CN111662152B - Method for extracting squalene from crude shark liver oil - Google Patents

Method for extracting squalene from crude shark liver oil Download PDF

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CN111662152B
CN111662152B CN202010642463.3A CN202010642463A CN111662152B CN 111662152 B CN111662152 B CN 111662152B CN 202010642463 A CN202010642463 A CN 202010642463A CN 111662152 B CN111662152 B CN 111662152B
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oil
shark liver
liver oil
squalene
molecular distillation
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CN111662152A (en
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戴志远
宋恭帅
朱蓓薇
沈清
王加斌
郑平安
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Zhejiang Gongshang University
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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
    • C11B3/006—Refining fats or fatty oils by extraction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C7/00—Purification; Separation; Use of additives
    • C07C7/10—Purification; Separation; Use of additives by extraction, i.e. purification or separation of liquid hydrocarbons with the aid of liquids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C7/00—Purification; Separation; Use of additives
    • C07C7/04—Purification; Separation; Use of additives by distillation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C7/00—Purification; Separation; Use of additives
    • C07C7/12—Purification; Separation; Use of additives by adsorption, i.e. purification or separation of hydrocarbons with the aid of solids, e.g. with ion-exchangers
    • 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
    • C11B3/001—Refining fats or fatty oils by a combination of two or more of the means hereafter
    • 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
    • C11B3/02—Refining fats or fatty oils by chemical reaction
    • C11B3/04—Refining fats or fatty oils by chemical reaction with acids
    • 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
    • C11B3/02—Refining fats or fatty oils by chemical reaction
    • C11B3/06—Refining fats or fatty oils by chemical reaction with bases
    • 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
    • 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
    • C11B3/12—Refining fats or fatty oils by distillation
    • 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
    • C11B3/16—Refining fats or fatty oils by mechanical means
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/50—Reuse, recycling or recovery technologies
    • Y02W30/74—Recovery of fats, fatty oils, fatty acids or other fatty substances, e.g. lanolin or waxes

Abstract

The invention discloses a method for extracting squalene from crude shark liver oil, which comprises the following steps: 1) Respectively degumming, deacidifying, decoloring and deodorizing the crude shark liver oil to obtain refined shark liver oil; 2) Adding a potassium hydroxide-ethanol solution into the refined shark liver oil to react to obtain an unsaponifiable matter; 3) Molecular distillation is carried out on the unsaponifiable matters in a wiped film type molecular distillation device to obtain molecular distillation products; 4) The molecular distillation product was passed through a column, eluent dichloromethane: methanol =9, and the resulting eluate contains enriched relief squalene.

Description

Method for extracting squalene from crude shark liver oil
Technical Field
The invention belongs to the field of oil processing, and particularly relates to a method for extracting (enriching) squalene from shark liver oil.
Background
Squalene is a highly unsaturated linear triterpenoid with a molecular formula of C, which is a compound linked by 6 isoprenes and is named as 2,6,10,15,19, 23-hexamethyl-2, 6,10,14,18, 22-tetracosahexaene 30 H 50 And is a light yellow or colorless oily liquid at normal temperature. The squalene has biological activity of improving anoxia tolerance, inhibiting microorganism growth, resisting bacteria, relieving inflammation, and regulating cholesterol metabolism. Is clinically used for preventing and treating heart diseases, cancers, hepatitis and various hypoxic diseases, and can relieve rhinitis, tonsillitis, gout, asthma and other diseases when being externally applied. It is a nontoxic intensifying agent with anti-aging and anticancer effects, and can be widely used in food, medicine and cosmetic industries. The squalene has wide sources and exists in microorganisms, plant seeds, microalgae, shark liver and human sebum. However, the content of squalene in the plant source is too low, so that the actual production and use difficulty is higher, and the development and utilization of squalene are further limited. Due to the outstanding physiological activity function of squalene, the application of squalene is more and more extensive, and the corresponding market demand is also expanding day by day. In order to meet the actual industrial production requirements, shark liver oil is still the main raw material for producing high-purity squalene.
At present, the methods for separating and purifying squalene by using crude shark liver oil mainly comprise the following steps: esterification separation method, solvent extraction method, supercritical fluid extraction method, ultrasonic assisted extraction method, water vapor extraction method, lipase selective hydrolysis method, molecular distillation method, adsorption separation method, etc. The supercritical fluid extraction and molecular distillation method has high equipment investment, relatively complex operation and high requirement on operators. The squalene prepared by the esterification separation method and the steam extraction method has low purity, and is often combined with other methods. The lipase selective hydrolysis method has high requirements on a reaction system and is not suitable for mass production. Therefore, a preparation method of squalene with low production cost, simple operation and high purity still needs to be provided.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for enriching squalene from crude shark liver oil, and the method can be used for preparing high-purity (more than or equal to 42%) squalene so as to meet the demand of the market on the squalene.
In order to solve the above technical problems, the present invention provides a method for extracting squalene from crude shark liver oil (a method for preparing high-purity squalene from crude shark liver oil), comprising the following steps:
1) Refining crude shark liver oil:
degumming, deacidifying, decolorizing, and deodorizing crude shark liver oil to obtain refined shark liver oil;
2) And extraction of unsaponifiable matter:
according to the refining shark liver oil: potassium hydroxide-ethanol solution =1: (5 plus or minus 0.5) adding 0.5-2.5 mol/L potassium hydroxide-ethanol solution into the refined shark liver oil, and reacting at 60-80 ℃ for 40-80 min;
carrying out post-treatment on the obtained reaction solution to obtain an unsaponifiable matter;
3) Molecular distillation:
molecular distilling the unsaponifiable matter in a wiped film type molecular distillation device to obtain molecular distillation products;
4) And column passing:
selecting a silica gel chromatographic column;
after dissolving the molecular distillation product with dichloromethane, loading the molecular distillation product on a column, wherein the loading amount of the molecular distillation product is 3g, and the eluent is dichloromethane: methanol =9, and the elution flow rate is 2mL/min;
the resulting eluate contained enriched angular squalene (high purity squalene).
As an improvement of the method for extracting squalene from crude shark liver oil, the step 3) is as follows:
in a wiped film molecular distillation device, the feeding rate of unsaponifiable matter (100 g) is 1.5ml/min, the pressure is 0.1pa, the preheating temperature is 65 ℃, the evaporation temperature is 180 ℃, and the wiped film rotating speed is 200rpm; obtaining a molecular distillation product at a light phase of the molecular distillation device.
As a further improvement of the process for extracting squalene from crude shark liver oil according to the present invention, the step 1) is:
1.1 And (2) degumming:
adding degelling agent 1% of the crude shark liver oil weight into the crude shark liver oil, and stirring at 80 deg.C and magnetic stirring speed of 500rpm for 30min, wherein the degelling agent is phosphoric acid water solution with volume concentration of 85%;
then, centrifugation (10 000g 10min); obtaining degummed oil (degummed shark liver oil);
1.2 Acid removal by alkaline process:
adding alkali liquor into the degummed oil, stirring at 40 deg.C and 500rpm for 20min; alkali solution is NaOH aqueous solution with the mass concentration of 20 percent;
weight W of lye Total addition amount ＝W Theoretical value +W Excess base ；W Theoretical value ＝7.13×10 -4 ×W Degumming oil ×AV，W Excess base ＝4％×W Degumming oil ，
AV is the acid value of the degummed oil, W Degumming oil Weight of degummed oil, W Total addition amount Is the weight of the alkali liquor;
centrifuging (10 000g, 10 min), washing with hot water (95 deg.C hot water), to obtain degummed deacidified oil;
1.3 Adsorption decoloring:
adding activated clay which accounts for 10 percent of the weight of the degummed deacidified oil into the degummed deacidified oil, and stirring for 20min at the temperature of 70 ℃ and the rotating speed of 500 rpm;
then, centrifugation (10 000g 10min); obtaining degummed deacidified destaining oil;
1.4 And (3) performing rotary evaporation deodorization:
stirring the degummed deacidified destained oil at 220 deg.C, rotation speed of 70rpm and pressure of 0.06Mpa for 60min to obtain refined shark liver oil.
As a further improvement of the method for extracting squalene from crude shark liver oil, the post-treatment of the step 2) is as follows:
adding water into the obtained reaction solution, extracting with n-hexane, washing the obtained extractive solution with 10% ethanol water solution, and rotary steaming to obtain unsaponifiable matter.
As a further improvement of the process for extracting squalene from crude shark liver oil according to the present invention, in said step 4), 1 collecting unit per 40mL is obtained, and 20 collecting units are obtained; preferably the 10 th collection unit.
As a further improvement of the process for extracting squalene from crude shark liver oil according to the invention, in step 2):
in the potassium hydroxide-ethanol solution, the concentration of potassium hydroxide is 1.5mol/L;
the reaction temperature is 70 ℃, and the reaction time is 60min.
In the invention, the chromatographic column is prepared by adopting the following method:
weighing silica gel with the particle size of 100-200 meshes and distilled water according to the weight ratio of 1:5, adding concentrated hydrochloric acid, adjusting the pH to about 2, keeping the pH for 10min, and washing the eluate with distilled water until the eluate is neutral. Then the mixture is activated for 24 hours at 105 ℃. And (3) filling the column by adopting a dry method, washing and removing impurities in the column by using dichloromethane after the interface of the silica gel does not descend any more, balancing for 2 hours, and standing for later use.
Carrying out GC analysis (tracking by using gas chromatography quantitative analysis) on the enriched sample (eluent obtained by passing through the column), and determining the content of squalene in the enriched sample; the method comprises the following specific steps:
weighing a certain amount of sample, dissolving in n-hexane, and filtering to be tested.
Gas chromatography conditions: HP-5 capillary chromatography column (30 m.times.0.32mm, 0.25 μm); temperature rising procedure: the initial temperature is 160 ℃, the temperature is increased to 220 ℃ at the speed of 15 ℃/min, and the temperature is kept for 2min; then raising the temperature to 280 ℃ at a speed of 5 ℃/min, and keeping the temperature for 20min; finally, the temperature is raised to 300 ℃ at the speed of 5 ℃/min, and the temperature is kept for 2min. The injection port temperature is 250 ℃, and the split ratio is 1. The injection port temperature is 300 ℃, the injection amount is 1 mu L, and the flow rate of carrier gas is 1mL/min. Each set of samples was tested in 3 replicates.
In the present invention, the parameters and results of the experimental design for "extraction of unsaponifiable matter" in step 2) are shown in table 1.
TABLE 1 test design and results
Figure BDA0002571691420000031
Figure BDA0002571691420000041
By analyzing the experimental results, the squalene purity is 42.65% under the conditions that the alkali concentration is 1.5mol/L, the reaction temperature is 70 ℃ and the reaction time is 60min.
The invention has the following technical advantages:
1. the crude shark liver oil is refined, so that the influence of impurities in the oil on the purity of the squalene is reduced, the quality of the squalene can be effectively improved, the squalene has an active effect on the subsequent preparation of the high-purity squalene, and the total amount of the squalene is not reduced before and after refining.
2. The refined shark liver oil is used as raw material, and the solvent extraction method is adopted to prepare unsaponifiable matter.
3. Further separating and purifying squalene in the molecular distillate by using 100-200 mesh silica gel.
The method adopts the combination of a solvent extraction method, a molecular distillation method and an adsorption separation method to separate and enrich squalene in the crude shark liver oil, and compared with the traditional ester exchange method, a supercritical fluid method, TLC and the like, the method is simpler and more convenient to operate, has lower requirements on equipment and can obviously improve the purity, and is expected to be put into industrial production. The invention provides a certain theoretical basis for the industrial preparation of high-purity squalene.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a flow chart of a process for preparing high purity squalene from crude shark liver oil;
FIG. 2 is a graph showing the effect of different concentrations of caustic wash on the angular squalene content;
FIG. 3 is a graph showing the effect of different temperatures on squalene content;
FIG. 4 is a graph showing the effect of different reaction times on squalene content
FIG. 5 is a gas chromatogram of a squalene standard;
FIG. 6 is a squalene standard curve.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, the present invention is further described in detail with reference to examples, but the scope of the present invention is not limited to the scope of the embodiments.
Example 1, a process for the preparation of high purity squalene from crude shark liver oil, sequentially following steps:
1) Refining crude shark liver oil:
degumming, deacidifying, decoloring and deodorizing respectively:
1.1 And (2) degumming:
adding degelling agent 1% of the weight of the crude shark liver oil into the crude shark liver oil, and stirring at 80 ℃ and a magnetic stirring speed of 500rpm for 30min, wherein the degelling agent is phosphoric acid water solution with the volume concentration of 85%;
then, 10 000g was centrifuged for 10min; obtaining degummed oil (degummed shark liver oil);
1.2 Acid removal by alkaline process:
adding alkali liquor into the degummed oil, and stirring for 20min at the temperature of 40 ℃ and the rotation speed of 500 rpm; alkali liquor is NaOH aqueous solution with the mass concentration of 20 percent;
weight W of lye Total addition amount ＝W Theoretical value +W Excess base ；W Theoretical value ＝7.13×10 -4 ×W Degumming oil ×AV，W Excess base ＝4％×W Degumming oil ，
AV is the acid value of the degummed oil, W Degumming oil Weight of degummed oil, W Total addition amount Is the weight of the alkali liquor;
then, after 10 000g is centrifuged for 10min, hot water at the temperature of 95 ℃ is added for washing, the amount of the hot water used for each washing is about 10 percent of the weight of the degummed oil, and the degummed oil is washed until the eluent is neutral; obtaining the degummed deacidified oil.
1.3 Adsorption decoloring:
adding activated clay accounting for 10% of the weight of the degummed deacidified oil into the degummed deacidified oil, and stirring for 20min at the temperature of 70 ℃ and the rotating speed of 500 rpm;
then, 10 000g was centrifuged for 10min; obtaining the degummed deacidified destaining oil.
1.4 ) and deodorizing by rotary evaporation:
stirring degummed deacidified destained oil at 220 deg.C and 70rpm under 0.06Mpa for 60min to obtain refined shark liver oil.
2) And extraction of unsaponifiable matter:
weighing refined shark liver oil in a three-neck flask, adding potassium hydroxide-ethanol solution with certain concentration (0.5, 1.0, 1.5, 2.0 and 2.5 mol/L), and refining the shark liver oil: potassium hydroxide-ethanol solution =1:5, at 70 ℃ for 60min.
After the reaction is finished, adding distilled water from the top of the condensation pipe and cooling to room temperature, wherein the using amount of the distilled water is approximately equal to the volume using amount of the refined shark liver oil; pouring into a separating funnel, extracting for 3 times by using normal hexane, and combining the extract liquor; then, washing the extract with 10% ethanol water solution until the eluent is neutral, and if the emulsion is opacified in the washing process, adding a small amount of absolute ethanol for demulsification; the dosage of the absolute ethyl alcohol only needs to ensure that the opacification phenomenon disappears; and finally, dehydrating the extract liquor by using anhydrous sodium sulfate, carrying out rotary evaporation (the rotary evaporation temperature is 65 ℃, and the pressure is 0.1 Pa) until the weight is constant to obtain an unsaponifiable matter, and storing at a low temperature for later use after nitrogen charging.
3) Molecular distillation:
weighing 100g of unsaponifiable matter, and putting the unsaponifiable matter into a wiped film type molecular distillation device, wherein the feeding rate is 1.5ml/min, the pressure is 0.1pa, the preheating temperature is 65 ℃, the evaporation temperature is 180 ℃, and the wiped film rotating speed is as follows: 200rpm.
Obtaining a molecular distillation product at a light phase of the molecular distillation device.
4) And manufacturing a chromatographic column:
weighing silica gel with the particle size of 100-200 meshes and distilled water according to the weight ratio of 1:5, adding concentrated hydrochloric acid, adjusting the pH to about 2, keeping the pH for 10min, and washing the eluate with distilled water until the eluate is neutral.
Then the mixture is activated for 24 hours at 105 ℃. And (3) filling the column by adopting a dry method, washing impurities in the column by using dichloromethane after the interface of the silica gel does not descend any more, balancing for 2 hours, and standing for later use.
5) Column passing: weighing the molecular distillation product obtained in the step 4), dissolving the molecular distillation product with dichloromethane, and loading the solution on a column.
The eluent is dichloromethane compounded with methanol (9,v/v), the loading amount of the molecular distillation product is 3g, and the elution flow rate is 2mL/min. 1 collection unit per 40mL (i.e., 1 tube per 40 mL) was followed by gas chromatography quantitation.
A total of 20 collection units of eluate were obtained.
6) GC analysis of squalene: and (3) carrying out GC analysis on the enriched sample (eluent) obtained in the step 5) to determine the content of squalene. Weighing a certain amount of sample dissolved in n-hexane (namely, 0.1g of sample of the 10 th collection unit in the step 5) is dissolved in 2ml of n-hexane, and filtering (passing through a filter membrane with the filter diameter of 0.22 mu m) to be detected.
Gas chromatography conditions: HP-5 capillary chromatography column (30 m.times.0.32mm, 0.25 μm); temperature rising procedure: the initial temperature is 160 ℃, the temperature is raised to 220 ℃ at a speed of 15 ℃/min, and the temperature is kept for 2min; then heating to 280 ℃ at the speed of 5 ℃/min, and keeping the temperature for 20min; finally, the temperature is raised to 300 ℃ at a speed of 5 ℃/min and is kept for 2min. The injection port temperature is 250 ℃, and the split ratio is 1. The injection port temperature is 300 ℃, the injection amount is 1 mu L, and the flow rate of the carrier gas is 1mL/min.
Obtaining the peak area when the peak-off time is 15.017 min; each set of samples was tested in duplicate 3 times.
7) Drawing of squalene standard curve
The gas phase analysis result of the squalene standard product is shown in FIG. 5, and the peak-off time of squalene is about 15.017min.
Replacing the enriched sample obtained in the step 5) with a pure squalene, setting 5 concentrations of 0, 50, 100, 200, 400 and 800 μ g/μ L, and detecting according to the gas chromatography conditions of the step 6), wherein a squalene standard curve is shown in fig. 6, the abscissa is the squalene concentration (μ g/mL), the ordinate is the peak area, and the linear regression equation is as follows: y =1.9214x +6.089 2 ＝0.9999。
And substituting the peak area obtained by detecting the enriched sample into the curve equation to obtain the squalene concentration in the enriched sample.
Substituting the enriched sample obtained in the step 5) with the crude shark liver oil, detecting according to the gas chromatography condition of the step 6), and substituting the peak area obtained by detection into the curve equation to obtain the concentration of squalene in the crude shark liver oil.
The results are as follows: the squalene purity in the enriched sample is shown in fig. 2. The crude shark liver oil had a squalene content of 8.50%.
As can be seen from fig. 2, the squalene purity tends to increase first and then decrease as the alkali concentration increases. At low concentration, the refined shark liver oil cannot be completely saponified, so that excessive fatty acid residues can be caused, and the refined shark liver oil cannot be removed in the water washing process, so that the content of squalene is low; with the continuous increase of the alkali concentration, the saponification reaction is more completely carried out, and squalene can be better enriched. However, an excess of base may destroy the squalene content of the sample. At a base concentration of 1.5mol/L, the squalene purity is at a maximum of 42.65%. Therefore, the alkali concentration is selected to be 1.5mol/L for the next optimization.
Therefore, the method of the invention can be used for effectively preparing high-purity squalene.
Example 2 preparation of high purity from crude shark liver oil:
in the step 2) 'extraction of unsaponifiable matter', the following steps are carried out: selecting 1.5mol/L potassium hydroxide-ethanol solution, and setting the reaction temperature at 60, 65, 70, 75 and 80 ℃;
the rest is equivalent to example 1.
The results are shown in FIG. 3.
As can be seen from FIG. 3, the squalene purity increases with increasing temperature at a reaction temperature in the range of 60 ℃ to 70 ℃, but shows a tendency to gradually decrease with increasing temperature. The reason for this analysis may be that, when the temperature is low, the saponification reaction rate is slow, resulting in incomplete saponification and excessive fatty acid residue, thereby reducing the squalene content; however, when the temperature is too high, the squalene structure is easily damaged by a high-temperature and alkaline reaction environment, and the purity of squalene is reduced. At a temperature of 70 ℃, the squalene purity is reaching the highest. Therefore, the reaction temperature was 70 ℃ for subsequent optimization.
Example 3 preparation of high purity shark liver oil from crude shark liver oil:
in the step 2) 'extraction of unsaponifiable matter', the following steps are carried out: 1.5mol/L potassium hydroxide-ethanol solution is selected to react for a certain time (40, 50, 60, 70 and 80 min) at the temperature of 70 ℃. The rest is equivalent to example 1.
The results are shown in FIG. 4.
As can be seen from FIG. 4, the squalene purity tends to increase first and then decrease with increasing reaction time. The analytical reasons may be that the reaction time is too short and the saponification reaction is incomplete; and the squalene is damaged in a high-temperature and alkaline environment for a long time, so that the purity of the squalene is reduced. In 60min, the squalene purity is at its highest. Therefore, the reaction time was 60min for subsequent optimization.
Embodiment 4, preferred technical solution:
in the step 2), the concentration of potassium hydroxide in the potassium hydroxide-ethanol solution is 1.5mol/L; the reaction temperature is 70 ℃, and the reaction time is 60min.
The rest is the same as example 1. The squalene purity was 42.65%.
Comparative examples 1,
Step 1) is eliminated;
the step 2) "extraction of unsaponifiable" is changed into the following steps: weighing crude shark liver oil, placing in a three-neck flask, adding 2.5mol/L potassium hydroxide-ethanol solution, and reacting at 90 deg.C for 80min. Crude shark liver oil: potassium hydroxide-ethanol solution =1:5 by volume.
After the reaction is finished, distilled water is added from the top of the condensation tube and cooled to room temperature, then the reaction liquid is poured into a separating funnel, and extraction is carried out for 3 times by using ethyl acetate, and then extraction liquid is combined. The rest is equivalent to embodiment 1.
The highest purity of squalene in the obtained product is 30.67%.
Comparative examples 2,
The "molecular distillation" of step 3) of example 4 was changed to: the dosage of unsaponifiable matter is 100g, the feeding rate is 2.0ml/min, the pressure is 0.1pa, the preheating temperature is 70 ℃, the evaporation temperature is 200 ℃, and the film scraping rotating speed is as follows: 200rpm. The rest is equivalent to example 4.
The highest purity of squalene in the obtained product is 35.98%.
Comparative example 3, the "column through" of step 5) of example 4 was changed to: the eluent is n-hexane: compounding ethyl acetate (100, v/v), wherein the loading amount is 2.5g, and the elution flow rate is 3mL/min; 1 collection unit per 10 mL. The rest is equivalent to example 4.
The highest purity of squalene in the obtained product is 38.05%.
Comparative example 4, step 1) of example 4 was changed to "degum" which was: the degumming agent is 85 percent of citric acid, and the addition amount is 1 percent of the weight of the oil; deacidifying is the weight W of alkali liquor Total addition amount ＝W Theoretical value (ii) a The 'decolorization' is changed into the condition that the adding amount of activated clay accounts for 1 percent of the weight of the oil. The rest is equivalent to example 4.
The highest purity of squalene in the obtained product is 32.11%.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Claims (3)

1. A method for extracting squalene from crude shark liver oil is characterized by comprising the following steps:
1) Refining crude shark liver oil:
degumming, deacidifying, decolorizing, and deodorizing crude shark liver oil to obtain refined shark liver oil;
2) And extraction of unsaponifiable matter:
according to the refining method of shark liver oil: potassium hydroxide-ethanol solution =1: (5 +/-0.5) adding 1.5mol/L potassium hydroxide-ethanol solution into the refined shark liver oil, and reacting at 70 ℃ for 60min;
carrying out post-treatment on the obtained reaction solution to obtain an unsaponifiable matter;
the post-treatment comprises the following steps: adding water into the liquid obtained by the reaction, extracting by using normal hexane, and cleaning and carrying out rotary evaporation on the obtained extract liquor to obtain an unsaponifiable matter;
3) And molecular distillation:
molecular distillation of the unsaponifiable matter in a wiped film molecular distillation apparatus to obtain molecular distillation products:
in a wiped film type molecular distillation device, the feeding rate of unsaponifiable matters is 1.5ml/min, the pressure is 0.1pa, the preheating temperature is 65 ℃, the evaporation temperature is 180 ℃, and the wiped film rotating speed is 200rpm; obtaining a molecular distillation product at a light phase of a molecular distillation device;
4) And column passing:
selecting a silica gel chromatographic column;
after dissolving the molecular distillation product with dichloromethane, loading the molecular distillation product on a column, wherein the loading amount of the molecular distillation product is 3g, and the eluent is dichloromethane: methanol =9, and the elution flow rate is 2mL/min;
the resulting eluate contains enriched angular squalene.
2. The process for extracting squalene from crude shark liver oil of claim 1, wherein said step 1) is:
1.1 B), degumming:
adding degelling agent 1% of the crude shark liver oil weight into the crude shark liver oil, and stirring at 80 deg.C and magnetic stirring speed of 500rpm for 30min, wherein the degelling agent is phosphoric acid water solution with volume concentration of 85%;
then, centrifuging; obtaining the degummed oil;
1.2 Acid removal by alkaline process:
adding alkali liquor into the degummed oil, stirring at 40 deg.C and 500rpm for 20min; alkali solution is NaOH aqueous solution with the mass concentration of 20 percent;
weight W of lye The total addition amount ＝W Theoretical value +W Excess base ；W Theoretical value ＝7.13×10 -4 ×W Degumming oil ×AV，W Excess base ＝4％×W Degumming oil ，
AV is the acid value of the degummed oil, W Degumming oil Weight of degummed oil, W The total addition amount Is the weight of the alkali liquor;
then centrifuging and washing with hot water to obtain degummed deacidified oil;
1.3 Adsorption decoloring:
adding activated clay which accounts for 10 percent of the weight of the degummed deacidified oil into the degummed deacidified oil, and stirring for 20min at the temperature of 70 ℃ and the rotating speed of 500 rpm;
then, centrifuging; obtaining degummed deacidified destaining oil;
1.4 ) and deodorizing by rotary evaporation:
stirring the degummed deacidified destained oil at 220 deg.C, rotation speed of 70rpm and pressure of 0.06Mpa for 60min to obtain refined shark liver oil.
3. A process for extracting squalene from crude shark liver oil according to claim 2,
in the step 4), each 40mL of the solution is 1 collecting unit, and 20 collecting units are obtained; the 10 th collection unit was selected.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4692280A (en) * 1986-12-01 1987-09-08 The United States Of America As Represented By The Secretary Of Commerce Purification of fish oils
CN101830770A (en) * 2010-06-02 2010-09-15 天津大学 Method for extracting squalene from vegetable oil deodorized distillate
CN103525876A (en) * 2013-09-20 2014-01-22 中国海洋大学 Method for enriching alkoxy glycerol in shark liver oil
CN110668945A (en) * 2019-09-29 2020-01-10 青岛利和生物科技有限公司 Method for extracting DHA and EPA in fish oil by combining supercritical fluid

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4692280A (en) * 1986-12-01 1987-09-08 The United States Of America As Represented By The Secretary Of Commerce Purification of fish oils
CN101830770A (en) * 2010-06-02 2010-09-15 天津大学 Method for extracting squalene from vegetable oil deodorized distillate
CN103525876A (en) * 2013-09-20 2014-01-22 中国海洋大学 Method for enriching alkoxy glycerol in shark liver oil
CN110668945A (en) * 2019-09-29 2020-01-10 青岛利和生物科技有限公司 Method for extracting DHA and EPA in fish oil by combining supercritical fluid

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
橄榄油中角鲨烯的分离纯化工艺研究;戴宇航;《中国优秀博硕士学位论文全文数据库(硕士)工程科技Ⅰ辑》;20111215;B018-47 *
鲨鱼肝油中角鲨烯和烷氧基甘油分析方法的建立及应用;唐小红;《中国优秀博硕士学位论文全文数据库(硕士)工程科技Ⅰ辑》;20120615;B024-241 *

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