CN112210437B - Method for purifying algae oil - Google Patents
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B3/00—Refining fats or fatty oils
- C11B3/10—Refining fats or fatty oils by adsorption
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- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
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- C11B3/00—Refining fats or fatty oils
- C11B3/001—Refining fats or fatty oils by a combination of two or more of the means hereafter
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- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
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- C11B3/00—Refining fats or fatty oils
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- C11B3/00—Refining fats or fatty oils
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- C11B3/00—Refining fats or fatty oils
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- C11B3/06—Refining fats or fatty oils by chemical reaction with bases
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- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
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Abstract
The application relates to a method for purifying algae oil, which comprises the following steps: 1) Contacting the algae oil with an alcohol-water solution in the presence of a grease adsorbent, wherein the volume ratio of the alcohol in the alcohol-water solution to the water is 1:5 to 1:1 so that the algae oil is distributed in an oil drop shape after contacting with the alcohol-water solution; 2) Separating a fat adsorbent from the alcohol-water solution; and 3) separating the algae oil from the oil adsorbent.
Description
The application relates to a patent application of a new algae oil purification method, which is divided into patent applications with the application date of 2018, 07, 26, the application number of CN201810837473.5 and the application name of the patent application.
Technical Field
The application relates to a method for purifying algae oil.
Background
Polyunsaturated fatty acids (PUFAs), such as omega-3 fatty acids, are of great importance for human health. Omega-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), can reduce the amount of triglycerides in the blood. The polyunsaturated fatty acids also have cardiovascular protective effects, and can be used for preventing and treating inflammation and neurodegenerative diseases, and promoting cognitive function (Sugano, michihiro, balanced intake of polyunsaturated fatty acids for health beans.J. Oleo Sci.2001,50 (5): 305-311). More studies have shown that EPA, but not DHA, is the major component of the above action (Martins JG. EPA but not DHA appears to be responsible for the efficacy of omega-3long chain polyunsaturated fatty acid supplementation in depression:evidence from ameta-analysis of randomized controlled trials. J Am Coll Nutr.2009Oct;28 (5): 525-42; eclov JA et al, EPA, non DHA, prevents fibrosis in pressure overload-induced heart failure: potential role of free fatty acid receptor 4.J Lipid Res.2015Dec;56 (12): 2297-308.Doi:10.1194/jlr.M062034.Epub 2015Oct 4).
Omega-3 fatty acids are typically derived from sea fish. However, with the problems of reduction of fish resources, enrichment of fish pollutants caused by the environment and the like, and more people are on vegetarian sense, the demand for omega-3 fatty acids from plant sources is increasing. In plant resources, algal polyunsaturated fatty acids show great potential, whether used as dietary supplements or as pharmaceuticals. Algae have the advantage that the area occupied by algae cultivation is smaller than that of common land crops, and seawater or fresh water resources can be widely utilized.
But algae oil extracted from algae using ethanol contains a large amount of impurities including salts, carbohydrates, proteins, pigments, etc., due to low selectivity of ethanol. The presence of these impurities, together with the omega-3 fatty acids in the algae oil, mainly in the form of triglycerides, glycolipids and phospholipids, makes the algae oil very viscous, causes great impediments to subsequent purification and concentration, and also makes the appearance of the algae oil poor. In large-scale industrial production, the above-mentioned triglycerides, glycolipids and phospholipids are generally converted into the more flowable ethyl ester form by transesterification, followed by subsequent purification and concentration. If necessary, the purified and concentrated ethylated omega-3 fatty acids are converted back to the natural form of triglycerides by transesterification.
Disclosure of Invention
The present disclosure has been made in view of the above-described conventional art, and an object thereof is to provide a method for purifying an algal oil, which is an esterified algal oil obtained by transesterification with a lower alcohol after ethanol extraction, capable of improving the purity of a polyunsaturated fatty acid contained therein. Specifically, the application provides a method for purifying algae oil, which comprises the following steps:
1) Contacting algae oil with an alcohol-water solution in the presence of an oil adsorbent, wherein the volume ratio of alcohol to water in the alcohol-water solution is 1:5 to 1:1 so that the algae oil is distributed in an oil drop shape after contacting with the alcohol-water solution;
2) Separating the fat adsorbent from the alcohol-water solution; and is also provided with
3) Separating algae oil from the oil adsorbent.
In the purification method according to the present disclosure, algae oil is in the form of oil droplets in an alcohol-water solution having an alcohol-to-water volume ratio of 1:5 to 1:1, working fluidity is improved, and water-soluble impurities can be removed better by being separated from the alcohol-water solution by being adsorbed on an oil adsorbent, while being in sufficient contact with the alcohol-water solution. After being purified by the purification method, the algae oil has greatly improved fluidity and can improve the purity of the polyunsaturated fatty acid.
In addition, in the purification method according to the present disclosure, the algae oil may be crude algae oil extracted by treating natural dry algae with ethanol or esterified algae oil obtained by transesterification with lower alcohol after ethanol extraction.
In addition, in the purification method according to the present disclosure, optionally, the volume ratio of the alcohol to water in the alcohol-water solution is 1:1, 1:2, 1:3, 1:4, or 1:5. In this case, the algae oil is contacted with the alcohol-water solution of the specific ratio and distributed in the form of oil droplets, so that the contact area of the algae oil with the alcohol-water solution is increased, thereby enabling water-soluble impurities contained in the algae oil to better enter the alcohol-water solution.
In addition, in the purification method according to the present disclosure, the fat adsorbent is optionally selected from paper towels or activated carbon.
In addition, in the purification method related to the present disclosure, optionally, the weight ratio of the algae oil to the tissue is 1:1 to 10:1.
In addition, in the purification method according to the present disclosure, step 2) may be achieved by collecting the grease adsorbent by filtration or centrifugation.
In addition, in the purification method according to the present disclosure, the oil adsorbent may be washed with water to remove water-soluble impurities after the oil adsorbent is collected.
In addition, in the purification method according to the present disclosure, optionally, before step 1), the algae oil is contacted with dilute acid, calcium carbonate, calcium hydroxide or sodium hydroxide is added to neutralize the acid, and the precipitate is removed by filtration or centrifugation. In this case, impurities can be removed better by separating some kinds of impurities from the effective components such as fatty acids in the algae oil.
In addition, in the purification method according to the present disclosure, optionally, the algal oil is contacted with the dilute acid in the presence of a lower alcohol.
Further, in the purification method to which the present disclosure relates, optionally, the alcohol-water solution is selected from a methanol-water solution, an ethanol-water solution, a propanol-water solution, or a combination thereof.
According to the purification method, algae oil is placed in alcohol-water solution with a specific proportion, so that the algae oil is distributed in an oil drop shape, the working fluidity of the algae oil is increased, the contact area between the algae oil and the alcohol-water solution is increased, and water-soluble impurities are better dissolved in the alcohol-water solution; and then the algae oil is adsorbed on the oil adsorbent, and the oil adsorbent is separated from the alcohol-water solution in a filtering or centrifuging mode, so that the effect of fully removing water-soluble impurities in the algae oil is achieved. The method is not only suitable for natural algae oil with polyunsaturated fatty acid in the forms of triglyceride, glycolipid and phospholipid, but also suitable for esterified algae oil obtained by transesterification of lower alcohol such as methanol or ethanol. After the treatment by the method of the application, the content of total fatty acids, especially useful polyunsaturated fatty acids such as EPA, in the algae oil is increased, and the fluidity of the algae oil is greatly improved.
Detailed Description
The algae oil extracted from microalgae via ethanol contains a large amount of impurities including salt, carbohydrate, protein, pigment, etc., and fatty acids in the algae oil mainly exist in three forms of triglyceride, glycolipid and phospholipid. The superposition of the two factors makes the algae oil present a viscous near-solid state, which causes certain difficulties for subsequent purification, concentration and the like.
The inventors of the present application found that when algae oil is placed in an alcohol-water solution having a volume ratio of alcohol to water of 1:1 or less, the algae oil in a nearly solid state will appear as oil droplets, so that fluidity in operation is improved, and the contact area of the algae oil with the alcohol-water solution is increased, so that water-soluble impurities in the algae oil can be better dissolved in the alcohol-water solution. This makes it possible to purify algal oil directly and efficiently without transesterification, and greatly reduces the flow and cost of the purification process.
By adding a fat adsorbent, for example, a cellulose product such as paper towel, activated carbon, or the like, to an alcohol-water solution, fatty acids such as EPA in algae oil are adsorbed to the fat adsorbent. The oil adsorbent is collected by filtration or centrifugation, so that the separation of the effective components of the algae oil, such as polyunsaturated fatty acids, the alcohol-water solution and water-soluble impurities contained in the alcohol-water solution is realized. After removing a large amount of impurities, the fluidity of the algae oil is improved and the purity of the polyunsaturated fatty acid is increased. The better fluidity means that the purity of the grease in the algae oil is high, and better appearance feeling is brought to the algae oil product.
The method is also suitable for the esterified algae oil after the transesterification treatment of lower alcohols such as methanol or ethanol. The esterified algae oil also presents an oil drop shape in an alcohol-water solution with the alcohol-water volume ratio of less than 1:1, so that the working fluidity of the algae oil can be further increased, the contact surface of the algae oil and the alcohol-water solution is increased, and water-soluble impurities are better removed.
The algae oil can be extracted by treating the dried algae with ethanol, and the extraction parameters can be adjusted as required by a person skilled in the art. In one embodiment, the dried algae having a moisture content of less than 5wt% is placed in absolute ethanol, water bath 80 ℃, rotational speed 20rpm, and treated for 2 hours, wherein the ratio of dried algae to absolute ethanol may be 1:20g/mL.
The ethanol extracted algae oil may be purified by the following method steps:
1) Optionally, the algae oil is contacted with dilute acid, calcium carbonate and the like are added to neutralize the acid, and sediment is removed by filtration or centrifugation and the like;
2) Contacting algae oil with an alcohol-water solution in the presence of a grease adsorbent;
3) Separating the fat adsorbent from the alcohol-water solution;
4) Optionally, washing the fat adsorbent with water; and
5) Separating algae oil from the oil adsorbent.
The algae oil extracted by ethanol contains impurities such as salt, carbohydrate, protein, and pigment. The addition of low concentrations of acid to the algae oil can separate certain classes of impurities from the active ingredients in the algae oil, such as fatty acids, and thus can better remove the impurities. The low concentration acid may be dilute sulfuric acid, dilute hydrochloric acid, dilute nitric acid, etc. Hydrochloric acid and nitric acid are volatile, the acidity of hydrochloric acid is too weak, and the residue of nitric acid can cause nitrogen compounds to be contained in the final product. Therefore, dilute sulfuric acid is the best choice. In order to use sufficient amounts of sulfuric acid while avoiding the occurrence of excessively high concentrations, the algae oil may be contacted with sulfuric acid in the presence of a lower alcohol such as ethanol, and the working concentration of sulfuric acid may be in the range of 0.01-0.1mol/L, for example in the range of 0.036-0.048 mol/L. Because of the low concentration of sulfuric acid, and the control of temperature, transesterification of polyunsaturated fatty acids with lower alcohols does not occur even in the presence of alcohols. The original concentration of the added dilute sulfuric acid is not too low, so that more water is brought in, and the preparation of the proportion of the alcohol-water solution in the subsequent step is difficult. In one embodiment, 45ml of dilute sulfuric acid having a concentration of 3.6mol/L is added to 4.5L of ethanol solution containing about 100g of algae oil, and the mixture is treated at room temperature and 20rpm for 2 hours. After dilute acid treatment, excess calcium carbonate, calcium hydroxide, etc. are added to neutralize the dilute acid and stop the acid action. Insoluble materials can be removed by filtration or centrifugation.
After dilute acid treatment, the algae oil is contacted with an alcohol-water solution in the presence of an oil adsorbent, wherein the volume ratio of the alcohol to the water in the alcohol-water solution can be below 1:1. When the algae oil is placed in the alcohol-water solution with the alcohol-water volume ratio, the algae oil is distributed in an oil drop shape, so that the contact area of the algae oil and the alcohol-water solution is increased, water-soluble impurities contained in the algae oil better enter the alcohol-water solution, the oil drops are gradually adsorbed in the oil adsorbent, and then the adsorbent is separated from the alcohol-water solution by a physical method, so that the effect of removing the water-soluble impurities contained in the algae oil is realized.
The contacting of the algae oil of step 2) with the alcohol-water solution may be carried out with stirring for e.g. 2 hours. The alcohol-water solution may be selected from methanol-water solution and ethanol-water solution. In the alcohol-water solution, the volume ratio of the alcohol to the water is below 1:1. In some embodiments, the volume ratio of alcohol to water in the alcohol-water solution may be 1:1 or less, 2:3 or less, or 1:2 or less, and 1:5 or more, 1:4 or more, or 1:3 or more. In some embodiments, the volume ratio of alcohol to water in the alcohol-water solution is 1:1, 1:2, 1:3, 1:4, or 1:5. The volume ratio of alcohol to water in the alcohol-water solution should not be too low, otherwise, the recovery of ethanol is not favored. Therefore, in actual operation, the volume ratio of alcohol to water may be set in the range of 1:1 to 1:2.
The ratio of algae oil to alcohol-water solution should be within a proper range. When this ratio is too high, it may cause the alcohol-water solution to be insufficient to dissolve all the water-soluble impurities; when the ratio is too low, the amount of alcohol and water used is too large, which increases the cost. The weight to volume ratio of algae oil to alcohol in step 2) may be in the range of 0.2-0.4g/mL, for example 0.225-0.354g/mL, based on the crude algae oil extracted with ethanol. Since the algae oil is extracted by ethanol, if the extraction step is directly carried out to the purification step of the present application without removing ethanol, there may be a problem in that the ratio of algae oil to the alcohol-water solution is too low, the volume of ethanol may be compressed by distillation or the like, and then water may be added according to the amount of ethanol after compression.
The grease adsorbent can be cellulose products, activated carbon and the like, and the type and the amount of the grease adsorbent can be determined by a person skilled in the art according to actual needs. In the present application, the cellulosic product may be a shredded tissue, such as any conventional household tissue. Cellulose products such as tissues contain a large amount of cellulose, and the cellulose can absorb grease and is insoluble in water and common organic solvents, thereby being beneficial to separating algae oil from alcohol-water solution. In one embodiment, the weight ratio of algae oil to paper towel may be in the range of 1:1-10:1.
The oil adsorbent having adsorbed the algae oil may be separated from the alcohol-water solution by filtration such as reduced pressure filtration or centrifugation. The fat adsorbent may be washed with water to further remove water-soluble impurities. The oil adsorbed in the oil adsorbent can be dissolved in absolute ethyl alcohol, the adsorbent is removed by filtration or centrifugation, and the absolute ethyl alcohol is removed by distillation, so that the purified algae oil can be obtained.
As described above, the method of the present application is also applicable to esterified algal oils after transesterification with lower alcohols such as methanol or ethanol. The algae oil extracted by ethanol treatment of dry algae can be subjected to transesterification reaction with lower alcohol at 70 ℃ under the catalysis of concentrated sulfuric acid. The transesterification conditions may be set by those skilled in the art according to the actual circumstances. The lower alcohol may be methanol, ethanol, etc., and is typically ethanol. The esterification reaction may be stopped by neutralizing sulfuric acid by adding an excessive amount of calcium carbonate or the like.
The transesterified algae oil may be purified by the following method steps:
1) Contacting the transesterified algae oil with an alcohol-water solution in the presence of a lipid adsorbent;
2) Separating the fat adsorbent from the alcohol-water solution;
3) Optionally, washing the fat adsorbent with water; and
4) Separating the grease from the grease adsorbent.
In step 1), the transesterified algae oil is contacted with an alcohol-water solution in the presence of a grease adsorbent, wherein the volume ratio of the alcohol to the water in the alcohol-water solution can be 1:1 or less. When the transesterified algae oil is placed in the alcohol-water solution with the alcohol-water volume ratio, the transesterified algae oil is distributed in an oil drop shape, so that the contact area of the algae oil and the alcohol-water solution is increased, water-soluble impurities contained in the algae oil better enter the alcohol-water solution, the oil drops are gradually adsorbed in the oil adsorbent, and then the adsorbent is separated from the alcohol-water solution by a physical method, so that the effect of removing the water-soluble impurities contained in the transesterified algae oil is realized. This step may be carried out with stirring for example for 2 hours. The alcohol-water solution may be selected from water solutions of lower alcohols such as methanol or ethanol, etc. In the alcohol-water solution, the volume ratio of the alcohol to the water is below 1:1. In some embodiments, the volume ratio of alcohol to water in the alcohol-water solution may be 1:1 or less, 2:3 or less, or 1:2 or less, and 1:5 or more, 1:4 or more, or 1:3 or more. In some embodiments, the volume ratio of alcohol to water in the alcohol-water solution is 1:1, 1:2, 1:3, 1:4, or 1:5. Considering the recycling of ethanol, the volume ratio of the ethanol to water may be set in the range of 1:1 to 1:2.
The ratio of transesterified algae oil to alcohol-water solution should be within a suitable range. When this ratio is too high, it may cause the alcohol-water solution to be insufficient to dissolve all the water-soluble impurities; when the ratio is too low, the amount of alcohol and water used is too large, which increases the cost. The weight to volume ratio of algae oil to alcohol in step 2) may be in the range of 0.2-0.4g/mL, for example 0.225-0.354g/mL, based on the crude algae oil extracted with ethanol. Since the algae oil is extracted by ethanol, if the extraction step directly goes into the purification step of the present application without ethanol removal, there may be a problem in that the ratio of algae oil to the alcohol-water solution is too low, the volume of ethanol may be compressed by distillation or the like, and then water may be added according to the amount of ethanol after compression.
The grease adsorbent can be a cellulose product such as paper towel, activated carbon, etc., and the type and the amount of the grease adsorbent can be determined by a person skilled in the art according to actual needs. In one embodiment, the fat absorbent is a cellulosic product, such as a tissue, and the weight ratio of algae oil to tissue may be in the range of 1:1 to 10:1.
In step 2), the fat adsorbent having the transesterified algae oil adsorbed thereto may be separated from the alcohol-water solution by filtration such as reduced pressure filtration or centrifugation.
In step 3), the fat adsorbent may be washed with water to further remove water-soluble impurities.
In the step 4), the transesterified algae oil adsorbed in the oil adsorbent can be dissolved in absolute ethyl alcohol, the adsorbent is removed by filtration or centrifugation, and the absolute ethyl alcohol is removed by distillation, so that the purified transesterified algae oil can be obtained.
EXAMPLE 1 preparation of dried algae
The nannochloropsis (Nannochloropsis salina) was grown in an open pond of artificial seawater containing 22g/L NaCl and 2.44g/L MgSO for 7 days 4 ·7H 2 O, 0.6g/L KCl, 0.25g/L urea, 0.3g/L CaCl 2 ·2H 2 O、4.4mg/L NaH 2 PO 4 ·2H 2 O、0.1g/L NaHCO 3 、0.75mg/L Na 2 EDTA·2H 2 O、0.097mg/L FeCl 3 ·6H 2 O、0.5mg/L H 3 BO 3 、1mg/LMnSO 4 、0.05mg/LZnSO 4 、0.02mg/LCoCl 2 ·6H 2 O, and 0.1mg/L Na 2 MoO 4 ·2H 2 O。
1 ton of algae liquid is taken, 6M NaOH solution is added, the pH is adjusted to 10.5, and the algae begins to settle at the pH value. After three hours, the upper aqueous layer was discarded and the residue was filtered through a Miracloth filter cloth having a pore size of 25 μm to give about 1kg of algal mud having a water content of about 80wt%. The algae mud was treated with a ZPG-G spray dryer for 2 hours to give 146G of dried algae having a water content of 5wt% or less.
The method for measuring the water content comprises the following steps: baking the algae mud or the algae powder in an oven at 80 ℃ for more than 2 hours until reaching constant weight; the weight before being put into the oven was compared with the final constant weight, and the moisture content was calculated as moisture content= (weight before baking-constant weight)/weight before baking ×100%.
EXAMPLE 2 extraction of algae oil
250g of the dried algae prepared in example 1, containing 6.86wt% eicosapentaenoic acid (EPA) and 18.50% Total Fatty Acids (TFA), was taken and placed in 2.5L of 94% ethanol, and treated in a water bath at 80℃for 2 hours, and a magnetic stirrer was set at 20rpm. Thereafter, filtration was performed under reduced pressure (-0.1 MPA) using a cloth funnel connected to a suction pump with filter paper (pore size 15-20 μm, new Star, qualitative filter paper). The filtered algae were added to 2.5L of 94% ethanol and the above steps were repeated. The two filtrates were combined to a total volume of about 4.5L containing about 106.2g crude algae oil.
1.5L of the filtrate was taken and placed in a rotary evaporator at 70℃until no liquid was present in the condenser. 35.4g of crude algae oil was obtained, with little flowability, containing 14.74wt% EPA and 33.63wt% TFA.
EXAMPLE 3 purification of algae oil
1.5L of the filtrate of example 2 was taken, 15mL of 3.6mol/L dilute sulfuric acid was added thereto, and the magnetic stirrer was set at 20rpm, followed by stirring at room temperature for 2 hours. 10g CaCO was added 3 To neutralize the sulfuric acid and stir for an additional 2h. Filtration was performed with filter paper (pore size 15-20 μm, new Star, qualitative filter paper) to give about 1.5L of filtrate. The filtrate was concentrated to 100mL by rotary evaporation (70 ℃, -0.1 MPA). To the resulting suspension 5g of shredded paper towels (Vida, each paper towel bar was approximately 2cm long and 1cm wide) were added. With stirring at 20rpm of the magnetic stirrer, 300mL of water was added and stirring continued for 2h. The paper towel was collected by filtration with filter paper (as above) under reduced pressure (-0.1 MPA), and washed with 200mL water to remove water-soluble components in the algae oil. The washed paper towel was placed in 200mL of 100% ethanol, and then filtered with filter paper (same as above) under reduced pressure (-0.1 MPA), to obtain a filtrate. The filtered towel was rinsed with 100mL of 100% ethanol and filtered again. The two filtrates were combined and rotary evaporated at 70℃to give 15.3g of purified crude oil with good flowability containing 31.50wt% EPA and 64.52wt% TFA.
Compared with the unpurified algae oil, the fluidity of the algae oil in this example is greatly improved, and the purity of polyunsaturated fatty acids is improved.
EXAMPLE 4 transesterification and purification of algae oil
The filtrate of example 2, 1.5L, was taken and ethanol was removed by rotary evaporation at 70℃to give 35.4g of crude algae oil. 70mL of 100% ethanol and 5mL of 98% concentrated sulfuric acid were added to the crude algae oil. The magnetic stirrer is set at 20rpm and the transesterification reaction is carried out at 70 ℃ for 2 hours. 20g CaCO was added to the reaction 3 To neutralize the sulfuric acid and stir for an additional 2h.
Ethanol was added to the reaction solution until the total volume reached 500mL to facilitate subsequent filtration. Filtration through filter paper (pore size 15-20 μm, new Star, qualitative filter paper) gave about 500mL of filtrate. The filtrate was concentrated to 100mL by rotary evaporation (70 ℃, -0.1 MPA). To the resulting suspension was added 5g of paper towels (Vida, each towel bar was approximately 2cm long and 1cm wide). With stirring at 20rpm of the magnetic stirrer, 300mL of water was added and stirring continued for 2h. The paper towel was collected by filtration with filter paper (as above) under reduced pressure (-0.1 MPA), and washed with 200mL water to remove water-soluble components. The washed paper towel was placed in 200mL of 100% ethanol, and filtered with filter paper (same as above) under reduced pressure (-0.1 MPA), to obtain a filtrate. The filtered towel was rinsed with 100mL of 100% ethanol and filtered again. The two filtrates were combined and rotary evaporated at 70℃to give 12.5g of purified fatty acid ethyl ester with good flowability containing 38.65wt% of ethylated EPA and 83.45wt% of ethylated TFA.
The polyunsaturated fatty acids of the algae oil in this example are of increased purity compared to the unpurified algae oil.
EXAMPLE 5 preparation of dried algae
Nannochloropsis was cultured and dried was prepared as described in example 1.
The quality of microalgae is greatly different due to the variation of growth conditions such as illumination. Specifically, the amounts of EPA and TFA contained in the prepared dried algae are shown in Table 1.
TABLE 1 amounts of EPA and TFA contained in dried algae
EXAMPLE 6 extraction of algae oil
250g of each of the dried algae prepared in example 5 was taken, 5L of absolute ethyl alcohol was added, and the mixture was treated in a water bath at 80℃for 2 hours, and the magnetic stirrer was set at 20rpm. Thereafter, filtration was carried out with filter paper (pore size 15-20 μm, new Star, qualitative filter paper) under reduced pressure (-0.1 MPA) using a cloth funnel connected to a suction pump to obtain about 4.5L of each filtrate, which contained 98.9g, 78g and 67.6g of crude algae oil, respectively, containing EPA and TFA as shown in Table 2.
TABLE 2 amounts of EPA and TFA contained in unpurified crude algae oil
| EPA wt％ | TFA wt％ | |
| Algae 1 | 13.67 | 35.68 |
| Algae 2 | 6.51 | 41.40 |
| Algae 3 | 12.01 | 60.20 |
EXAMPLE 7 purification of algae oil
4.5L of the filtrate obtained in example 6 was taken, two parts of alga 1, and one part of alga 2 and one part of alga 3 each. To each filtrate, 60mL, 45mL and 45mL of 3.6mol/L dilute sulfuric acid were added, and the magnetic stirrer was set at 20rpm, and stirred at room temperature for 2 hours. 30g CaCO was added separately 3 To neutralize the sulfuric acid and stir for an additional 2h. Filtration was performed with filter papers (pore size 15-20 μm, novelties, qualitative filter papers) to give a volume of about 4.5L of each filtrate. The filtrate was concentrated to 300mL by rotary evaporation (70 ℃, -0.1 MPA). To each of the obtained suspensions, 50g, 20g and 15g of tissues (Vida, each tissue strip was approximately 2cm long and 1cm wide) were added, respectively. 900mL, 600mL, 1500mL and 300mL of water were added with stirring at 20rpm with a magnetic stirrer. With filter paper (same kind)Above) was filtered under reduced pressure (-0.1 MPA) and the paper towels were collected. The towel was rinsed with 900mL of water to remove water soluble components. The washed paper towel was placed in 600mL of 100% ethanol, and then filtered with filter paper (same as above) under reduced pressure (-0.1 MPA), to obtain a filtrate. The filtered towel was rinsed with 600mL of 100% ethanol and filtered. The two filtrates were combined and rotary evaporated (70 ℃, -0.1 MPA) to give 45.9g, 40.60g, 39.0g and 59.4g of purified crude oil with good flowability containing EPA and TFA as indicated in table 3.
TABLE 3 purification of EPA and TFA amounts contained in algae oil
Compared with the unpurified algae oil, the fluidity of the algae oil in this example is greatly improved, and the purity of polyunsaturated fatty acids is improved.
Example 8 esterification and purification of algae oil
Taking 4.5L of filtrate obtained in example 6, two parts of algae 1, one part of algae 2 and one part of algae 3, performing rotary evaporation at 70 ℃, concentrating to 500mL, adding 14mL of 98% concentrated sulfuric acid, and performing transesterification reaction for 3h at 70 ℃ with a magnetic stirrer set at 20rpm. 40g CaCO was added to the reaction 3 To neutralize the sulfuric acid and stir for an additional 2h.
Filtration through filter paper (pore size 15-20 μm, new Star, qualitative filter paper) gave about 500mL of filtrate. The filtrate was concentrated to 300mL by rotary evaporation (70 ℃, -0.1 MPA). To the resulting suspension, 10g, 15g, 25g and 15g of paper towels (Vida, each approximately 2cm long and 1cm wide) were added, respectively. 900mL, 1500mL, 1200mL and 300mL of water were added with stirring at 20rpm with a magnetic stirrer. The resulting mixture was filtered through a filter paper (the same as above) under reduced pressure (-0.1 MPA), and the paper towel was collected. The towel was rinsed with 900mL of water to remove water soluble components. The washed paper towel was placed in 600mL of 100% ethanol, and then filtered with filter paper (same as above) under reduced pressure (-0.1 MPA), to obtain a filtrate. The filtered towel was rinsed with 600mL of 100% ethanol and filtered. The two filtrates were combined and rotary evaporated at 70℃to give 37.5g, 34.4g, 50.4g and 50.4g of purified fatty acid ethyl esters with good flowability, which contained EPA ethyl esters and TFA ethyl esters as shown in Table 4.
TABLE 4 amounts of EPA Ethyl and TFA Ethyl esters contained in purified ethylated algae oil
The polyunsaturated fatty acids of the algae oil in this example are of increased purity compared to the unpurified algae oil.
EXAMPLE 9 extraction of algae oil
3000g of the dried algae 4 prepared in example 5 was taken, added with 24L of absolute ethanol, treated in a water bath at 80℃for 2 hours, and the magnetic stirrer was set at 20rpm. Thereafter, filtration was performed with filter paper (pore size 15-20 μm, new Star, qualitative filter paper) under reduced pressure (-0.1 MPA) using a cloth funnel connected to a suction pump to give about 20L of filtrate containing 930g of crude algae oil containing 10.67wt% EPA and 60.37wt% TFA.
EXAMPLE 10 purification of algae oil
20L of the filtrate obtained in example 9 was taken, 200mL of 3.6mol/L dilute sulfuric acid was added to the filtrate, and the magnetic stirrer was set at 20rpm and stirred at room temperature for 2 hours. 100g CaCO was added 3 To neutralize the sulfuric acid and stir for an additional 2h. Filtration was performed with filter paper (pore size 15-20 μm, new Star, qualitative filter paper) to give a volume of about 20L of filtrate. The filtrate was concentrated to 4mL by rotary evaporation (70 ℃, -0.1 MPA). To the resulting suspension 250g of paper towels (Vida, each paper towel bar was approximately 2cm long and 1cm wide) were added. 4L of water were added with stirring at 20rpm of the magnetic stirrer. The resulting mixture was filtered through a filter paper (the same as above) under reduced pressure (-0.1 MPA), and the paper towel was collected. The towel was rinsed with 4L of water to remove water soluble components. Placing the washed paper towel in 4L of 100% ethanol, and filtering with filter paper (same as above) under reduced pressure (-0.1 MPA)To a filtrate. The filtered towel was rinsed with 4L of 100% ethanol and filtered. The two filtrates were combined and rotary evaporated (70 ℃, -0.1 MPA) to give 900g of purified crude oil with good flowability containing 12.83wt% EPA and 67.11wt% TFA.
Compared with the unpurified algae oil, the fluidity of the algae oil in this example is greatly improved, and the purity of polyunsaturated fatty acids is improved.
Determination of fatty acid component in Dry algae
0.100g of the dried algae of example 1 or 5 was weighed, placed in a 50mL glass hydrolysis tube (with lid), 21mL chloroform methanol mixture (2:1, v/v) was added, and placed on a shaker 200r/min and shaken for 12h. The hydrolysis tube was removed from the shaker, placed in a 60℃water bath, blown dry with nitrogen, then 10mL of 5% sulfuric acid methanol solution (95 mL methanol+5 mL sulfuric acid) was added, and the mixture was placed in a 100℃water bath for 1h. The hydrolysis tube was removed from the water bath, cooled, 2mL of n-hexane was added, mixed well, and then an appropriate amount of deionized water was added to separate the solution into layers. The upper organic phase was passed through a 0.22 μm organic membrane (nylon, brand: agilent) and placed in a sample bottle, onto a GC sample tray, and awaited detection.
The GC detector was agilent 7890b, fid detector, chromatographic column: agilent 19091J-413HP-5 30m*0.32mm*0.25 μm. Sample injection volume: 1 μl, sample inlet temperature: 250 ℃, without split flow. Programming temperature: 60 ℃ (0 min) -270 ℃ (31.25 min) @8 ℃/min, holding for 5min,270 ℃ (31.25 min) -280 ℃ (38.25 min) @2 ℃/min, holding for 2min. The detector temperature is 280 ℃, the air flow rate is 400mL/min, the hydrogen gas flow rate is 30mL/min, and the tail gas flow rate is 31.5mL/min.
Determination of fatty acid component in non-transesterified algae oil
After distilling off ethanol which may be present, 0.05g of the algae oil of example 2, 3, 6, 7, 9 or 10 was weighed, 10ml of 5% sulfuric acid methanol solution was then added thereto, and the mixture was subjected to water bath at 100℃for 1 hour. Taking out the hydrolysis tube, cooling, adding 5mL of n-hexane, mixing, adding proper amount of deionized water for layering, putting the upper organic phase into a sample injection bottle after passing through a 0.22 mu m organic film, and putting the sample injection bottle on a GC sample injection disc for waiting for detection.
The same instrument and parameters as described above were used for the determination of the composition.
Determination of fatty acid component in transesterified algae oil
After removing ethanol which may be present by distillation, 0.05g of the algae oil in example 4 or 8 was weighed, and then dissolved with n-hexane to a volume of 10mL. After passing through an organic film of 0.22 mu m, the mixture is filled into a sample injection bottle and put on a GC sample injection disc to wait for detection.
The same instrument and parameters as described above were used for the determination of the composition.
The application has been described with reference to the above specific embodiments, but these embodiments do not limit the scope of the application. The scope of the application is defined by the appended claims.
Claims (6)
1. A method of purifying algae oil, the method comprising:
1) Contacting algae oil with an alcohol-water solution in the presence of a grease adsorbent, wherein the volume ratio of alcohol to water in the alcohol-water solution is 1:5 to 1:1 so that the algae oil is distributed in an oil drop shape after being contacted with the alcohol-water solution, the weight-volume ratio of the algae oil to alcohol in the alcohol-water solution is 0.2-0.4g/mL, and the grease adsorbent is selected from paper towels and activated carbon;
2) Separating a fat adsorbent from the alcohol-water solution; and is also provided with
3) Separating the algae oil from the oil adsorbent;
the algae oil is esterified algae oil obtained by transesterification of lower alcohol after ethanol extraction.
2. The method of claim 1, wherein the volume ratio of alcohol to water in the alcohol-water solution is 1:1, 1:2, 1:3, 1:4, or 1:5.
3. The method of claim 1, wherein the grease adsorbent is a tissue, and the weight ratio of the algae oil to the tissue is 1:1-10:1.
4. The method of claim 1, wherein step 2) is accomplished by collecting the fat adsorbent by filtration or centrifugation.
5. The method of claim 4, wherein after collecting the fat adsorbent, the fat adsorbent is washed with water to remove water-soluble impurities.
6. The method of claim 1, wherein the alcohol-water solution is selected from the group consisting of methanol-water solution, ethanol-water solution, propanol-water solution, or a combination thereof.
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| CN102746947A (en) * | 2012-07-18 | 2012-10-24 | 福建华尔康生物科技有限公司 | Method for separating and purifying DHA (docosahexaenoic acid) and saturated fatty acid from schizochytrium limacinum oil |
| CN103937604A (en) * | 2014-04-08 | 2014-07-23 | 北京化工大学 | Method for extracting oil in microalgae |
| CN104059773A (en) * | 2014-07-02 | 2014-09-24 | 华南理工大学 | Method for wet extraction of purified microalgae oil |
| CN107760440A (en) * | 2017-12-07 | 2018-03-06 | 河西学院 | A kind of method of purification of microalgae grease |
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| CN102746947A (en) * | 2012-07-18 | 2012-10-24 | 福建华尔康生物科技有限公司 | Method for separating and purifying DHA (docosahexaenoic acid) and saturated fatty acid from schizochytrium limacinum oil |
| CN103937604A (en) * | 2014-04-08 | 2014-07-23 | 北京化工大学 | Method for extracting oil in microalgae |
| CN104059773A (en) * | 2014-07-02 | 2014-09-24 | 华南理工大学 | Method for wet extraction of purified microalgae oil |
| CN107760440A (en) * | 2017-12-07 | 2018-03-06 | 河西学院 | A kind of method of purification of microalgae grease |
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