CN108191638A - A kind of method of GLA in the algae oil using urea purification - Google Patents

A kind of method of GLA in the algae oil using urea purification Download PDF

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CN108191638A
CN108191638A CN201711308785.9A CN201711308785A CN108191638A CN 108191638 A CN108191638 A CN 108191638A CN 201711308785 A CN201711308785 A CN 201711308785A CN 108191638 A CN108191638 A CN 108191638A
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urea
gla
sample
petroleum ether
ethanol solution
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CN108191638B (en
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王强
陈辉
何晨柳
朱曦
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Institute of Hydrobiology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/09—Preparation of carboxylic acids or their salts, halides or anhydrides from carboxylic acid esters or lactones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/48—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/487—Separation; Purification; Stabilisation; Use of additives by treatment giving rise to chemical modification
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/007—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids using organic solvents
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/02—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils
    • C11C1/025—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by saponification and release of fatty acids

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  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Fats And Perfumes (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The present invention relates to the methods of the GLA in algae oil using urea purification a kind of, include the following steps：S1：Free fatty sample is prepared using the algae oil；S2：The free fatty sample is added in urea liquid, the first inclusion reaction is carried out at 04 DEG C, filtered, removed filter residue, aliphatic acid is extracted from filtrate with petroleum ether, obtains unrighted acid sample；S3：The unrighted acid sample is added in urea liquid, the second inclusion reaction is carried out at 40 30 DEG C, filtered, collect filter residue；S4：GLA is extracted from the filter residue.By using the dual urea adduct method of the present invention, the GLA that DNA purity in 15% grease is up to more than 95% can be only accounted for from original GLA, realizes the purpose for the GLA that high-purity is obtained under conditions of low cost.

Description

A kind of method of GLA in the algae oil using urea purification
Technical field
The present invention relates to microalgae components to extract field, more specifically it relates to the GLA in a kind of algae oil using urea purification Method.
Background technology
Gamma-Linolenic acid (gamma linolenic acid, GLA), belongs to one in n-6 series polyunsaturated fatty acids Kind, its chemical name is all-cis-Δ 6, Δ 9, Δ 12- octatecatrienoic acids, molecular formula C18H28O2, it is colorless oil liquid Body is easily aoxidized in air.GLA is one of essential fatty acid, it passes through complicated metabolic process in vivo, Generate a variety of secondary metabolites.Dihomo-gamma-linolenic acid or arachidonic acid are primarily formed, and then is converted into prostaglandin, white Triolefin and thromboxane etc..GLA and its range of metabolic object are in immune system, cardiovascular system, reproductive system, internal system All there is important and extensive physiological action.Clinically, GLA can be used for certain geriatric diseases for example diabetes, high fat of blood, Atherosclerosis, thrombotic cardiovascular and cerebrovascular disease, cancer and gastric ulcer, obesity, schizophrenia, atopic eczema, Rheumatic arthritis, vasculitis etc., it may also be used for the precursor of synthesis of prostaglandins has the work for adjusting blood pressure and cholesterol biosynthesis With, and the effect of controlling inflammation and promoting cell Proliferation can be played.
Linolenic comparision contents are few, and contain the alpha-linolenic acid than higher concentration, the object toward contact in general plant Matter is the isomer of GLA, is not readily separated in the purification process of GLA.The study found that some algae such as scenedesmus, spiral In algae etc., GLA accounts for the content of total fatty acids up to 15-25%, and by algae selection and training systern, the content of GLA is very To up to 30-40%, far above the GLA contents in higher plant.Therefore, GLA can be extracted from these algae for science Research or clinical application.
Urea adduct method is one of method of enriching polyunsaturated fatty acid from animal and plant fat.The original of urea adduct method Reason is that urea molecule forms relatively stable crystal inclusion compound precipitation in crystallization process with saturated fatty acid or single unsaturated fatty acid, And polyvalent unsaturated fatty acid is since double bond is more, carbochain bending, has certain steric configuration, is not easy by urea clathrate.It adopts Saturated fatty acid and monounsaturated fatty acids and the inclusion compound of urea formation are removed with the method for filtering, so that it may obtain higher degree Polyvalent unsaturated fatty acid.
Urea clathrate technology has small investment, and simple for process, operation temperature is low, can more fully retain unsaturated fat The advantages that nutrition of acid and physiological activity.But when in algae oil there are DHA, EPA when polyunsaturated fatty acids when, use urea Inclusion method can not isolated high-purity GLA.The reason is that DHA, EPA can not be removed using this method, and retain GLA. Therefore, it is necessary to a kind of new methods to purify GLA.
Invention content
We are had found when including temperature reduction to a certain extent, fatty acid mixed sample when studying urea adduct method GLA largely reduce, and the amount of DHA and EPA is almost unchanged, this purifies GLA for us and provides new thinking.
Found based on above, the present invention provides a kind of methods for purifying the GLA in algae oil, which is characterized in that including with Lower step：
S1：Free fatty sample is prepared using the algae oil；
S2：The free fatty sample is added in urea liquid, the first inclusion reaction is carried out at 0-4 DEG C, filtered, Filter residue is removed, aliphatic acid is extracted from filtrate with petroleum ether, obtains unrighted acid sample；
S3：The unrighted acid sample is added in urea liquid, the second inclusion reaction is carried out at -40--30 DEG C, Filter residue is collected in filtering；
S4：GLA is extracted from the filter residue.
In a specific embodiment, S1 includes the following steps：
S11：The algae oil is added in the KOH/methanol solution of 0.5M, obtain saponification system；
S12：The saponification system at 55 DEG C is flowed back, until oil droplet disappears, obtains reaction product；
S13：Free fatty is extracted from the reaction product with petroleum ether, the free fat is obtained after evaporating petroleum ether Fat acid sample.
In a specific embodiment, S2 includes the following steps：
S21：The urea ethanol solution of 0.1g/ml is prepared at 60 DEG C；
S22：The urea ethanol solution is added in the container equipped with the free fatty sample, is stirred to room temperature, Obtain the first inclusion reaction solution；
S23：The first inclusion reaction solution at 0-4 DEG C is incubated 12 hours, carries out inclusion reaction；
S24：Filtering extracts aliphatic acid from filtrate with petroleum ether, obtains the unrighted acid sample.
In a specific embodiment, S3 includes the following steps：
S31：The urea ethanol solution of 0.1g/ml is prepared at 60 DEG C；
S32：The urea ethanol solution is added in the container equipped with the unrighted acid sample, is stirred to room Temperature obtains the second inclusion reaction solution；
S33：The second inclusion reaction solution at -40--30 DEG C is incubated 12 hours, carries out inclusion reaction；
S34：Filter residue is collected in filtering.
In a specific embodiment, S4 includes the following steps：
S41：The filter residue is dissolved in ethanol solution, obtains the ethanol solution containing GLA and urea；
S42：It is extracted using petroleum ether from described containing the ethanol solution of GLA and urea, takes petroleum ether layer extract；
S43：After the petroleum ether layer extract is washed with deionized, then dry moisture therein is evaporated petroleum ether, Obtain GLA.
In a preferred embodiment, in S2, the weight of the free fatty sample and the urea ethanol solution Volume ratio is 0.2-0.4g/ml.
In a preferred embodiment, in S3, the weight of the unrighted acid sample and the urea ethanol solution Amount volume ratio is 0.05-0.1g/ml.
By using the dual urea adduct method of the present invention, DNA purity in 15% grease can be only accounted for from original GLA and is up to More than 95% GLA realizes the purpose for the GLA that high-purity is obtained under conditions of low cost.
Specific embodiment
The principle of the present invention and feature are described below in conjunction with example, the given examples are served only to explain the present invention, and It is non-to be used to limit the scope of the present invention.
1. algae oil
The algae oil used in detailed description below is to extract from the algae oil of scenedesmus, other than containing saturated fatty acid, Also containing 15% or so GLA, and also contain DHA, EPA.
2. the preparation of free fatty sample
25g algae oils are added in the KOH/methanol solution of 250ml 0.5M, flow back 1h under 55 DEG C of water-baths, until oil droplet disappears, Obtain reaction product.20ml distilled water and 100ml petroleum ethers are added in extract unsaponifiable matter, stratification.A layer solution is removed, Concentrated hydrochloric acid is added dropwise to ≈ 1, adds in 150ml petroleum ethers in two times, standing takes ether layer.Ether layer is rotated to solvent-free and is steamed, is obtained Free fatty pours into freezen protective in ground glass stoppered bottle.
3. first time urea clathrate reacts
5g urea is taken in 50ml ethyl alcohol, 60 DEG C of heating water baths are stirred to dissolving.By the free fatty in above-mentioned experiment It is in beaker (w/v of free fatty sample and urea ethanol solution is 0.2-0.4g/ml), urea ethyl alcohol is molten Liquid is rapidly added in the beaker containing free fatty, is stirred until room temperature with glass bar, until entering refrigerator, is incubated at 0-4 DEG C 12 hours.After take out inclusion compound, it is rapid to filter, retain the filtrate for being enriched and not including aliphatic acid.Filtrate is evaporated, adds in 20ml Water, it is neutral that concentrated hydrochloric acid, which is added dropwise, to pH.20ml petroleum ethers are added in, stratification takes ether layer, is washed with deionized 3 times.5 wash Anhydrous sodium sulfate, dry 5h are added in petroleum ether extract after washing, filtering, revolving obtain polyunsaturated fatty acid.Pass through liquid Phase chromatographic determination shows that the content of GLA is 65% or so.
4. second of urea clathrate reaction
The polyunsaturated fatty acid in above-mentioned experiment is taken in beaker, urea ethanol solution is rapidly added containing free fat In the beaker of fat acid (w/v of polyunsaturated fatty acid sample and urea ethanol solution is 0.05-0.1g/ml), glass is used Glass stick is stirred until room temperature, is cooled at -40--30 DEG C and is incubated 20 hours.Filtering, takes filter residue.Filter residue is dissolved in ethyl alcohol, Petroleum ether is added in, stratification takes ether layer, is washed with deionized 3 times.It is added in petroleum ether extract after 5 washings anhydrous Sodium sulphate, dry 5h, filtering, revolving obtain polyunsaturated fatty acid.It is shown by liquid chromatogram measuring, the content of GLA is 96.23%.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.

Claims (7)

  1. A kind of 1. method for purifying the GLA in algae oil, which is characterized in that include the following steps：
    S1：Free fatty sample is prepared using the algae oil；
    S2：The free fatty sample is added in urea liquid, the first inclusion reaction is carried out at 0-4 DEG C, filtered, removal Filter residue extracts aliphatic acid with petroleum ether from filtrate, obtains unrighted acid sample；
    S3：The unrighted acid sample is added in urea liquid, the second inclusion reaction, mistake are carried out at -40--30 DEG C Filter residue is collected in filter；
    S4：GLA is extracted from the filter residue.
  2. 2. according to the method described in claim 1, it is characterized in that, S1 includes the following steps：
    S11：The algae oil is added in the KOH/methanol solution of 0.5M, obtain saponification system；
    S12：The saponification system at 55 DEG C is flowed back, until oil droplet disappears, obtains reaction product；
    S13：Free fatty is extracted from the reaction product with petroleum ether, the free fatty is obtained after evaporating petroleum ether Sample.
  3. 3. according to the method described in claim 1, it is characterized in that, S2 includes the following steps：
    S21：The urea ethanol solution of 0.1g/m l is prepared at 60 DEG C；
    S22：The urea ethanol solution is added in the container equipped with the free fatty sample, stirs to room temperature, obtains First inclusion reaction solution；
    S23：The first inclusion reaction solution at 0-4 DEG C is incubated 12 hours, carries out inclusion reaction；
    S24：Filtering extracts aliphatic acid from filtrate with petroleum ether, obtains the unrighted acid sample.
  4. 4. according to the method described in claim 1, it is characterized in that, S3 includes the following steps：
    S31：The urea ethanol solution of 0.1g/m l is prepared at 60 DEG C；
    S32：The urea ethanol solution is added in the container equipped with the unrighted acid sample, stirs to room temperature, obtains To the second inclusion reaction solution；
    S33：The second inclusion reaction solution at -40--30 DEG C is incubated 20 hours, carries out inclusion reaction；
    S34：Filter residue is collected in filtering.
  5. 5. according to the method described in claim 1, it is characterized in that, S4 includes the following steps：
    S41：The filter residue is dissolved in ethanol solution, obtains the ethanol solution containing GLA and urea；
    S42：It is extracted using petroleum ether from described containing the ethanol solution of GLA and urea, takes petroleum ether layer extract；
    S43：After the petroleum ether layer extract is washed with deionized, then dry moisture therein is evaporated petroleum ether, obtains GLA。
  6. 6. method according to any one of claims 1-5, which is characterized in that in S2, the free fatty sample with The w/v of the urea ethanol solution is 0.2-0.4g/m l.
  7. 7. method according to any one of claims 1-5, which is characterized in that in S3, the unrighted acid sample W/v with the urea ethanol solution is 0.05-0.1g/m l.
CN201711308785.9A 2017-12-11 2017-12-11 Method for purifying GLA in algae oil by using urea Active CN108191638B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
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