃.

In step d), the mixture in step c) is filtered through a filter paper, wherein the pore diameter of the filter paper can be less than 50 microns as long as the effect of filtering the activated carbon can be achieved. After suction filtration, ethanol is removed by distillation or the like.

Examples

Example 1 preparation of algal polyunsaturated fatty acid Ethyl esters

Nannochloropsis salina (Nannochloropsis salina) is grown in artificial seawater containing 22g/L NaCl and 2.44g/L MgSO for 7 days in open pond₄·7H₂O, 0.6g/L KCl, 0.25g/L urea, 0.3g/L CaCl₂·2H₂O、4.4mg/L NaH₂PO₄·2H₂O、0.1g/L NaHCO₃、0.75mg/L Na₂EDTA·2H₂O、0.097mg/L FeCl₃·6H₂O、0.5mg/L H₃BO₃、1mg/L MnSO₄、0.05mg/L ZnSO₄、0.02mg/L CoCl₂·6H₂O, and 0.1mg/L Na₂MoO₄·2H₂O。

One ton of the algal culture was collected, and 6M NaOH was added thereto to bring the pH to 10.5, whereby the algae began to settle. After three hours, the upper layer of water was removed and the bottom sediment was filtered using Miracloth with a pore size of 25 microns to give about 1 kg of algal mud with a water content of about 80 wt%. The algal puree was Spray dried in a drying cabinet (ZPG-G organic Spray Dryer) for 2 hours with an air temperature at the inlet of about 180 ℃ and an air temperature at the outlet of about 90 ℃. Thus, about 146 g of dried algal powder with a water content of about 5 wt% was obtained.

The method for measuring the water content is as follows: baking the algae mud or algae powder in an oven at 80 ℃ for more than 2 hours until constant weight is achieved; the weight before placing in the oven was compared to the final constant weight and the water content was calculated as (weight before baking-constant weight)/weight before baking 100%.

25g of the obtained dried algal powder was weighed and mixed with 250 g of 99.5% ethanol. The resulting suspension was stirred in a three-necked round-bottomed flask connected to a reflux condenser at 78 ℃ for 30 minutes by a magnetic stirrer to extract algal oil. Thereafter, suction filtration was performed in a buchner funnel connected to a suction pump using a filter paper with a pore size of 50 μm to remove the solid therefrom, and another 250 g of 99.5% ethanol was added to the solid to repeat the algal oil extraction process. The extracted algae oil-containing liquids were combined to yield 641 ml of dark green algae oil-containing liquid. The dark green liquid was then suction filtered in a distributor funnel connected to a suction pump using a filter paper with a pore size of 50 microns to remove the solids therefrom and the ethanol was then evaporated off in a vacuum rotary evaporator at 55 ℃ under a vacuum of-0.07 MPa to give 9.73 g of algal oil as a viscous oil.

To the resulting algal oil were added 51 ml of 99.5% ethanol and 3.57 ml of 100% sulfuric acid. The resulting mixture was subjected to transesterification at 70 ℃ for 2 hours. The reaction product was poured into a separatory funnel, and 20 ml of hexane and 20 ml of water were added. After several minutes, the liquids separated into an upper hexane phase, a lower aqueous phase and an interposed emulsified phase layer. The hexane phase containing the ethyl esters of the fatty acids formed by the reaction was collected and washed 4 to 5 times with water until the pH became neutral, the hexane phase: water 1:1, v/v. The intermediate emulsion layer was re-extracted with 4 ml of hexane and washed with water until the pH became neutral. Due to the inevitable loss of the hexane phase in the water washing, about 14.7 ml of hexane phase containing about 2.63 g/2.94 ml of crude fatty acid ethyl ester (referred to as crude EE for short, which is called crude algal oil in the present invention) is finally obtained.

After blowing off the hexane with flowing nitrogen, the crude EE appeared as a dark brown liquid, 2ml of which was taken and the content of EPA in ethanol was determined with a gas chromatograph.

The gas chromatograph used was an Ultra Trace (Thermo Scientific, usa) equipped with a hydrogen Flame Ionization Detector (FID); DB-23 quartz capillary columns (0.25 mm. times.60 m, film layer thickness 250 nm; Agilent Technologies, USA) were used; gas chromatography analysis procedure: the initial column temperature is 50 deg.C, and is maintained for 1min, the temperature is raised to 170 deg.C per minute at 40 deg.C, and is maintained at 170 deg.C for 1 min; heating to 210 deg.C at 18 deg.C per minute, and maintaining for 28 min; the temperature of a sample inlet is 270 ℃, and the split ratio is 50: 1; n is a radical of₂Is used as carrier gas and is in a constant flow mode, and the flow rate is 2 ml/min; the detector temperature is 280 ℃, the air flow is 350ml/min, H₂It is 35 ml/min.

Example 2 decolorization of algal PUFA ethyl esters

13.52 g of crude EE, prepared as described in example 1, are weighed out, admixed with 135.2 g of absolute ethanol and 7 g of activated carbon (200 mesh, iodine number (Mg/g) not less than 1210, methylene blue not less than 17, ash content not more than 5%, moisture not more than 10%, iron content 0.05%, pH 4, from Nanhai Barsan carbon Co., Ltd., Fushan City), and stirred with a digital display thermostatic magnetic stirrer at 68 ℃ for 1 hour (rotation speed 20 r/s). Then, suction filtration was carried out in a Buchner funnel connected to a suction pump using a filter paper with a pore size of 50 μm, the vacuum degree of the suction pump was set to-0.07 MPa, and about 135 g of the filtrate was collected. 7 g of activated carbon was added to the filtrate, and the mixture was stirred at 68 ℃ for 1 hour. Then, the filtrate was filtered, and the obtained filtrate was evaporated with a rotary evaporator (Shanghai Yangrong Biochemical Instrument factory, RE-5203) to remove ethanol, and further blown off with a nitrogen blower (Shanghai' an Spectroscopy, science and technology Co., Ltd., DC-12), wherein the parameters of the rotary evaporator were set as follows: heating in water bath at 50 deg.C under-0.07 MPa, and rotating at 80 r/min; the setting parameters of the nitrogen blowing instrument are as follows: 99.99% nitrogen purity, 10L/min nitrogen flow, and heating in 70 deg.C water bath. 10.96 g of crude EE was obtained, and 2ml of the crude EE was analyzed by gas chromatography to determine the EPA ethanol content.

The crude EE changed color from dark brown to light yellow upon two activated carbon treatments. As shown in table 1 below, the EPA ethyl ester content increased from 15.63 wt% to 17.56 wt% after decolorization, with a recovery of 90.96% from the decolorization step.

TABLE 1 quality, EPA-EE content and recovery of crude EE before and after decolorization