CN114574280B

CN114574280B - Preparation method of high-content polyunsaturated fatty acid grease

Info

Publication number: CN114574280B
Application number: CN202011378740.0A
Authority: CN
Inventors: 汪志明; 陆姝欢; 杨刚; 马凡提; 李翔宇
Original assignee: Cabio Biotech Wuhan Co Ltd
Current assignee: Cabio Biotech Wuhan Co Ltd
Priority date: 2020-12-01
Filing date: 2020-12-01
Publication date: 2023-08-08
Anticipated expiration: 2040-12-01
Also published as: CN114574280A

Abstract

The invention provides a preparation method of high-content polyunsaturated fatty acid grease. The preparation method comprises a method for removing chlorophyll in algae microbial oil, and the method comprises the following steps: 1) Reacting the lower monohydric alcohol esterified algae oil with an acid solution to obtain first grease; 2) Reacting the first grease with alkali to obtain second grease; 3) Decolorizing the second oil with activated carbon and/or clay, and collecting the decolorized oil. The invention also provides a method for enriching polyunsaturated fatty acid glyceride from the algae microbial oil. The method provided by the invention can effectively remove the residual chlorophyll in the grease through the simple reaction steps. The method for enriching polyunsaturated fatty acid glyceride from algae microbial oil provided by the invention can directly obtain high-purity triglyceride, the content can be more than 90%, the operation is simple, and the industrial production is easy.

Description

Preparation method of high-content polyunsaturated fatty acid grease

Technical Field

The invention relates to the technical field of microbial oil purification and synthesis, in particular to a preparation method of high-content polyunsaturated fatty acid oil.

Background

Polyunsaturated fatty acids such as omega-3, omega-6 and the like, such as eicosapentaenoic acid (EPA, C20:5 n-3), docosahexaenoic acid (DHA), eicosatetraenoic acid (AA) and the like, have important physiological significance in nutrition and medicine, for example, EPA can promote brain development, improve brain functions, promote the health of a circulatory system and has positive effects on preventing and treating diseases such as rheumatoid arthritis, hypertension, diabetes and the like.

Algae are currently used as one of the main raw materials for extracting polyunsaturated fatty acids, and one problem about extraction is that part of algae lipid contains a large amount of chlorophyll components, so that the algae oil is brown or dark green, and the chlorophyll can be partially removed from the results by adopting a common adsorption mode such as activated carbon, clay and silica gel, but a small amount of chlorophyll is still dissolved in the grease, so that the grease still presents green; if the amount of the adsorbent is increased, a larger cost burden is caused. In addition, algae contain a large amount of phospholipids and glycolipids, and when common adsorption and distillation processes are used for removing chlorophyll and purifying grease from algae oil, raw materials are often too viscous to operate, so that the purification purpose cannot be realized. Since high contents of eicosapentaenoic acid, docosahexaenoic acid and the like have high values in the fields of health foods and pharmaceuticals, it is required to develop a method capable of industrially and effectively removing chlorophyll and purifying algal oil and obtaining polyunsaturated fatty acid esters, particularly polyunsaturated fatty acid glycerides, in high contents.

Disclosure of Invention

A first object of the present invention is to provide a method for removing chlorophyll in algal microbial oil, the method comprising the steps of:

1) Reacting the lower monohydric alcohol esterified algae oil with an acid solution to obtain first grease;

2) Reacting the first grease in the step 1) with alkali to obtain second grease;

3) Further decolorizing the second oil obtained in the step 2) by using activated carbon and/or clay, and collecting a decolorized oil.

The invention can effectively remove most of residual chlorophyll in the lower monohydric alcohol esterified algae oil by only the simple reaction steps (specific sequence: firstly reacting with acid solution, then reacting with alkali, and then decoloring by using active carbon and/or clay), wherein at least 80% of chlorophyll can be effectively removed.

The lower monohydric alcohol esterified algae oil is obtained by the chemical method or enzymatic method of algae microbial grease and the lower monohydric alcohol transesterification. The microalgae is obtained by culturing microalgae, the microalgae can perform photosynthesis, the extract contains a large amount of chlorophyll, the chlorophyll content exceeds 2%, the microalgae can be blue algae, green algae, golden algae and red algae, typical algae are nannochloropsis, spirulina, chlorella and the like, the grease can be obtained from algae powder or algae mud by using a method commonly used in the field, and the invention is described in detail by taking nannochloropsis as an example in the embodiment of the invention.

Wherein the lower monohydric alcohol is a monohydric alcohol having less than 6 carbon atoms as is well known in the art, and typical alcohols are methanol, ethanol, propanol, butanol, pentanol, hexanol, etc., and in the examples of the present invention, ethanol is taken as an example for the detailed description of the present invention.

Wherein, the chemical method and the enzymatic method are conventional methods in the art, and typical chemical methods are that lower monohydric alcohol and sulfuric acid are added into algae oil, transesterification reaction is carried out at about 80 ℃, the reacted mixture is fully extracted by using ethane and water, hexane phase is collected, and water is washed until pH is neutral, thus obtaining the lower monohydric alcohol esterified algae oil. The enzyme method is to add ethanol and lipase into the algae oil, react for 4-24 hours at 35-65 ℃ to obtain the lower monohydric alcohol esterified algae oil, and preferably 500-2000 ppm soap is added into the system. Although the obtained lower monohydric alcohol esterified algae oil converts part of fatty acid into ethyl ester, the fluidity is improved to a certain extent, and the problem that raw materials are sticky and difficult to process is solved, the lower monohydric alcohol esterified algae oil is still a sticky mixture of ethyl ester, glycolipid and phospholipid, wherein the glycolipid content is about 8-20wt% and the phospholipid content is about 3-10wt%, and the sticky mixture still brings great difficulty to the subsequent processing steps if the subsequent purification treatment is carried out according to the prior disclosed technology.

In a preferred embodiment of the present invention, a distillation step is further included, which may be before step 1) or after the decoloring step 3), with the aim of increasing the content of polyunsaturated fatty acids in the lower monohydric alcohol esterified algal oil and removing chlorophyll. Research shows that the distillation step has better effect after being subjected to the decoloring step 3), namely the method also comprises the step 4), the decoloring oil obtained in the step 3) is distilled, and the unsaturated fatty acid alkyl ester is collected. After the step, chlorophyll can be removed by more than 95%, even 99%, and polyunsaturated fatty acid with higher purity can be obtained. The distillation conditions are available according to routine experience of the person skilled in the art, and are selected according to different equipment and methods, and the typical range of distillation conditions is 10 to 100Pa, and the distillation temperature is 100 to 200 ℃. Wherein the typical unsaturated fatty acid comprises one or more of eicosapentaenoic acid, eicosatetraenoic acid, docosahexaenoic acid, octadecadienoic acid, octadecatrienoic acid, and stearidonic acid.

In a preferred embodiment of the invention, the unsaturated fatty acid alkyl ester is a fatty acid ethyl ester, and the conditions of distillation are: and collecting the fraction at 160-190 ℃ under the condition of 50-80 pa to obtain the ethyl ester oil rich in polyunsaturated fatty acid.

In a preferred embodiment of the present invention, in step 1), the acid is used in an amount of 1.5 to 5wt% based on the weight of the lower monohydric alcohol esterified algal oil. The mass fraction of the acid solution is preferably 50-70%. The reaction temperature in step 1) is preferably 70 to 90℃and the reaction time is preferably 60 to 200 minutes. In the reaction, in order to improve the chlorophyll removing effect, the lower monohydric alcohol esterified algae oil is preferably reacted with the acid solution for 30 to 60 minutes, and then the pure water is added for 30 to 140 minutes. Wherein, the addition amount of the pure water is preferably 4 to 5 weight percent of the lower monohydric alcohol esterified algae oil. And after the reaction is finished, collecting an upper layer to obtain the first grease. The acid in the acid solution may be any acid commonly used in the art, including but not limited to hydrochloric acid, citric acid, etc., preferably citric acid that is food-grade and has no corrosive effect on equipment. The acid solution may be an aqueous acid solution.

In a preferred embodiment of the invention, in step 2), the amount of base is such that the free fatty acids in the ethyl ester oil and the acid value of the first grease in step 1) are neutralized. The reaction temperature in step 2) is preferably 50 to 60 ℃. The reaction time is related to the amount of base used, and it is sufficient to neutralize the free fatty acid in the ethyl ester oil and the unreacted acid in step 1). In the step 2), after the reaction of the first oil and the alkali is finished, the second oil can be obtained by centrifuging or filtering to separate impurities. The base may be used as commonly used in the art, including, but not limited to, sodium hydroxide, potassium hydroxide, sodium ethoxide, potassium ethoxide, sodium methoxide, sodium ethoxide, solid base, and the like. Wherein in this step, the alkali is an aqueous alkali solution, and the mass fraction is preferably 20 to 40wt%.

In a preferred embodiment of the present invention, in step 3), the amounts of the activated carbon and clay are respectively 5 to 10% by weight of the lower monohydric alcohol esterified algae oil. Preferably, in the step 3), activated carbon and clay are sequentially used for decoloring the second grease, and the decolored oil is collected, wherein the decoloring reaction temperature is preferably 50-70 ℃, and the reaction time is preferably 60-120 min. In the step 3), specifically, at 50-70 ℃, the active carbon according to the proportion is firstly added for reaction for 30-60 min, then the clay according to the proportion is continuously added for reaction for 30-60 min, and the colourless oil is obtained by filtration.

In the above steps 1), 2) and 3), the stirring may be performed under stirring, and the stirring speed is preferably 100 to 500rpm.

In a preferred embodiment of the present invention, in order to make the chlorophyll removal effect better, it further comprises adding one or more solvents such as n-hexane, ethanol, acetone, butane, ethyl acetate, methanol, isopropanol, ethanol, n-hexane, etc., preferably ethanol, to the reaction system of step 1), the reaction system of step 2), the decoloring step of step 3), or the distillation process of step 4). Wherein, the addition amount of the organic solvent is preferably 100 to 200 weight percent of the weight of the lower monohydric alcohol esterified algae oil. It is further preferable to add the above organic solvent to the reaction system of step 1).

In a preferred embodiment of the invention, the method consists of the steps of:

2) Reacting the first grease with alkali to obtain second grease;

3) Decolorizing the second grease by using activated carbon and/or clay, and collecting a decolorized oil;

4) Distilling the decolorized oil, and collecting unsaturated fatty acid alkyl ester.

Wherein the unsaturated fatty acid comprises one or more of eicosapentaenoic acid, eicosatetraenoic acid, docosahexaenoic acid, octadecadienoic acid, octadecatrienoic acid and stearidonic acid.

According to the preferred embodiment, chlorophyll remained in the lower monohydric alcohol esterified algae oil can be effectively removed by the simple reaction steps (firstly, the reaction with an acid solution, then the reaction with alkali, and then the decolorization and the gradient distillation are carried out by using active carbon and/or clay), and preferably more than 95% of chlorophyll can be effectively removed.

The invention also aims to provide a method for enriching polyunsaturated fatty acid glyceride from algae microbial oil, which can directly obtain high-purity triglyceride with the content of 90 percent. Among them, polyunsaturated fatty acids described in the present invention include polyunsaturated fatty acids which are omega-3 fatty acids, omega-6 fatty acids, typical fatty acids of which include one or more of docosahexaenoic acid, eicosapentaenoic acid, docosapentaenoic acid, arachidonic acid, linolenic acid, linoleic acid, or stearidonic acid.

The method comprises the following steps: the unsaturated fatty acid alkyl ester obtained by the method is subjected to transesterification with glycerin, and then molecular distillation is carried out to obtain the unsaturated fatty acid alkyl ester. The method comprises the following steps: the unsaturated fatty acid alkyl ester obtained by the method is subjected to transesterification with glycerin under the action of enzyme, and then is subjected to molecular distillation to remove impurities under the pressure of less than 100Pa and the temperature of 150-180 ℃, and absolute ethyl alcohol is eluted, so that the product is obtained.

In a preferred embodiment of the invention, the enzyme is a lipase. The enzyme is used in an amount of 1-5% by weight of the light phase ethyl ester oil. Wherein the dosage of the glycerol is 4-5 times of the weight of the light phase ethyl ester oil. The temperature of the transesterification is 35-65 ℃ and the time is 4-24 h. In this reaction, in order to increase the exchange rate and further increase the enrichment method, it is preferable that a base is added in an amount to control the soap content in the system within a range of 100 to 2000 ppm. Wherein the alkali can be sodium hydroxide, potassium hydroxide, sodium ethoxide, potassium ethoxide, sodium methoxide, sodium ethoxide, solid alkali, etc.

And (3) removing impurities (free fatty acid, mono-and diglycerides) by molecular distillation under the pressure of less than 100pa and the temperature of 150-180 ℃, collecting substrate glycerol and polyunsaturated fatty acid glyceride, eluting by using absolute ethanol with the concentration of 1-5 times, dissolving the glycerol in the ethanol, and collecting the polyunsaturated fatty acid glyceride.

The method provided by the invention can effectively remove chlorophyll of microbial oil through a specific sequence and simple reaction steps (at least more than 80% of chlorophyll can be effectively removed), and has the advantages of high yield, low cost and easiness in industrialization. The method for enriching polyunsaturated fatty acid glyceride from algae microbial oil provided by the invention can directly obtain high-purity triglyceride, the content can be more than 90%, the process is simple, the operation is simple, the industrial production is easy, the cost is low, the application range is wide, and the market demand is solved. The product obtained by the invention has wide application field and can be used in edible oil, health products, infant foods and medical products.

Drawings

In fig. 1, the crude EE oil (lower monohydric alcohol esterified algae oil) of example 1, the second grease obtained in step 2 of example 3, the dehydrated oil obtained in step 3 of example 3, and the unsaturated fatty acid ethyl ester oil obtained in step 4 of example 3 are shown in this order from left to right;

the unsaturated fatty acid ethyl ester oils obtained in example 4 and example 3 are shown in fig. 2 in order from left to right;

the unsaturated fatty acid ethyl ester oils obtained in example 3 and comparative example 1 are shown in fig. 3 in this order from left to right.

Detailed Description

The following describes the embodiments of the present invention in further detail with reference to examples. The following examples are illustrative of the present invention, but are not intended to limit the scope of the invention.

Example 1

Crude algae oil is extracted from nannochloropsis and transesterification is performed by using a chemical method:

(1) Mixing dried nannochloropsis powder 25g with ethanol 250g, stirring at 80deg.C for 30min to extract lipid, separating the residue, repeatedly extracting residue with ethanol, mixing filtrates to obtain dark green lipid-containing liquid (i.e. crude algae oil), and desolventizing.

(2) To the crude algal oil was added 50mL of ethanol and 4mL of 100% sulfuric acid, and transesterification was performed at 80℃for 6 hours. Pouring the reaction product into a separating funnel, adding 40mL of hexane and water (1:1), collecting a hexane phase containing fatty acid ethyl ester generated by the reaction, washing for 4 to 5 times until the pH becomes neutral, and steaming out hexane to obtain crude EE, namely the lower monohydric alcohol esterified algae oil, wherein the phospholipid content of the crude algae oil is 8.75%, the glycolipid content is 14.62%, the ethyl ester is 61.24%, and the chlorophyll content is 4.02%.

Example 2

The crude algae oil is transesterified by an enzymatic method:

taking 100g of crude algae oil obtained in the step (1) in the example 1 and 100g of 95% ethanol or absolute ethanol, wherein the using amount of lipase is 5g, adding sodium hydroxide to control the soap using amount to be 500ppm, stirring for 300r, and reacting at the temperature of 55 ℃ for 12 hours. After the reaction is finished, removing residual ethanol by negative pressure desolventizing to obtain crude EE, namely the lower monohydric alcohol esterified algae oil.

Example 3

The embodiment provides a method for removing chlorophyll in lower monohydric alcohol esterified algae oil, which comprises the following steps:

1. 100g of the crude EE oil obtained in example 1 and 100g of ethanol were mixed and heated to 80℃for reflux, 2.5g of citric acid was added, the volume was fixed to 5ML of aqueous solution, and the mixture was stirred for 30 minutes at 300r, and then 5g of pure water was further added for reaction for 30 minutes. Centrifuging or filtering to collect upper clear oil (i.e. first oil), metering ethyl ether with ethyl ether to volume, and detecting chlorophyll content at 667nm with spectrophotometry to obtain acid value of 8.9.

2. The first grease in the step 1 is stirred at 50 ℃ and 300r, 5.4g of sodium hydroxide is added into 18ML of pure water with constant volume, and the reaction is carried out for 60min. And (3) separating and removing soap and part of chlorophyll through centrifugation or filtration to obtain second grease, wherein chlorophyll is detected in the second grease by 1.4%.

3. Adding active carbon (wherein the addition amount of the active carbon is 5% of that of the second grease, w/v) into the second grease obtained in the step 2 at 50 ℃, reacting for 30min, continuously adding carclazyte (wherein the addition amount of the carclazyte is 5% of that of the second grease, w/v) to react for 30min, adsorbing impurities such as chlorophyll and soap, filtering to obtain a dehydrated oil, and detecting 0.8% of chlorophyll.

4. Taking 500g of the decolorized oil, pumping negative pressure to 50pa, heating to 185 ℃ for distillation, and collecting light phase ethyl ester oil, namely unsaturated fatty acid ethyl ester oil, wherein the color of the obtained unsaturated fatty acid ethyl ester oil is clear, and detecting chlorophyll to be 0.01%.

Example 4

1. 100g of the crude EE oil obtained in example 1 was heated to 80℃and refluxed, 2.5g of citric acid was added, the volume was fixed to 5ML of aqueous solution, stirred for 30min at 300r, and then 5g of pure water was further added to react for 30min. And centrifuging or filtering to collect upper clear oil (first grease), and detecting chlorophyll content of 3.0% by using ethyl ester oil, wherein the acid value is 8.9.

2. Stirring the first grease in the step 1 at 50 ℃ and 300r, adding 5.405g of sodium hydroxide into 18ML of pure water with constant volume, and reacting for 60min. And (3) separating and removing soap and part of chlorophyll through centrifugation or filtration to obtain second grease, wherein chlorophyll is detected in the second grease by 2.0%.

3. Adding active carbon (wherein the addition amount of the active carbon is 5% of that of the second grease, w/v) into the second grease obtained in the step 2 at 50 ℃, reacting for 30min, continuously adding carclazyte (wherein the addition amount of the carclazyte is 5% of that of the second grease, w/v) to react for 30min, adsorbing impurities such as chlorophyll and soap, filtering to obtain a dehydrated oil, and detecting 1.5% of chlorophyll.

4. Taking 500g of the decolorized oil, pumping negative pressure to 50pa, heating to 145 ℃ for distillation, and preserving heat for 1 hour until no light phase flows out; and then continuously heating to 185 ℃ for distillation, collecting light phase ethyl ester oil, namely unsaturated fatty acid ethyl ester oil, wherein the color of the obtained unsaturated fatty acid ethyl ester oil is clear, and detecting chlorophyll to be 0.20%. EPA content 80.2% and AA content 9.5% were measured.

Example 5

The embodiment provides a method for enriching polyunsaturated fatty acid glyceride from algae microbial oil, which comprises the following steps:

1. 100g of the unsaturated fatty acid ethyl ester oil obtained in step 4 of example 3 were taken and 400g of glycerol were added for random transesterification with the aid of a TLIM enzyme, wherein: the enzyme amount was 5% by weight of the total substrate, 25g of enzyme was added, and the reaction temperature was 65 ℃. The reaction time was 8 hours and the product was collected. Wherein a small amount of sodium hydroxide was added to the mixture prior to the reaction to control the production of 500ppm of soap and water, the substrate moisture content being 0.1%.

2. The reaction system is subjected to molecular distillation at a negative pressure of 30pa and a temperature of 185 ℃ to remove free fatty acid, mono-and di-glyceride (recyclable) and collect substrates glycerol and Gan San ester. Then adding 2 times of absolute ethyl alcohol for eluting, dissolving the glycerol in the ethyl alcohol, and collecting all the components (recycling the glycerol). And desolventizing the collected triglyceride to obtain the triglyceride, wherein the content of unsaturated fat is detected to obtain the triglyceride, the EPA content is 82.3%, the AA content is 10.5%, and the chlorophyll content is not detected. The obtained triglyceride has yellow color Y=5.2 and red light R=0.5, and reaches the national standard of grease.

Example 6

1. 100g of the unsaturated fatty acid ethyl ester oil obtained in step 4 of example 3 were taken and 400g of glycerol were added for random transesterification with the aid of a TLIM enzyme, wherein: the enzyme amount was 5% by weight of the total substrate, 25g of enzyme was added, and the reaction temperature was 65 ℃. The reaction time was 8 hours.

2. The reaction system is subjected to molecular distillation at a negative pressure of 30pa and a temperature of 185 ℃ to remove free fatty acid, mono-and di-glyceride (recyclable) and collect substrates glycerol and Gan San ester. Then adding 2 times of absolute ethyl alcohol for eluting, dissolving the glycerol in the ethyl alcohol, and collecting all the components (recycling the glycerol). And desolventizing the collected triglyceride to obtain the triglyceride, and detecting the unsaturated fatty acid to measure the EPA content of 75.5% and the AA content of 6.8%. The obtained triglyceride has yellow color y=3.5 and red light r=0.4, and reaches the national standard of grease.

Comparative example 1

The comparative example provides a method for removing chlorophyll in lower monohydric alcohol esterified algae oil, comprising the following steps:

1. 100g of the crude EE oil obtained in example 1 and 100g of ethanol were mixed, stirred at 50℃and 300r, 5g of activated carbon was added and reacted for 30min, and further 5g of clay was added and reacted for 30min. Filtering to obtain a colorless oil, and detecting chlorophyll 2.8%.

2. Distilling the decolorized oil, taking 500g of the oil obtained by the process, pumping negative pressure to 50pa, heating to 135 ℃ for distillation, preserving heat and distilling for 1 hour, and collecting light phase 1 (mainly distilling saturated fatty acid ethyl ester C12-C18 at the temperature) independently without light phase outflow; and continuously heating to 165 ℃ for distillation, collecting unsaturated fatty acid ethyl ester oil (polyunsaturated fatty acids such as C20 and C22, DHA, EPA, AA), removing chlorophyll, and collecting light phase ethyl ester oil, namely unsaturated fatty acid ethyl ester oil, wherein the light phase ethyl ester oil is clear in color and is detected to be 0.43 percent of chlorophyll. EPA content 81.6% and AA content 9.3% were measured.

Comparative example 2

The comparative example provides a method for enriching polyunsaturated fatty acid glycerides from algal microbial oil, comprising the steps of:

1. 100g of the unsaturated fatty acid ethyl ester oil obtained in step 4 of example 3 was taken, 400g of glycerin was added thereto and stirred for 300r, and the reaction temperature was 65 ℃. The reaction time was 8 hours. Wherein a small amount of sodium hydroxide is added to the mixture before the reaction to control the soap content to 500ppm, and water is added to control the water content of the substrate to be 0.1% -0.2%.

2. The reaction system is subjected to molecular distillation at a negative pressure of 30pa and a temperature of 185 ℃ to remove free fatty acid, mono-and di-glyceride (recyclable) and collect substrates glycerol and Gan San ester. Then adding 2 times of absolute ethyl alcohol for eluting, dissolving the glycerol in the ethyl alcohol, and collecting all the components (recycling the glycerol). No triglyceride was collected after ethanol elution.

Comparative example 3

1. mixing 100g of crude EE oil obtained in example 1 with 100g of ethanol, heating to 50 ℃, adding active carbon (wherein the addition amount of the active carbon is 5% of that of the lower monohydric alcohol esterified algae oil, w/v), reacting for 30min, continuously adding clay (wherein the addition amount of the clay is 5% of that of the lower monohydric alcohol esterified algae oil), reacting for 30min, adsorbing impurities such as chlorophyll, and filtering to obtain third grease, wherein the chlorophyll content is 3.1%.

2. And (3) heating the third grease in the step (1) to 80 ℃, adding 2.5g of citric acid, stirring for 30min at 300r, and continuing to add 5g of pure water for reaction for 30min. The upper ethyl ester oil was collected by centrifugation.

3. The upper ethyl ester oil in the step 2 is stirred at 50 ℃ and 300r, 5.4g of sodium hydroxide is added into 18ML of pure water with constant volume, and the reaction is carried out for 60min. And (3) separating and removing soap and part of chlorophyll through centrifugation or filtration to obtain fourth grease, wherein the chlorophyll content in the fourth grease is 1.1%.

4. Taking 500g of the fourth grease, pumping negative pressure to 50pa, heating to 135 ℃ for distillation, and preserving heat for 1 hour until no light phase flows out; and then continuously heating to 185 ℃ for distillation, and collecting light phase ethyl ester oil, namely unsaturated fatty acid ethyl ester oil, wherein the obtained unsaturated fatty acid ethyl ester oil detects 0.32% of chlorophyll.

The purity comparisons of the products obtained in each example and comparative example are shown in the following table:

it can be seen from fig. 1 that the color of the algae oil is gradually reduced by the step of treating chlorophyll, and finally a satisfactory removal effect is achieved. In fig. 2, it can be seen that the addition of the organic solvent can effectively improve the treatment effect, and in fig. 3, it can be seen that the treatment mode adopting the method has obvious difference in color and luster from the treatment mode of chlorophyll in the prior art.

The nannochloropsis adopted in the embodiment of the invention is taken as an object to be enumerated, the purpose and the effect of the invention are realized, in fact, the method has general adaptability to the treatment of algae oil, no specificity exists, and no obstacle exists for the technical personnel in the field to adopt the invention to realize the method on other algae such as chlorella and spirulina. The nannochloropsis in this example contains a large amount of unsaturated fatty acids EPA (eicosapentaenoic acid) and ARA (eicosatetraenoic acid), and other algae may contain DHA (docosahexaenoic acid) or linoleic acid and linolenic acid according to species, which do not affect the implementation of the method of the present invention. In addition, the embodiment of the invention adopts the ethanol to esterify with crude algae oil for the purpose of food safety, but the esterification of other lower monohydric alcohols for other purposes does not affect the achievement of the purpose of the invention.

Finally, the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for removing chlorophyll in algae microbial oil, which is characterized by comprising the following steps:

2) Reacting the first grease with alkali to obtain second grease;

in the step 1), the dosage of acid in the acid solution is 1.5-5 wt% of the weight of the lower monohydric alcohol esterified algae oil; the mass fraction of the acid solution is 50-70%; the reaction temperature is 70-90 ℃ and the reaction time is 60-200 min;

in step 2), the amount of the base is based on neutralization of the free fatty acids in the lower monohydric alcohol esterified algal oil and the acid value of the first grease in step 1).

2. The method of claim 1, further comprising step 4) distilling the decolorized oil to collect the unsaturated fatty acid alkyl esters.

3. The method according to claim 1 or 2, wherein the acid solution is a citric acid solution.

4. The method according to claim 1 or 2, wherein in step 3), the amount of the activated carbon and clay is 5-10% of the weight of the lower monohydric alcohol esterified algae oil, the temperature of the decoloring reaction is 50-70 ℃, and the reaction time is 60-120 min.

5. The method according to claim 2, further comprising adding an organic solvent in the reaction system of step 1), the reaction system of step 2), the decoloring step of step 3), or the distillation step of step 4); the organic solvent is one or more of n-hexane, ethanol, acetone, butane, ethyl acetate, methanol and isopropanol.

6. The process according to claim 5, wherein an organic solvent is added in the reaction of step 1).

7. The method according to claim 1 or 2, characterized in that it consists of the following steps:

2) Reacting the first grease with alkali to obtain second grease;

4) Distilling the decolorized oil, and collecting unsaturated fatty acid alkyl ester;

8. A method for enriching polyunsaturated fatty acid glycerides from algal microbial oils, comprising the steps of:

the unsaturated fatty acid alkyl ester obtained by the method of any one of claims 2 to 7 is subjected to transesterification with glycerin under the action of enzyme, and then the impurities are removed by molecular distillation under the pressure of less than 100pa and the temperature of 150-180 ℃, and the unsaturated fatty acid alkyl ester is obtained after absolute ethanol is eluted.

9. The method of claim 8, wherein the enzyme is a lipase, the enzyme is used in an amount of 1 to 5% by weight of the unsaturated fatty acid alkyl esters, and the glycerol is used in an amount of 4 to 5 times by weight of the unsaturated fatty acid alkyl esters; the temperature of the transesterification is 35-65 ℃ and the time is 4-24 h.

10. The process according to claim 8 or 9, wherein a base is further added to the transesterification reaction in an amount to control the soap content in the transesterification system to 100 to 2000 ppm.