CN102219866A - Method for extracting and separating ganoderma lucidum polysaccharide from ganoderma lucidum sporocarp - Google Patents
Method for extracting and separating ganoderma lucidum polysaccharide from ganoderma lucidum sporocarp Download PDFInfo
- Publication number
- CN102219866A CN102219866A CN 201110161167 CN201110161167A CN102219866A CN 102219866 A CN102219866 A CN 102219866A CN 201110161167 CN201110161167 CN 201110161167 CN 201110161167 A CN201110161167 A CN 201110161167A CN 102219866 A CN102219866 A CN 102219866A
- Authority
- CN
- China
- Prior art keywords
- enzymolysis
- polysaccharide
- prozyme
- ganoderma sporophore
- ganoderma lucidum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 150000004676 glycans Chemical class 0.000 title claims abstract description 64
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 64
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 44
- 240000008397 Ganoderma lucidum Species 0.000 title abstract description 17
- 235000001637 Ganoderma lucidum Nutrition 0.000 title abstract description 17
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 34
- 238000000605 extraction Methods 0.000 claims abstract description 26
- 238000000926 separation method Methods 0.000 claims abstract description 26
- 108090000790 Enzymes Proteins 0.000 claims abstract description 17
- 102000004190 Enzymes Human genes 0.000 claims abstract description 17
- 229940088598 enzyme Drugs 0.000 claims abstract description 17
- 239000012528 membrane Substances 0.000 claims abstract description 17
- 238000000746 purification Methods 0.000 claims abstract description 14
- 238000010828 elution Methods 0.000 claims abstract description 7
- 238000001035 drying Methods 0.000 claims abstract description 3
- 241000222336 Ganoderma Species 0.000 claims description 40
- 239000012530 fluid Substances 0.000 claims description 30
- 239000000843 powder Substances 0.000 claims description 25
- 108010059892 Cellulase Proteins 0.000 claims description 17
- 229940106157 cellulase Drugs 0.000 claims description 17
- 238000009295 crossflow filtration Methods 0.000 claims description 16
- 230000007062 hydrolysis Effects 0.000 claims description 11
- 238000006460 hydrolysis reaction Methods 0.000 claims description 11
- 150000001450 anions Chemical class 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 239000003957 anion exchange resin Substances 0.000 claims description 6
- 239000012141 concentrate Substances 0.000 claims description 6
- 239000011347 resin Substances 0.000 claims description 6
- 229920005989 resin Polymers 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 5
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 4
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 4
- 238000011049 filling Methods 0.000 claims 1
- 238000004108 freeze drying Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000001914 filtration Methods 0.000 abstract description 3
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- 238000007670 refining Methods 0.000 abstract description 2
- 239000003480 eluent Substances 0.000 abstract 2
- 230000014759 maintenance of location Effects 0.000 abstract 2
- 108090000526 Papain Proteins 0.000 abstract 1
- 239000004365 Protease Substances 0.000 abstract 1
- 229920002678 cellulose Polymers 0.000 abstract 1
- 239000001913 cellulose Substances 0.000 abstract 1
- 229940055729 papain Drugs 0.000 abstract 1
- 235000019834 papain Nutrition 0.000 abstract 1
- 239000008213 purified water Substances 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 47
- 238000005516 engineering process Methods 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 239000012153 distilled water Substances 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- 238000013375 chromatographic separation Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000003321 amplification Effects 0.000 description 4
- 238000011097 chromatography purification Methods 0.000 description 4
- 239000000945 filler Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 229920006395 saturated elastomer Polymers 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000003637 basic solution Substances 0.000 description 1
- 229940088623 biologically active substance Drugs 0.000 description 1
- OAABHEHWRQAHEJ-UHFFFAOYSA-N butan-1-ol;chloroform Chemical compound ClC(Cl)Cl.CCCCO OAABHEHWRQAHEJ-UHFFFAOYSA-N 0.000 description 1
- 239000003610 charcoal Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000874 microwave-assisted extraction Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 239000001814 pectin 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
- 150000004804 polysaccharides Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 235000015961 tonic Nutrition 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 229960000716 tonics Drugs 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Images
Landscapes
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for extracting and separating ganoderma lucidum polysaccharide from ganoderma lucidum sporocarp. The method comprises the steps of carrying out enzymolysis and extraction with complex enzyme on the ganoderma lucidum sporocarp to obtain enzymolysis solution, concentrating and separating the enzymolysis solution through a tangential flow filtering membrane to obtain ultrafiltration retention solution, carrying out elution, separation and purification on the ultrafiltration retention solution with purified water through radial flow chromatograph to obtain polysaccharide eluent, and conducting reduced pressure concentration and drying on the polysaccharide eluent to obtain ganoderma lucidum polysaccharide, wherein the papain and and cellulose are mixed in a weight ratio of 1:2-4:1 to form complex enzyme. The invention builds a set of polysaccharide production process of ganoderma lucidum sporocarp from raw materials to products, the process is environment-friendly, fast and efficient and suitable for large-scale production and the process cost for refining ganoderma lucidum polysaccharide can be greatly reduced.
Description
Technical field
The present invention relates to a kind of from Ganoderma sporophore the method for extraction separation ganoderan.
Background technology
Modern pharmacology and clinical practice confirm that ganoderan is that glossy ganoderma is strengthened the body resistance to consolidate the constitution, strengthening by means of tonics, the major ingredient of promoting longevity.Ganoderan has characteristics such as the non-special resistance of stimulation of host, immune specific reaction and inhibition transplantation tumor physiologically active, energy enhance immunity power, and hypoxia-bearing capability, the market requirement is increasing.
Traditional ganoderan extracting method is: Ganoderma sporophore pulverizing-water is carried/soda acid extraction-alcohol precipitation-oven dry, it is easy to scale operation, but the polysaccharide impurity that obtains is many, active constituent content is low, technology content is very low, though especially the soda acid extraction method can improve extraction yield, high and destructible polysaccharide structures also can cause environmental pollution simultaneously to equipment requirements; The decoloring method of ganoderan is based on charcoal absorption and oxidative decoloration, but these two kinds of method polysaccharide loss rates are bigger; Remove ganoderma lucidum crude polysaccharide protein and use the Savag method mostly, and this method is not only used a large amount of organic solvents, have big toxicity, and complicated operation, cost is higher, is not suitable for mass-producing.
For the modern biotechnology product, separation is the basic fundamental link during Bio-engineering Products are produced, and separation costs can account for 70%～90% of product total cost.In the separation and purification link of ganoderan, adopt ion exchange resin to handle ganoderma lucidum crude polysaccharide and can carry out effective separation and purification, but, in the mass-producing amplification is produced, have bigger difficulty because ion column is an axial columns.Radial flow chromatogram method (Radial Flow Chromatography, RFC) be a kind of novel chromatographic separation analytical technology that grows up over past ten years, compare with axial chromatogram, have that working pressure is low, flow velocity is high, linear amplification reaches outstanding advantages such as treatment capacity is big easily.
Summary of the invention
The method that the purpose of this invention is to provide a kind of separation and purification ganoderma lucidum fruitbody polysaccharide, it has environmental protection, efficient, the advantage that is easy to large-scale production.The present invention has not only kept the activity of ganoderan to greatest extent in preparation process, do not use any organic solvent, and can realize ganoderan scale preparation fast and efficiently.
The technical solution used in the present invention is:
A kind of from Ganoderma sporophore the method for extraction separation ganoderan, described method is: the Ganoderma sporophore powder carries out enzymolysis and extraction with prozyme and gets enzymolysis solution, and the gained enzymolysis solution separates to such an extent that ultrafiltration trapped fluid, gained ultrafiltration trapped fluid get polysaccharide elutriant, gained polysaccharide elutriant concentrating under reduced pressure, drying by radial flow chromatogram with the separation and purification of pure water wash-out and make ganoderan through the tangential flow filtration membrane concentration; Described prozyme is papoid and 1: 2～4: 1 mixing of cellulase mass ratio.
Described Ganoderma sporophore powder carries out enzymolysis and extraction with prozyme and can carry out as follows: the Ganoderma sporophore of 100 eye mesh screens is crossed in the broken back of selection, get the Ganoderma sporophore powder, adding quality is in the water of 10～30 times in Ganoderma sporophore powder, adjust pH is 4.5-7, add prozyme, carry out enzymolysis and extraction under stirring, enzymatic hydrolysis condition is: hydrolysis temperature is 40 ℃-65 ℃, enzymolysis time is 1h-3h, the add-on of prozyme is the 1%-5% of Ganoderma sporophore powder quality, and described prozyme is papoid and 1: 2～4: 1 mixing of cellulase mass ratio; Enzymolysis and extraction finishes the back centrifugation, gets supernatant liquor and promptly gets enzymolysis solution; The enzyme of described papoid unit alive is 80-100 ten thousand U/g, and the enzyme of described cellulase unit alive is 5-10 ten thousand U/g.
Described centrifugation under the 4000rpm rotating speed centrifugal 10～15 minutes usually.
Described adjust pH is used basic solution usually, as sodium hydroxide solution, saturated aqua calcis etc.
Papoid in the used prozyme or cellulase all can adopt commercial commercial enzyme, and the effect of papoid is the hydrolysis foreign protein, and the effect of cellulase is the dissociated cell wall, is convenient to the polysaccharide stripping.
Described enzymolysis solution carries out as follows through the separation of tangential flow filtration membrane concentration: enzymolysis solution enters the tangential flow filtration system, selecting molecular weight cut-off for use is the daltonian ultra-filtration membranes of 100,000 dalton-1,000,000, pressure is 0～30psi, room temperature, the volumetric flow rate of enzymolysis solution is 125-700mL/min, cycles of concentration is 10-20 times, gets the ultrafiltration trapped fluid.By tangential flow filtration, can remove a large amount of protein in the enzymolysis solution, remove the free pigment in the enzymolysis solution simultaneously.
Described tangential flow filtration system is the filtering system of the separation and purification macromole biologically active substance used always, and different with the conventional filtration mode, the flow direction of liquid to be filtered is tangentially parallel with filter membrane, is a kind of filter operation mode that has been widely adopted in the pharmaceutical technology.The all commercially available acquisition of employed system, known its using method of those skilled in the art.The tangential flow filtration system that adopts in the embodiment of the invention is Congent M, U.S. Millipore Corp..
Described ultrafiltration trapped fluid carries out with the separation and purification of pure water wash-out as follows by radial flow chromatogram: the ultrafiltration trapped fluid adds in the radial flow chromatographic column, adopt weak base anion-exchange resin to fill radial flow chromatographic column, use the pure water wash-out, separation and purification gets the polysaccharide elutriant.The ultrafiltration trapped fluid removes most protein and pigment after by radial flow chromatogram.
Described radial flow chromatogram and patent application 200810163568
A kind of radial flow chromatogram to remove Method of protein in the Crude polysaccharidesWhat adopt is identical method, the radial flow chromatogram separation method adopts the radial flow particular design, compare with traditional axial chromatogram, this method has that flow velocity height, working pressure are low, linear amplification reaches outstanding advantages such as quantity of sample handling is big easily, employed radial flow chromatographic column can be by commercially available, and using method also is known.
Further, the method for the invention is preferably carried out according to the following steps:
(1) Ganoderma sporophore of 100 eye mesh screens is crossed in the broken back of selection, get the Ganoderma sporophore powder, adding quality is in the water of 10～30 times in Ganoderma sporophore powder, adjust pH is 4.5-7, adds prozyme, carries out enzymolysis and extraction under stirring, enzymatic hydrolysis condition is: hydrolysis temperature is 40 ℃-65 ℃, enzymolysis time is 1h-3h, and the add-on of prozyme is the 1%-5% of Ganoderma sporophore powder quality, and described prozyme is papoid and 1: 2～4: 1 mixing of cellulase mass ratio; Enzymolysis and extraction finishes the back centrifugation, gets supernatant liquor and promptly gets enzymolysis solution; Described papoid the enzyme unit that lives be 80-100 ten thousand U/g, the enzyme of the described cellulase unit that lives is 5-10 ten thousand U/g;
(2) enzymolysis solution that obtains of step (1) enters the tangential flow filtration system, and selecting molecular weight cut-off for use is the daltonian ultra-filtration membranes of 100,000 dalton-1,000,000, and pressure is 0～30psi, room temperature, the volumetric flow rate of enzymolysis solution is 125-700mL/min, and cycles of concentration is 10-20 times, gets the ultrafiltration trapped fluid; The concentration of gained ultrafiltration trapped fluid is generally 2mg/mL-12mg/mL;
(3) carry out separation and purification in the ultrafiltration trapped fluid adding radial flow chromatographic column that step (2) obtains, get the polysaccharide elutriant; Adopt weak base anion-exchange resin to fill radial flow chromatographic column, use the pure water wash-out, applied sample amount is 30-120mL, and last sample flow velocity is 1mL/min-10mL/min, and the pure water elution flow rate is 20mL/min-70mL/min; Described weak base anion-exchange resin is A103S resin anion(R.A), A100 resin anion(R.A) or A105 resin anion(R.A);
(4) step (3) gained polysaccharide elutriant concentrates 5-15 doubly in 40～90 ℃ of scopes, and the lyophilize of gained concentrated solution makes ganoderan.
Advantage of the present invention is:
1, set up a cover from the former ganoderma lucidum fruitbody polysaccharide production technique of expecting product, this technology has environmental protection, characteristics fast and efficiently, and is applicable to scale operation, can reduce the technology cost of refining ganoderam lucidum polysaccharide significantly.
2, thoroughly break away from the production technique of traditional ganoderan, do not related to the process of highly energy-consumings such as alcohol precipitation, organic solvent deproteinated, unit decolouring, consumption material, when reducing cost, prepared ganoderan quickly and efficiently.
3, adopt the prozyme solution to extract ganoderma lucidum fruitbody polysaccharide, thereby make the easier extraction of effective constituent reach the purpose of raising extraction yield or reduction solvent-oil ratio by the degrading plant cell walls on the one hand; On the other hand can be at most of impurity (starch, pectin, protein etc.) selectivity degraded wherein, to be more conducive to the carrying out of later separation purifying; Compare with extracting method such as traditional water extraction and ultrasonic, microwaves, can improve polysaccharide yield significantly, the extraction process mild condition is energy-saving and cost-reducing simultaneously.
But 4, adopt that tangential flow filtration membrane concentration enzymolysis solution has that the activity do not damaged, separation efficiency height, energy consumption are low, the simple continuous production of equipment, advantage such as pollution-free; Can ganoderma lucidum fruitbody polysaccharide is carried out concentrated and purified in the screening different molecular weight polysaccharide, be enriched with active polysaccharide; Adopting tangential flow filtration to concentrate the enzymolysis solution required time only is 1/6 of rotary evaporation.
5, adopt radial flow chromatogram that the film trapped fluid is carried out separation and purification and can greatly improve output and speed, also can guarantee the activity of ganoderan; Because radial flow chromatogram has the characteristic of linear amplification, can set up chromatographic condition simultaneously, directly amplify production by lab scale.
Description of drawings
The process flow sheet of Fig. 1 extraction separation ganoderan from Ganoderma sporophore
Fig. 2 different methods extracts glossy ganoderma seed polysaccharide yield comparison diagram
Embodiment
The present invention will be further described with specific embodiment below, but protection scope of the present invention is not limited thereto.
Embodiment 1:
(1) complex enzyme hydrolysis extracts ganoderma lucidum fruitbody polysaccharide: the Ganoderma sporophore of 100 eye mesh screens is crossed in the broken back of selection, get 50.0g Ganoderma sporophore powder, add in the water of 1000g, with saturated aqua calcis regulator solution pH to 6.8, add the 1.5g prozyme, prozyme is that papoid (is gone up sea blue season development in science and technology company limited, 1,000,000 U/g) with cellulase (gold source, Zhangjiagang biochemical industry company limited, 80,000 U/g) 2: 1 mixing of mass ratio, be positioned over 55 ℃ of thermostat water baths, at the uniform velocity stir 2h, centrifugal 10 minutes of 4000rpm gets supernatant liquor and promptly gets enzymolysis solution;
(2) cross-flow ultrafiltration membrane concentration enzymolysis solution: enzymolysis solution enters (the Congent M of tangential flow filtration system, U.S. Millipore Corp.), choosing molecular weight is 1,000,000 daltonian ultra-filtration membranes, the enzymolysis solution volumetric flow rate is 500mL/min, 25 ℃ of room temperatures, pressure is 23-26psi, is concentrated into 17 times, gets the ultrafiltration trapped fluid;
(3) radial flow chromatographic separation and purification trapped fluid: the ultrafiltration trapped fluid adds in the radial flow chromatographic column, the radial flow chromatographic column model is Superflo-250Column, filler is weakly-basic anion A103S, sample size is 100mL, the sample introduction flow velocity is 2mL/min, use the pure water wash-out, elution flow rate is 50mL/min, gets the polysaccharide elutriant;
(4) concentrating under reduced pressure polysaccharide elutriant concentrates 8 times down at 50 ℃, and the lyophilize of gained concentrated solution gets ganoderan product 0.88g, and wherein polysaccharide content is 85%, total sugar content 98%.。
The enzymolysis yield is 3.10%, and polysaccharide content is 80.7% in the preparation gained ganoderan elutriant, and protein content is 0.76%, and comparing with enzymolysis solution and calculating the albumen decreasing ratio is 99.24%.The polysaccharide elutriant is compared with the ultrafiltration trapped fluid, and the polysaccharide recovery in the radial flow process is 89.52%.
To step (1) gained enzymolysis solution, step (2) gained ultrafiltration trapped fluid, step (3) gained polysaccharide elutriant detect total sugar content, DNS method mensuration reducing sugar content, Folin-phenol method mensuration protein content, gained data such as following table 1 with the phenol sulfuric acid process respectively.
The comparison of table 1 different process stage ganoderan
Embodiment 2:
(1) complex enzyme hydrolysis extracts ganoderma lucidum fruitbody polysaccharide: the Ganoderma sporophore of 100 eye mesh screens is crossed in the broken back of selection, get 50.0g Ganoderma sporophore powder, add in the water of 500g, with saturated aqua calcis regulator solution pH to 6.0, add the 1.2g prozyme, prozyme is that papoid (is gone up sea blue season development in science and technology company limited, 1,000,000 U/g) with cellulase (gold source, Zhangjiagang biochemical industry company limited, 80,000 U/g) 1: 1 mixing of mass ratio, be positioned over 40 ℃ of thermostat water baths, at the uniform velocity stir 1h, centrifugal 12 minutes of 4000rpm gets supernatant liquor and promptly gets enzymolysis solution;
(2) cross-flow ultrafiltration membrane concentration enzymolysis solution: enzymolysis solution enters the tangential flow filtration system, and choosing molecular weight is 100,000 daltonian ultra-filtration membranes, and the enzymolysis solution volumetric flow rate is 700mL/min, 25 ℃ of room temperatures, and pressure is 28psi, is concentrated into 10 times, gets the ultrafiltration trapped fluid;
(3) radial flow chromatographic separation and purification trapped fluid: the ultrafiltration trapped fluid adds in the radial flow chromatographic column, the radial flow chromatographic column model is Superflo-250Column, filler is weakly-basic anion A103S, sample size is 120mL, the sample introduction flow velocity is 5mL/min, use the pure water wash-out, elution flow rate is 40mL/min, gets the polysaccharide elutriant;
(4) concentrating under reduced pressure polysaccharide elutriant concentrates 10 times down at 55 ℃, and the lyophilize of gained concentrated solution gets ganoderan product 0.91g, and wherein polysaccharide content is 82%, total sugar content 96%.
The enzymolysis yield is 3.02%, and polysaccharide content is 78.1% in the preparation gained ganoderan elutriant, and protein content is 0.62%.
Embodiment 3
(1) complex enzyme hydrolysis extracts ganoderma lucidum fruitbody polysaccharide: the Ganoderma sporophore of 100 eye mesh screens is crossed in the broken back of selection, get 50.0g Ganoderma sporophore powder, add in the water of 1500g, with saturated aqua calcis regulator solution pH to 4.5, add the 0.5g prozyme, prozyme is that papoid (is gone up sea blue season development in science and technology company limited, 1,000,000 U/g) with cellulase (gold source, Zhangjiagang biochemical industry company limited, 80,000 U/g) 4: 1 mixing of mass ratio, be positioned over 65 ℃ of thermostat water baths, at the uniform velocity stir 3h, centrifugal 12 minutes of 4000rpm gets supernatant liquor and promptly gets enzymolysis solution;
(2) cross-flow ultrafiltration membrane concentration enzymolysis solution: enzymolysis solution enters the tangential flow filtration system, and choosing molecular weight is 300,000 daltonian ultra-filtration membranes, and the enzymolysis solution volumetric flow rate is 400mL/min, 25 ℃ of room temperatures, and pressure is 26psi, is concentrated into 20 times, gets the ultrafiltration trapped fluid;
(3) radial flow chromatographic separation and purification trapped fluid: the ultrafiltration trapped fluid adds in the radial flow chromatographic column, the radial flow chromatographic column model is Superflo-250Column, filler is weakly-basic anion A103S, sample size is 50mL, the sample introduction flow velocity is 1mL/min, use the pure water wash-out, elution flow rate is 70mL/min, gets the polysaccharide elutriant;
(4) concentrating under reduced pressure polysaccharide elutriant concentrates 15 times down at 60 degrees centigrade, and the lyophilize of gained concentrated solution gets ganoderan product 1.2g, and wherein polysaccharide content is 83%, total sugar content 96%.
The enzymolysis yield is 2.85%, and the polysaccharide content in the preparation gained ganoderan elutriant is 72.1%, and protein content is 2.8%.
Comparative example 1: boiling water refluxing extraction polysaccharide method: take by weighing the Ganoderma sporophore powder that 20g crosses 100 eye mesh screens, press solid-liquid ratio and add 400mL distilled water at 1: 20, boiling water refluxing extraction 1.5h, gained extracting solution 380mL detects its polysaccharide yield 2.07%, sees Fig. 2.
Comparative example 2: supersound extraction polysaccharide method: take by weighing the Ganoderma sporophore powder that 20g crosses 100 eye mesh screens, press solid-liquid ratio and add 400mL distilled water at 1: 20, ultrasonic power is 800W, pulse 8s extracts 1h, gained extracting solution 385mL, detecting its polysaccharide yield is 2.61%, sees Fig. 2.
Comparative example 3: microwave extraction polysaccharide method: take by weighing the Ganoderma sporophore powder that 20g crosses 100 eye mesh screens, press solid-liquid ratio and add 400mL distilled water at 1: 20, microwave power is 800W, extracting temperature is 90 ℃, extracts 1.5h, gained extracting solution 375mL, detecting its polysaccharide yield is 2.54%, sees Fig. 2.
Comparative example 4: papoid extracting method: take by weighing the Ganoderma sporophore powder that 20g crosses 100 eye mesh screens, press solid-liquid ratio and add 400mL distilled water at 1: 20, enzyme concentration is 3%, hydrolysis temperature is 55 ℃, enzymolysis time is 1.5h, and pH is 5.7, gained enzymolysis solution 390mL, detecting its polysaccharide yield is 2.05%, sees Fig. 2.
Comparative example 5: cellulase extracting method: take by weighing the Ganoderma sporophore powder that 20g crosses 100 eye mesh screens, press solid-liquid ratio and add 400mL distilled water at 1: 20, enzyme concentration is 3%, hydrolysis temperature is 55 ℃, enzymolysis time is 1.5h, and pH is 4.8, gained enzymolysis solution 380mL, detecting its polysaccharide yield is 1.88%, sees Fig. 2.
Comparative example 6: prozyme solution: take by weighing the Ganoderma sporophore powder that 20g crosses 100 eye mesh screens, press solid-liquid ratio and add 400mL distilled water at 1: 20, add prozyme, enzyme concentration is 3%, papoid in the prozyme: cellulase=2: 1 (mass ratio), hydrolysis temperature is 55 ℃, enzymolysis time is 1.5h, and pH is 6.8, gained enzymolysis solution 380mL, detecting its polysaccharide yield is 3.15%, sees Fig. 2.
Comparative example 7 (radial flow chromatogram)
Step (1) operation according to embodiment 1 obtains enzymolysis solution,
The gained enzymolysis solution is carried out radial flow chromatographic separation and purification, condition is: enzymolysis solution adds in the radial flow chromatographic column, the radial flow chromatographic column model is Superflo-250Column, filler is weakly-basic anion A103S, sample size is 100mL, and the sample introduction flow velocity is 2mL/min, uses the pure water wash-out, elution flow rate is 50mL/min, gets the polysaccharide elutriant; The albumen decreasing ratio is 80.59%, and polysaccharide recovery is 89.21%, sees Table 2.
Comparative example 8
Sevag method deproteinated
Sevag reagent: it is 4: 1 mixed solution that chloroform-propyl carbinol is mixed with volume ratio;
Step (1), (2) operation according to embodiment 1 obtain the ultrafiltration trapped fluid, get 50mL ultrafiltration trapped fluid mixes with volume ratio with Sevag reagent at 3: 1,30min fully vibrates in the separating funnel, leave standstill 3h, get the upper strata polysaccharide soln and measure protein and polysaccharide content, calculate its albumen decreasing ratio and polysaccharide recovery, as shown in table 2.
Table 2 different methods removes ganoderan protein test result contrast
Claims (5)
1. the method for an extraction separation ganoderan from Ganoderma sporophore, it is characterized in that described method is: the Ganoderma sporophore powder carries out enzymolysis and extraction with prozyme and gets enzymolysis solution, and the gained enzymolysis solution separates to such an extent that ultrafiltration trapped fluid, gained ultrafiltration trapped fluid get polysaccharide elutriant, gained polysaccharide elutriant concentrating under reduced pressure, drying by radial flow chromatogram with the separation and purification of pure water wash-out and make ganoderan through the tangential flow filtration membrane concentration; Described prozyme is papoid and 1: 2～4: 1 mixing of cellulase mass ratio.
2. the method for claim 1, it is characterized in that described Ganoderma sporophore powder carries out enzymolysis and extraction with prozyme and carries out as follows: the Ganoderma sporophore of 100 eye mesh screens is crossed in the broken back of selection, get the Ganoderma sporophore powder, adding quality is in the water of 10～30 times in Ganoderma sporophore powder, adjust pH is 4.5-7, add prozyme, carry out enzymolysis and extraction under stirring, enzymatic hydrolysis condition is: hydrolysis temperature is 40 ℃-65 ℃, enzymolysis time is 1h-3h, the add-on of prozyme is the 1%-5% of Ganoderma sporophore powder quality, and described prozyme is papoid and 1: 2～4: 1 mixing of cellulase mass ratio; Enzymolysis and extraction finishes the back centrifugation, gets supernatant liquor and promptly gets enzymolysis solution; The enzyme of described papoid unit alive is 80-100 ten thousand U/g, and the enzyme of described cellulase unit alive is 5-10 ten thousand U/g.
3. the method for claim 1, it is characterized in that described enzymolysis solution carries out as follows through the separation of tangential flow filtration membrane concentration: enzymolysis solution enters the tangential flow filtration system, selecting molecular weight cut-off for use is the daltonian ultra-filtration membranes of 100,000 dalton-1,000,000, pressure is 0～30psi, room temperature, the volumetric flow rate of enzymolysis solution is 125-700mL/min, and cycles of concentration is 10-20 times, gets the ultrafiltration trapped fluid.
4. the method for claim 1, it is characterized in that described ultrafiltration trapped fluid carries out with the separation and purification of pure water wash-out as follows by radial flow chromatogram: the ultrafiltration trapped fluid adds employing weak base anion-exchange resin filling radial flow chromatographic column in the radial flow chromatographic column, use the pure water wash-out, separation and purification gets the polysaccharide elutriant.
5. the method for claim 1 is characterized in that said method comprising the steps of:
(1) Ganoderma sporophore of 100 eye mesh screens is crossed in the broken back of selection, get the Ganoderma sporophore powder, adding quality is in the water of 10～30 times in Ganoderma sporophore powder, adjust pH is 4.5-7, adds prozyme, carries out enzymolysis and extraction under stirring, enzymatic hydrolysis condition is: hydrolysis temperature is 40 ℃-65 ℃, enzymolysis time is 1h-3h, and the add-on of prozyme is the 1%-5% of Ganoderma sporophore powder quality, and described prozyme is papoid and 1: 2～4: 1 mixing of cellulase mass ratio; Enzymolysis and extraction finishes the back centrifugation, gets supernatant liquor and promptly gets enzymolysis solution; Described papoid the enzyme unit that lives be 80-100 ten thousand U/g, the enzyme of the described cellulase unit that lives is 5-10 ten thousand U/g;
(2) enzymolysis solution that obtains of step (1) enters the tangential flow filtration system, and selecting molecular weight cut-off for use is the daltonian ultra-filtration membranes of 100,000 dalton-1,000,000, and pressure is 0～30psi, room temperature, the volumetric flow rate of enzymolysis solution is 125-700mL/min, and cycles of concentration is 10-20 times, gets the ultrafiltration trapped fluid;
(3) carry out separation and purification in the ultrafiltration trapped fluid adding radial flow chromatographic column that step (2) obtains, get the polysaccharide elutriant; Adopt weak base anion-exchange resin to fill radial flow chromatographic column, use the pure water wash-out, applied sample amount is 30-120mL, and last sample flow velocity is 1mL/min-10mL/min, and the pure water elution flow rate is 20mL/min-70mL/min; Described weak base anion-exchange resin is A103S resin anion(R.A), A100 resin anion(R.A) or A105 resin anion(R.A);
(4) step (3) gained polysaccharide elutriant concentrates 5-15 doubly in 40～90 ℃ of scopes, gained concentrated solution obtained by freeze drying ganoderan.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110161167 CN102219866B (en) | 2011-06-15 | 2011-06-15 | Method for extracting and separating ganoderma lucidum polysaccharide from ganoderma lucidum sporocarp |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110161167 CN102219866B (en) | 2011-06-15 | 2011-06-15 | Method for extracting and separating ganoderma lucidum polysaccharide from ganoderma lucidum sporocarp |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102219866A true CN102219866A (en) | 2011-10-19 |
| CN102219866B CN102219866B (en) | 2012-12-12 |
Family
ID=44776591
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110161167 Active CN102219866B (en) | 2011-06-15 | 2011-06-15 | Method for extracting and separating ganoderma lucidum polysaccharide from ganoderma lucidum sporocarp |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102219866B (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102649823A (en) * | 2012-04-28 | 2012-08-29 | 浙江工业大学 | Method for preparing copper polysaccharide through radial flow coupling ultra-filtering separation |
| CN102718880A (en) * | 2012-06-23 | 2012-10-10 | 上海市农业科学院 | Ganoderma lucidum polysaccharide of high molecular weight and detection method thereof |
| CN104292357A (en) * | 2014-11-11 | 2015-01-21 | 济南凯因生物科技有限公司 | Extraction method of ganoderma lucidum polysaccharide |
| CN105367675A (en) * | 2015-11-20 | 2016-03-02 | 临沂大学 | Method for extraction of polysaccharide from ganoderma lucidum karst |
| CN105919093A (en) * | 2016-05-11 | 2016-09-07 | 福州东星生物技术有限公司 | Qi-replenishing and spleen-tonifying compound lingzhi mushroom preparation and preparation method thereof |
| CN105995966A (en) * | 2016-06-20 | 2016-10-12 | 张勇 | Preparation method of enzymolysis ganoderma lucidum powder |
| CN108341890A (en) * | 2018-05-09 | 2018-07-31 | 四川岚晟生物科技有限公司 | A kind of extracting method of ganoderma lucidum polysaccharide |
| CN108707200A (en) * | 2018-08-13 | 2018-10-26 | 辽宁大学 | The preparation method of polysaccharide in narrow vicia amoena |
| CN110016086A (en) * | 2019-03-14 | 2019-07-16 | 合肥工业大学 | A kind of refining process of Ganoderma lucidum polysaccharide |
| CN113121714A (en) * | 2021-03-16 | 2021-07-16 | 浙江工业大学 | Ganoderma lucidum oligosaccharide with probiotic activity and preparation method and application thereof |
| CN114558040A (en) * | 2022-02-25 | 2022-05-31 | 河南省纳普生物技术有限公司 | Ganoderma lucidum saccharide extract for relieving visual fatigue and preparation method thereof |
| CN115785300A (en) * | 2022-11-30 | 2023-03-14 | 上海市农业科学院 | Ganoderma lucidum water-extracted ultrafiltrate rich in β-(1→3,1→6)-glucan and preparation method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1297994A (en) * | 2000-12-08 | 2001-06-06 | 曾文伟 | Production process of fungus powder and fungus polysaccharide for food and medicine |
| US6613754B1 (en) * | 2000-09-22 | 2003-09-02 | National Yang-Ming University | Polysaccharide-based extract from ganoderma, pharmaceutical use thereof, and process for preparing the same |
| KR20040067175A (en) * | 2003-01-22 | 2004-07-30 | 엔제피아 주식회사 | Method for preparing immune enhancing polysaccharides |
| CN101649000A (en) * | 2009-07-21 | 2010-02-17 | 上海市农业科学院 | Preparation method of high purity ganoderma polysaccharide |
| CN101724088A (en) * | 2009-11-24 | 2010-06-09 | 浙江工业大学 | Method for removing proteins and pigments in ganoderma lucidum crude polysaccharide |
| CN101935358A (en) * | 2010-09-10 | 2011-01-05 | 上海市农业科学院 | A kind of ganoderma polysaccharide and preparation method thereof |
-
2011
- 2011-06-15 CN CN 201110161167 patent/CN102219866B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6613754B1 (en) * | 2000-09-22 | 2003-09-02 | National Yang-Ming University | Polysaccharide-based extract from ganoderma, pharmaceutical use thereof, and process for preparing the same |
| CN1297994A (en) * | 2000-12-08 | 2001-06-06 | 曾文伟 | Production process of fungus powder and fungus polysaccharide for food and medicine |
| KR20040067175A (en) * | 2003-01-22 | 2004-07-30 | 엔제피아 주식회사 | Method for preparing immune enhancing polysaccharides |
| CN101649000A (en) * | 2009-07-21 | 2010-02-17 | 上海市农业科学院 | Preparation method of high purity ganoderma polysaccharide |
| CN101724088A (en) * | 2009-11-24 | 2010-06-09 | 浙江工业大学 | Method for removing proteins and pigments in ganoderma lucidum crude polysaccharide |
| CN101935358A (en) * | 2010-09-10 | 2011-01-05 | 上海市农业科学院 | A kind of ganoderma polysaccharide and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 《分析化学》 20100315 何晋渐等 灵芝多糖的结构特征分析 第372-376页 1,4 第38卷, 第3期 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102649823B (en) * | 2012-04-28 | 2014-06-11 | 浙江工业大学 | Method for preparing copper polysaccharide through radial flow coupling ultra-filtering separation |
| CN102649823A (en) * | 2012-04-28 | 2012-08-29 | 浙江工业大学 | Method for preparing copper polysaccharide through radial flow coupling ultra-filtering separation |
| CN102718880A (en) * | 2012-06-23 | 2012-10-10 | 上海市农业科学院 | Ganoderma lucidum polysaccharide of high molecular weight and detection method thereof |
| CN104292357B (en) * | 2014-11-11 | 2017-01-25 | 牛睦元 | Extraction method of ganoderma lucidum polysaccharide |
| CN104292357A (en) * | 2014-11-11 | 2015-01-21 | 济南凯因生物科技有限公司 | Extraction method of ganoderma lucidum polysaccharide |
| CN105367675A (en) * | 2015-11-20 | 2016-03-02 | 临沂大学 | Method for extraction of polysaccharide from ganoderma lucidum karst |
| CN105919093A (en) * | 2016-05-11 | 2016-09-07 | 福州东星生物技术有限公司 | Qi-replenishing and spleen-tonifying compound lingzhi mushroom preparation and preparation method thereof |
| CN105995966A (en) * | 2016-06-20 | 2016-10-12 | 张勇 | Preparation method of enzymolysis ganoderma lucidum powder |
| CN108341890A (en) * | 2018-05-09 | 2018-07-31 | 四川岚晟生物科技有限公司 | A kind of extracting method of ganoderma lucidum polysaccharide |
| CN108707200A (en) * | 2018-08-13 | 2018-10-26 | 辽宁大学 | The preparation method of polysaccharide in narrow vicia amoena |
| CN110016086A (en) * | 2019-03-14 | 2019-07-16 | 合肥工业大学 | A kind of refining process of Ganoderma lucidum polysaccharide |
| CN110016086B (en) * | 2019-03-14 | 2021-03-19 | 合肥工业大学 | Refining process of ganoderma lucidum polysaccharide |
| CN113121714A (en) * | 2021-03-16 | 2021-07-16 | 浙江工业大学 | Ganoderma lucidum oligosaccharide with probiotic activity and preparation method and application thereof |
| CN114558040A (en) * | 2022-02-25 | 2022-05-31 | 河南省纳普生物技术有限公司 | Ganoderma lucidum saccharide extract for relieving visual fatigue and preparation method thereof |
| CN115785300A (en) * | 2022-11-30 | 2023-03-14 | 上海市农业科学院 | Ganoderma lucidum water-extracted ultrafiltrate rich in β-(1→3,1→6)-glucan and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102219866B (en) | 2012-12-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102219866A (en) | Method for extracting and separating ganoderma lucidum polysaccharide from ganoderma lucidum sporocarp | |
| CN106008645B (en) | A kind of method that momordica grosvenori glycoside V is extracted from Momordica grosvenori | |
| CN102276679B (en) | Method for extracting high-purity tea saponin from oil-tea-cake by decompression boiling | |
| CN101244119A (en) | Method for extracting polysaccharide and effective components from Cyclocarya paliurus | |
| CN105294790A (en) | Method for extracting high-purity steviol glycosides from stevia rebaudiana | |
| CN101962486B (en) | Industrial production method for extracting natural purple sweet potato coloring matter for food from edible purple sweet potato | |
| CN104861079A (en) | Microwave assisted lentinan extraction equipment and technology | |
| CN102516325A (en) | Method for producing crocin with higher than 95% purity from gardenia | |
| CN101875703A (en) | Method for extracting pectin and synephrine by utilizing peels and marc | |
| CN106544372A (en) | A kind of method that gamma aminobutyric acid is purified from zymotic fluid | |
| CN102633900A (en) | Method for extracting and purifying polysaccharide from Cordyceps militaris | |
| CN114213500A (en) | Tea saponin, tea polysaccharide and tea protein purification process based on comprehensive extraction of camellia oleifera | |
| EP3505525B1 (en) | Method for preparing rubusoside | |
| CN101475620B (en) | Efficient energy-saving extraction and production method for high-purity cordycepin | |
| CN102002072A (en) | Process for extracting flavone from date pit | |
| CN102558902A (en) | Process for extracting gardenia yellow from gardenia | |
| CN109180457A (en) | A kind of process for separating and purifying of biosynthesis phloretin | |
| CN107594472A (en) | A kind of Suaeda salsa biogenic salt and Betacyanins combined extraction method | |
| CN107217080A (en) | A kind of method that utilization immobilised enzymes prepares jerusalem artichoke FOS | |
| CN102808046A (en) | Method for extracting fructose from apple pomace | |
| CN101531646B (en) | Salting-out method for extracting gibberellin | |
| CN204752588U (en) | Microwave -assisted draws lentinan's equipment | |
| CN109134178A (en) | A method of it is synchronous from watermelon to extract lycopene and citrulling | |
| CN104876986B (en) | A kind of method of anthocyanin in extraction purple sweetpotato | |
| CN102850470A (en) | Method for separating and purifying water-soluble hemicellulose from green tea leaves |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240325 Address after: 313200 No.892, Changhong East Street, Fuxi street, Deqing County, Huzhou City, Zhejiang Province Patentee after: ZHEJIANG JOHNCAN BIOTECHNOLOGY Co.,Ltd. Country or region after: China Address before: The city Zhaohui six districts Chao Wang Road Hangzhou City, Zhejiang province 310014 18 Patentee before: JIANG University OF TECHNOLOGY Country or region before: China |
|
| TR01 | Transfer of patent right |