CN107778337B - Method for extracting glycolipid from spirulina by supercritical carbon dioxide - Google Patents
Method for extracting glycolipid from spirulina by supercritical carbon dioxide Download PDFInfo
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Abstract
The invention relates to a method for extracting glycolipid from spirulina, which comprises the following steps: collecting spirulina raw materials; subjecting the Spirulina raw material to supercritical CO2Extraction, wherein the supercritical CO2In the extraction process, an organic solvent is used as an entrainer, and a porous material is added; collecting the extract; the extract is further extracted to obtain glycolipids. The method of the invention has high extraction rate and extraction selectivity for extracting the spirulina.
Description
Technical Field
The present application relates to the field of industrial microalgae application, and in particular to a method for obtaining glycolipids, in particular glycolipids rich in gamma-linolenic acid, from spirulina by supercritical carbon dioxide extraction.
Background
Spirulina (Spirulina) also called "Arthrospira", belongs to Cyanophyceae, Oscillatoriaceae, and is a prokaryote. Spirulina is receiving attention because it is rich in various nutrients such as carotenoids, proteins, lipids, etc. Among many nutrients contained in spirulina, glycolipids are one of the nutrients of high interest. Most of the lipids in spirulina are glycolipids, which have the effects of preventing cardiovascular diseases, reducing blood pressure, improving lipid metabolism, reducing cholesterol and the like. Moreover, the monogalactosyldiacetyl glyceride and the digalactosyldiacetylglyceride in the glycolipid contain a gamma-linolenic acid part, and the gamma-linolenic acid has obvious effects on reducing blood fat and blood sugar. At present, spirulina is artificially cultured in large scale at home and abroad, and people expect to obtain required nutrients from the spirulina, particularly glycolipids rich in part of gamma-linolenic acid (hereinafter referred to as GLA for short, and the gamma-linolenic acid and the GLA are used interchangeably herein).
Commonly used methods for obtaining nutrients including glycolipids from spirulina include, for example, accelerated solvent extraction, organic solvent extraction, soxhlet extraction, and the like. The method has the defects of high extraction temperature, large solvent consumption, high toxicity, low glycolipid proportion in the extract, unsuitability for large-scale industrial production of bioactive substances and the like. Supercritical CO2Extraction of nutrients from spirulina has also been reported. For example, Sajilata, M.G. et al (J.food Engineering 84, 321-326; 2008) report the use of supercritical CO2Method for extracting gamma-linolenic acid from spirulina, but the method has low extraction rate of the gamma-linolenic acid (similar to Bligh)&Compared with a Dyer method, the extraction efficiency of the gamma-linolenic acid is only 78 percent)The carbon dioxide consumption is large (200--1) And the like.
It is desired to develop a method suitable for industrial application and capable of efficiently obtaining nutrients, particularly glycolipids such as gamma-linolenic acid-rich glycolipids, from spirulina.
Disclosure of Invention
In one aspect, the present invention provides a method for extracting glycolipids from Spirulina (Spirulina), comprising the steps of: (i) collecting spirulina raw materials; (ii) subjecting the Spirulina raw material to supercritical CO2Extraction, wherein the supercritical CO2In the extraction process, an organic solvent is used as an entrainer, and a porous material is added; (iii) collecting the extract; and (iv) further extracting the extract to obtain glycolipids.
In another aspect, the present invention provides a method for extracting a GLA-rich fraction of glycolipids from spirulina comprising the steps of: (i) collecting spirulina raw materials; (ii) subjecting the Spirulina raw material to supercritical CO2Extraction, wherein the supercritical CO2In the extraction process, an organic solvent is used as an entrainer, and a porous material is added; (iii) collecting the extract; and (iv) further separating the extract to obtain glycolipids enriched in the GLA fraction.
In one embodiment, the glycolipid is a GLA moiety-rich glycolipid.
In one embodiment, the glycolipid is selected from one or more of the following groups: MGDG, DGDG and SGDG, preferably mixtures of MGDG, DGDG and SGDG.
In one embodiment, the porous material is selected from the group consisting of: diatomite, macroporous resin and molecular sieve, preferably diatomite.
In one embodiment, the method further comprises separating a mixture of remaining spirulina and diatomaceous earth in the extract and using the mixture as the supercritical CO2Porous material in the extraction process.
In one embodiment, the porous material, preferably diatomaceous earth, comprises 0.11% to 66.7% moisture.
In one embodiment, the organic solvent is selected from the group consisting of: lower alcohols such as methanol, ethanol, isopropanol, etc.; lower esters such as ethyl acetate, propyl acetate, butyl acetate, etc.; acetone; n-hexane; or mixtures of one or more of the foregoing; preferably selected from methanol, ethanol and ethyl acetate or mixtures thereof; more preferably ethanol; more preferably food grade ethanol.
In one embodiment, the porous material is added in an amount of 0.25 to 1.0 times (g/g), preferably 0.5 times (g/g) the mass of the spirulina raw material.
In one embodiment, in the supercritical CO2In the extraction process, the static extraction time is 15-60min, preferably 30 min; the dynamic extraction and collection time is 10-60min, preferably 30 min; the extraction pressure is 4000-; the extraction temperature is 30-50 ℃, preferably 40 ℃; CO 22The flow rate is 8-20 ml.min-1Preferably 20 ml/min-1。
In one embodiment, the liquid-to-material ratio of the amount of entrainer added to the mass of the spirulina raw material is 0.5-3.0(ml/g), preferably 3 (ml/g).
In one embodiment, the spirulina is selected from the group consisting of: spirulina platensis (Spirulina platensis), Spirulina maxima (Spirulina maxima) and Spirulina subsalsa (Spirulina subsalsa).
In another aspect, the present invention also provides a glycolipid, preferably a glycolipid enriched in a GLA moiety, which is prepared by the above method.
The method of the present invention can efficiently obtain glycolipids, particularly glycolipids rich in GLA moiety, from spirulina. Moreover, the method of the invention is suitable for industrial application.
Drawings
FIG. 1: structures of monogalactosyldiacetyl glyceride (MGDG), digalactosyl diacetylglyceride (DGDG), and sulfoisorhamnese diacetylglyceride (SQDG);
FIG. 2: the structure of gamma-linolenic acid;
FIG. 3: the structure of molecules comprising a GLA moiety in MGDG and DGDG;
FIG. 4: super clinical medicineBoundary CO2Experimental flow charts for fluid extractors;
1-CO2a steel cylinder; 2-a cooler; 3, a booster pump; 4-entrainer tank; 5-entrainer pump; 6-extraction kettle; 7-collecting bottle; and
FIG. 5: supercritical CO2And (3) analyzing the results of the thin layer chromatography of the crude fat obtained by the extraction method.
Detailed Description
In one aspect, the present invention provides a method for extracting glycolipids from spirulina, comprising the steps of: (i) collecting spirulina raw materials; (ii) subjecting the Spirulina raw material to supercritical CO2Extraction, wherein the supercritical CO2In the extraction process, an organic solvent is used as an entrainer, and a porous material is added; (iii) collecting the extract; and (iv) further separating the extract to obtain glycolipids.
Herein, the term "Spirulina (Spirulina)" refers to a prokaryote belonging to the Oscillatoriaceae of the Cyanophyceae, and is further divided into three species of Spirulina platensis (Spirulina platensis), Spirulina maxima (Spirulina maxima) and Spirulina subsalsa (Spirulina subsalsa). The spirulina material used in the present invention may be, for example, spirulina powder or spirulina wet algae mud. In one embodiment, the spirulina is selected from one or more of the following: spirulina platensis, Spirulina maxima and Spirulina indica. In one embodiment, the spirulina is spirulina platensis.
Spirulina contains various nutrients including chlorophyll, carotenoid, etc., pigments, carbohydrate, phospholipid, glycolipid, etc., and protein. The term "extract" is a generic term for the various nutrients described above, and "extract" and "total nutrient" are used interchangeably herein. Most of the lipids in spirulina are glycolipids, which are substances formed by the combination of saccharides and fatty acids. Glycolipids in spirulina are classified into Monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoisoglyeryl diacylglycerol (SQDG), each representing a mixture of esters formed from different fatty acids and sugars. The molecular structures of MGDG, DGDGDG and SQDG are shown in FIG. 1, wherein R represents different fatty acids. Theoretically, glycolipids contained in spirulina usually represent 3.6-5.5 wt% of the dry weight of spirulina. The glycolipid has beneficial effects in preventing and treating cardiovascular diseases, and improving lipid metabolism. In one embodiment, the glycolipid is selected from one or more of the following groups: monogalactosyldiacylglycerolipids, digalactosyldiacetylglycerolipids, and thioglycerol diacetylglycerolipids. In one embodiment, the glycolipid is a mixture of monogalactosyldiacylglycerolipid, digalactosyldiacylglycerolipid, and thioisorhamnolipid diacetylglycerolipid.
The principle of the supercritical carbon dioxide extraction process is that supercritical carbon dioxide has a special dissolving effect on certain specific substances, and in a supercritical state, supercritical carbon dioxide fluid is in contact with substances to be extracted to selectively extract expected components.
The present inventors have found that the extraction rate of glycolipids, particularly MGDG and DGDG, enriched with GLA moieties in spirulina nutrients can be greatly increased compared to some known methods by adding a porous material such as diatomaceous earth in the supercritical carbon dioxide extraction process in combination with an entrainer such as ethanol. The method of the invention has high extraction rate for the extraction of glycolipids in spirulina nutrients, particularly the extraction of glycolipids MGDG and DGDGDG rich in GLA part. The GLA enriched glycolipids MGDG and DGDG (calculated as GLA) obtained by the method of the invention as shown in example 1 have extraction rates approaching 100%.
Furthermore, in the usual case, CO is extracted during supercritical carbon dioxide extraction2Is typically in the order of hundreds, or even hundreds, of milliliters per minute. Can adsorb CO with expected porous material2In contrast, and surprisingly, the CO used in the supercritical carbon dioxide extraction process of the present invention2The flow rate of (2) is greatly reduced and can be only 8-20ml per minute.
The porous material used in the present invention is a material having a network structure of interconnected or closed pores and a high specific surface area, and includes, but is not limited to, diatomaceous earthMacroporous resins, molecular sieves, and the like. In addition, the extract of the present method contains a mixture of spirulina and diatomaceous earth remaining after extraction. The mixture itself is also a substance with a porous structure and can therefore also be used as supercritical CO2Porous material in the extraction process. The recycling of this raw material is advantageous for the industrialization of the process of the invention. Thus, in one embodiment, the porous material is selected from the group consisting of: diatomite, macroporous resin and molecular sieve. In one embodiment, the porous material is supercritical CO2The remaining mixture of spirulina and diatomaceous earth after extraction. In one embodiment, the method further comprises separating a mixture of spirulina and diatomaceous earth remaining in the extract, the mixture of spirulina and diatomaceous earth being used as the supercritical CO2Porous material in the extraction process.
The present inventors have further found that when a porous material such as diatomaceous earth used in the present invention is made to contain a certain amount of moisture, it will also contribute to the improvement of the extraction rate of glycolipids in spirulina nutrients, particularly the extraction rate of glycolipids MGDG and DGDG rich in GLA moiety. Generally, the extraction of nutrients from dried spirulina is common. As demonstrated in example 3, the extraction rate of glycolipids in spirulina nutrients, particularly the extraction rate of glycolipids MGDG and DGDG rich in the GLA moiety, is significantly increased after increasing the water content of porous materials such as diatomaceous earth. This finding makes it possible to use the wet algal sludge having a certain water content directly as a raw material in the process of the present invention. The direct supercritical extraction of the wet algae mud with certain water content can reduce the additional processing steps of dehydration, drying and the like in the microalgae harvesting link, save energy consumption and is beneficial to the industrialization of the method. In addition, porous materials commonly used in laboratories are often porous solid particles. Since the spirulina raw material and the porous material such as diatomaceous earth are in the same system during the carbon dioxide supercritical extraction, it is expected that the extraction rate of glycolipids in spirulina nutrients, particularly the extraction rates of glycolipids MGDG and DGDG rich in GLA moiety, can be improved also when the spirulina raw material or the whole of the spirulina raw material and the porous material is controlled to have a certain water content.
Thus, in one embodiment, the porous material, preferably diatomaceous earth, has a water content of 0.1 wt% to 70 wt%, more preferably comprises 50 wt%, 51 wt%, 52 wt%, 53 wt%, 54 wt%, 55 wt%, 56 wt%, 57 wt%, 58 wt%, 59 wt%, 60 wt%, 61 wt%, 62 wt%, 63 wt%, 64 wt% and 65 wt% water, more preferably 60 wt%, based on the mass of the diatomaceous earth feedstock.
The entrainer used in the present invention may be an organic solvent including, but not limited to, lower alcohols such as methanol, ethanol, isopropanol, etc.; lower esters such as ethyl acetate, propyl acetate, butyl acetate, etc.; acetone; n-hexane; or mixtures of one or more of the foregoing. The use of entrainer is helpful to improve the extraction rate of the method of the invention to the glycolipid in the spirulina nutrient, in particular to the extraction rate of the glycolipids MGDG and DGDGDG rich in GLA part. In addition, compared with an entrainer such as petroleum ether (with high boiling point and flammability and explosiveness) used in some supercritical carbon dioxide extraction processes, the solvent is selected as the entrainer, so that the subsequent extraction and solvent separation is facilitated, the solvent residue is reduced, and the product performance is improved.
Thus, in one embodiment, the entrainer is selected from the group consisting of: lower alcohols such as methanol, ethanol, isopropanol, etc.; lower esters such as ethyl acetate, propyl acetate, butyl acetate, etc.; acetone; n-hexane; or mixtures of one or more of the foregoing. In one embodiment, the entrainer is selected from methanol, ethanol and ethyl acetate. In one embodiment, the entrainer is ethanol, preferably food grade ethanol.
After the extract is obtained from spirulina by the supercritical carbon dioxide extraction method of the present invention, the extract may be further separated and purified to obtain glycolipids in the extract. The results of using macroporous resin HP20 to separate and purify glycolipids in crude spinach leaf extracts such as Naoki Maeda (J. Current medical chemistry 14(9), 955-967; 2007) show that when gradient elution is carried out by using 70% and 95% ethanol, polar esters, phospholipids and the like in ethanol extracts can be removed, and meanwhile, glycolipids can be primarily enriched. Changhu Xue (J.food Chemistry 77(1), 9-13; 2002) and the like, obtained glycolipids from extracts by changing the elution system using silica gel column chromatography. It will be appreciated by those skilled in the art that the extract may be isolated and purified by methods known in the art to obtain glycolipids.
The fatty acid chains in the lipids of spirulina are mostly unsaturated fatty acids, in particular unsaturated fatty acids such as gamma-linolenic acid and linoleic acid. Gamma-linolenic acid (C)18H30O2) Also known as all-cis 6,9, 12-octadecatrienoic acid, belongs to the omega-6 series of polyenoic fatty acids (see fig. 2). The main classes of esters in MGDG and DGDG are esters formed from GLA and sugars, i.e. the main ester molecules in both MGDG and DGDG comprise a GLA moiety (see fig. 3). GLA in Spirulina is mainly present at Sn-1 or Sn-2 position of MGDG and DGDGDG. Theoretically, the GLA fraction contained in spirulina is usually 0.92-1.39 wt% based on the dry weight of spirulina. The current research proves that GLA has the effects of reducing blood fat, reducing blood sugar, resisting cardiovascular diseases and the like. As a reservoir for many beneficial fatty acids, it is desirable to be able to further obtain beneficial fatty acids, particularly gamma-linolenic acid, from spirulina "extracts".
As described above, the method of the present invention can not only increase the extraction rate of glycolipids in spirulina nutrients, but also increase the extraction rate of glycolipids MGDG and DGDGDG rich in GLA. Thus, the process of the invention can also be used to extract glycolipids enriched in GLA fractions.
In another aspect, the present invention also relates to a method for extracting GLA-rich fractions of glycolipids from spirulina comprising the steps of: (i) collecting spirulina raw materials; (ii) subjecting the Spirulina raw material to supercritical CO2Extraction, wherein the supercritical CO2In the extraction process, an organic solvent is used as an entrainer, and a porous material is added; (iii) collecting the extract; and (iv) further separating the extract to obtain glycolipids enriched in the GLA fraction.
In order to obtain the best extraction efficiency, the invention also researches the dosage of the porous material, the dosage of the entrainer, the extraction time, the temperature, the pressure and the CO2These factors including flux were for total nutrients, glycolipids (based on total fatty acids) and MGDG in spirulinaInfluence of DGDG (in GLA) on the extraction efficiency.
In one embodiment, the porous material is added in an amount of 0.25 to 1.0 times, for example 0.25 times, 0.33 times, 0.5 times and 1 time, preferably 0.5 times (g/g) the mass of the spirulina raw material. In one embodiment, the entrainer is added in an amount of 0.5-4.0 times (ml/g), for example 0.5 times, 1.0 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times and 4.0 times, preferably 3.0 times (ml/g) the mass of the spirulina raw material. In one embodiment, in the supercritical CO2In the extraction process, the static extraction time is 15-60min, such as 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min and 60min, preferably 30 min; the dynamic extraction and collection time is 10-60min, preferably 30 min; the extraction pressure is 4000-; the extraction temperature is 30-50 deg.C, such as 35 deg.C, 40 deg.C, 45 deg.C and 50 deg.C, preferably 40 deg.C, CO2The flow rate is 8-25 ml/min-1Preferably 8 to 20 ml.min-1E.g. 8ml min-1、9ml·min-1、10ml·min-1、11ml·min-1、12ml·min-1、13ml·min-1、14ml·min-1、15ml·min-1、16ml·min-1、17ml·min-1、18ml·min-1、19ml·min-1And 20 ml. min-1Preferably 20 ml/min-1。
Examples
The present invention will be described in more detail with reference to the following examples. However, the following examples are provided only for explaining the present invention and should not be construed as limiting the scope and spirit of the present invention.
Experimental materials and apparatus
Supercritical CO2Extraction apparatus (SFT-110 XW SFE, USA), vacuum rotary evaporator (BUCHI), gas chromatography-mass spectrometer (Agilent 7890B/5977A GC/MS, chromatographic column HP-88: 60m 0.25mm 0.2 um). Absolute ethanol (food grade), high purity CO2(99.999%, Beijing Chengweiseng technologies Co.). Spirulina platensis (Spirulina platensis), purchased fromFrom the century bioengineering limited company of east tai city. TLC Silica Gel 60(Merck kGaA, Germany). Analytically pure chloroform, methanol (national pharmaceutical group chemical reagents, ltd.) for thin layer chromatography, chromatographically pure chloroform (Honeywell, international), methanol, n-hexane (Sigma-Aldrich) for GC/MS sample treatment and analytical determination.
Summary of the Experimental procedures
1. Supercritical CO2Basic process for extracting nutrient substances in spirulina:
s1: weighing a certain amount of spirulina powder, and uniformly mixing a porous material which is 0.25-1.0 time of the mass of the raw material and the spirulina raw material in advance for later use;
s2: according to the weight percentage of ethanol: adding entrainer into the raw material (0.5: 1-3:1(ml/g volume mass ratio)), mixing with the mixture obtained in step S1, loading into material bag, loading into extraction kettle, installing extraction kettle sealing end cap and screwing thread to prevent leakage;
s3: opening the inlet and outlet valves of the extraction kettle, and using CO2Purging the whole system pipeline to remove air;
s4: opening a switch of a refrigerator, precooling for 15min, and setting the extraction conditions of the system through an equipment control panel, wherein the temperature of the extraction kettle is 30-50 ℃ (the temperature of an outlet valve is 40-60 ℃), the pressure is 4000-2The flow rate of (A) is 8-20 ml.min-1；
S5: after the temperature of the extraction kettle is stabilized, pressing CO2RUN/STOP bond of pump, CO2Inputting the mixture into an extraction kettle through a high-pressure pump;
s6: opening the outlet valve of the extraction kettle after ensuring the extraction residence time of 15-60min, and mixing entrainer and supercritical CO2The mixed fluid is throttled and expanded by a pressure reducing valve, then is discharged, is collected and concentrated, is frozen and dried in vacuum and is weighed. The experimental operation was stopped near extraction equilibrium (i.e., a state where no extract flowed out after the extraction was continued for a certain period of time);
s7: concentrating the collected substance, vacuum freeze-drying to obtain extract, wherein the main component of the extract is a mixture of pigment and glycolipid (SQDG, MGDG and DGDGDG), Thin Layer Chromatography (TLC) is adopted due to different polarities of each component, iodine vapor staining is adopted, corresponding points are scraped, and the content of different fatty acid methyl esters is determined by means of trans-methylation according to the in-situ transesterification reaction of Lievee M.L. and the like (Analytical and biological Chemistry,403: 167-.
S8: the total fatty acids and their GLA content were calculated and converted to the corresponding total glycolipid amount (based on total fatty acids) and the total MGDG and DGDG amount (based on GLA) in the glycolipids.
Since fatty acids are not volatile, the fatty acid methyl ester which is not easily volatilized is converted into fatty acid methyl ester which is easily volatilized for accurate quantification by GC/MS.
The Supercritical Fluid extraction equipment for experiments is manufactured by Supercritical Fluid Technologies, Inc. of America, the volume of a stainless steel extraction column is 100ml, the highest working pressure of the equipment is 10,000psi, the fluctuation of the pressure up and down is not more than 2%, and the temperature control deviation is +/-3 ℃.
Supercritical CO2The extraction apparatus is shown in FIG. 4 and mainly comprises CO2The device comprises a steel cylinder system, a cooling system, a pressure boosting system, an extraction system and a separation and collection system. CO coming out of the cylinder2Enters the refrigeration system and is cooled to the subzero temperature. Cooled CO2The mixture is conveyed to a preheater through a high-pressure pump for preheating, and enters an extraction kettle to reach the set extraction pressure and the set extraction temperature after being preheated to the set temperature. CO in the extraction kettle2Contacting with raw material to dissolve the extracted component in CO2And entrainer, extracting. Subsequently, the extract is subjected to supercritical CO2The extract and CO enter a separation kettle at the set temperature of the separation kettle2Separated, and CO is received by the receiving system2And (5) emptying.
2. Analytical characterization of glycolipids
Analysis of Spirulina supercritical CO by high performance thin layer chromatography2Extract with CHCl as developing agent3：CH3OH：H2O ═ 4:1:0.1(v/v/v), iodine vapor stain. As shown in FIG. 5, other components in the extract can be separated from glycolipid by high performance thin layer chromatography, and three components SQDG, MGDG in glycolipid,DGDG was also clearly isolated.
Scraping silica gel containing corresponding glycolipid in a silica gel plate, placing the silica gel plate into a sample injection vial for methyl esterification, and measuring the content of fatty acid methyl ester in the silica gel plate by GC/MS (gas chromatography/mass spectrometry), thereby converting the content into the content of MGDG, DGDGDG and SQDG.
Example 1 use of porous Material for supercritical CO2Influence of extraction rate of glycolipid and GLA-rich fraction from Spirulina
Supercritical CO2Preparing the extraction instrument for starting up:
and opening a gas cylinder in advance to purge the whole system for several minutes, and discharging water and gas in a pipeline.
Weighing a certain amount of spirulina powder, uniformly mixing diatomite (with the water content of 60 percent and the particle size of 20-40 meshes) which is 0-1.0 time of the mass of the raw materials with the materials for later use, and comparing the mixture with a sample without the diatomite.
Thirdly, according to the spirulina raw materials: adding entrainer into ethanol at a ratio of 1:3(g/ml), uniformly mixing with the mixture obtained in the step S2, putting into a material bag, putting into an extraction kettle, installing a sealing end cover of the extraction kettle, and screwing threads to prevent leakage.
Opening the inlet and outlet valves of the extraction kettle and using CO2Purging the whole system pipeline to remove air.
Opening a refrigerant switch (carbon dioxide pump can be started after precooling for 15 min), setting the extraction pressure to be 6000psi (alarm pressure is 0-8000psi), and setting CO2The flow rate is 20 ml/min-1(ii) a And opening a switch of a temperature controller of the mainframe box, and respectively setting the temperature of the extraction kettle to be 40 ℃ and the temperature of the gas outlet valve to be 50 ℃.
After the temperature of the extraction kettle is stable, pressing CO2RUN/STOP bond of pump, CO2And is input into an extraction kettle through a high-pressure pump.
Seventhly, opening an outlet valve of the extraction kettle, the entrainer and the supercritical CO after ensuring the extraction retention time of 30min2The mixed fluid is throttled and expanded by a pressure reducing valve, discharged, concentrated after 30min collection, vacuum freeze-dried and weighed.
Eighthly, concentrating the collected substance, performing vacuum freeze drying to obtain an extract, analyzing by adopting a thin layer chromatography, and developing a developing agent which is chloroform: methanol: water 4:1:0.1(v/v/v), iodine vapor staining, scraping the corresponding spot, performing direct methyl esterification reaction according to the in-situ transesterification reaction of Lieve and the like and optimizing the reaction, and measuring the content of glycolipid in the crude fat by GC/MS.
Ninthly, directly carrying out methyl esterification reaction on the crude fat, and determining the fatty acid composition and content of the crude fat by GC/MS, wherein the results are shown in the following table 1.
TABLE 1
DW% represents weight% based on dry weight of spirulina raw material;
the amount of glycolipids in the extract is calculated as total fatty acids and the amount of glycolipids enriched with GLA moieties is calculated as GLA.
As shown in table 1, the extraction rate of MGDG and DGDG (calculated as GLA) in glycolipids (calculated as total fatty acids), in particular glycolipids, can be greatly improved by adding a certain amount of porous material such as diatomaceous earth compared to when no porous material is used in the supercritical carbon dioxide extraction. When the mass ratio of the porous material to the spirulina raw material is 0.5, the extraction rate of MGDG and DGDGDG (calculated as GLA) in the glycolipid is close to 100%. This shows that the addition of porous materials such as diatomaceous earth can greatly improve the extraction rate of glycolipids in spirulina nutrients, particularly the extraction rate of glycolipids MGDG and DGDGDG rich in GLA. In addition, the data in table 1 also indicate that the higher the amount of porous material added, the better, the best extraction results when the mass ratio of porous material to spirulina raw material is 0.5.
Example 2 use of entrainer for supercritical CO2Effect of extraction of Total fatty acids from Spirulina and GLA extraction yield
The specific procedure was essentially as described in example 1. In this example, the ratio of the entrainer (food grade ethanol) to the spirulina raw material is in the range of 0-4 (volume to mass ratio ml/g, abbreviated as "liquid to material ratio"), and the other conditions are the same and are as follows: diatomite/spirulina raw material is 0.5 percent, the water content of diatomite is 60 percent, T is 60min, T is 40 ℃, P is 6000psi, CO2A flow rate of20ml·min-1. The results are shown in table 2 below.
TABLE 2
As shown in table 2, the use of entrainers, particularly ethanol, in the carbon dioxide supercritical extraction also helps to increase the extraction rate of glycolipids in spirulina nutrients, particularly the extraction rate of glycolipids MGDG and DGDG rich in GLA moieties. The data in Table 2 also show that the extraction yield is also affected by the amount of entrainer added as a single factor without changing other conditions, with the best entrainer usage being a liquid to feed ratio of 3:1 (ml/g).
To further validate the extraction rate of glycolipids by entrainers alone, the inventors repeated the above experiments, except that no porous material was used. The results are shown in Table 3.
TABLE 3
The results in table 3 show that the entrainer itself is also helpful in increasing the extraction of glycolipids from spirulina nutrients, particularly the extraction of the GLA-rich glycolipids MGDG and DGDG. The invention uses porous material and entrainer in the process of supercritical carbon dioxide extraction, and the action of the porous material and the entrainer can improve the extraction rate of glycolipid in spirulina nutriment, especially the extraction rate of glycolipid MGDG and DGDGDG rich in GLA part.
Example 3 Effect of Water content of Spirulina raw Material on extraction yield
The specific procedure was as in example 1. In this example, the water content of the porous diatomite is controlled within the range of 0.11% to 66.7%, and the other conditions are the same and are as follows: diatomaceous earth/spirulina raw material 05, liquid-to-material ratio is 3, T is 60min, T is 40 ℃, P is 6000psi, CO2The flow rate is 20 ml/min-1. The results are shown in Table 4.
TABLE 4
As shown in table 4, when the porous material diatomaceous earth used in the present invention is made to contain a certain amount of moisture, it is also helpful to increase the extraction rate of glycolipids in spirulina nutrients, particularly the extraction rates of glycolipids MGDG and DGDG rich in GLA moiety. The data show that the extraction was highest when the diatomaceous earth had a water content of 60%. Compared with the method that the dried spirulina powder is generally adopted as the raw material at present, the result shows that the method can directly use the wet spirulina mud with certain water content as the raw material. This will greatly save unnecessary steps such as drying treatment in the process of harvesting spirulina, and is very beneficial to industrialized production.
In addition, since the spirulina raw material and the porous material are in the same system during the carbon dioxide supercritical extraction, the above results also suggest that the extraction rate can be improved when the overall water content of the spirulina raw material and the porous material is controlled to 60%.
Example 4 Effect of different porous materials on extraction yield
The specific procedure was as in example 1 except that the diatomaceous earth was replaced with macroporous adsorbent resins (HP20 and D101) and molecular sieves (HND A-15), respectively, and the extraction efficiency was measured at 0.5 and 1 for the porous material/Spirulina material, respectively.
The results show that when macroporous adsorption resin and molecular sieve are used as porous materials, the extraction rate of glycolipid in spirulina nutrient can be improved, especially the extraction rate of glycolipid MGDG and DGDGDG rich in GLA part. When the mass ratio of the macroporous adsorption resin to the molecular sieve to the spirulina raw material is controlled to be 0.5 respectively, the extraction rate of the macroporous adsorption resin to the total fatty acid and the extraction rate of the GLA to the spirulina raw material are lower than the corresponding extraction rate when the mass ratio of the diatomite to the spirulina raw material is 0.5. When the mass ratios of the macroporous adsorption resin, the molecular sieve and the spirulina raw material are respectively improved to 1.0, the yield of total fatty acid is 3.30% DW and the yield of GLA is 1.03% DW for the macroporous adsorption resin; for molecular sieves, the total fatty acid yield was 3.28% DW and the GLA yield was 1.01% DW. The above values are very close to the data obtained using diatomaceous earth as the porous material.
Without being bound by any theory, the above results indicate that the achievement of the high selectivity of the present invention for the extraction of glycolipids, in particular MGDG and DGDG, may be related to certain common properties of porous materials. For example, it is possible that the porous material can effectively increase glycolipids in spirulina, particularly MGDG and DGDG and CO in glycolipids2The contact area of the fluid is increased, thereby increasing the extraction ratio of glycolipids, particularly MGDG and DGDGDG in the spirulina nutrient.
Example 5 supercritical CO2Effect of other parameters in extraction on extraction yield
After the above-mentioned important factors influencing the extraction selectivity of glycolipids, especially MGDG and DGDGDG in spirulina nutrients, such as porous material and entrainer, etc., are determined, in order to further optimize supercritical CO2The extraction conditions, including extraction time, temperature, pressure and CO, are further studied2The effect of these factors including flux on total nutrients, glycolipids (based on total fatty acids) and extraction efficiency of MGDG and DGDG (based on GLA) in spirulina.
5.1 Effect of extraction time on extraction efficiency
When the optimal usage amount of the entrainer is determined, the discharged material is collected at a certain speed, and no solute flows out after 30min, so that 30min is the optimal time for dynamic extraction collection, and the factors influencing the extraction efficiency are mainly the duration of static extraction.
In this example, the specific operation steps are as in example 1, compared with the extraction effect when the static extraction time is 15-90min, and the other conditions are the same and are as follows: diatomite/spirulina material (0.5 wt.%), diatomite (60 wt.%), diatomite (3 wt.%), T (40 deg.C), P (6000 psi), and CO (carbon monoxide)2The flow rate is 20 ml/min-1。
As shown in Table 5, the extraction efficiency was not high by simply increasing the extraction time. For example, at 60min of static extraction, the total fatty acid yield is 3.80. + -. 0.16% DW, and the GLA yield is 1.16. + -. 0.07% DW; at 90min, the total fatty acid yield was 3.48. + -. 0.28% DW, and the GLA yield was 1.01. + -. 0.09% DW. In order to obtain the best selectivity and meet the industrial requirements, the method of the invention preferably adopts static extraction for 60 min.
TABLE 5
5.2 Effect of temperature on extraction efficiency
In this example, the specific operation steps are as in example 1, comparing the effect of the temperature in the range of 30-60 ℃ on the extraction efficiency, and the other conditions are the same and as follows: diatomite/spirulina raw material is 0.5 percent, the water content of diatomite is 60 percent, the liquid-material ratio is 3, t is 60min, P is 6000psi, CO2The flow rate is 20 ml/min-1。
As shown in table 6, the increase in temperature did not significantly affect the extraction rate of MGDG and DGDG from glycolipids, including glycolipids, with increasing temperature. In order to obtain the best selectivity and meet the industrial requirements, the extraction condition of 40 ℃ is preferred in the method of the invention.
TABLE 6
5.3 Effect of pressure on extraction efficiency
In this example, the specific operation steps are as in example 1, comparing the effect of the temperature in the range of 30-60 ℃ on the extraction efficiency, and the other conditions are the same and as follows: diatomite/spirulina raw material is 0.5 percent, the water content of diatomite is 60 percent, the liquid-material ratio is 3, T is 60min, T is 40 ℃, and CO is added2The flow rate is 20 ml/min-1。
As shown in Table 7, the fatty acid and GLA contents of the resulting extract increased as the pressure increased from 4000psi to 6000 psi. At 6000psi, the extraction rates of fatty acid and GLA were essentially in equilibrium. There was no significant increase in GLA extraction when raised to 7000 psi. For optimum selectivity and for commercial reasons, extraction conditions at 6000psi are preferred for the process of the invention.
TABLE 7
5.4 CO2Effect of flow on extraction efficiency
In this example, the flow range of carbon dioxide is compared to 8-20 ml.min-1The specific operation steps of the method are as in example 1, and the other conditions are the same and are as follows: diatomite/spirulina raw material is 0.5 percent, the water content of diatomite is 60 percent, the liquid-material ratio is 3, T is 60min, T is 40 ℃, and CO is added2The flow rate is 20 ml/min-1。
The results in Table 8 show CO2The flow rate does not have a significant effect on the extraction efficiency. In order to obtain the best selectivity and meet the industrial requirements, the method of the invention preferably selects CO2The flow rate is 20 ml/min-1The extraction conditions of (1).
TABLE 8
Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
Claims (9)
1. A process for extracting glycolipids enriched with GLA moieties from Spirulina (Spirulina) comprising the steps of:
(i) collecting spirulina raw materials;
(ii) Subjecting the Spirulina raw material to supercritical CO2Extraction, wherein the supercritical CO2In the extraction process, organic solvent is used as entrainer, diatomite porous material is added,
wherein the diatomaceous earth comprises 60% moisture; in the supercritical CO2In the extraction process, static extraction time is 30min, dynamic extraction collection time is 30min, extraction pressure is 6000psi, extraction temperature is 40 deg.C, and CO is added2The flow rate is 20 ml/min-1(ii) a The adding amount of the porous material is 0.5 time of the mass of the spirulina raw material, and the weight ratio is calculated; the liquid-material ratio of the addition amount of the entrainer to the mass of the spirulina raw material is 3 in terms of ml to g;
(iii) collecting the extract; and
(iv) the extract is further extracted to obtain glycolipids.
2. The method of claim 1, wherein the glycolipid is selected from one or more of the group consisting of: MGDG, DGDG and SGDG.
3. The method of claim 1, wherein glycolipid is a mixture of MGDG, DGDG and SGDG.
4. The method of claim 1, wherein the method further comprises separating a mixture of remaining spirulina and diatomaceous earth in the extract and using the mixture as the supercritical CO2Porous material in the extraction process.
5. The method of claim 1, wherein the organic solvent is selected from the group consisting of: methanol, ethanol, isopropanol, ethyl acetate, propyl acetate, butyl acetate, acetone, n-hexane, or a mixture of one or more of the foregoing.
6. The method of claim 1, wherein the organic solvent is selected from the group consisting of: methanol, ethanol and ethyl acetate or mixtures thereof.
7. The method of claim 1, wherein the organic solvent is ethanol.
8. The method of claim 1, wherein the organic solvent is food grade ethanol.
9. The method of claim 1, wherein the spirulina is selected from the group consisting of: spirulina platensis (Spirulina platensis), Spirulina maxima (Spirulina maxima) and Spirulina subsalsa (Spirulina subsalsa).
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