LU500388B1

LU500388B1 - Method for extracting squalene from crude shark liver oil

Info

Publication number: LU500388B1
Application number: LU500388A
Authority: LU
Inventors: Ping'an Zheng; Jiabin Wang; Beiwei Zhu; Qing Shen; Gongshuai Song; Zhiyuan Dai
Original assignee: Univ Zhejiang Gongshang
Priority date: 2020-07-06
Filing date: 2021-07-05
Publication date: 2022-01-06
Also published as: CN111662152B; CN111662152A

Abstract

The present disclosure discloses a method for extracting squalene from crude shark liver oil. The method includes the following steps: 1) respectively degumming, deacidifying, decolorizing and deodorizing crude shark liver oil to obtain refined shark liver oil; 2) adding a potassium hydroxide- ethanol solution into the refined shark liver oil for reaction to obtain an unsaponifiable matter; 3) conducting molecular distillation on the unsaponifiable matter in a wiped-film molecular distillation device to obtain a molecular distillation product; and 4) passing the molecular distillation product through a column, wherein an eluent is a mixed solution of dichloromethane and methanol at a volume ratio of 9:1 and an obtained eluate contains enriched squalene.

Description

BL-5269 METHOD FOR EXTRACTING SQUALENE FROM CRUDE SHARK LIVER IL 509988

TECHNICAL FIELD

[01] The present disclosure belongs to the technical field of oil processing and in particular, relates to a method for extracting (enriching) squalene from crude shark liver oil.

BACKGROUND ART

[02] Squalene, also known as triacontahexaene, is a highly unsaturated straight-chain triterpenoid formed by connecting 6 isoprenes. The squalene is systematically named as 2,6,10,15,19, 23- hexamethyl-2, 6,10,14,18, 22-tetracosahexaene, has a molecular formula of CsoHso, and is light yellow or colorless oily liquid at a normal temperature. The squalene has biological activities of improving anoxia tolerance, inhibiting microorganism growth, resisting microorganisms, relieving inflammation, and regulating cholesterol metabolism. It is clinically used for preventing and treating heart diseases, cancers, hepatitis and hypoxic diseases. When externally applied, the squalene can relieve rhinitis, tonsillitis, gout, asthma and other diseases. The squalene is a nontoxic intensifying agent, has 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 the squalene in the plant source is too low, such that actual production and use are difficult and development and utilization of the squalene are limited. Due to outstanding physiological activities of the squalene, the use of the squalene is more extensive and a corresponding market demand is also expanding. In order to meet a demand of actual industry production, shark liver oil is still a main raw material for producing high-purity squalene.

[03] At present, the squalene is mainly separated and purified from crude shark liver oil by means of esterification separation, solvent extraction, supercritical fluid extraction, ultrasound-assisted extraction, water vapor extraction, lipase selective hydrolysis, molecular distillation, adsorption separation, etc. The supercritical fluid extraction and molecular distillation have a high equipment investment, relatively complex operations and a high requirement on operators. The squalene prepared by the esterification separation and water vapor extraction has a low purity, thus the two methods often need to be combined with other methods. The lipase selective hydrolysis has a relatively high requirement on a reaction system, but is not suitable for mass production. Therefore, a preparation method of squalene with low production cost, simple operation and high purity is still needed.

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SUMMARY LU500388

[04] The present disclosure aims to solve the technical problem by providing a method for enriching squalene from crude shark liver oil. The method can be used for preparing high-purity (more than or equal to 42%) squalene so as to meet a market demand on the squalene.

[05] To solve the above-mentioned technical problem, the present disclosure provides a method for extracting squalene from crude shark liver oil (a method for preparing high-purity squalene from crude shark liver oil ) and the method includes the following steps:

[06] 1) refining of crude shark liver oil

[07] respectively degumming, deacidifying, decolorizing and deodorizing the crude shark liver oil to obtain refined shark liver oil

[08] 2) extraction of unsaponifiable matter

[09] according to a volume ratio of the refined shark liver oil to a potassium hydroxide-ethanol solution at 1:(5+0.5), adding the potassium hydroxide-ethanol solution at a concentration of 0.5-2.5 mol/L into the refined shark liver oil for reaction at 60-80°C for 40-80 min; and

[10] post-treating obtained liquid from the reaction to obtain the unsaponifiable matter;

[11] 3) molecular distillation

[12] conducting molecular distillation on the unsaponifiable matter in a wiped-film molecular distillation device to obtain a molecular distillation product; and

[13] 4) column chromatography

[14] selecting a silica gel chromatographic column; and

[15] dissolving the molecular distillation product in dichloromethane, loading an obtained sample and passing it through the column, where 3 g of the molecular distillation product is loaded, an eluent is a mixed solution of dichloromethane and methanol at a volume ratio of 9:1, an elution flow rate is 2 mL/min;

[16] and an obtained eluate contains enriched squalene (high-purity squalene).

[17] As an improvement of the method for extracting squalene from crude shark liver oil of the present disclosure, step 3) may specifically include the following steps:

[18] conducting the molecular distillation in the wiped-film molecular distillation device at a feeding rate of the unsaponifiable matter (100 g) of 1.5 ml/min, a pressure of 0.1 Pa, a preheating temperature of 65°C, an evaporation temperature of 180°C, and a film-wiping rotating speed of 200 rpm; and obtaining the molecular distillation product in a light phase of the molecular distillation device.

[19] As an improvement of the method for extracting squalene from crude shark liver oil of the present disclosure, step 1) may specifically include the following steps:

[20] 1.1) degumming 2

[21] adding a degumming agent at 1% of a weight of the crude shark liver oil to the crude Shark 00 liver oil and stirring at 80°C with a magnetic stirrer at 500 rpm for 30 min, where the degumming agent is a phosphoric acid aqueous solution at a volume concentration of 85%; and

[22] conducting a centrifugation at 10,000 g for 10 min to obtain degummed oil (degummed shark liver oil);

[23] 1.2) deacidifying by alkaline method

[24] adding an alkaline solution to the degummed oil and stirring at 40°C and a rotating speed of 500 rpm for 20 min, where the alkaline solution is a NaOH aqueous solution at a mass concentration of 20%,

[25] a weight of the alkaline solution is calculated as follows: Wiotal added amount = Wtheoretical value + Wexcess alkali; Wherein Wineoretical vame = 7.13% 107 x Waegummed oil X AV, Wexcess alkali = 4% x Waegummed oil,

[26] AV is an acid value of the degummed oil, Waegummed oi 1s a weight of the degummed oil, and Wootal added amount 18 the weight of the alkaline solution; and

[27] conducting a centrifugation at 10,000 g for 10 min and hot-water washing with hot water at 95°C to obtain degummed and deacidified oil;

[28] 1.3) decolorizing by adsorption

[29] adding activated clay 10% of a weight of the degummed and deacidified oil to the degummed and deacidified oil, and stirring at 70°C and a rotating speed of 500 rpm for 20 min; and

[30] conducting a centrifugation at 10,000 g for 10 min to obtain degummed, deacidified and decolorized oil; and

[31] 1.4) deodorizing by rotary evaporation

[32] stirring the degummed, deacidified and decolorized oil at 220°C, a rotating speed of 70 rpm and at a pressure of 0.06 Mpa for 60 min to obtain the refined shark liver oil.

[33] As an improvement of the method for extracting squalene from crude shark liver oil of the present disclosure, post-treating of step 2) may specifically include the following steps:

[34] adding water to the obtained liquid from the reaction, conducting an extraction with n-hexane and washing (with a 10% ethanol-water solution) and rotary-evaporating an obtained extract to obtain the unsaponifiable matter.

[35] As an improvement of the method for extracting squalene from crude shark liver oil of the present disclosure, in step 4), every 40 mL is taken as one collection unit and a total of 20 collection units are obtained; and preferably the 10th collection unit may be selected.

[36] As an improvement of the method for extracting squalene from crude shark liver oil of the present disclosure, in step 2),

[37] in the potassium hydroxide-ethanol solution, a concentration of potassium hydroxide is 1.5 mol/L; and

[38] the reaction is conducted at 70°C for 60 min.

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[39] In the present disclosure, the used chromatographic column is prepared by the following method:

[40] weighing 100-200 mesh silica gel, mixing the weighed silica gel with distilled water at a weight ratio of 1:5, adding concentrated hydrochloric acid, adjusting a pH to about 2 and keep it for min, and washing with distilled water until an eluate is neutral; placing the silica gel at 105°C for an activation for 24 h; and packing a column by a dry method until a silica gel interface no longer drops, eluting with dichloromethane to remove impurities in the column and conducting an equilibration for 2 h, and standing for later use.

[41] An enriched sample (an eluate obtained from the column) is analyzed by GC (quantitative analysis and tracking by gas chromatography) and the content of squalene is determined; and specific steps are as follows:

[42] weighing a certain amount of the sample, dissolving it in n-hexane, and filtering the dissolved sample for testing.

[43] Gas chromatographic conditions: HP-5 capillary chromatographic column (30 mx0.32 mm,

0.25 um); heating program: initial temperature at 160°C, rising to 220°C at 15°C/min and keeping for 2 min; heating to 280°C at 5°C/min and keeping for 20 min; heating to 300°C at 5°C/min and keeping for 2 min; a temperature of an injection port at 250°C and a split ratio at 1:10; a temperature of an injection port at 300°C, an injection amount at 1 uL and a flow rate of carrier gas at 1 ml/min; and each group of the sample is repeatedly tested for 3 times.

[44] In the present disclosure, aiming at "extraction of unsaponifiable matter" in step 2), parameters and results of an experimental design are shown in Table 1.

[46] Through an analysis of the above experimental results, when an alkali concentration is 1.5 mol/L, a reaction temperature is 70°C, and time is 60 min, a purity of squalene is 42.65%.

[47] The present disclosure has the following technical advantages:

[48] 1. Crude shark liver oil is refined to reduce an influence of impurities in the oil on a purity of squalene, which can effectively improve its quality and has a positive effect on a subsequent 4 preparation of high-purity squalene. Besides, the total amount of the squalene is not reduced after the 9 refining.

[49] 2. Refined shark liver oil is used as a raw material and an unsaponifiable matter is prepared by a solvent extraction.

[50] 3. 100-200 mesh silica gel is used to further separate and purify the squalene in a molecular distillation product.

[51] The present disclosure adopts a combination of a solvent extraction, a molecular distillation and an adsorption separation to separate and enrich squalene in crude shark liver oil. Compared with a traditional ester exchange, a supercritical fluid extraction, TLC, etc., the method of the present disclosure is simpler and more convenient to operate, has a lower equipment requirement, can significantly improve purity and is expected to be used in industrial production. The present disclosure provides a certain theoretical basis for an industrial preparation of high-purity squalene.

BRIEF DESCRIPTION OF DRAWINGS

[52] The specific implementations of the disclosure will be further described in detail below with reference to the accompanying drawings.

[53] FIG. 1 is a production flow chart for preparing high-purity squalene from crude shark liver oil;

[54] FIG. 2 is a graph showing an influence of different alkali concentrations on the content of squalene;

[55] FIG. 3 is a graph showing an influence of different temperatures on the content of squalene;

[56] FIG. 4 is a graph showing an influence of different reaction time on the content of squalene;

[57] FIG. 5 is a gas chromatogram of a squalene standard; and

[58] FIG. 6 is a standard curve of squalene.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[59] To make the foregoing objective, features, and advantages of the present disclosure clearer and more comprehensible, the present disclosure is further described in detail below with reference to the examples, but the protection scope required by the present disclosure is not limited to the scope described in the examples.

[60] Example 1 A method for preparing high-purity squalene from crude shark liver oil successively included the following steps:

[61] 1) refining of crude shark liver oil

[62] The crude shark liver oil was respectively degummed, deacidified, decolorized and deodorized;

[63] 1.1) degumming

[64] A degumming agent at 1% of a weight of the crude shark liver oil was added to the erde 9 shark liver oil and stirring was conducted at 80°C with a magnetic stirrer at 500 rpm for 30 min, where the degumming agent was a phosphoric acid aqueous solution at a volume concentration of 85%; and

[65] a centrifugation was conducted at 10,000 g for 10 min to obtain degummed oil (degummed shark liver oil);

[66] 1.2) deacidifying by alkaline method

[67] an alkaline solution was added to the degummed oil and stirring was conducted at 40°C and a rotating speed of 500 rpm for 20 min, where the alkaline solution was a NaOH aqueous solution at a mass concentration of 20%;

[68] a weight of the alkaline solution was calculated as follows: Wiotal added amount = Wiheoretical value + Wexcess alkali; Wherein Wineoretical van = 7.13% 10 x Waegummed oil X AV, Wexcess alkali = 4% x Wegummed oil,

[69] AV was an acid value of the degummed oil, Wdegummed oil Was a weight of the degummed oil, and Wiotal added amount Was the weight of the alkaline solution; and

[70] a centrifugation was conducted at 10,000 g for 10 min, hot water at 95°C was added for washing, the amount of the hot water used for each washing accounted for about 10% of the weight of the degummed oil, washing was conducted until an eluate was neutral; and the degummed and deacidified oil was obtained;

[71] 1.3) decolorizing by adsorption

[72] activated clay 10% of a weight of the degummed and deacidified oil was added to the degummed and deacidified oil, and stirring was conducted at 70°C and a rotating speed of 500 rpm for 20 min; and

[73] a centrifugation was conducted at 10,000 g for 10 min to obtain degummed, deacidified and decolorized oil; and

[74] 1.4) deodorizing by rotary evaporation

[75] the degummed, deacidified and decolorized oil was stirred at 220°C, a rotating speed of 70 rpm and at a pressure of 0.06 Mpa for 60 min to obtain the refined shark liver oil;

[76] 2) extraction of unsaponifiable matter

[77] the refined shark liver oil was weighed into a three-necked flask, a certain concentration (0.5,

1.0, 1.5, 2.0 and 2.5 mol/L) of a potassium hydroxide-ethanol solution was added at a volume ratio of the refined shark liver oil to the potassium hydroxide-ethanol solution of 1:5 and reaction was conducted at 70°C for 60 min; and

[78] after the reaction was over, distilled water was added from a top part of a condenser tube to cool to a room temperature and the volume amount of the distilled water was about equal to the volume amount of the refined shark liver oil; an obtained product was poured into a separatory funnel and extracted 3 times with n-hexane, and extracts were combined; a combined extract was washed 6 with a 10% ethanol-water solution until an eluate was neutral, if turbidity occurred during the washing 0 process, a small amount of absolute ethanol must be added for demulsification; the amount of absolute ethanol only needed to ensure that the turbidity disappeared; and the extract was dehydrated by anhydrous sodium sulfate, a rotary evaporation (at a rotary evaporation temperature of 65°C and a pressure of 0.1 Pa) was conducted until constant weight to obtain an unsaponifiable matter, and after nitrogen filling, the unsaponifiable matter was stored at a low temperature for later use;

[79] 3) molecular distillation

[80] 100 g of the unsaponifiable matter was weighed and loaded into a wiped-film molecular distillation device at a feeding rate of 1.5 ml/min, a pressure of 0.1 Pa, a preheating temperature of 65°C, an evaporation temperature of 180°C and a film-wiping rotating speed of 200 rpm; and

[81] a molecular distillation product was obtained in a light phase of the molecular distillation device.

[82] 4) manufacture of chromatographic column

[83] 100-200 mesh silica gel was weighed, the weighed silica gel was mixed with distilled water at a weight ratio of 1:5, concentrated hydrochloric acid was added, a pH was adjusted to about 2 and kept for 10 min, and distilled water was used for washing until an eluate was neutral;

[84] the silica gel was placed at 105°C for an activation for 24 h; and a column was packed by a dry method until a silica gel interface no longer dropped, impurities in the column were eluted with dichloromethane, the column was equilibrated for 2 h, and standing of the column was conducted for later use;

[85] 5) column chromatography: the molecular distillation product obtained in step 4) was weighed and dissloved with dichloromethane, and an obtained sample was loaded and passed through the column,

[86] where an eluent was a mixed solution of dichloromethane and methanol (9:1, v/v), a sample loading amount of the molecular distillation product was 3 g, an elution flow rate was 2 mL/min; each 40 mL was taken as one collection unit (i.e., every 40 mL was collected into one tube) and quantitative analysis and tracking by gas chromatography were used; and

[87] a total of 20 collection units of an eluate were obtained;

[88] 6) GC analysis of squalene: an enriched sample (an eluate) obtained in step 5) was analyzed by GC and the content of squalene was determined; a certain amount of the sample was weighed and dissolved in n-hexane (that is, 0.1g sample of the 10th collection unit in step 5) was dissolved in 2 ml of n-hexane) and the dissolved sample was filtered (through a filter membrane with a filter diameter of 0.22 um) for testing;

[89] gas chromatographic conditions: HP-5 capillary chromatographic column (30 mx0.32 mm,

0.25 um); heating program: an initial temperature was 160°C, the temperature rose to 220°C at 15°C/min and kept for 2 min; the temperature was heated to 280°C at 5°C/min and kept for 20 min; 7 the temperature was heated to 300°C at 5°C/min and kept for 2 min; a temperature of an inj ection 0 port was 250°C and a split ratio was 1:10; a temperature of an injection port was 300°C, an injection amount was 1 uL and a flow rate of carrier gas was 1 ml/min; and

[90] a peak area at peak appearance time of 15.017 min was obtained; and each group of the sample was repeatedly tested for 3 times; and

[91] 7) plotting of standard curve of squalene

[92] a gas phase analysis result of a squalene standard product was shown in FIG. 5 and the peak appearance time of the squalene was about 15.017 min;

[93] a pure squalene product was used to replace the enriched sample obtained in step 5), 5 concentrations of 0, 50, 100, 200, 400 and 800 ug/uL were set, detection was conducted according to the gas chromatographic conditions of step 6), a standard curve of squalene was shown in FIG. 6, an x-coordinate was concentration of squalene (ug/mL), a y-coordinate was a peak area and a linear regression equation was: y=1.9214x+6.089, R? =0.9999;

[94] the peak area obtained from the detection of the enriched sample was brought into the above equation to obtain the concentration of the squalene in the enriched sample; and

[95] the crude shark liver oil replaced the enriched sample obtained in step 5), a detection was conducted according to the gas chromatographic conditions of step 6), and the peak area obtained from the detection of the enriched sample was brought into the above equation to obtain the concentration of the squalene in the crude shark liver oil.

[96] Results were as follows: the purity of the squalene in the enriched sample was as shown in FIG. 2. In the crude shark liver oil, the content of the squalene was 8.50%.

[97] It can be seen from FIG. 2 that the purity of the squalene increased first and decreased as the alkali concentration increased. At a low concentration, the refined shark liver oil cannot be completely saponified, resulting in excessive fatty acid residues which cannot be removed during water washing and low squalene content; and as the alkali concentration continued to increase, the saponification reaction was more complete and can better enrich the squalene. However, excessive alkali may destroy the squalene content in the sample. When the alkali concentration was 1.5 mol/L, the purity of the squalene reached the highest, 42.65%. Therefore, the alkali concentration was selected as 1.5 mol/L for subsequent optimization.

[98] Thus, the method of the present disclosure can effectively prepare high-purity squalene.

[99] Example 2 A method for preparing high-purity squalene from crude shark liver oil:

[100] in step 2) “extraction of unsaponifiable matter”, some operations were changed that 1.5 mol/L potassium hydroxide-ethanol solution was selected and a reaction temperature was set to 60, 65, 70, 75 and 80°C; and

[101] the remaining operations were the same as those of Example 1.

[102] Obtained results were shown in FIG. 3.

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[103] It can be seen from FIG. 3 that the purity of the squalene increased with the increase of tha 0 temperature in the reaction temperature range of 60°C to 70°C, but as the temperature continued to increase, the purity of the squalene showed a gradual decrease trend. A reason may be that when the temperature was low, a saponification reaction rate was slow, resulting in incomplete saponification and excessive fatty acid residues, thereby reducing the content of the squalene; but when the temperature was too high, high-temperature and alkaline reaction environment was easy to cause damage to a structure of the squalene, thereby reducing the purity of the squalene. When the temperature was 70°C, the purity of the squalene reached the highest. Therefore, the reaction temperature was 70°C for subsequent optimization.

[104] Example 3 A method for preparing high-purity squalene from crude shark liver oil:

[105] in step 2) “extraction of unsaponifiable matter”, some operations were changed that 1.5 mol/L potassium hydroxide-ethanol solution was selected and reaction was conducted at 70°C for certain time (40, 50, 60, 70 and 80 min); and the remaining operations were the same as those of Example 1.

[106] Results are shown in FIG. 4.

[107] It can be seen from FIG. 4 that the purity of the squalene increased first and decreased as the reaction time increased. A reason may be that when the reaction time was too short, saponification reaction was incomplete; and too long reaction time and high-temperature and alkaline reaction environment would destroy a large amount of the squalene, thereby reducing the purity of the squalene. When the reaction time was 60 min, the purity of the squalene reached the highest. Therefore, the reaction time was set as 60 min for subsequent optimization.

[108] Example 4 A preferred technical scheme:

[109] in step 2), in the potassium hydroxide-ethanol solution, a concentration of potassium hydroxide is 1.5 mol/L; and the reaction was conducted at 70°C for 60 min; and

[110] the remaining operations were the same those of Example 1. The purity of the squalene was

42.65%.

[111] Comparative example 1

[112] Step 1) was canceled,

[113] in step 2) “extraction of unsaponifiable matter”, some operations were changed that the crude shark liver oil was weighed into a three-necked flask, 2.5 mol/L potassium hydroxide-ethanol solution was added, and reaction was conducted at 90°C for 80 min; a volume ratio of the crude shark liver oil to the potassium hydroxide-ethanol solution was 1:5;

[114] after the reaction was over, distilled water was added from a top part of a condenser tube to cool to a room temperature, an obtained product was poured into a separatory funnel and extracted 3 times with n-hexane, and extracts were combined; the remaining operations were the same as those of Example 1.

[115] In the obtained product, the highest purity of the squalene was 30.67%.

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[116] Comparative example 2 7900589

[117] In step 3) of example 4 “molecular distillation”, some operations were changed that the use amount of the unsaponifiable matter was 100 g, a feeding rate was 2.0 ml/min, a pressure was 0.1 Pa, a preheating temperature was 70°C, an evaporation temperature was 200°C, and a film-wiping rotating speed was 200 rpm; and the remaining operations were the same as those of Example 4.

[118] In the obtained product, the highest purity of the squalene was 35.98%.

[119] Comparative example 3 In step 5) of example 4 “column chromatography”, some operations were changed that the eluent was a mixed solution of n-hexane:ethyl acetate (100:1, v/v), the loading amount was 2.5g, and the elution flow rate was 3mL/min; each 10 mL was 1 collection unit; and the remaining operations were the same as those of Example 4.

[120] In the obtained product, the highest purity of the squalene was 38.05%.

[121] Comparative example 4 In step 1) of example 4, some operations were changed that in the “degumming”, the degumming agent was 85% citric acid and the addition amount was 1% of the weight of the oil; in the “deacidifying”, a weight of the alkaline solution was calculated as follows: Wiotal added amount = Wheoretical value; and in the “decolorizing”, the addition amount of the activated clay accounted for 1% of the weight of the oil; and the remaining operations were the same as those of Example 4.

[122] In the obtained product, the highest purity of the squalene was 32.11%.

[123] Finally, it should be noted that the examples listed above are merely a few specific examples of the present disclosure. Apparently, the present disclosure would not be limited to the above examples, and many variations are possible. All modifications that can be directly derived or conceived by a person of ordinary skill in the art from the specification of the present disclosure should be regarded as falling into the protection scope of the present disclosure.

Claims

WHAT IS CLAIMED IS: HU500358

1. A method for extracting squalene from crude shark liver oil, comprising the following steps: 1) refining of crude shark liver oil respectively degumming, deacidifying, decolorizing and deodorizing the crude shark liver oil to obtain refined shark liver oil 2) extraction of unsaponifiable matter according to a volume ratio of the refined shark liver oil to a potassium hydroxide-ethanol solution at 1:(5+0.5), adding the potassium hydroxide-ethanol solution at a concentration of 0.5-2.5 mol/L into the refined shark liver oil for reaction at 60-80°C for 40-80 min; and post-treating obtained liquid from the reaction to obtain the unsaponifiable matter; 3) molecular distillation conducting molecular distillation on the unsaponifiable matter in a wiped-film molecular distillation device to obtain a molecular distillation product; and 4) column chromatography selecting a silica gel chromatographic column; and dissolving the molecular distillation product in dichloromethane, loading an obtained sample and passing it through the column, wherein 3 g of the molecular distillation product is loaded, an eluent is a mixed solution of dichloromethane and methanol at a volume ratio of 9:1, an elution flow rate is 2 mL/min; and an obtained eluate contains enriched squalene.

2. The method for extracting squalene from crude shark liver oil according to claim 1, wherein step 3) specifically comprises: conducting the molecular distillation in the wiped-film molecular distillation device at a feeding rate of the unsaponifiable matter of 1.5 ml/min, a pressure of 0.1 Pa, a preheating temperature of 65°C, an evaporation temperature of 180°C, and a film-wiping rotating speed of 200 rpm; and obtaining the molecular distillation product in a light phase of the molecular distillation device.

3. The method for extracting squalene from crude shark liver oil according to claim 2, wherein step 1) specifically comprises:

1.1) degumming adding a degumming agent at 1% of a weight of the crude shark liver oil to the crude shark liver oil and stirring at 80°C with a magnetic stirrer at 500 rpm for 30 min, wherein the degumming agent is a phosphoric acid aqueous solution at a volume concentration of 85%; and conducting a centrifugation to obtain degummed oil; 11

1.2) deacidifying by alkaline method HU500388 adding an alkaline solution to the degummed oil and stirring at 40°C and a rotating speed of 500 rpm for 20 min, wherein the alkaline solution is a NaOH aqueous solution at a mass concentration of 20%, a weight of the alkaline solution is calculated as follows: Wiotal added amount = Whheoretical value + Wexcess alkali; Wherein Wineoretical vaue = 7.133107 x Waegummed oit X AV, Wexcess alkali = 4% x Wdegummed oil, AV is an acid value of the degummed oil, Wdegummed oil 15 a weight of the degummed oil, and Wootal added amount 18 the weight of the alkaline solution; and conducting a centrifugation and hot-water washing to obtain degummed and deacidified oil;

1.3) decolorizing by adsorption adding activated clay 10% of a weight of the degummed and deacidified oil to the degummed and deacidified oil, and stirring at 70°C and a rotating speed of 500 rpm for 20 min; and conducting a centrifugation to obtain degummed, deacidified and decolorized oil; and

1.4) deodorizing by rotary evaporation stirring the degummed, deacidified and decolorized oil at 220°C, a rotating speed of 70 rpm and at a pressure of 0.06 Mpa for 60 min to obtain the refined shark liver oil.

4. The method for extracting squalene from crude shark liver oil according to claim 3, wherein the post-treating in step 2) specifically comprises: adding water to the obtained liquid from the reaction, conducting an extraction with n-hexane and washing and rotary-evaporating an obtained extract to obtain the unsaponifiable matter.

5. The method for extracting squalene from crude shark liver oil according to claim 4, wherein in step 4), every 40 mL is taken as one collection unit and a total of 20 collection units are obtained; and the 10th collection unit is selected.

6. The method for extracting squalene from crude shark liver oil according to any one of claim 1-5, wherein in step 2), in the potassium hydroxide-ethanol solution, a concentration of potassium hydroxide is 1.5 mol/L; and the reaction is conducted at 70°C for 60 min.

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