TW201808277A - Method for extracting and purifying conjugated triene linolenic acid comprising extracting, esterfying and purifying - Google Patents

Abstract

The present invention relates to a method for extracting and purifying conjugated triene linolenic acid (CLN). The method comprises the following steps: (a) extracting step: placing a bitter gourd seed sample in an extraction vessel and extracting the crude extract of the bitter gourd seed with a supercritical fluid; (b)esterifying step: esterifying the crude extract of the bitter gourd seed obtained in (a) with an esterifying agent to form an esterified bitter gourd seed oil; and (c) purifying step: in a simulated moving bed, separating esterified unsaturated fatty acids from the esterified bitter gourd seed oil obtained in (b) to obtain a bitter gourd seed oil having more than 50% by weight of conjugated triene leneoleic acid (CLN).

Description

Extraction and purification method of conjugated triene linolenic acid (CLN)

The present invention relates to a method for effectively extracting bitter gourd seed oil and separating esterified unsaturated fatty acids from bitter gourd seeds, in particular to a method for extracting and separating from bitter gourd seeds to obtain high purity by using supercritical fluid and simulated moving bed Extraction and purification of conjugated triene linolenic acid (CLN).

Bitter melon has been considered a natural vegetable and fruit that lowers blood sugar. Many medical studies have clearly pointed out: Momordica charantia saponins, which are cucurbitane-type triterpenoids in momordica charantia, and momordica seed oil in momordica charantia seeds, have the effects of reducing body fat, lowering blood fat, lowering blood sugar, and inducing apoptosis of cancer cells. On the other hand, bitter melon also has the title of "plant insulin" because there are many compounds of polypeptide-P similar to insulin in bitter melon.

The fatty acid compounds present in bitter melon seed oil mainly include conjugated triene linolenic acid (9c, 11t, 13t-congugated linolenic acid, CLN) and its isomers, stearic unsaturated fatty acids, and conjugated diene Linoleic acid, lutein, etc. Among the fatty acid compounds of bitter melon seed oil, the content of CLN is the highest. Overall, the content of linoleic acid or linoleic acid of conjugated double bonds is higher than 50%, and the content of stearic acid About 10 to 35%; although the above contents will vary slightly with the maturity of bitter gourd, from the published literature, the content differences due to different varieties are generally not obvious.

Usually, the fatty acid compounds are generally analyzed by GC / MS or HPLC in research. The extraction method of bitter gourd seed oil generally uses an alkane-based solvent, for example, hexane. As for the main oil content composition of bitter gourd seed oil, the details are shown below:

In terms of traditional separation and purification technologies, the method mainly uses direct multi-step extraction and purification from pulp or whole fruit, for example, using a supercritical fluid, a resin adsorption method, or a urea crystallization method for separation. However, because these conventional technical methods are batch rather than continuous production, and auxiliary processing procedures such as solvents and high temperature processing are often required, the production cost will increase, the time will be too long, the purity of the product will not be easy to control, and it will not be conducive to industrialization. The problems of production, etc. may even cause adverse effects on the human body due to the remaining harmful solvents.

Therefore, there is an urgent need to develop an extraction and purification CLN method that does not have the above-mentioned problems of traditional technologies and can reduce production costs, increase production time, accurately control product purity, and facilitate industrial mass production.

In view of this, through intensive research and searching for various possible solutions to solve traditional technical problems, the inventors have developed a method that can not only solve the above problems of conventional extraction technology, but also reduce production costs and increase production time. , Precise control of product purity, and can facilitate the industrialized continuous production of conjugated unsaturated fatty acid extraction and purification methods, especially the problem of poor extraction effect and high manufacturing cost, especially a kind of low temperature and low pressure from bitter melon seeds The novel extraction and refining method of continuous extraction and purification of conjugated triene linolenic acid (CLN) has completed the present invention.

That is, the present invention provides a method for the extraction and purification of conjugated triene linolenic acid (CLN), which comprises: (a) an extraction step: placing a bitter gourd seed sample in an extraction container, and extracting the bitter gourd using a supercritical fluid; Crude seed oil extract; (b) Esterification step: the crude bitter melon seed oil obtained in (a) above is esterified with an esterifying agent to form an esterified bitter melon seed oil; (c) a purification step: Using a simulated moving bed, from the esterified balsam pear seed oil obtained in (b) above, an esterified unsaturated fatty acid having a Henry constant of 3 or less with the carbon 18 solid adsorbent was separated, and then further purified to obtain balsam pear seed oil containing Conjugated triene linolenic acid (CLN). More specifically, by the extraction and purification method of the present invention, bitter gourd seed oil having a total content of conjugated triene linolenic acid (CLN) exceeding 50% by weight can be obtained.

According to one aspect of the present invention, the method for extracting and purifying conjugated triene linolenic acid (CLN) according to the present invention preferably includes: before performing (a) the extraction step, heat-treating balsam pear seeds 4 to 8 The water content of this bitter gourd seed is less than 8%.

According to another aspect of the present invention, in the extraction and purification method of conjugated triene linolenic acid (CLN) of the present invention, the conditions for the supercritical fluid extraction are preferably: a pressure of 10 MPa to 60 MPa, a temperature It is 20 ℃ ~ 60 ℃.

According to another aspect of the present invention, the extraction and purification method of conjugated triene linolenic acid (CLN) according to the present invention is preferably further adding a co-solvent of 0 wt% to 40 wt% in the supercritical fluid; The solvent is preferably at least one selected from the group consisting of organic alcohols, short carbon chain organic alcohols containing 1 to 6 carbons, and mixtures thereof.

According to another aspect of the present invention, in the extraction and purification method of conjugated triene linolenic acid (CLN) of the present invention, the esterifying agent is preferably an organic alcohol containing 1 to 6 carbons. The short carbon chain organic alcohols and their mixtures constitute at least one selected from the group.

In addition, according to one aspect of the present invention, in the conjugated triene linolenic acid (CLN) extraction and purification method of the present invention, the esterification reaction is preferably a base-catalyzed reaction or an ion-exchange resin-catalyzed reaction.

Furthermore, according to another aspect of the present invention, in the method for extracting and purifying conjugated triene linolenic acid (CLN) according to the present invention, the stationary phase of the simulated moving bed is made of silicon dioxide with 4-30 carbon atoms. The surface-modified particles of alkanes are preferably 18-carbon surface-modified particles.

According to still another aspect of the present invention, in the extraction and purification method of conjugated triene linolenic acid (CLN) of the present invention, the mobile phase of the simulated moving bed is preferably 100% to 80% by weight of ethanol, and 0% to 20% by weight of deionized water.

In the following, different specific embodiments are listed and described in more detail for the implementation aspects of the present invention in order to make the spirit and content of the present invention more complete and easy to understand; however, those with ordinary knowledge in this technology should understand The invention is of course not limited to these examples, and other identical or equal functions and sequence of steps can be used to achieve the invention.

In addition, through the following specific examples, the scope of the present invention can be further proved, but it is not intended to limit the scope of the present invention in any form.

In the following, the definitions of technical terms and terms used in this article are explained first.

The "bitter gourd seed" herein refers to, for example, including but not limited to wild bitter gourd seed, or bitter gourd seed obtained by artificial cultivation. In addition, the water content of bitter gourd seeds used in the present invention is not particularly limited, and for example, bitter gourd seeds dried to a moisture content of less than 10% can be used. Secondly, the size of bitter gourd seed is not particularly limited as long as it does not affect the extraction efficiency; for example, bitter gourd seed broken into small particles with a particle size of less than 5 mm can be used. In addition, the variety of bitter gourd seed used in the present invention is not particularly limited, for example, including but not limited to using Hualian No. 4 bitter gourd seed provided by Huanyu Biotech Co., Ltd.

"Extraction" herein refers to, for example, a process that includes, but is not limited to, extraction of bitter melon seed oil using a supercritical fluid.

"Esterification" herein refers to, for example, the addition of alcoholic compounds in a process step to cause esterification of the active ingredients in balsam pear seeds, such as, but not limited to, "methylation", "ethyl esters" "And so on.

"Purification" in this article refers to the effect of separating and removing crude extracts and impurities containing impurities to form high-purity products by various means. For example, by using Simulated Moving Bed (SMB) to remove bitter gourd Procedure for separation and purification of ethylated conjugated unsaturated fatty acids in seed oil.

Hereinafter, the extraction and purification method of the conjugated triene linolenic acid (CLN) of the present invention will be described.

As shown in FIG. 2, the implementation steps of the method for extracting and purifying conjugated triene linolenic acid (CLN) according to the present invention include: an extraction step S1, a transesterification step S2, and a separation step S3.

In a specific example of the extraction step S1, for example, a dried bitter gourd seed sample may be first placed in an extraction solvent, so that the conjugated unsaturated fatty acid component in the bitter gourd seed is dissolved in the extraction solvent to form a bitter gourd Seed extract. The extraction solvent used in the extraction step S1 of the present invention is not particularly limited, and may be, for example, water, an organic solvent, or a supercritical fluid. In general, organic solvents and supercritical fluids are more conducive to the extraction of active ingredients with low polarity or organic affinity, and water is more conducive to the extraction of active ingredients with high polar properties.

Secondly, the extraction step S1 of the present invention is also suitable for adopting a supercritical fluid extraction method. In addition, the equipment applicable to the supercritical fluid extraction method of the present invention is not particularly limited; for example, a supercritical carbon dioxide extraction device as shown in FIG. 3 may be used. Generally speaking, a supercritical carbon dioxide extraction device generally includes a carbon dioxide storage tank 1 and a co-solvent storage tank 2, which are respectively connected to an extraction tank 3, and a back pressure valve 4 is used to adjust the internal pressure of the extraction tank 3, and A temperature regulator 5 regulates the internal temperature of the supercritical carbon dioxide extraction device, so that the carbon dioxide flowing into the extraction tank 3 can be converted into a supercritical fluid for extraction when the critical pressure and the critical temperature are reached. The extraction tank 3 and a gas-liquid The separation tank 6 communicates with the carbon dioxide flowing back into the gaseous state when decompressed, and is recovered to the carbon dioxide storage tank 1, wherein the supercritical carbon dioxide extraction device is provided with a plurality of liquid pumps 7 and a plurality of valves 8 to regulate the The flow of carbon dioxide or co-solvent in the supercritical carbon dioxide extraction device. The carbon dioxide storage tank 1 and the gas-liquid separation tank 6 preferably include an adsorption column 9 to remove impurities in the carbon dioxide gas.

According to an aspect of the present invention, the extraction solvent used in the extraction and purification method of the conjugated triene linolenic acid (CLN) of the present invention may be, for example, an organic (or low polarity) The supercritical fluid is preferably carbon dioxide in a supercritical state. In more detail, according to some embodiments, the supercritical carbon dioxide fluid used in the present invention is preferably a carbon dioxide that can form a supercritical state under conditions of greater than a critical pressure of 35 MPa and greater than a critical temperature of 50 ° C or more. The best is that supercritical carbon dioxide can be formed under the conditions of pressure of 20 ~ 35 MPa and temperature of 40 ~ 60 ℃.

The supercritical fluid extraction method can also be mixed. In addition, according to an aspect of the present invention, in the extraction method of the present invention, a co-solvent capable of promoting the physicochemical properties of the supercritical fluid and improving the extraction efficiency can be further used.

Hereinafter, the esterification step S2 in the extraction and purification method of the conjugated triene linolenic acid (CLN) of the present invention will be described in more detail with reference to FIG. 2.

According to an aspect of the present invention, the esterification means in the esterification step S2 of the present invention is not particularly limited; for example, it can be achieved by a base-catalyzed reaction or an ion-exchange resin-catalyzed reaction. When the esterification is achieved by a base-catalyzed reaction, for example, a base catalyst such as NaOH, ethanol, or the like can be used. In the present invention, the amount of the alkali catalyst used is not particularly limited. For example, it may be in the range of about 1% to 50% by weight relative to the total weight of bitter gourd seed oil. In addition, as the base catalyst suitable for the method of the present invention, NaOH is preferably used.

Hereinafter, the separation step S3 of the present invention will be described in more detail with reference to FIG. 2.

According to one aspect of the present invention, in the method for extracting and purifying conjugated triene linolenic acid (CLN) according to the present invention, the separation step S3 is mainly used to separate conjugated unsaturated in the esterified balsam pear seed oil. Fatty acids, such as stearic acid, can be achieved by using, but not limited to, a simulated moving bed, or by using less efficient batch chromatography.

The so-called simulated moving bed chromatography (SMB) is a continuous chromatography process based on the principle of high performance liquid chromatography (HPLC), which changes the flow at a specific switching time. Phase (mobile phase), feed solution and extraction solution flow into / out of the chromatographic bed body constructed by the solid adsorption phase, and make the stationary phase generate a simulated mobile solid adsorption that is opposite to the flow direction of the mobile phase The phase promotes the countercurrent flow contact between the stationary phase and the mobile phase, thereby improving the resolution of the chromatography, which has the advantages of high product concentration, good separation effect and high recovery rate. In some specific embodiments, the present invention is based on this technical principle and modified to be used in the method for the separation and purification of conjugated triene linolenic acid (CLN).

In addition, according to some embodiments, the simulated moving bed of the present invention preferably includes at least three separate sections such as a first section, a second section, a third section, and the like. A first extraction liquid outlet (QE), a feed inlet (QF) for recovering the washing liquid, and a second extraction liquid outlet (QR) are respectively provided at the positions. In addition, the three separation sections are provided with mutually connected separation columns; and the separation columns are each filled with a stationary phase that allows a mobile phase to flow in the same direction in sequence, and particles of the stationary phases It is preferable to have a pore through which a mobile phase can pass.

According to some embodiments, in the simulated moving bed used in the present invention, a stationary phase (SP) may be used as a solid object having adsorption properties with a low-polarity solute. In addition, according to an aspect of the present invention, in the purification and separation step S3 of the present invention, it is preferred that the stationary phase uses surface-modified silica solid particles. Octadecyl is bonded to the surface of silicon dioxide.

In addition, in addition to being able to dissolve the esterified balsam pear seed oil, the mobile phase (MP) in the simulated moving bed must also be able to facilitate the purification of CLN by the simulated moving bed; for example, in some aspects of the invention In the examples, the mobile phase can be water, a polar solvent such as ethanol, or a mixture thereof. According to some embodiments of the invention, for example, the mobile phase is preferably an aqueous solution containing 95% ethanol.

In addition, according to the extraction and purification method of conjugated triene linolenic acid (CLN) according to the present invention, the ratio of the mobile phase mixed liquid is not particularly limited. For example, in some embodiments, it is preferably 0: 100 to 10 : 90, more preferably 5: 95.

In addition, the triangular theoretical formula (1) and formula (2) of the simulated moving bed tomography are as follows: (1) (2) In the formula, K _A is the Henry constant (isothermal adsorption constant) of component _A to be separated; K _B is the Henry constant (isothermal adsorption constant) of component B to be separated; t ₀ is a substance that is not adsorbed by the stationary phase The retention time flowing through the column; t _A is the retention time of the component to be separated _{A that} is not adsorbed by the stationary phase flowing through the column; t _B is the residence time of the component to be separated _{B that} is not adsorbed by the stationary phase flowing through the column; t _d is The retention time of the component to be purified; ε _t is the porosity of the column used; m1, m2, and m3 are the relative volume flow rates of each section.

Therefore, the first extraction liquid discharge port between the first section, the second section, and the third section can be calculated according to the above-mentioned triangular theoretical formula (1) and formula (2) of the simulated moving bed chromatography. The specific switching time of the feed inlet of the flushing liquid and the outlet of the second extraction liquid, so as to achieve the goals of promoting separation efficiency, increasing the concentration of the separated product, and increasing the recovery rate.

For example, in the simulated moving bed of some embodiments of the present invention, the stationary phase is a carbon 18 solid adsorbent; the Henry constant of the conjugated unsaturated fatty acid of one of the components to be separated is 1.89; The Henry constant of fatty acid is 3.59; when the net mass flux of the conjugated unsaturated fatty acid in each separation section is FA and the net mass flux of the stearic acid A is FB; and the volume flow rate of each separation section When the ratios are m1, m2, and m3, the specific switching time can be obtained by calculating according to the above formula (1) and formula (2).

In the following, several examples of extraction, separation and purification of palmitic acid, stearic acid, oleic acid, linoleic acid, and conjugated linolenic acid are given to describe the conjugated triene linolenic acid of the present invention more specifically. (CLN) extraction purification method. "Examples 1 to 9" (extraction of fatty acids contained in balsam pear)

First, a carbon dioxide supercritical fluid extraction device (bed porosity: 0.582) as shown in FIG. 3 was used, and the temperatures were 40 ° C, 50 ° C, and 60 ° C as shown in Table 1 below; 20MPa, 25MPa, and 35 MPa, respectively. Pressure; 30 g / mL, 45 g / mL, 60 g / mL flow rate; and the operating conditions with or without the addition of co-solvents; the water content of the 8 mesh particle size provided by Universal Biotech 8.65% dried bitter gourd seed powder (bitter gourd seed density is 1.10 g / mL) was subjected to extraction under nine different operating conditions of Examples 1 to 9. In order to completely recover the extractable components in bitter gourd seeds, first extract for 6 hours Then, the ethanol was mixed with carbon dioxide before the extraction tank for 2.0 ~ 2.5 hours, and then adjusted again to mix with carbon dioxide at the outlet end of the extraction tank for 0.5 hours to completely dry the bitter gourd seed residue in the extraction tank, and then A bitter melon seed oil extract was obtained.

Table 1 List of operating conditions for supercritical fluid extraction of bitter gourd seed oil

In Examples 1 to 7, no co-solvent was added, and in Examples 8 and 9, ethanol was added as a co-solvent, and the concentrations of ethanol added were 7.31 wt% and 13.63 wt%, respectively. In addition, during the experiments of Examples 1 to 9, and using the extraction kinetic model of Sovova at the same time, the extraction experiments were fitted, and the kinetic parameters obtained by the fitting are arranged in Table 1.

In addition, although the auxiliary solvent was not used in Examples 1 to 7, at the same time, another HPLC pump was used to inject 6 mL / min of ethanol (100%) and carbon dioxide at the outlet end of the extraction tank, and then drained together. After pressing, it enters the separation tank to avoid the extracted high-viscosity grease from sticking between the tube walls. In addition, in Examples 8 and 9, the ethanol co-solvent was mixed with carbon dioxide at the inlet end of the extraction tank at the beginning, and after the extraction was completed, it was adjusted to mix at the outlet end of the extraction tank to dry the bitter gourd seed residue.

In the process of supercritical fluid extraction, samples are collected every half an hour, the entire sample weight is weighed and the collected liquid volume is calculated. The samples collected every half an hour are shaken and mixed uniformly, and then naturally dried, so as to estimate the weight of all the fats extracted every half an hour. The fat obtained after drying was re-dissolved in n-hexane, and then methylated, followed by analysis of the component content of the methylated bitter gourd seed oil sample using Agilent GC / MS.

The column used in the above GC / MS analysis was DB-5MS (30m × 250μm × 0.25 μm); helium was selected as the band gas; GC heating conditions: the initial 120 ° C was increased to 10 ° C / min to 220 The temperature was maintained at ℃ for 6 minutes, and then the temperature was increased to 10 ° C / min to 250 ° C for 12 minutes, and then the temperature was increased to 10 ° C / min to 320 ° C for 5 minutes. The analysis time was 43 minutes; the injection volume was 1 μL. In the analysis of GC / MS, pentadecane (Pentadecane) was used as the internal standard to establish the FAME (methylated fatty acid) content calibration curve.

The palmitic acid (C16: 0), stearic acid (C18: 0), and oleic acid (C18: 1) were calculated from the GC / MS spectrum of the methylated bitter melon seed oil sample and the equations shown below Concentration of five fatty acids, linolenic acid (C18: 2), and conjugated linolenic acid (c9, t11, t13-CLN; C18: 3). Among them, A is the area of the signal peak of the spectrum; C is the sample concentration; V is the sample volume; the subscripts FAME and IS represent methylated fatty acids and internal standards, respectively.

The standard reference products of methylated fatty acids used in Examples 1 to 9 were purchased from the following companies: methylated palmitic acid (C16: 0) (manufactured by Jingming Chemical Co., Ltd., purity> 97%); methylated stearic acid (C18: 0) (Youhe Trading Co., Ltd., purity> 99.5%); methylated oleic acid (C18: 1) (Youhe Trading Co., Ltd., purity> 99.3%) ; Methylated linoleic acid (C18: 2) (Youhe Trading Co., Ltd., purity> 99.3%); methylated conjugated linolenic acid (c9, t11, t13-CLN; C18: 3) (Cayman) (Purity> 98%); 99.5% absolute ethanol (made by Jingming Chemical Co., Ltd., purity is LC).

The bitter melon seed oil extract samples obtained in Examples 1 to 9 include palmitic acid (C16: 0), stearic acid (C18: 0), oleic acid (C18: 1), and linoleic acid (C18 : 2), the average content and concentration of five kinds of fatty acids of conjugated linolenic acid (c9, t11, t13-CLN; C18: 3) are shown in Table 2.

Table 2 Concentrations of five methylated fatty acids in balsam pear seed oil Note: Palmitic acid (C16: 0), stearic acid (C18: 0), oleic acid (C18: 1), linoleic acid (C18: 2), conjugated linolenic acid (c9, t11, t13-CLN C18: 3). "Example 10" Isolation and purification of conjugated triene linolenic acid (CLN)

First, according to the same equipment, methods and operation methods as those in the above embodiments 1 to 9, the carbon dioxide supercritical fluid equipment (bed porosity: 0.582) was used in the same way. The particle size provided by Huanyu Biotechnology Co. was 8 The dried Momordica charantia powder with a moisture content of 8.65% (the density of Momordica charantia seeds is 1.10 g / mL) was extracted to obtain a Momordica charantia seed oil extract.

Next, a sample of the obtained bitter melon seed oil extract was dissolved in ethyl acetate, followed by decolorization with activated carbon, and then ethyl acetate was removed to obtain a decolorized bitter melon seed oil. Next, the discolored bitter melon seed oil was dissolved in ethanol, and then the ethylation reaction of bitter melon seed oil was catalyzed by sodium hydroxide at 80 C. After the reaction was carried out for three hours, the reaction was terminated with ice water, and then the ethyl esterified product was extracted with n-hexane. After washing with hydrochloric acid aqueous solution and pure water in order, the n-hexane was removed to obtain ethyl esterified bitter gourd seed oil.

The ethylation reaction method is to sequentially add 0.2 g of NaOH fragments and 10 mL of absolute ethanol to 1.0 g of the crude bitter melon seed oil obtained from the above decolorization, and then place it in an 85 ° C oil bath to react with cold water reflux. After three hours, the stirring speed was maintained at 450 rpm. After the reaction, the liquid was placed in the freezer and left for one hour. Then, 1.0 mL of secondary water and 20 mL of n-hexane were added for stirring for 20 minutes at a speed of 450 rpm.

After the ethyl esterification reaction solution was stirred, it was allowed to stand and separate. The lower layer was removed. The upper layer was added to 0.6 mL of concentrated hydrochloric acid and mixed uniformly, and then left to separate. The lower salt layer was removed, leaving the upper organic layer. Add the same volume of secondary water as the upper organic layer, shake and wash, and remove the aqueous layer after standing and layering. After repeating the water washing step, anhydrous magnesium sulfate was added to remove water, and the liquid after drying was dried to obtain ethylated bitter melon seed oil.

Furthermore, the ELSD detector was used for the chromatographic separation test of bitter gourd seed oil with 95% ethanol as the mobile phase at a flow rate of 2.0 mL / min. The test results obtained two main signal peaks as shown in Fig. 4 (a), showing that the separation effect is good.

The chromatogram of the standard ethyl stearate showed that the peak retention time of ethyl stearate was 7.49 minutes, and the retention time of ethyl palmitate was 6.37 minutes. Therefore, from the chromatogram of the ethylated bitter melon seed oil as shown in FIG. 4 (a), it can be determined that the bitter melon seed oil contains a high amount of ethyl stearate (peak retention time: 7.49 minutes), and palm Ethyl acetate (peak retention time is 6.37 minutes), and fatty acids C18: 1 and C18: 2 (peak retention time: between 5.74 to 7.49 minutes).

In addition, according to one aspect of the present invention, in addition to using 95% alcohol as the mobile phase of the present invention as described above, other polar solvents and non-polar solvents may also be suitable; for example, pure water, deionized, 100% alcohol, etc. may be used. . For example, in some embodiments, 0-30% water, 70-100% alcohol, and / or mixtures thereof can be used to remove a large amount of unsaturated stearin in bitter gourd seed oil. Acid to further improve the separation of CLN-EE free fatty acids (FFA: Free Fatty Acids), 16- and 18-carbon saturated and / or unsaturated fatty acids, and ethyl stearate.

Next, for 1.0 g of the above-obtained ethyl ester bitter gourd seed oil, a preparative grade of YMC-AQ C18 (50 um) was selected using a stationary phase column and filled into 6 stainless steel filled column columns of 1.0 × 10 cm. As the mobile phase, 95% ethanol was selected, and a simulated moving bed (SMB: simulated moving bed) device was used to perform the reverse phase chromatography SMB test. The SMB equipment is equipped with six 1.0 x 10 cm stainless steel packed columns, and the six pipe columns are configured for the three sections of the open circuit. The configuration is generally recorded as 2/2/2. The SMB equipment is pressurized by a liquid pump (HITACHI L-2130) to make the mobile phase flow in the same direction in the column of each section.

In this embodiment, the specific switching time of the simulated moving bed (SMB) is based on the use of carbon 18 solid adsorbent of YMC-AQ C18 as the stationary phase; Henry of conjugated unsaturated fatty acids, one of the components to be separated The constant is 1.89; the Henry constant of stearic acid of the other component to be separated is 3.59; the net mass flux of the conjugated unsaturated fatty acid in each separation section is set to FA and the net mass flux of the stearic acid Let it be FB; and the volume flow rate ratios of each separation section are set to m1, m2, and m3, respectively, and calculated according to the following triangular theoretical formula (1) and formula (2) of the simulated moving bed tomography. Switch time.

(1) (2) In the formula, K _A is the Henry constant (isothermal adsorption constant) of component _A to be separated; K _B is the Henry constant (isothermal adsorption constant) of component B to be separated; t ₀ is a substance that is not adsorbed by the stationary phase The retention time flowing through the column; t _A is the retention time of the component to be separated _{A that} is not adsorbed by the stationary phase flowing through the column; t _B is the residence time of the component to be separated _{B that} is not adsorbed by the stationary phase flowing through the column; t _d is The retention time of the component to be purified; ε _t is the porosity of the column used; m1, m2, and m3 are the relative volume flow rates of each section.

In this example, the total porosity of the packed column: e = 0.73, the peak time when the column is not connected: t _d = 0.76 minutes, so at a flow rate of 2.0 mL / min, the non-retention of the packed column Peak time of component: t _o = 3.63 minutes. According to the chromatogram of the ethylated bitter gourd seed oil, two signal peaks representing the esterified CLN and stearic acid have retention times tA and tB of t _A = 5.74 and t _B = 7.49 minutes, respectively. The Henry's adsorption constants K _A and K _{B of} ethyl esterified CLN and stearic acid obtained from the calculation of the triangle theoretical formula (1) and formula (2) of the simulated moving bed chromatography are K _A = 1.89 and K _{B respectively.} = 3.59. Therefore, the Henry constant ratio of conjugated triene linolenic acid to stearic acid is 1 or more.

In this example, for each experiment with a different switching time, samples from the extraction end and the extraction end after the fourth circle are collected and analyzed by GC / MS, and then the most representative one is calculated using the calibration curve. The distribution of five fatty acids, C16: 0, C18: 2, C18: 1, C18: 0, CLN, and the CLN-EE recovery rate was calculated to explore the effectiveness of the separation. The CLN-EE recovery rate is defined as follows: (4) Among them, C represents the concentration analyzed by GC / MS; superscript R and E represent the raffinate end and the extraction end.

Results of GC / MS analysis of this example, palmitic acid (C16: 0), stearic acid (C18: 0), oleic acid (C18: 1), linoleic acid (C18: 2), and conjugated linseed oil The distribution of the five fatty acids of the acids (c9, t11, t13-CLN; C18: 3) is shown in Figure 5; the weight fractions of the five ethylated fatty acids were calculated and the CLN-EE recovery was recorded. In Table 3.

With reference to Table 3, it can be clearly seen that ethyl stearate appears at the extraction end, and CLN-EE, which has a weak retention, appears at the raffinate end. Also, as shown in Table 3, due to the strong retention of ethyl stearate, it can be seen that the weight fraction of CLN-EE in bitter melon seed oil is only 0.296; however, after SMB separation, most of the The ethyl stearate has been removed, so the CLN-EE weight fraction of the ethylated bitter melon seed oil collected at the raffinate end has been greatly increased to 0.530 ~ 0.714. In addition, the CLN-EE recovery rate Y calculated according to the above formula (4) also shows that most of the CLN-EE was recovered at the raffinate end. At the same time, high-purity ethyl stearate was collected at the extraction end. Except that the experiment with a switching time of 4.5 minutes contained approximately 6.5% wt of CLN-EE, most of the rest was ethyl stearate.

Table 3 Fatty acid distribution of ethylated balsam pear seeds before and after SMB separation as a function of switching time

From Table 3 above, it can be confirmed that under the condition that the switching time is 4.75 to 5.50 minutes, ethyl stearate and CLN-EE contained in bitter melon seed oil can be completely separated, and the switching time is 4.50 minutes. Conditions are classified as pure extractive operating conditions.

In summary, from Examples 1 to 10, it was confirmed that a simulated moving bed (SMB: simulated moving bed) using an adsorption column of carbon 18 such as YMC-AQ C18 as a stationary phase and an ethanol aqueous solution as a mobile phase can be used. The ethyl stearate was completely separated from the esterified bitter melon seed oil, and the ethylated bitter melon seed oil with high CLN-EE content was obtained.

In other words, by using the extraction and purification method of the conjugated triene linolenic acid (CLN) of the present invention, the conjugated triene linolenic acid can be effectively separated from the bitter melon seed oil extract, and can be continuously fed. Feed method, by simulating a moving bed to obtain an ultra-high content of conjugated triene linolenic acid from ethyl esterified balsam pear seed oil from 53.0 wt% to 71.4 wt%, that is, according to the present invention, the promotion of balsam pear seeds can be realized The efficiency of oil separation, increasing the concentration of the product of balsam pear seed oil, and increasing the recovery rate, thereby achieving the industrial mass production of extracting conjugated triene linolenic acid (CLN) from balsam pear seeds.

In summary, the content of the present invention has been described in the examples listed above by way of example, but the present invention is not limited to these embodiments. Those with ordinary knowledge in the technical field to which the present invention belongs should understand that, without departing from the spirit and scope of the present invention, various changes and modifications can be made; for example, combining the technical contents illustrated in the foregoing embodiments or Changes have been made to new embodiments, and such embodiments are of course considered to be part of the present invention. Therefore, the scope of protection in this case also includes the scope of patent application and its defined scope.

S1‧‧‧Extraction steps
S2‧‧‧Esterification step
S3‧‧‧Separation steps
1‧‧‧CO2 storage tank
2‧‧‧Co-solvent storage tank
3‧‧‧extraction tank
4‧‧‧Back pressure valve
5‧‧‧Temperature Regulator
6‧‧‧Gas-liquid separation tank
7‧‧‧ liquid pump
8‧‧‧ Valve
9‧‧‧ adsorption column
FM‧‧‧ Flowmeter
W‧‧‧ level gauge

FIG. 1 is a photograph showing dried bitter gourd seeds and pulverized bitter gourd seeds used in the present invention. FIG. 2 is a block diagram showing the specific implementation steps of the extraction and purification method of conjugated triene linolenic acid (CLN) according to the present invention. FIG. 3 is a photograph showing the appearance of a supercritical carbon dioxide extraction device used in an embodiment of the present invention. Figure 4a shows a single column chromatogram of an embodiment of the present invention. Figure 4b shows the effect of ethanol / water concentration in one embodiment of the present invention.

S1‧‧‧Extraction steps

S2‧‧‧Esterification step

S3‧‧‧Separation steps

Claims (10)

A method for extracting and purifying conjugated triene linolenic acid (CLN), comprising: (a) an extraction step: placing a bitter gourd seed sample in an extraction container, and extracting a crude extract of bitter gourd seed oil using a supercritical fluid; (b) Esterification step: the crude bitter melon seed oil obtained in (a) above is esterified with an esterifying agent to form an esterified bitter melon seed oil; (c) a purification step: to simulate a moving bed, from The esterified saturated fatty acid is separated from the esterified bitter melon seed oil obtained in the above (b), and a bitter melon seed oil mainly composed of conjugated triene linolenic acid is obtained. The method for extracting and purifying conjugated triene linolenic acid (CLN) according to claim 1, comprising: before performing the (a) extraction step, performing heat treatment for 4 to 8 hours to make the bitter gourd The moisture content of the seeds is below 8%. The extraction and purification method of conjugated triene linolenic acid (CLN) as described in claim 1, when performing the (c) purification step, the Henry constant ratio of conjugated triene linolenic acid to stearic acid is 1 or more. The extraction and purification method of conjugated triene linolenic acid (CLN) according to claim 1, wherein the conditions for the extraction of the supercritical fluid are a pressure of 10 MPa to 60 MPa and a temperature of 20 ° C to 60 ° C. The method for extracting and purifying conjugated triene linolenic acid (CLN) according to claim 1, further adding a co-solvent of 0 wt% to 40 wt% to the supercritical fluid; the co-solvent is organic Alcohols, short carbon chain organic alcohols containing 1 to 6 carbons, and mixtures thereof constitute at least one selected from the group. The method for extracting and purifying conjugated triene linolenic acid (CLN) according to claim 1, wherein the esterifying agent is from an organic alcohol, a short carbon chain organic alcohol containing 1 to 6 carbons, And their mixtures constitute at least one selected from the group. The conjugated triene linolenic acid (CLN) extraction and purification method according to claim 1, wherein the esterification reaction is a base-catalyzed reaction or an ion-exchange resin-catalyzed reaction. The conjugated triene linolenic acid (CLN) extraction and purification method as described in claim 1, wherein the stationary phase of the simulated moving bed is particles of silicon dioxide modified by an alkane surface of 4 to 30 carbons, Surface modified particles of 18 carbons are preferred. The conjugated triene linolenic acid (CLN) extraction and purification method as described in claim 1, wherein the switching time of the simulated moving bed is 4.75 to 5.50 minutes. The extraction and purification method of conjugated triene linolenic acid (CLN) according to claim 1, wherein the mobile phase of the simulated moving bed is 100% to 80% by weight of ethanol, and 0% to 20% by weight Go away from the water. The method for extracting and purifying conjugated triene linolenic acid (CLN) according to claim 1, which is used for separating and purifying palmitic acid, stearic acid, oleic acid, linolenic acid contained in bitter melon seed oil, and At least one of conjugated linolenic acid.