CN114874349B - Method for separating and purifying ganoderan based on field flow separation technology - Google Patents
Method for separating and purifying ganoderan based on field flow separation technology Download PDFInfo
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- 238000000926 separation method Methods 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 title claims abstract description 55
- 150000004676 glycans Chemical class 0.000 claims abstract description 69
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 65
- 239000005017 polysaccharide Substances 0.000 claims abstract description 65
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 58
- 240000008397 Ganoderma lucidum Species 0.000 claims abstract description 42
- 235000001637 Ganoderma lucidum Nutrition 0.000 claims abstract description 42
- 239000012528 membrane Substances 0.000 claims abstract description 39
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 30
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 30
- 239000007788 liquid Substances 0.000 claims abstract description 28
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 27
- 239000012498 ultrapure water Substances 0.000 claims abstract description 27
- 229920002678 cellulose Polymers 0.000 claims abstract description 17
- 239000001913 cellulose Substances 0.000 claims abstract description 17
- 239000000243 solution Substances 0.000 claims description 20
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 14
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 14
- 238000002390 rotary evaporation Methods 0.000 claims description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims description 12
- 241000222336 Ganoderma Species 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 10
- 238000002347 injection Methods 0.000 claims description 7
- 239000007924 injection Substances 0.000 claims description 7
- 230000010355 oscillation Effects 0.000 claims description 7
- 239000002244 precipitate Substances 0.000 claims description 7
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- 238000004108 freeze drying Methods 0.000 claims description 4
- 150000004804 polysaccharides Polymers 0.000 claims description 4
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- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims 1
- 238000000746 purification Methods 0.000 abstract description 13
- 238000000502 dialysis Methods 0.000 abstract description 6
- 238000010612 desalination reaction Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 230000000052 comparative effect Effects 0.000 description 36
- 235000018102 proteins Nutrition 0.000 description 27
- 239000000523 sample Substances 0.000 description 24
- 230000007423 decrease Effects 0.000 description 5
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- 238000006703 hydration reaction Methods 0.000 description 5
- 238000010828 elution Methods 0.000 description 4
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- 229920012266 Poly(ether sulfone) PES Polymers 0.000 description 3
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- 150000003839 salts Chemical class 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 235000004252 protein component Nutrition 0.000 description 2
- 241001391944 Commicarpus scandens Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 241001489091 Ganoderma sinense Species 0.000 description 1
- 241000222341 Polyporaceae Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
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- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000011034 membrane dialysis Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
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- 239000012488 sample solution Substances 0.000 description 1
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- 238000005728 strengthening Methods 0.000 description 1
- 238000005556 structure-activity relationship Methods 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention provides a method for separating and purifying ganoderan based on a field flow separation technology. The method firstly adopts Sevag method to remove protein for 4 times for extracted ganoderma lucidum crude polysaccharide, then passes through an AF4 pool channel, adopts ultrapure water as carrier liquid, adopts a 10kDa renewable cellulose membrane, carries out ultrafiltration for a period of time at an ultrafiltration flow rate of 0.2mL/min, and finally carries out asymmetric field flow separation, wherein ultrapure water is adopted as carrier liquid during asymmetric field flow separation, and the initial cross flow rate exponential attenuation is reduced to 0.05mL/min. The invention reduces the times of protein removal in the process of separating and purifying the ganoderma lucidum polysaccharide, and the carrier liquid is ultrapure water in the whole process, so that the step of dialysis and desalination is not needed, the separation and purification efficiency is improved on the basis of protecting the activity of the ganoderma lucidum polysaccharide, and the high-purity ganoderma lucidum polysaccharide is obtained.
Description
Technical Field
The invention relates to the technical field of separation and purification of ganoderma lucidum polysaccharide, in particular to a method for separating and purifying ganoderma lucidum polysaccharide based on a field flow separation technology.
Background
Ganoderma (Ganoderma lucidum) is a dried fruiting body of Ganoderma lucidum or Ganoderma sinense belonging to Polyporaceae. In China, ganoderma lucidum has more than 2000 years of medicinal history. According to records in Shen nong Ben Cao, ganoderma has effects of strengthening body resistance, consolidating constitution, tonifying, and prolonging life. The polysaccharide is one of the main active ingredients of ganoderma lucidum, so that the preparation of high-purity ganoderma lucidum polysaccharide is a necessary condition for researching the structure-activity relationship of the ganoderma lucidum polysaccharide.
The separation and purification of natural plant polysaccharide is a time-consuming and tedious process, which comprises the steps of crude polysaccharide extraction, protein removal, salt removal, purification and the like. At present, the Sevag method and the trichloroacetic acid method commonly used for removing protein from polysaccharide have low efficiency, and the protein is removed repeatedly (often more than ten times), so that the glycosidic bond of the polysaccharide is easy to break, the biological activity of the polysaccharide is influenced, and a large amount of organic solvent is used. Dialysis desalination is also a time consuming process (greater than 24 h). Membrane separation technology is an important component of polysaccharide purification, and requires replacement of running water to maintain its membrane dialysis efficiency. Currently, ion exchange chromatography is generally adopted for purifying polysaccharide, a mobile phase is a salt solution, and the purified polysaccharide solution needs further dialysis for desalting. Asymmetric flow field-flow separation (AF 4) is a type of chromatography, with no stationary phase in the AF4 cell-line, reducing the risk of polysaccharide degradation during separation. In the AF4 separation sample process, the carrier liquid generates external force through an ultrafiltration membrane in the pool channel, and when the external force and the sample diffusion reach balance, the balance layer is closer to the center of the pool channel due to the large diffusion coefficient of the components with small hydration diameter, and then is eluted out firstly. Salts are often added to the AF4 carrier liquid to adjust its ionic strength and improve the separation of sample components. Therefore, the collected AF4 sample fragments require dialysis for desalting.
Disclosure of Invention
The invention aims to provide a method for separating and purifying ganoderma lucidum polysaccharide based on a field flow separation technology, which can realize separation and purification of ganoderma lucidum polysaccharide without dialysis and desalination.
The invention is realized by the following steps: a method for separating and purifying ganoderan based on field flow separation technology comprises the following steps:
(a) Weighing defatted Ganoderma fruiting body powder, adding 25 times of ultrapure water, mixing, extracting with ultrasound-assisted hot water method at 70 deg.C for 34min, and vacuum filtering to remove Ganoderma residue;
(b) Performing rotary evaporation and concentration on the filtrate, adding a Sevag reagent, shaking, removing protein for 4 times, and standing to remove precipitate;
(c) Carrying out rotary evaporation and concentration on the supernatant obtained in the step (b) to 10% for later use;
(d) Subjecting the concentrate obtained in step (c) to a period of time (T) by means of an asymmetric field flow separation system u ) Ultrafiltration with T being less than or equal to 30min u Less than or equal to 45min, the carrier liquid is ultrapure water, the ultrafiltration membrane is a 10kDa Renewable Cellulose (RC) membrane, and the ultrafiltration flow rate is 0.2mL/min;
(e) Subjecting the sample of step (d) to asymmetrySeparating field flow with sample volume of 100 μ L and carrier liquid of ultrapure water, eluting with exponential decay cross flow velocity, and initial cross flow velocity (V) c ) Reduced to 0.05mL/min and V is less than or equal to 2.6mL/min c Less than or equal to 3.0mL/min, half life period is 0.8min;
(f) Collecting polysaccharide fragments separated by the asymmetric field flow in the step (e), carrying out rotary evaporation and concentration to 1%, and carrying out freeze drying to obtain the high-purity ganoderma lucidum polysaccharide.
Preferably, sevag reagent is added in step (b), chloroform and n-butanol (V: V) =4:1, sample solution and reagent (V: V) =4:1, oscillation time 10min.
Preferably, step (d) uses asymmetric field flow separation technology in combination with an ultraviolet-visible light detector and a refractive index detector (AF 4-UV-dRI), and the carrier liquid used for separation monitoring is ultrapure water.
The invention relates to a method for separating and purifying ganoderan, which is formed by combining an ultrafiltration membrane based on the separation principle of an asymmetric field flow separation technology. The method firstly adopts Sevag method to remove protein for 4 times to extract ganoderma lucidum crude polysaccharide, and removes protein components with similar hydration diameter to polysaccharide sample. Next, a period of time (T) was run through the AF4 cell line using ultrapure water as the carrier liquid, a 10kDa Regenerable Cellulose (RC) membrane, an ultrafiltration flow rate of 0.2mL/min u ) And T is less than or equal to 30min u Less than or equal to 45min for removing salt and partial hydrated protein components with small diameter, thereby improving the separation degree of polysaccharide components. Finally, using ultrapure water as the carrier liquid, a 10kDa Regenerable Cellulose (RC) membrane, initial cross-flow velocity (V) c ) The exponential decay is reduced to 0.05mL/min, and V is less than or equal to 2.6mL/min c Less than or equal to 3.0mL/min, and the half-life period of 0.8min, so that the protein with smaller hydration diameter is separated from the polysaccharide.
The invention utilizes AF4 separation principle, adopts ultrapure water as carrier liquid, removes micromolecular impurities through ultrafiltration, collects AF4 polysaccharide fragments after polysaccharide components are separated from proteins with smaller hydration diameter, and obtains high-purity ganoderma lucidum polysaccharide through concentration and freeze drying. The invention reduces the times of protein removal in the process of separating and purifying the ganoderma lucidum polysaccharide, and the carrier liquid is ultrapure water in the whole process, so that the step of dialysis and desalination is not needed, the separation and purification efficiency is improved on the basis of protecting the activity of the ganoderma lucidum polysaccharide, and the high-purity ganoderma lucidum polysaccharide is obtained.
Drawings
FIG. 1 is an AF4-UV spectrum of ganoderma lucidum polysaccharide isolated and purified by the method of comparative examples 1-5.
FIG. 2 is an AF4-dRI spectrum of the separation and purification of ganoderan using the method of comparative examples 1 to 5.
FIG. 3 is an AF4-UV spectrum of the separation and purification of ganoderan using the method of comparative examples 6-10.
FIG. 4 is an AF4-dRI spectrum obtained by separating and purifying ganoderan using the method of comparative examples 6 to 10.
FIG. 5 is AF4-UV spectrum of separation and purification of ganoderan using the methods of examples 1-2 and comparative examples 11-13.
FIG. 6 is an AF4-dRI spectrum obtained by separating and purifying ganoderan according to the methods of examples 1 to 2 and comparative examples 11 to 13.
FIG. 7 is AF4-UV spectrum of separation and purification of ganoderan using the method of example 1 and comparative example 14.
FIG. 8 is a AF4-dRI spectrum of the separation and purification of ganoderan using the methods of example 1 and comparative example 14.
Detailed Description
The invention is further illustrated by the following examples, which are given by way of illustration only and are not intended to limit the scope of the invention in any way.
Procedures and methods not described in detail in the following examples are conventional methods well known in the art, and reagents used in the examples are all analytically pure and are either commercially available or prepared by methods well known to those of ordinary skill in the art. The following examples all achieve the objects of the present invention.
In the following examples, ganoderan was prepared using the following method: weighing 10g of defatted ganoderma lucidum fruiting body powder, placing the powder into a 500mL beaker, and adding 250mL of ultrapure water; placing in 70 deg.C water bath, and extracting with ultrasonic power of 200W for 34min. And after extraction, naturally cooling to room temperature, performing suction filtration by using a vacuum pump to remove ganoderma lucidum residues, and performing rotary evaporation and concentration on the filtrate.
Removing protein from the ganoderma lucidum crude polysaccharide by adopting a Sevag method. The crude polysaccharide solution was mixed with Sevag reagent (chloroform: n-butanol = 4:1) 4:1 into a 200mL Erlenmeyer flask, and shaken well for 10min. And (3) after oscillation, quickly transferring to a separating funnel, standing for 10min for layering, removing the lower-layer precipitate, and performing rotary evaporation and concentration on the supernatant to 10% to obtain a ganoderma lucidum crude polysaccharide aqueous solution for later use.
Adopting asymmetric field flow separation technology to combine ultraviolet-visible light detector and refractive index detector (AF 4-UV-dRI), using ultrapure water as carrier liquid, and using 10kDa Renewable Cellulose (RC) membrane as ultrafiltration membrane, and performing ultrafiltration at ultrafiltration flow rate of 0.2mL/min for a period of time (T) u ) And T is less than or equal to 30min u Less than or equal to 45min; performing asymmetric field flow separation after ultrafiltration with spacer thickness of 350 μm and sample introduction volume of 100 μ L, eluting with exponential decay cross flow velocity, and initial cross flow velocity (V) c ) Reduced to 0.05mL/min and V is less than or equal to 2.6mL/min c Less than or equal to 3.0mL/min, half-life period of 0.8min, and wavelength lambda of ultraviolet detector =280nm. Collecting the Ganoderma polysaccharide fragments separated by the asymmetric field flow separation system according to the separation patterns of the ultraviolet-visible light detector and the refractive index detector, concentrating the collected liquid by volume rotation until 1% is transferred to an EP tube, standing overnight at-20 deg.C, taking out, and freeze-drying to obtain purified Ganoderma polysaccharide.
Comparative example 1
Detecting and analyzing the ganoderma lucidum crude polysaccharide solution, wherein the step of removing protein is carried out for 0 time, the carrier liquid is ultrapure water, the ultrafiltration membrane is a 10kDa Renewable Cellulose (RC) membrane, the ultrafiltration flow rate is 0.2mL/min, and the ultrafiltration time is 0min. The AF4 separation conditions were: the injection volume was 50 μ L, the detector flow rate was 1.0mL/min, the initial cross-flow rate was 1.4mL/min, the exponential decay was to 0.05mL/min, and the half-life was 0.8min.
Comparative example 2
Deproteinizing the crude polysaccharide of Ganoderma lucidum by Sevag method, mixing the crude polysaccharide solution with Sevag reagent (chloroform: n-butanol = 4:1) 4:1 into a 200mL Erlenmeyer flask, and shaken well for 10min. And (3) quickly transferring to a separating funnel after oscillation is finished, standing for 10min for layering, discarding a lower-layer precipitate, carrying out volume rotary evaporation and concentration on a supernatant to 10%, and carrying out a protein removal step for 1 time. The carrier liquid is ultrapure water, the ultrafiltration membrane is a 10kDa Renewable Cellulose (RC) membrane, the ultrafiltration flow rate is 0.2mL/min, and the ultrafiltration time is 0min. The AF4 separation conditions were: the injection volume was 50 μ L, the detector flow rate was 1.0mL/min, the initial cross-flow rate was 1.4mL/min, the exponential decay was to 0.05mL/min, and the half-life was 0.8min.
Comparative example 3
Deproteinizing the crude polysaccharide of Ganoderma lucidum by Sevag method according to the ratio of crude polysaccharide solution to Sevag reagent (chloroform: n-butanol = 4:1) 4:1 into a 200mL Erlenmeyer flask, and shaken well for 10min. And (3) quickly transferring to a separating funnel after oscillation is finished, standing for 10min for layering, discarding a lower-layer precipitate, carrying out rotary evaporation and concentration on the volume of a supernatant to 10%, and carrying out a protein removal step for 2 times. The carrier liquid is ultrapure water, the ultrafiltration membrane is a 10kDa Renewable Cellulose (RC) membrane, the ultrafiltration flow rate is 0.2mL/min, and the ultrafiltration time is 0min. The AF4 separation conditions were: the injection volume was 50 μ L, the detector flow rate was 1.0mL/min, the initial cross-flow rate was 1.4mL/min, the exponential decay was to 0.05mL/min, and the half-life was 0.8min.
Comparative example 4
Deproteinizing the crude polysaccharide of Ganoderma lucidum by Sevag method according to the ratio of crude polysaccharide solution to Sevag reagent (chloroform: n-butanol = 4:1) 4:1 into a 200mL Erlenmeyer flask, and shaken well for 10min. And (3) quickly transferring to a separating funnel after oscillation is finished, standing for 10min for layering, discarding a lower-layer precipitate, carrying out volume rotary evaporation and concentration on a supernatant to 10%, and carrying out protein removal for 3 times. The carrier liquid is ultrapure water, the ultrafiltration membrane is a 10kDa Renewable Cellulose (RC) membrane, the ultrafiltration flow rate is 0.2mL/min, and the ultrafiltration time is 0min. The AF4 separation conditions were: the injection volume was 50 μ L, the detector flow rate was 1.0mL/min, the initial cross-flow rate was 1.4mL/min, the exponential decay was to 0.05mL/min, and the half-life was 0.8min.
Comparative example 5
Deproteinizing the crude polysaccharide of Ganoderma lucidum by Sevag method according to the ratio of crude polysaccharide solution to Sevag reagent (chloroform: n-butanol = 4:1) 4:1 into a 200mL Erlenmeyer flask, and shaking well for 10min. And (3) quickly transferring to a separating funnel after oscillation is finished, standing for 10min for layering, discarding a lower-layer precipitate, carrying out volume rotary evaporation and concentration on a supernatant to 10%, and carrying out a protein removal step for 4 times. The carrier liquid is ultrapure water, the ultrafiltration membrane is a 10kDa Renewable Cellulose (RC) membrane, the ultrafiltration flow rate is 0.2mL/min, and the ultrafiltration time is 0min. The AF4 separation conditions were: the injection volume was 50 μ L, the detector flow rate was 1.0mL/min, the initial cross-flow rate was 1.4mL/min, the exponential decay was to 0.05mL/min, and the half-life was 0.8min.
The results of the tests obtained in comparative examples 1 to 5 are shown in FIGS. 1 and 2. Since the polysaccharide has no ultraviolet absorption, the ultraviolet signal is mainly protein. As can be seen from FIG. 1, the signal intensity of the elution peak of the sample decreases with the increase of the protein removal times, which indicates that the protein in the sample has gradually decreased, and the UV signal (2-5 min) is substantially decreased to 0 after the ganoderma lucidum polysaccharide removes the protein for 4 times. The signal still remains at 2-5 min for the differential detector in FIG. 2, indicating that proteins with larger hydration diameters can be removed by 4 protein removals. The hydrated smaller diameter proteins (1-2 min) can be separated from the polysaccharide component in subsequent ultrafiltration and AF4 separation processes.
Comparative example 6
And (3) deproteinizing the crude polysaccharide solution of Ganoderma according to the method of comparative example 5, wherein the carrier liquid is ultrapure water, the ultrafiltration membrane is a 10kDa Renewable Cellulose (RC) membrane, the ultrafiltration flow rate is 0.2mL/min, and the ultrafiltration time is 0min. The AF4 separation conditions were: the sample volume was 100. Mu.L, the detector flow rate was 1.0mL/min, the initial cross-flow rate was 1.4mL/min, the exponential decay was to 0.05mL/min, and the half-life was 0.8min.
Comparative example 7
And (3) deproteinizing the crude polysaccharide solution of Ganoderma according to the method of comparative example 5, wherein the carrier liquid is ultrapure water, the ultrafiltration membrane is a 10kDa Renewable Cellulose (RC) membrane, the ultrafiltration flow rate is 0.2mL/min, and the ultrafiltration time is 15min. The AF4 separation conditions were: the sample volume was 100. Mu.L, the detector flow rate was 1.0mL/min, the initial cross-flow rate was 1.4mL/min, the exponential decay was to 0.05mL/min, and the half-life was 0.8min.
Comparative example 8
And (3) deproteinizing the ganoderma lucidum crude polysaccharide solution according to the method of the comparative example 5, wherein the carrier liquid is ultrapure water, the ultrafiltration membrane is a 10kDa Renewable Cellulose (RC) membrane, the ultrafiltration flow rate is 0.2mL/min, and the ultrafiltration time is 30min. The AF4 separation conditions were: the sample volume was 100. Mu.L, the detector flow rate was 1.0mL/min, the initial cross-flow rate was 1.4mL/min, the exponential decay was to 0.05mL/min, and the half-life was 0.8min.
Comparative example 9
And (3) deproteinizing the crude polysaccharide solution of Ganoderma according to the method of comparative example 5, wherein the carrier liquid is ultrapure water, the ultrafiltration membrane is a 10kDa Renewable Cellulose (RC) membrane, the ultrafiltration flow rate is 0.2mL/min, and the ultrafiltration time is 45min. The AF4 separation conditions were: the sample volume was 100. Mu.L, the detector flow rate was 1.0mL/min, the initial cross-flow rate was 1.4mL/min, the exponential decay was to 0.05mL/min, and the half-life was 0.8min.
Comparative example 10
The method of comparative example 5 is used for deproteinizing the ganoderma lucidum crude polysaccharide solution, the carrier liquid is ultrapure water, the ultrafiltration membrane is a 10kDa Renewable Cellulose (RC) membrane, the ultrafiltration flow rate is 0.2mL/min, and the ultrafiltration time is 60min. The AF4 separation conditions were: the sample introduction volume is 100 μ L, the detector flow rate is 1.0mL/min, the initial cross flow rate is 1.4mL/min, the exponential decay is 0.05mL/min, and the half-life is 0.8min.
The results of the tests obtained in comparative examples 6 to 10 are shown in FIGS. 3 and 4. To increase the amount of purified sample and further improve the purification efficiency, the injection volume was increased from 50. Mu.L to 100. Mu.L. As can be seen from FIGS. 3 and 4, as the ultrafiltration time increases, the intensity of the sample elution peak signal decreases, which indicates that part of the small molecular impurities in the ganoderma lucidum crude polysaccharide solution pass through the ultrafiltration membrane, when the ultrafiltration time is 30min, the ultraviolet signal significantly decreases, and when the ultrafiltration time is 60min, the ultraviolet signal does not significantly decrease.
Comparative example 11
The method of comparative example 9 is used for removing protein and ultra-filtering the ganoderma lucidum crude polysaccharide solution, the ultra-filtering time is 45min, and the AF4 separation conditions are as follows: the sample volume was 100. Mu.L, the detector flow rate was 1.0mL/min, the initial cross-flow rate was 1.8mL/min, the exponential decay was to 0.05mL/min, and the half-life was 0.8min.
Comparative example 12
The method of comparative example 9 was used to remove protein and ultrafilter the ganoderma lucidum crude polysaccharide solution, the ultrafiltration time was 45min, and the AF4 separation conditions were: the sample volume was 100 μ L, the detector flow rate was 1.0mL/min, the initial cross-flow rate was 2.2mL/min, the exponential decay was to 0.05mL/min, and the half-life was 0.8min.
Comparative example 13
The method of comparative example 9 is used for removing protein and ultra-filtering the ganoderma lucidum crude polysaccharide solution, the ultra-filtering time is 45min, and the AF4 separation conditions are as follows: the sample volume was 100 μ L, the detector flow rate was 1.0mL/min, the initial cross-flow rate was 3.2mL/min, the exponential decay was to 0.05mL/min, and the half-life was 0.8min.
Example 1
The method of comparative example 9 is used for removing protein and ultra-filtering the ganoderma lucidum crude polysaccharide solution, the ultra-filtering time is 45min, and the AF4 separation conditions are as follows: the sample volume was 100 μ L, the detector flow rate was 1.0mL/min, the initial cross-flow rate was 2.6mL/min, the exponential decay was to 0.05mL/min, and the half-life was 0.8min.
Example 2
The method of comparative example 9 is used for removing protein and ultra-filtering the ganoderma lucidum crude polysaccharide solution, the ultra-filtering time is 45min, and the AF4 separation conditions are as follows: the sample volume was 100. Mu.L, the detector flow rate was 1.0mL/min, the initial cross-flow rate was 3.0mL/min, the exponential decay was to 0.05mL/min, and the half-life was 0.8min.
The results of the tests obtained in examples 1 to 2 and comparative examples 11 to 13 are shown in FIGS. 5 and 6. As can be seen from FIG. 5, after removing protein for 4 times, the carrier liquid is ultrapure water, the ultrafiltration membrane is a 10kDa Renewable Cellulose (RC) membrane, the ultrafiltration flow rate is 0.2mL/min, the separation degree of polysaccharide components gradually becomes good along with the increase of the cross-flow rate after the ultrafiltration time is 45min, and the ganoderma lucidum polysaccharide can achieve a good separation effect when the initial cross-flow rate of exponential decay is 2.6-3.0 mL/min. When the initial cross flow velocity is 3.2mL/min, there is a weak difference signal at 4-6 min in FIG. 6, which indicates that there is a small amount of polysaccharide aggregates.
Comparative example 14
The crude polysaccharide solution of Ganoderma lucidum was deproteinized, ultrafiltered and separated according to the method of example 1, the ultrafiltration membrane was a 10kDa Polyethersulfone (PES) membrane.
The results of the tests obtained in example 1 and comparative example 14 are shown in FIGS. 7 and 8. As can be seen from fig. 7 and 8, the elution peak signal of the Polyethersulfone (PES) membrane sample is significantly reduced, with poor resolution. This is due to the cross-linking of the polysaccharide sample with the surface of a Polyethersulfone (PES) ultrafiltration membrane, resulting in a decrease in the signal intensity of the sample elution peak. The target fragment of example 1 was collected, and the polysaccharide content of the collected AF4 fragment was 108.37% and RSD =0.45% as determined by phenol-sulfuric acid method.
Claims (4)
1. A method for separating and purifying ganoderan based on field flow separation technology is characterized by comprising the following steps:
a. weighing defatted Ganoderma fruiting body powder, adding ultrapure water, mixing, extracting with ultrasound-assisted hot water method, and vacuum filtering to remove Ganoderma residue;
b. b, carrying out rotary evaporation and concentration on the filtrate obtained in the step a, adding a Sevag reagent, oscillating, removing protein for 4 times, standing and removing precipitate;
c. c, carrying out rotary evaporation and concentration on the supernatant obtained in the step b for later use;
d. d, carrying out ultrafiltration on the concentrated solution obtained in the step c by using an asymmetric field flow separation system, wherein the carrier liquid is ultrapure water, and the ultrafiltration membrane is a renewable cellulose membrane;
e. carrying out asymmetric field flow separation on the sample in the step d, wherein the carrier liquid is ultrapure water, and the sample is eluted by adopting exponential decay cross flow velocity;
f. collecting polysaccharide fragments separated by the asymmetric field flow in the step e, performing rotary evaporation concentration, and performing freeze drying to obtain purified ganoderma lucidum polysaccharide;
in the step d, the ultrafiltration time is 30-45 min, and the ultrafiltration flow rate is 0.2mL/min;
in the step e, the sample injection volume is 100 mu L, the initial cross flow velocity is 2.6-3.0 mL/min, the initial cross flow velocity exponential decay is reduced to 0.05mL/min, and the half-life period is 0.8min.
2. The method for separating and purifying ganoderan based on field flow separation technology as claimed in claim 1, wherein in step f, concentration is performed by rotary evaporation to 1%.
3. The method for separating and purifying ganoderan based on field flow separation technology as claimed in claim 1, wherein Sevag reagent is added in step b, and the volume ratio of chloroform to n-butanol is 4:1, the volume ratio of sample liquid to reagent is 4:1, oscillation time 10min.
4. The method for separating and purifying ganoderan based on field flow separation technique as claimed in claim 1, wherein step d employs asymmetric field flow separation technique in combination with UV-visible detector and refractometer detector.
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Application publication date: 20220809 Assignee: Hebei Guangxun Biotechnology Co.,Ltd. Assignor: HEBEI University Contract record no.: X2024990000042 Denomination of invention: A method for separating and purifying Ganoderma lucidum polysaccharides based on field flow separation technology Granted publication date: 20230310 License type: Common License Record date: 20240117 Application publication date: 20220809 Assignee: Hanpai Traditional Chinese Medicine Hebei Co.,Ltd. Assignor: HEBEI University Contract record no.: X2024990000039 Denomination of invention: A method for separating and purifying Ganoderma lucidum polysaccharides based on field flow separation technology Granted publication date: 20230310 License type: Common License Record date: 20240117 Application publication date: 20220809 Assignee: Hangzhou Yiju Juqi Biotechnology Co.,Ltd. Assignor: HEBEI University Contract record no.: X2024990000038 Denomination of invention: A method for separating and purifying Ganoderma lucidum polysaccharides based on field flow separation technology Granted publication date: 20230310 License type: Common License Record date: 20240117 |