Separation and purification method of phytosterol ester

Abstract

The invention discloses a separation and purification method of phytosterol ester, and particularly relates to the field of fine chemical engineering. The method comprises the following steps: firstly removing a phytosterol crude product from a plant deodorized distillate, then carrying out solvent crystallization on a phytosterol ester concentrated solution from which natural vitamin E is removed, carrying out column chromatography on a crystallized product, and separating to obtain a phytosterol ester product, wherein the filler of the chromatographic column is a mixture of modified silica gel and porous graphene; wherein the modified silica gel is prepared by the following steps: firstly, putting silica gel into a hydrochloric acid aqueous solution for activation to obtain activated silica gel; and carrying out reflux reaction on the activated silica gel, anhydrous toluene and 3-(trimethoxysilyl) propyl methacrylate, and drying to obtain the silica gel modified by the silane coupling agent. According to the separation method disclosed by the invention, the phytosterol ester with high purity and stable content can be separated.

Description

Technical Field

[0001] The invention relates to the field of fine chemicals, and in particular to a method for separating and purifying phytosterol esters. Background Art

[0002] Plant deodorized distillate (also known as DD oil, hereinafter referred to as DD oil) mainly contains free fatty acids (FFA), vitamin E, phytosterols, fatty acid glycerides (FAG), phytosterol esters and a small amount of saturated hydrocarbon compounds; DD oil includes various sources, such as soybean DD oil, corn DD oil, rapeseed DD oil, etc. The levels of compounds contained in DD oil from different sources are also different. Taking soybean DD oil as an example, it usually contains 40-50% FFA, 5-10% natural vitamin E (VE), 5-10% phytosterols, 15-20% FAG (including monoglycerides, diglycerides and triglycerides), 3-5% phytosterol esters and a small amount of saturated hydrocarbon compounds.

[0003] Phytosterol esters are generally prepared by esterification or transesterification of plant sterols and fatty acids. Since there are many types of sterols and fatty acids that can be used to make plant sterol esters, a variety of plant sterol esters with different physical and chemical properties can be obtained. The three main plant sterol esters are β-sitosterol esters, stigmasterol esters, and campesterol esters. Sterol esters can be converted into sterols and fatty acids in the human body, so their physiological functions include the physiological functions of both plant sterols and fatty acids. They have the same effect of reducing plasma total cholesterol and low-density lipoprotein cholesterol as free plant sterols, and in some aspects, the effect is even better.

[0004] Chinese patent CN103045664A discloses a method for producing biodiesel, sterols and vitamin E using deodorized distillates. This patent is actually a separation method for producing phytosterols, but does not separate natural phytosterol esters.

[0005] Patent WO 01 / 75083 discloses a method for selective alcoholysis or esterification (ester exchange) of free sterols (including phytosterols) in fat-based products, wherein an immobilized, selectively surfactant-coated lipase is used. The reaction occurs in the presence of triglycerides contained in the fat-based product, and at least one carboxylic fatty acid or its ester derivative can be optionally added. Modified milk fat compositions and low-cholesterol food products obtained by the disclosed method, and further discloses fat-based products rich in phytosterol esters obtained in situ, and the patent also uses phytosterol esters obtained by the reaction of phytosterols and fatty acids, rather than natural separation.

[0006] Chinese patent CN1668631A, fractionation of phytosterol esters in oil, although the patent mentions the use of fractionation to separate phytosterol esters synthesized from fatty acids and phytosterols, the fractionation method of the patent is quite wasteful, and although the separated phytosterol esters are relatively pure, the unseparated portion still contains a large amount of phytosterol esters, which is quite wasteful.

[0007] That is to say, the existing plant sterol esters are usually obtained by esterifying free fatty acids such as oleic acid with plant sterols using an acidic catalyst. The esterification reaction is a reversible reaction. If the reaction product cannot be separated and removed in time, the esterification reaction will not be thorough, thereby affecting the purity of the plant sterol esters. The utilization rate of raw materials will also be affected, and the acidic catalyst needs to be removed after the esterification is completed, which may cause environmental pollution and other problems. The plant sterol esters synthesized by esterification reaction have a relatively single component, and usually a certain free fatty acid is used to esterify plant sterols, and the obtained plant sterol esters are the corresponding fatty acid plant sterol esters, such as oleic acid sterol esters, linoleic acid sterol esters or stearic acid sterol esters, etc.

[0008] It can be seen that the existing phytosterol esters are generally prepared by the esterification reaction of phytosterols and fatty acids, but there is no report on the separation of natural phytosterol esters in plant DD oil. Summary of the invention

[0009] To this end, the present invention provides a method for separating and purifying phytosterol esters to solve the above-mentioned problems.

[0010] At present, the separation and removal of FFA in DD oil is carried out by esterifying it into fatty acid methyl esters using strong acid catalysis, which has no effect on FAG. However, the high boiling point of FAG causes it to be enriched in the final concentrate, thereby reducing the enrichment of phytosterol esters and further increasing the difficulty of column separation of phytosterol esters.

[0011] In order to achieve the above object, the present invention provides the following technical solutions:

[0012] A method for separating and purifying phytosterol esters provided by the present invention comprises:

[0013] Step 1, firstly, separating free phytosterols by bio-enzyme catalyzed esterification and transesterification and cold precipitation crystallization to obtain crude phytosterols and a third esterification liquid;

[0014] Step 2, using a multi-stage molecular distillation method to separate the esterified liquid into a concentrated solution of natural vitamin E and phytosterol esters;

[0015] Step 3, using isopropanol solvent to perform solvent cold precipitation crystallization on the concentrated solution of phytosterol ester to obtain a crude phytosterol ester;

[0016] Step 4, dissolving the crude sterol ester after cold analysis in a non-polar solvent, adding it to a chromatography column, eluting it in sequence using eluents of different polarities, collecting the plant sterol ester components by TLC qualitative judgment, and finally removing the solvent under reduced pressure to obtain the plant sterol ester product.

[0017] The filler of the chromatography column is modified silica gel mixed with porous graphene; wherein the modified silica gel comprises firstly placing silica gel into a hydrochloric acid aqueous solution for activation to obtain activated silica gel; then refluxing the activated silica gel, anhydrous toluene and 3-(trimethoxymethylsilyl)propyl methacrylate and drying to obtain silica gel modified with a silane coupling agent.

[0018] As an example: the filling material of the chromatography column is to first put silica gel into a hydrochloric acid aqueous solution, reflux for 1-24 hours under the protection of an inert gas, wash with high-purity water until neutral, and vacuum dry to obtain activated silica gel; then add the activated silica gel, anhydrous toluene, and silane coupling agent 3-(trimethoxymethylsilyl)propyl methacrylate into a container, stir magnetically, reflux for 1-20 hours, then wash with an organic solvent A, and vacuum dry to obtain silica gel modified with a silane coupling agent; the silica gel modified with a silane coupling agent is stirred and evenly mixed with graphene to obtain a silica gel mixture.

[0019] Furthermore, in step 1, the method of bio-enzyme catalyzed esterification and transesterification includes: adding lipase solution to DD oil, adding anhydrous methanol under temperature control, stirring the reaction, washing out glycerol produced by the reaction, and obtaining an initial esterification solution.

[0020] Furthermore, the amount of the lipase is 0.1-0.5% (m / m) of the DD oil; the amount of the anhydrous methanol is 10-30% (m / v) of the DD oil.

[0021] As an example, the temperature is controlled at 40-45° C., and the stirring reaction time is 20-40 h; the glyceride conversion in the esterification liquid A is qualitatively determined by TLC.

[0022] Furthermore, the acid value of the initial esterification liquid is less than 5 mgKOH / g.

[0023] Furthermore, in the step 1, the cold crystallization separation of free phytosterols includes reducing the pressure of the initial esterification liquid to remove methanol and water to obtain a second esterification liquid, transferring the second esterification liquid to a cold crystallization container, and slowly crystallizing and precipitating the free phytosterols in the liquid after programmed cooling, and filtering to obtain crude phytosterols and a third esterification liquid.

[0024] As an example, the temperature condition for removing methanol and water under reduced pressure is 80-90°C; the temperature is programmed to drop to 4°C.

[0025] Furthermore, in the step 2, the multi-stage molecular distillation includes the third esterified liquid passing through the first-stage short-path evaporator, removing the residual solvent and water, and then entering the second-stage short-path evaporator, and then distilling to obtain fatty acid methyl ester and then entering the third-stage short-path evaporator, and the remaining liquid after distilling to obtain natural vitamin E is a concentrated liquid enriched with phytosterol esters.

[0026] Further, the first stage short-path evaporator has a temperature of 120-130°C and a vacuum of 300-350Pa; the second stage short-path evaporator has a temperature of 130-160°C and a vacuum of 30-40Pa; the third stage short-path evaporator has a temperature of 180-230°C and a vacuum of 1Pa. As an example, the first stage temperature is preferably 140°C and the vacuum is 320Pa; the second stage is preferably 150°C and the vacuum is 35Pa; the third stage is preferably 200°C and the vacuum is 1Pa.

[0027] Furthermore, in the step 4, the eluents are, in sequence, a non-polar solvent, a non-polar solvent: a polar solvent = 98:2 (v / v), and a polar solvent.

[0028] Furthermore, in step 4, the non-polar solvent refers to a solvent with a relatively low dielectric constant, including but not limited to one or more of n-hexane, petroleum ether, and cyclohexane as examples.

[0029] Furthermore, in step 4, the polar solvent has a strong polarity and a large dielectric constant, and examples thereof include but are not limited to one or more of ethyl acetate, isobutyl acetate, and dichloromethane.

[0030] The present invention uses DD oil (plant deodorization distillate) as a raw material, converts free fatty acids (FFA) and glycerides (FAG) therein into fatty acid methyl esters by a bioenzymatic method, separates free phytosterols by cold crystallization, and then separates fatty acid methyl esters and VE by multi-stage molecular distillation, so that the phytosterol esters in the raw material are enriched from 3-5% to 50-60%, and finally the enriched phytosterol ester material is purified by column separation to obtain a phytosterol ester product with a content of more than 90%.

[0031] The present invention has the following advantages:

[0032] The present invention does not adopt conventional esterification and transesterification reactions, but uses biological enzymes to replace traditional strong acids and strong bases; and the lipase selected in the present invention has both esterification and transesterification functions, so that the FAG in the raw material is converted into fatty acid methyl esters in the first step, so that the phytosterol ester content in the concentrated solution after molecular distillation can reach 50-60%.

[0033] The separation method of the present invention can separate plant sterol esters with high purity and stable content. DETAILED DESCRIPTION

[0034] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Silicone mixture:

[0036] First, 10.0 g of silica gel was placed in a hydrochloric acid aqueous solution, refluxed for 6 hours under nitrogen protection, washed with high-purity water until neutral, and vacuum dried to obtain activated silica gel; then 10.0 g of activated silica gel, 60.0 g of anhydrous toluene, and 8.0 g of 3-(isobutyleneoxy)propyltrimethoxysilane were added to a three-necked flask, magnetically stirred, refluxed for 4 hours, then washed with toluene and acetone, and vacuum dried to obtain silane coupling agent modified silica gel; the silane coupling agent modified silica gel was mixed with porous graphene (XF269, 7440-44-0) in a ratio of 2:1 to obtain a silica gel mixture.

[0037] Example 1

[0038] This embodiment provides a method for separating and purifying phytosterol esters from plant deodorization distillates:

[0039] Step 1, weigh 129.5g DD oil (wherein the acid value is 96.5mgKOH / g, the VE content is 4.92%, the free phytosterol content is 6.67%, and the phytosterol ester content is 4.20%) and preheat to 45°C, add 0.13g lipase solution, start stirring, add a total of 25mL of anhydrous methanol dropwise, react for 28h and then wash with water to obtain esterified liquid A128.7g (wherein the acid value is 4.6mgKOH / g, the VE content is 4.92%, the free phytosterol content is 6.70%, and the phytosterol ester content is 4.21%), and TLC shows no glyceride (i.e., the ester exchange is completed);

[0040] Step 2: 128.7 g of esterification liquid A is decompressed to remove excess methanol and water, and then transferred to a cold precipitation container, and the temperature is lowered to 4° C. by program, and the free phytosterol is crystallized by cold precipitation. After filtering, 9.9 g of crude phytosterol (including 83.59% free phytosterol) is obtained, and then 118.5 g of esterification liquid C (including 5.33% VE content, 0.26% free phytosterol, and 4.54% phytosterol ester content) is obtained;

[0041] Step 3, 118.5g of esterified liquid C enters a first-stage short-path evaporator (temperature 124°C, vacuum degree 331Pa) for dehydration and degassing, and then enters a second-stage short-path evaporator (temperature 155°C, vacuum degree 37Pa) to evaporate 97.81g of fatty acid methyl ester, and enters a third-stage short-path evaporator (temperature 226°C, vacuum degree 1Pa) to evaporate VE 10.0g (wherein the VE content is 62.15%), and finally obtains 9.7g of concentrated liquid D (wherein the phytosterol ester content is 53.88%);

[0042] Step 4: Crystallization

[0043] Using isopropanol as solvent, performing solvent cold precipitation crystallization on the concentrated solution of phytosterol ester to obtain a crude phytosterol ester;

[0044] Step 5, dissolve 8.4 g of crude sterol ester in 10 mL of n-hexane, transfer to the top of a chromatography column containing 50 g of a silica gel mixture (100-200 mesh), and elute with 50 mL of n-hexane, 150 mL of n-hexane: isobutyl acetate = 98:2 (v / v), and 100 mL of isobutyl acetate, collect 138 mL of the phytosterol ester fraction, and remove the solvent under reduced pressure to obtain 5.4 g of phytosterol ester (with a phytosterol ester content of 94.65% and a total yield of 93.81%).

[0045] Example 2

[0046] This embodiment provides a method for separating and purifying phytosterol esters from plant deodorization distillates:

[0047] Step 1, weigh 250.4g DD oil (with an acid value of 88.6mgKOH / g, a VE content of 5.17%, a free phytosterol content of 5.54%, and a phytosterol ester content of 4.61%) and preheat to 45°C, add 0.50g lipase solution, start stirring, dropwise add 50mL of anhydrous methanol, react for 23h, and then wash with water to obtain esterification liquid A 252.2g (with an acid value of 3.5mgKOH / g, a VE content of 5.13%, a free phytosterol content of 5.56%, and a phytosterol ester content of 4.57%), and TLC shows no glyceride (i.e., the ester exchange is completed);

[0048] Step 2: After removing excess methanol and water from 252.2 g of esterification liquid A under reduced pressure, transfer it to a cold precipitation container, cool it to 4 ° C through a program, and crystallize free phytosterols through cold precipitation. After filtering to obtain 16.4 g of crude phytosterols (including 79.74% free phytosterols), 233.7 g of esterification liquid C (including 5.39% VE content, 0.33% free phytosterols, and 4.69% phytosterol ester content) is obtained;

[0049] Step 3, 233.7g of esterified liquid C enters a first-stage short-path evaporator (temperature 126°C, vacuum degree 322Pa) for dehydration and degassing, and then enters a second-stage short-path evaporator (temperature 151°C, vacuum degree 31Pa) to evaporate 192.1g of fatty acid methyl ester, and enters a third-stage short-path evaporator (temperature 226°C, vacuum degree 1Pa) to evaporate VE 20.7g (wherein the VE content is 58.95%), and finally obtains 20.6g of concentrated liquid D (wherein the phytosterol ester content is 51.43%);

[0050] Step 4: Crystallization

[0051] Using isopropanol as solvent, performing solvent cold precipitation crystallization on the concentrated solution of phytosterol ester to obtain a crude phytosterol ester;

[0052] Step 5, dissolve 16.3g of crude sterol ester in 20mL of n-hexane, transfer to the top of a chromatography column containing 100g of a silica gel mixture (100-200 mesh), and elute with 100mL of n-hexane, 300mL of n-hexane: ethyl acetate = 98:2 (v / v), and 200mL of ethyl acetate in sequence, collect the phytosterol ester fraction, and remove the solvent under reduced pressure to obtain 11.3g of phytosterol ester (including 93.70% phytosterol ester content and 91.07% total yield).

[0053] Comparative Example 1

[0054] This comparative example provides a method for separating and purifying phytosterol esters from plant deodorization distillates:

[0055] Except that the multi-stage molecular distillation in step 3 was replaced by three fractional distillations, the rest was exactly the same as in Example 1. Finally, 5.8 g of phytosterol esters (with a phytosterol ester content of 90.69% and a total yield of 84.33%) were obtained from 148.3 g of DD oil.

[0056] It can be seen that multi-stage molecular distillation can increase the yield of phytosterol esters.

[0057] Comparative Example 2

[0058] This comparative example provides a method for separating and purifying phytosterol esters from plant deodorization distillates:

[0059] This comparative example does not have step 4, and the rest is completely consistent with Example 1. Finally, 5.6 g of phytosterol esters (with a phytosterol ester content of 87.55% and a total yield of 92.79%) were obtained from 124.8 g of DD oil.

[0060] It can be seen that the process of adding solvent to cold crystallization before column chromatography can purify sterol esters to more than 60% before column chromatography, thereby increasing the processing capacity of silica gel column chromatography, and the impurities removed after cold crystallization will avoid irreversible adsorption on silica gel.

[0061] Comparative Example 3

[0062] This comparative example provides a method for separating and purifying phytosterol esters from plant deodorization distillates:

[0063] Except that the silica gel mixture in the chromatography column in step five was replaced with ordinary 50g silica gel (100-200 mesh), the rest was exactly the same as Example 1. Finally, 6.9g of phytosterol esters (with a phytosterol ester content of 81.94% and a total yield of 88.41%) were obtained from 153.2g DD oil.

[0064] It can be seen that the silica gel mixture can increase the purity of phytosterol esters.

[0065] Comparative Example 4

[0066] This comparative example provides a method for separating and purifying phytosterol esters from plant deodorization distillates:

[0067] Except that the silica gel mixture in the chromatography column in step five is replaced by silica gel (100-200 mesh) modified with 50g of silane coupling agent without adding graphene, the rest is exactly the same as Example 1. Finally, 5.6g of phytosterol esters (with a phytosterol ester content of 87.07% and a total yield of 89.84%) are obtained from 130.3g of DD oil.

[0068] It can be seen that the addition of graphene to the silica gel mixture can increase the purity of phytosterol esters.

[0069] Comparative Example 5

[0070] This comparative example provides a method for separating and purifying phytosterol esters from plant deodorization distillates:

[0071] Except that the silica gel mixture in the chromatography column in step five was replaced by 50 g of non-coupled silica gel + graphene mixture (100-200 mesh), the rest was exactly the same as Example 1. Finally, 5.3 g of phytosterol esters (with a phytosterol ester content of 85.28% and a total yield of 90.02%) were obtained from 120.6 g of DD oil.

[0072] It can be seen that the silica gel adsorption effect after coupling is better and the purity of phytosterol esters can be increased.

[0073] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for separating and purifying phytosterol esters, characterized in that: The method comprises: Step 1, firstly, separating free phytosterols by bio-enzyme catalyzed esterification and transesterification and cold precipitation crystallization to obtain crude phytosterols and a third esterification liquid; Step 2, using a multi-stage molecular distillation method to separate the esterified liquid into a concentrated solution of natural vitamin E and phytosterol esters; Step 3, using isopropanol solvent to perform solvent cold precipitation crystallization on the concentrated solution of phytosterol ester to obtain crystals of phytosterol ester; Step 4, dissolving the crude sterol ester after cold analysis in a non-polar solvent, adding it to a chromatography column, eluting it in sequence with eluents of different polarities, collecting the phytosterol ester components by TLC qualitative judgment, and finally removing the solvent under reduced pressure to obtain the phytosterol ester product; The filler of the chromatography column is modified silica gel mixed with porous graphene; wherein the modified silica gel comprises firstly placing silica gel into a hydrochloric acid aqueous solution for activation to obtain activated silica gel; then refluxing the activated silica gel, anhydrous toluene and 3-(trimethoxymethylsilyl)propyl methacrylate and drying to obtain silica gel modified with a silane coupling agent.

2. The method for separating and purifying phytosterol esters according to claim 1, characterized in that: In the step 1, the method of enzyme-catalyzed esterification and transesterification includes: adding lipase solution into DD oil, adding anhydrous methanol under temperature control, stirring for reaction, washing out glycerol produced by the reaction, and obtaining an initial esterification solution.

3. The method for separating and purifying phytosterol esters according to claim 2, characterized in that: The amount of the lipase is 0.1-0.5% (m / m) of the DD oil; the amount of the anhydrous methanol is 10-30% (m / v) of the DD oil.

4. The method for separating and purifying phytosterol esters according to claim 2, characterized in that: The acid value of the initial esterification liquid is less than 5 mgKOH / g.

5. The method for separating and purifying phytosterol esters according to claim 1, characterized in that: In the step 1, the separation of free phytosterols by cold crystallization includes reducing the pressure on the initial esterification liquid to remove methanol and water to obtain a second esterification liquid, transferring the second esterification liquid to a cold precipitation container, and slowly crystallizing and precipitating the free phytosterols in the liquid after programmed cooling, and filtering to obtain crude phytosterols and a third esterification liquid.

6. The method for separating and purifying phytosterol esters according to claim 1, characterized in that: In the step 2, the multi-stage molecular distillation includes the third esterified liquid passing through the first stage short-path evaporator, removing the residual solvent and water, and then entering the second stage short-path evaporator, and then distilling to obtain fatty acid methyl ester and entering the third stage short-path evaporator, and the remaining liquid after distilling to obtain natural vitamin E is a concentrated liquid enriched with phytosterol esters.

7. The method for separating and purifying phytosterol esters according to claim 6, characterized in that: The first stage short-path evaporator has a temperature controlled at 120-130°C and a vacuum degree of 300-350Pa; the second stage short-path evaporator has a temperature controlled at 130-160°C and a vacuum degree of 30-40Pa; the third stage short-path evaporator has a temperature controlled at 180-230°C and a vacuum degree of 1Pa.

8. The method for separating and purifying phytosterol esters according to claim 1, characterized in that: In the step 3, the eluents are non-polar solvent, non-polar solvent: polar solvent = 98:2 (v / v), and polar solvent.

9. The method for separating and purifying phytosterol esters according to claim 8, characterized in that: In the step 3, the non-polar solvent is one or more of n-hexane, petroleum ether and cyclohexane.

10. The method for separating and purifying phytosterol esters according to claim 9, characterized in that: In the step 3, the polar solvent is one or more of ethyl acetate, isobutyl acetate and dichloromethane.