CN106748615B - Method for extracting squalene from oil - Google Patents

Abstract

The invention discloses a method for extracting squalene from grease, which utilizes glycerol and lipase to carry out glycerolysis on the grease, converts most triglyceride in the grease into monoglyceride and diglyceride, and then utilizes a process technology of cyclodextrin inclusion technology to extract the squalene, thereby avoiding the complex operation of one-step column chromatography or saponification extraction method, greatly reducing the use of organic solvent, and also avoiding the defects of high cost and low efficiency of supercritical extraction and high-efficiency countercurrent chromatography. Because the impurities are few, the aim of further purifying the squalene can be achieved only by subsequently utilizing an alumina column to adsorb and separate the squalene and the residual sterol.

Description

Method for extracting squalene from oil

Technical Field

The invention belongs to the field of chemical engineering, and particularly relates to a method for extracting squalene from grease.

Background

Squalene, also known as squalene, triacontahexaene, cod liver oil terpene, has the chemical name 2,6,10, 15,19, 23-hexamethyl-2, 6,10,14,18, 22-tetracosahexaene, is a highly unsaturated hydrocarbon compound originally found in black shark liver oil in 1906 by the japanese chemist Tsujimoto. The squalene is colorless oily liquid at normal temperature, is insoluble in water, is insoluble in methanol, ethanol and glacial acetic acid, is soluble in organic solvents such as diethyl ether, petroleum ether, acetone, carbon tetrachloride and the like, and has polarity similar to that of triglyceride and sterol.

Squalene is often used as a base for pharmaceutical ointments (hydrophilic ointments, water-absorbing ointments) and also as a component of suppositories, and can promote sufficient absorption of drugs. It can also be used as moisturizer in cosmetics such as cream (cold cream, cleansing cream, and skin caring cream), lotion, hair oil, hair cream, lipstick, aromatic oil, and aromatic powder.

Many research results show that squalene has certain biological activity for treating tumors, for example, squalene has an anti-tumor effect when being used alone in mice, and the action mechanism of squalene is that squalene can inhibit the growth of tumor cells and enhance the immunity of the organism, so that the resistance to tumors is enhanced. On the other hand, squalene can inhibit the generation of carcinogen nitrosamine, thereby playing an anti-tumor role. In addition, clinical experiments show that the squalene can be used together with other anti-tumor drugs, so that the drug effects of the drugs are greatly improved, and the squalene is suitable for various tumors such as lymphoma and the like. Besides good anti-tumor effect, squalene also has certain curative effect on other diseases, such as ulcer, hemorrhoid, dermatitis, skin scald and other symptoms, and can be used for treating or assisting in treating hyperlipidemia.

The biological activity of squalene is also widely used in cosmetic drugs. The preparation containing squalene has remarkable therapeutic effect on dermatoses such as acne, and has no side effect. Since squalene is easily oxidized at high temperature and under ultraviolet irradiation, when applied to skin care products, it can protect skin from damage by high temperature and ultraviolet light. Squalene is a good carrier for active oxygen delivery, so that cosmetic containing squalene has effects of preventing pachylosis and enhancing skin immunity.

The application of squalene as a bioactive substance with multiple physiological functions is increasingly emphasized by people, and the market demand is more and more increased. However, due to limited resources, finding and developing new resources for squalene is a problem that we are currently facing. The main source of squalene is shark liver oil from deep sea, and small amount of squalene exists in unsaponifiable matter of oil, especially olive oil, fish oil, fructus Siraitiae Grosvenorii kernel oil, amaranth oil, pumpkin seed oil, etc. With the development of science and technology, a plurality of new methods for extracting and separating squalene are developed, including a solvent extraction method, a supercritical carbon dioxide extraction method, a high-efficiency counter-current chromatography method, a column chromatography method, a silver ion extraction method and the like, and the requirements for extracting squalene from deodorized distillate of oil and fat can be basically met.

However, the oil contains a relatively large amount of squalene, and is also an important squalene resource, such as deep sea fish liver oil, fructus momordicae kernel oil, olive oil and the like. However, since the main component of triglyceride is similar to squalene in nature, the current methods for extracting squalene from oils and fats are mainly by saponification and column chromatography. The main principle of the saponification method is that triglyceride has ester bond, and is hydrolyzed into components with larger polarity, such as glycerol, fatty acid or salts thereof, and then separated by extraction or chromatography. The method is easy to generate emulsification, needs a large amount of organic solvent, and has complex process operation, low yield and no environmental friendliness. The column chromatography method directly utilizes the tiny polarity difference between the triglyceride and the squalene for separation, and because the two substances have close properties and very different proportions, a large amount of chromatographic materials and organic solvents are required to be used for achieving a good separation effect. As can be seen, both of these conventional methods have not achieved satisfactory results in solving the problem of separating triglycerides from squalene.

Therefore, in order to fully explore important resources of squalene in oil and overcome the defects of the prior art, a method for efficiently extracting squalene from enriched oil and fat needs to be developed. Therefore, the invention creatively adopts the glycerolysis technology to organically combine with the cyclodextrin inclusion technology, realizes the purpose of extracting and enriching squalene from grease in an environment-friendly and high-efficiency way, and can obtain high-purity squalene by adopting a simple separation means.

The invention content is as follows:

the invention aims to provide a high-efficiency and environment-friendly process method aiming at the technical problem of large use amount of organic solvent in the process of extracting squalene from grease in the prior art. The method utilizes glycerol and lipase to carry out glycerolysis on the grease, converts most of triglyceride in the grease into monoglyceride and diglyceride, and then utilizes a process technology of extracting squalene by a cyclodextrin inclusion technology, thereby avoiding the complex operation of one-step column chromatography or saponification extraction method, greatly reducing the use of organic solvent, and also avoiding the defects of high cost and low efficiency of supercritical extraction and high-efficiency counter-current chromatography. Because the impurities are few, the aim of further purifying the squalene can be achieved only by subsequently utilizing an alumina column to adsorb and separate the squalene and the residual sterol.

The purpose of the invention is realized by the following scheme:

a method for extracting squalene from oil comprises the following steps:

(1) adding lipase into the grease raw material and glycerol to perform glycerolysis reaction in a reactor, removing the lipase, and separating redundant glycerol;

(2) performing cyclodextrin inclusion on the glycerolysis product;

(3) carrying out reflux resolution of the inclusion compound by using ethanol to obtain a squalene crude product;

(4) the squalene can be obtained by adsorbing and separating alumina.

In the above method for extracting squalene from oil and fat: the lipase in the step (1) is immobilized by adopting a carrier, wherein the immobilized carrier is silica gel, anion resin, activated carbon, alumina, diatomite, porous ceramic or porous glass.

In the above method for extracting squalene from oil and fat: the reactor in the step (1) comprises a stirred tank reactor, a bubbling reactor, a packed bed reactor, a fluidized bed reactor or a membrane reactor;

in the method for extracting squalene from oil and fat, the cyclodextrin in the step (2) comprises α -cyclodextrin, β -cyclodextrin or gamma-cyclodextrin;

in the above method for extracting squalene from oil and fat: the filler used by the alumina column in the step (2) comprises neutral alumina, acidic alumina or basic alumina.

In the above method for extracting squalene from oil and fat: the weight ratio range of the grease raw material to the lipase in the step (1) is 100:1-100: 20;

the weight ratio range of the oil raw material to the glycerol is 8: 1-1: 1.

in the above method for extracting squalene from oil and fat: the glycerolysis temperature range in the step (1) is 40-70 ℃; the glycerolysis time range is 4h to 24 h;

in the above method for extracting squalene from oil and fat: the mode for removing the lipase in the step (2) comprises normal pressure filtration, reduced pressure filtration or centrifugal separation; the weight ratio of the cyclodextrin to the squalene in the oil is 2:1 to 10: 1; the weight ratio of water to cyclodextrin ranges from 2:1 to 10: 1; the temperature range of the cyclodextrin inclusion reaction is 40 ℃ to 70 ℃; the time range of the cyclodextrin inclusion reaction is 0.5h to 2 h; the low-temperature standing time range is 20min to 90 min; the temperature range of the low-temperature standing is 4 ℃ to 15 ℃.

In the above method for extracting squalene from oil and fat: the ethanol in the step (3) comprises an aqueous solution with the ethanol content of 70-100%; the weight ratio range of the ethanol to the clathrate solid in the step (3) is 3:1 to 10: 1; the temperature range of the solution-inclusion reaction in the step (3) is 70 ℃ to 90 ℃; the time range of the de-clathration reaction in the step (3) is 0.5h to 2 h;

in the above method for extracting squalene from oil and fat: the organic solvent used by the alumina column in the step (4) is n-hexane or petroleum ether.

The characteristics of the invention and the prior conventional technology are described as follows: because the main component of the oil is triglyceride, the polarity of the oil is very close to that of squalene, and the content ratio of the oil relative to the squalene is high, the separation difficulty of the prior conventional technology is very high. The prior general method for extracting squalene from oil raw materials mainly comprises a saponification method and a column chromatography method. The main principle of the saponification method is that triglyceride is hydrolyzed into components with larger polarity, such as glycerin and fatty acid or salts thereof, by utilizing the difference that triglyceride has ester bonds but squalene does not have ester bonds, and then the components are separated by utilizing the method of organic solvent extraction or chromatographic adsorption elution. The method is easy to produce emulsification in the extraction process, so that the yield is low and the cost is high; the subsequent chromatographic adsorption elution also needs to use a large amount of environmentally-friendly low-polarity organic solvent for elution because the content ratio of the fatty acid to the fatty acid salt is too large. The column chromatography method is directly adopted, namely, the separation is carried out by utilizing the tiny polarity difference between the triglyceride and the squalene, the separation efficiency of the existing adsorption material is low, and a large amount of chromatography materials and organic solvents are needed. It can be seen that the prior art processes are costly in the separation stage of triglycerides from squalene. The invention creatively adopts the technology of hydrolyzing and combining the glycerol with the cyclodextrin to include the cyclodextrin, so that a large amount of triglyceride is converted into diglyceride and monoglyceride, and the inclusion efficiency of the cyclodextrin on the angular squalene is promoted. As the squalene cyclodextrin inclusion compound is not basically dissolved in lipid and water, the separation and enrichment can be realized by only utilizing a simple solid-liquid separation method. In addition, because the selectivity of cyclodextrin inclusion is high, the obtained squalene crude product has few impurities, and high-purity squalene can be obtained only by simple adsorption separation in the subsequent process.

Compared with the prior art, the invention has the following beneficial effects:

1. the method adopts a combined process of glycerolysis and cyclodextrin inclusion, realizes high-efficiency enrichment of squalene in grease, overcomes the defect of using a large amount of organic solvent in the prior art, and avoids the defect of reduced squalene yield caused by the emulsification of fatty acid or fatty acid salt;

2. the squalene crude product enriched by the method has few impurity types, the squalene can be obtained only by utilizing an alumina column for adsorption separation, and the operation is simple, convenient and effective;

3. compared with the prior art, the method reduces the use of organic solvents, especially low-polarity organic solvents; avoids the difficulty caused by the emulsification of the saponified substance, and has environment-friendly and high-efficiency process. And lays a solid foundation for converting most of the grease into diglyceride with health care function for further comprehensive utilization.

Detailed Description

Example 1 taking 10kg of olive oil (with 0.36% of squalene content), adding 5% of lipase 435 and 5kg of glycerol, reacting for 8h in a water bath at 60 ℃, centrifuging to remove lipase, separating excessive glycerol, adding 3L of water and 720g of β -cyclodextrin into oil, stirring for 1h in a water bath at 60 ℃, standing for 30min at 4 ℃, filtering to obtain an inclusion compound, washing the inclusion compound with a small amount of water at room temperature, performing reflux resolution on the inclusion compound by using 95% ethanol at 80 ℃ for 1h, filtering while hot, concentrating the filtrate to obtain 46.5g of squalene crude product, performing adsorption separation by using a neutral alumina column, and eluting by using n-hexane to obtain 31.1g of squalene, 98.4% of content and 85.4% of transfer rate.

Example 2, 10kg of cod liver oil (containing 29.6% of squalene) is taken, 15% of lipase TL IM and 1.25kg of glycerol are added, the mixture is reacted for 18h in a water bath at 40 ℃, the lipase is removed by filtration, excessive glycerol is separated, 30L of water and 5.9kg of β -cyclodextrin are added into grease, the mixture is stirred for 2h in a water bath at 50 ℃, the mixture is kept stand for 30min at 4 ℃, an inclusion compound is obtained by filtration, the inclusion compound is washed by a small amount of water at room temperature, the inclusion compound is subjected to reflux resolution at 70 ℃ by 90% of ethanol for 1.5h, the mixture is filtered while hot, 3.22kg of crude squalene is obtained by concentrating the filtrate, 2.71kg of squalene is obtained by adsorption and separation by a neutral alumina column, the content is 99.1%, and the total transfer rate is 90..

Example 3 deep sea fish oil 10kg (squalene content 13.6%) was taken, 10% lipase RM IM and 10kg glycerin were added, reaction was carried out for 12 hours in a 50 ℃ water bath, lipase was removed by filtration, excess glycerin was separated, 20L of water and 6.8kg β -cyclodextrin were added to oil and fat, stirring was carried out for 1.5 hours in a 60 ℃ water bath, standing was carried out for 60 minutes at 8 ℃, an inclusion compound was obtained by filtration, the inclusion compound was washed with a small amount of water at room temperature, the inclusion compound was taken and subjected to reflux resolution with 80% ethanol at 90 ℃ for 30 minutes, filtration was carried out while hot, the filtrate was concentrated to obtain 1.51kg of crude squalene, 1.23kg of squalene was obtained by adsorption and separation with a neutral alumina column, the content was 97.9%, and the total transfer rate was 88.4%.

Example 4 pumpkin seed oil 10kg (squalene content 0.34%) was taken, 1% lipase 435 and 8kg glycerol were added, the mixture was reacted in a water bath at 60 ℃ for 8 hours, the lipase was removed by centrifugation, excess glycerol was separated, 10L of water and 5.1kg α -cyclodextrin were added to oil and fat, the mixture was stirred in a water bath at 70 ℃ for 0.5 hour, the mixture was allowed to stand at 10 ℃ for 90 minutes, an inclusion compound was obtained by filtration, the inclusion compound was washed with a small amount of water at room temperature, the inclusion compound was subjected to resolution by anhydrous ethanol at 80 ℃ under reflux for 2 hours, filtration was carried out while hot, the filtrate was concentrated to obtain a squalene crude product 43.8g, the squalene was adsorbed and separated by an acidic alumina column, and eluted with petroleum ether to obtain squalene 28.6g, the squalene content was 97.7%, and the transfer rate was 82..

Example 5A, 10kg of momordica grosvenori kernel oil (the content of squalene is 11.9%) is taken, 20% of lipase TLIM and 4kg of glycerol are added, the mixture reacts for 4h in a water bath at 70 ℃, the lipase is removed by filtration, excessive glycerol is separated, 59L of water and 5.9kg of β -cyclodextrin are added into oil, the mixture is stirred for 1h in a water bath at 50 ℃, the mixture is kept stand for 90min at 15 ℃, an inclusion compound is obtained by filtration, a small amount of water is washed at room temperature, the inclusion compound is taken and is subjected to reflux resolution inclusion for 2h with 70% of ethanol at 90 ℃, the mixture is filtered when the mixture is hot, 1.63kg of crude squalene is obtained by concentrating the filtrate, and 1.06kg of squalene is obtained by adsorption and separation of a neutral alumina column, the content is 98.7.

Example 6: taking 10kg of amaranth oil (the content of squalene is 4.1%), adding 5% of lipase RM IM and 6kg of glycerol, reacting for 24h in a water bath at 40 ℃, filtering to remove lipase, and separating redundant glycerol. Adding 40L of water and 4.9kg of gamma-cyclodextrin into grease, stirring for 2h in water bath at 40 ℃, standing for 90min at 6 ℃, filtering to obtain an inclusion compound, washing the inclusion compound with a small amount of water at room temperature, taking the inclusion compound, performing reflux resolution inclusion on the inclusion compound by 90 ethanol at 70 ℃ for 0.5h, filtering while hot, concentrating the filtrate to obtain 541.7g of crude squalene, and performing adsorption separation by using an alkaline alumina column to obtain 352.6g of squalene, wherein the content is 97.8 percent, and the transfer rate is 84.1 percent.

Claims (1)

1. A method for extracting squalene from oil is characterized by comprising the following steps:

(1) adding lipase into the grease raw material and glycerol to perform glycerolysis reaction in a reactor, removing the lipase, and separating redundant glycerol;

(2) performing cyclodextrin inclusion on the glycerolysis product;

(3) carrying out reflux resolution of the inclusion compound by using ethanol to obtain a squalene crude product;

(4) obtaining squalene by alumina adsorption separation;

the lipase in the step (1) is immobilized by adopting a carrier, wherein the immobilized carrier is silica gel, anion resin activated carbon, alumina, diatomite, porous ceramic or porous glass;

the cyclodextrin in the step (2) is α -cyclodextrin, β -cyclodextrin or gamma-cyclodextrin;

the glycerolysis temperature range in the step (1) is 40-70 ℃; the glycerolysis time range is 4h to 24 h;

the reactor in the step (1) is a stirred tank reactor, a bubbling reactor, a packed bed reactor, a fluidized bed reactor or a membrane reactor;

the filler used by the alumina column in the step (4) is neutral alumina, acidic alumina or alkaline alumina;

the mode for removing the lipase in the step (1) is normal pressure filtration, reduced pressure filtration or centrifugal separation; the weight ratio range of the cyclodextrin to the squalene in the oil in the step (2) is 2:1 to 10: 1; the weight ratio of water to cyclodextrin ranges from 2:1 to 10: 1; the temperature range of the cyclodextrin inclusion reaction is 40 ℃ to 70 ℃; the time range of the cyclodextrin inclusion reaction is 0.5h to 2 h; the low-temperature standing time range is 20min to 90 min; the temperature range of low-temperature standing is 4-15 ℃;

the ethanol in the step (3) is an aqueous solution with the ethanol content of 70-100%; the weight ratio range of the ethanol to the clathrate solid in the step (3) is 3:1 to 10: 1; the temperature range of the solution-inclusion reaction in the step (3) is 70 ℃ to 90 ℃; the time range of the de-clathration reaction in the step (3) is 0.5h to 2 h; the organic solvent used by the alumina column in the step (4) is n-hexane or petroleum ether.