CN103319618B - The preparation method of active polysaccharide of male agaric mycelium - Google Patents
The preparation method of active polysaccharide of male agaric mycelium Download PDFInfo
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- CN103319618B CN103319618B CN201310247203.6A CN201310247203A CN103319618B CN 103319618 B CN103319618 B CN 103319618B CN 201310247203 A CN201310247203 A CN 201310247203A CN 103319618 B CN103319618 B CN 103319618B
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Abstract
The preparation method that the invention discloses a kind of active polysaccharide of male agaric mycelium, the method for the present invention utilizes wood flour and these cheap substrate of wheat bran to carry out solid fermentation on the one hand, and cost is low, and technology is simple, and general peasant household can carry out, it is easy to promote；On the other hand, adopting the method for the present invention can effectively improve the purity of phellinus igniarius mycelium crude polysaccharides, the phellinus igniarius mycelium crude polysaccharides obtained has good immunocompetence.
Description
Technical field
The present invention relates to edible fungi to cultivate and manufacture field, the preparation method being specifically related to a kind of active polysaccharide of male agaric mycelium.
Background technology
Phellinus baumii (PhellinusbaumiiPilat), is commonly called as Phellinus igniarius (L. ex Fr.) Quel., is the medicinal fungi of a kind of perennial, filemot preciousness, has the laudatory title of " forest gold ".Because its antitumous effect is notable, have become as the focus of research both at home and abroad, fashionable in Japan and Korea S, sell well.
As rare medicinal fungi, the main active of Phellinus igniarius (L. ex Fr.) Quel. is polysaccharide, concentrates on Polyose extraction and anticancer effect research thereof to the research of Phellinus igniarius (L. ex Fr.) Quel. is more at present.Great many of experiments shows, the good antitumous effect that phellinus igniarius mycelium and fruiting body extract have.Owing to the Phellinus igniarius (L. ex Fr.) Quel. sporophore growth cycle is long, expensive rareness, and the mycelial growth cycle is short, therefore extracting active substance from phellinus igniarius mycelium is good developing direction.Extract Phellinus igniarius (L. ex Fr.) Quel. active polysaccharide, it is necessary to the mycelium of enriched；Liquid fermentation needs fermentation tank, and power cost is relatively big, technology relative complex；And solid fermentation relative ease is easy, substrate can adopt the agricultural byproduct that can eat, and can comprehensively utilize.
But, the toxigenic capacity of existing Phellinus igniarius (L. ex Fr.) Quel. is high, and the purity of active polysaccharide is low, have impact on its further application pharmaceutically.
Summary of the invention
The preparation method that it is an object of the invention to provide a kind of active polysaccharide of male agaric mycelium, in order to realize the purpose of the present invention, intends adopting the following technical scheme that
The preparation method that one aspect of the present invention relates to a kind of active polysaccharide of male agaric mycelium, described method comprises the steps:
(1) cultured mycelia: be longer than on PDA inclined-plane by Phellinus igniarius (L. ex Fr.) Quel. strain, growth cycle is within one week, be Phellinus igniarius (L. ex Fr.) Quel. mother's kind；Taking the sawdust medium autoclaving mixed, cooling is followed by Phellinus igniarius (L. ex Fr.) Quel. mother and plants in culture medium, and namely 27-29 DEG C obtain Phellinus igniarius (L. ex Fr.) Quel. original seed in 8-12 days in the cultivation of constant incubator lucifuge；Being turned over by original seed receives in sawdust medium, cultivates 12-18 days amplification culture in mushroom house lucifuge for 27-29 DEG C；The original seed that will be enlarged by cultivating is inoculated on sawdust medium, is placed in 27-29 DEG C of mushroom house and cultivates, growth cycle 25-30 days；Described sawdust medium includes 50-70 weight portion wood flour, 15-25 weight portion wheat bran, 0.5-1.5 weight portion Gypsum Fibrosum and suitable quantity of water；
(3) extraction of mycelium polysaccharides
Removing bacterium bag, smashed to pieces together with compost by cultured mycelium, add distilled water boiling waterbath 1-3h, filtering residue repeats to extract 1-3 time, and united extraction liquid is evaporated to material ratio 1: 8-12 on a rotary evaporator；Take concentrated solution; the centrifugal 20-40min of 8000-12000 × g removes insoluble impurities; add ethanol precipitation more than 20h, precipitation separation, use the ethanol identical with alcohol precipitation concentration that precipitation is washed; then water dissolution is used; with 3200-3800 molecular weight bag filter dialysis 2-4d, remove small-molecule substance, lyophilization phellinus igniarius mycelium crude polysaccharides; optionally, the step that phellinus igniarius mycelium crude polysaccharides is further purified also is included.
In a preferred embodiment of the present invention kind, concentration when described ethanol precipitates is 30%-80%, it is preferred that for 60-80%.
In another preferred embodiment of the present invention kind, the content of described cryodesiccated phellinus igniarius mycelium crude polysaccharides is more than 30%, it is preferable that more than 33%.
In another preferred embodiment of the present invention kind, described phellinus igniarius mycelium crude polysaccharides includes fucose, rhamnose, arabinose, galactose, glucose, xylose and mannose.
Another aspect of the present invention further relates to adopt the preparation-obtained phellinus igniarius mycelium crude polysaccharides of said method.
In another aspect of this invention, further relating to the application of above-mentioned phellinus igniarius mycelium crude polysaccharides, described phellinus igniarius mycelium crude polysaccharides is for improving the immunity of human or animal body.
The method of the present invention utilizes wood flour and these cheap substrate of wheat bran to carry out solid fermentation on the one hand, and cost is low, and technology is simple, and general peasant household can carry out, it is easy to promote；On the other hand, adopting the method for the present invention can effectively improve the purity of phellinus igniarius mycelium crude polysaccharides, the phellinus igniarius mycelium crude polysaccharides obtained has good immunocompetence.
Detailed description of the invention
Embodiment 1
(1) preparation Bag Material culture medium
Configure 59% wood flour, 20% wheat bran, 1% Gypsum Fibrosum, use water mix, keep water content about 65%.Use polypropylene bag (17cm × 33cm × 0.0005cm) charging, every packed sawdust medium wet feed 1kg, 121 DEG C of autoclaving 120min.
(2) female kind, original seed, cultigen and mycelial cultivation
Being longer than on PDA inclined-plane by Phellinus igniarius (L. ex Fr.) Quel. strain, growth cycle is within one week, be Phellinus igniarius (L. ex Fr.) Quel. mother's kind.
Take the conical flask of the sawdust medium dress 500ml mixed to 1/2nd; about make 5 bottles (1 bottle of female kind can make 16 bags of original seeds); autoclaving 120min, cooling is followed by Phellinus igniarius (L. ex Fr.) Quel. mother and plants in culture medium, and namely 28 DEG C obtain Phellinus igniarius (L. ex Fr.) Quel. original seed in 10 days in the cultivation of constant incubator lucifuge.
Being turned over by 5 bottles of original seeds receives in Bag Material culture medium, cultivates 15 days amplification culture in mushroom house lucifuge for 28 DEG C and obtains 80 bag cultivating kinds.Take above-mentioned Phellinus igniarius (L. ex Fr.) Quel. bacteria cultivation kind to be inoculated in the Bag Material culture medium of embodiment 1, be placed in 28 DEG C of mushroom house and cultivate, growth cycle 25-30 days.
(3) mensuration of the extraction of mycelium polysaccharides and total sugar content
Removing bacterium bag, smashed to pieces together with compost by cultured mycelium, add distilled water boiling waterbath 2h, filtering residue repeats to extract 2 times, and united extraction liquid is evaporated to material ratio 1: 10 on a rotary evaporator；Taking concentrated solution, the centrifugal 30min of 10000 × g removes insoluble impurities, adds dehydrated alcohol respectively to ethanol final concentration of 30%, 50%, 70%, and static 24h, 10000 × g is centrifuged 30min.Then respectively by the ethanol cyclic washing precipitation of corresponding ethanol final concentration.Finally washing out precipitation, water-bath Back stroke alcohol with deionized water, with 3500 molecular weight bag filters (MD34, molecular cut off 3500) dialysis 3d, remove small-molecule substance, freeze drier lyophilizing obtains phellinus igniarius mycelium crude polysaccharides.Measuring polyoses content with sulfuric acid-phynol, after the ethanol precipitate with ethanol of 30%, 50%, 70% variable concentrations, dialysis obtains in crude polysaccharides polyoses content respectively 16.10%, and 33.18%, 37.83%.
(4) phellinus igniarius mycelium polysaccharide ion vitro immunization activity research
Phellinus igniarius mycelium crude polysaccharides is originated with in embodiment 4；Measure the antioxidant activity of phellinus igniarius mycelium crude polysaccharides, be substantially carried out stimulated in vitro macrophage RAW264.7 and discharge the test of NO amount.
NO is easily oxidized in vivo, and forms nitrite, and Griess method measures the concentration of culture supernatant nitrite, it is possible to reflection macrophage NO burst size indirectly.
Concrete operation step: take out the cell that ultra cold storage freezer is frozen, 1000rpm is centrifuged 3min, remove frozen stock solution, collect cell, it is evenly distributed to equipped with in the little culture bottle of DMEM complete medium, at 37 DEG C, cultivate when 5%CO2 saturated humidity, regularly change liquid, when cell covers at the bottom of culture bottle bottle 80～90%, with 0.25% trypsin solution digestion of 37 DEG C of heating, Digestive system is in the centrifugal 3min of 1000rpm, collect cell, with colourless 1640 complete mediums by cell dilution to 5 × 105/mL, calculate required hole count, in 96 porocyte culture plates, every hole adds 180 μ L cell diluents, it is subsequently adding 20 μ L testing samples, each sample concentration arranges 3 multiple holes, with LPS (10 μ g/mL) for positive control, PBS is negative control, 96 orifice plates are placed in 37 DEG C, 48h is cultivated when 5%CO2 saturated humidity, then every hole supernatant 100 μ L is drawn, add 50 μ LGriess reagent, place 10min, 543nm measures absorbance.
It is shown that final concentration of 30%, 50%, 70% precipitate with ethanol of the ethanol of 100ug/ml obtains phellinus igniarius mycelium crude polysaccharides stimulating expression of macrophage RAW264.7 discharges NO amount respectively 32.05%, 30.16%, 49.16.So we choose the ethanol final concentration choosing 70% of 70% carries out precipitate with ethanol, it is thus achieved that crude polysaccharides activity better, close to the burst size 52.64% of positive control LPS.
(5) analysis of phellinus igniarius mycelium monosaccharide constituent
Sample pre-treatments: take Phellinus igniarius (L. ex Fr.) Quel. crude polysaccharides sample, about 2mg is accurately weighed in tool plug test tube with ten thousand/balance, the TFA of 2mol/L is added according to the ratio of (3mL: 2mg), 110 DEG C of hydrolysis 4h, cooling, steam instrument low-temperature reduced-pressure with rotation and be evaporated hydrolyzed solution, it is subsequently added into 3mL methanol, evaporated under reduced pressure, repetitive operation 4-5 time, to remove the trifluoroacetic acid of residual, hydrolyzate deionized water is cleaned and is settled to 50mL, applied sample amount is 20 μ L, with monosaccharide standard substance Gal, Glu, Ara, Fuc, Rha, Man, Xyl, Fru, Rib, GluA and GalA is comparison, composition and the mol ratio of monosaccharide is measured by high-efficiency anion chromatograph (HPAEC).
It is shown that the principal monosaccharides constituent of phellinus igniarius mycelium crude polysaccharides is fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, and monosaccharide component ratio is 4.1: 1: 8.8: 11.3: 12.3: 15.1: 6.3.
The above is the preferred embodiments of the present invention; it should be pointed out that, for those skilled in the art, under the premise without departing from principle of the present invention; can also making some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (6)
1. a preparation method for active polysaccharide of male agaric mycelium, described method comprises the steps:
1) cultured mycelia: be longer than on PDA inclined-plane by Phellinus igniarius (L. ex Fr.) Quel. strain, growth cycle is within one week, be Phellinus igniarius (L. ex Fr.) Quel. mother's kind；Taking the sawdust medium autoclaving mixed, cooling is followed by Phellinus igniarius (L. ex Fr.) Quel. mother and plants in culture medium, and namely 27-29 DEG C obtain Phellinus igniarius (L. ex Fr.) Quel. original seed in 8-12 days in the cultivation of constant incubator lucifuge；Being turned over by original seed receives in sawdust medium, cultivates 12-18 days amplification culture in mushroom house lucifuge for 27-29 DEG C；The original seed that will be enlarged by cultivating is inoculated on sawdust medium, is placed in 27-29 DEG C of mushroom house and cultivates, growth cycle 25-30 days；Described sawdust medium includes 50-70 weight portion wood flour, 15-25 weight portion wheat bran, 0.5-1.5 weight portion Gypsum Fibrosum and suitable quantity of water；
2) extraction of mycelium polysaccharides
Removing bacterium bag, smashed to pieces together with compost by cultured mycelium, add distilled water boiling waterbath 1-3h, filtering residue repeats to extract 1-3 time, and united extraction liquid is evaporated to material ratio 1: 8-12 on a rotary evaporator；Take concentrated solution; the centrifugal 20-40min of 8000-12000 × g removes insoluble impurities; add ethanol precipitation more than 20h, precipitation separation, use the ethanol identical with alcohol precipitation concentration that precipitation is washed; then water dissolution is used; with 3200-3800 molecular weight bag filter dialysis 2-4d, remove small-molecule substance, lyophilization phellinus igniarius mycelium crude polysaccharides; optionally, the step that phellinus igniarius mycelium crude polysaccharides is further purified also is included.
2. preparation method according to claim 1, concentration when described ethanol precipitates is 30%-80%.
3. preparation method according to claim 2, described concentration of alcohol is 60-80%, and the content of described cryodesiccated phellinus igniarius mycelium crude polysaccharides is more than 30%.
4. preparation method according to claim 3, described phellinus igniarius mycelium crude polysaccharides includes fucose, rhamnose, arabinose, galactose, glucose, xylose and mannose.
5. the preparation-obtained phellinus igniarius mycelium crude polysaccharides of claim 1-4 any one preparation method.
6. the application of the phellinus igniarius mycelium crude polysaccharides described in claim 5, described phellinus igniarius mycelium crude polysaccharides is for improving the immunity of human or animal body.
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| CN106318826A (en) * | 2016-11-17 | 2017-01-11 | 青岛农业大学 | Preparation method for phellinus linteus healthcare wine |
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| CN104397699A (en) * | 2014-12-04 | 2015-03-11 | 郭士平 | Health care product for conditioning syndromes before and after menstruation and dysmenorrhea and preparation technology of health care product |
| CN104829742A (en) * | 2015-05-14 | 2015-08-12 | 江苏大学 | Phellinus linteus polysaccharide separation and purification method |
| CN104910289A (en) * | 2015-06-16 | 2015-09-16 | 江苏大学 | Method for continuously preparing phellinus igniarius mycelium polysaccharide |
| CN105542030A (en) * | 2015-12-17 | 2016-05-04 | 黑龙江众生生物工程有限公司 | Method for extracting water-soluble beta-glucan from Phellinus sporocarp |
| CN106010931A (en) * | 2016-06-30 | 2016-10-12 | 余林岚 | Preparation method of phellinus igniarius composite health vinegar |
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| CN110627917A (en) * | 2019-09-27 | 2019-12-31 | 苏州顺泰元虫草生物科技有限公司 | Method for extracting phellinus igniarius mycelium polysaccharide |
| CN112899109A (en) * | 2021-04-23 | 2021-06-04 | 长江师范学院 | Preparation method of white peach mulberry yellow wine |
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| CN1720785A (en) * | 2004-08-12 | 2006-01-18 | 陈全勇 | An artificial high-yield cultivation method of male agaric and application of fruit body thereof |
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| CN1720785A (en) * | 2004-08-12 | 2006-01-18 | 陈全勇 | An artificial high-yield cultivation method of male agaric and application of fruit body thereof |
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| CN106318826A (en) * | 2016-11-17 | 2017-01-11 | 青岛农业大学 | Preparation method for phellinus linteus healthcare wine |
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