CN101735329A - Pholiota adiosapose polysaccharide and preparation method thereof - Google Patents
Pholiota adiosapose polysaccharide and preparation method thereof Download PDFInfo
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- CN101735329A CN101735329A CN200810226056A CN200810226056A CN101735329A CN 101735329 A CN101735329 A CN 101735329A CN 200810226056 A CN200810226056 A CN 200810226056A CN 200810226056 A CN200810226056 A CN 200810226056A CN 101735329 A CN101735329 A CN 101735329A
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Abstract
The invention provides pholiota adiosapose polysaccharide, which is extracted from a pholiota adiosapose mycelium or fruiting body and prepared by purification, is a heteropolysaccharide comprising four monosaccharides, namely glucose, fructose, mannose and galactose, can improve body immunity and has functions of preventing cancers, resisting cancers, resisting ageing and the like, and can be used in combination with ganoderma lucidum polysaccharide, coriolus versicolor polysaccharide and the like. The invention also provides a method for preparing the pholiota adiosapose polysaccharide. The method is an improvement on water extract method and makes the yield of the polysaccharide reach over 23 percent, the content of the polysaccharide reach 55 percent, and the growth amount of the mycelium after the crude polysaccharide is purified up to 93.67 percent. Combined with a mycelium liquid culture technique, the method can increase the growth amount of the mycelium by over 10 percent so as to decrease the production cost, improve the production efficiency and in particular greatly improve bioactivity of polysaccharide products.
Description
Technical field
The present invention relates to a kind of pholiota adiosapose polysaccharide, it is to extract the polysaccharide that obtains from yellow umbrella, and the purified product of this polysaccharide can improve body immunity, the invention still further relates to the preparation method of this polysaccharide.
Background technology
Yellow umbrella formal name used at school Pholiota adiposa (Fr.) Qu é l. belongs to Mycophyta Eumycota on taxonomy, Basidiomycotina Basidiomycotina, Hymenomycetes Hymenomycetes, Agaricales Agaricales, the Strophariaceae of Stropharia rugoso-annulata section, ring rust umbrella belongs to GenusPholipta, is a kind of food and medicament dual-purpose bacterium." Chinese macro fungi " (fourth of the twelve Earthly Branches morning mist, 2000)
(1)Put down in writing yellow umbrella " there is one deck cement on the sporophore surface, can get the polysaccharide body through salt solution, warm water, alkaline solution or organic solvent extraction, and this polysaccharide body reaches 80%～90% to the inhibiting rate of small white mouse sarcoma 180 and ehrlich carcinoma ".Su Yanyou (2004)
(2)Utilization orthogonal experiment is tentatively studied pholiota adiosapose polysaccharide extraction process and the external evoked scavenger cell effect of relevant flooding polysaccharide, its result of study shows, pholiota adiosapose polysaccharide has the effect of immunostimulant, activating macrophage effectively, by strengthen cytokine secretion, strengthen NO the generation level, to strengthen its phagocytic function and external killing activity etc. multiple by way of regulating immunity system.Wang Qian (2006)
(3)Learn that Deng by mouse experiment yellow umbrella fermented product extract has the effect of auxiliary adjustment triglyceride level; The technology of aspects such as present wild domestication, biological characteristics and artificial cultivation technique to yellow umbrella, liquid culture also obtains certain progress
(4) (5)But the yellow destroying angel production cycle is long, not high, the unstable product quality of output, it is low that pholiota adiosapose polysaccharide extracts yield, purity is not high, at present the purifying of yellow umbrella extraction polysaccharide and the research of higher structure thereof be there is no report both at home and abroad, and the physiologically active of pholiota adiosapose polysaccharide is not seen yet behind the purifying report.
The present invention by improving extraction process, reaches multistage purification process by yellow umbrella liquid culture mycelium is carried out the polysaccharide extraction and purification, obtains the high purity polysaccharide.
Summary of the invention
The purpose of this invention is to provide a kind of pholiota adiosapose polysaccharide, it can improve the human immunological competence.
Another object of the present invention is to provide the method for preparing pholiota adiosapose polysaccharide.
Pholiota adiosapose polysaccharide of the present invention is to be raw material with yellow umbrella (mycelium or sporophore), obtains by extracting purifying, and it is a kind of a kind of mixed polysaccharide of being made up of glucose, fructose, seminose and four kinds of monose of semi-lactosi.
Pholiota adiosapose polysaccharide of the present invention prepares by the following method:
1. the extraction of pholiota adiosapose polysaccharide
1. the material pre-treatment is crushed to 120～160 orders with yellow umbrella (mycelium or sporophore) freeze-drying, and preferred powder is broken to 140 orders; Material-water ratio 1: 20～30, ultrasonication 5～15min, power 600～1000W, preferably treatment power 600W, treatment time 10min.
2. lixiviate refluxing extraction 1～4 hours is extracted 60～90 ℃ of temperature, gets vat liquor;
3. alcohol is analysed the dehydrated alcohol that adds 3～5 times of amounts in vat liquor, and alcohol was analysed 8～16 hours.
4. concentrated alcohol is analysed the centrifugal 15～25min of back 3500～5000rpm and is abandoned supernatant liquor, gets throw out, uses dehydrated alcohol (1 time), acetone (2 times) washing precipitation successively.
5. vacuum lyophilization gets yellow umbrella Crude polysaccharides.
2. the purifying of pholiota adiosapose polysaccharide
The pholiota adiosapose polysaccharide purifying is the process with floating preteins in the pholiota adiosapose polysaccharide and pigment removal, free proteic removal adopts iso-electric point in conjunction with the seveg method in the pholiota adiosapose polysaccharide, at first yellow umbrella Crude polysaccharides is made 1～5% polysaccharide soln, drip an amount of strong aqua and make its pH value reach 8～10, leave standstill behind 10～15min that centrifugal 10～15min removes albumen precipitation under 4500～5000rpm.With 1～3 times of polysaccharide soln dilution, (propyl carbinol: chloroform=1: 4) behind the thermal agitation 25-30min, centrifugal 10～15min under the 4500-5000rpm gets supernatant liquor repetition seveg and removes the albumen operation once to add isopyknic seveg reagent more then.Polysaccharide soln is concentrated postlyophilization.
Get that polysaccharide is dissolved in distillation after 20～28 ℃ of following purifying of the further room temperature of ion exchange column of being made up of D152 and D301T series connection, elutriant is a distilled water, behind the purifying, again through Sephadex G-100 gel filtration chromatography purifying (20～28 ℃ of room temperatures), as elutriant, can obtain the high purity polysaccharide product with distilled water.
More than extract in the purge process by lyophilize and can remove organic solvent in the polysaccharide soln that to remove fully in the concentration process, thereby reduce the destruction that organic solvent removes pillar in the subsequent step, simultaneously, the step be can calculate and the yield of polysaccharide and purity removed in the protein process.
The polysaccharide product that obtains according to the method described above adopts the phenol sulfuric acid process to measure its polysaccharide content and can reach 93.67%, adopts Folin-phenol method to measure albumen and does not detect.
The present invention also provides a kind of pholiota adiposa mycelium liquid cultivating method, can improve mycelial increment by this method, thereby enhance productivity, and reduces cost.
Pholiota adiposa mycelium liquid cultivating method provided by the invention is as follows:
1. liquid culture based formulas: glucose 15～25g/L, yeast soak powder 6～10g/L, vitamins B
1200～400 μ g/L, Mg
2SO
40.3～0.5g/L, KH
2PO
40.8～1.2g/L, K
2HPO
40.8～1.2g/L, water surplus, autoclaving is standby;
2. strain cultivation: by the aseptic technique method, get female mycelium of planting, by inoculum size 1～10% (V/V), in the liquid nutrient medium after the switching sterilization, culture temperature: at 24～26 ℃; Under shaking speed 120r/min～140r/min condition, incubation time 10～14 days;
3. fermentation culture: with the seed culture bacterial classification, the aseptic technique method, (V/V) is seeded in the fermentor tank by inoculum size 5～10%, fermention medium is: glucose 20～40g/L, corn steep liquor 15～25g/L, potassium primary phosphate 1.5～2.5g/L, pH value 6.5～7.0,24～26 ℃ of controlled temperature, mixing speed 150～180r/min, air flow are 0.8～2 (V/Vmin), fermentation culture 7～10 days.
Wherein step is got female the kind 2. preferably by the aseptic technique method with diameter 0.5cm punch tool, gets inoculum size and be 3 solid spawn, in the liquid nutrient medium after switching is sterilized (inoculum size is about 3%).Culture temperature: at 25 ℃; Under shaking speed 120r/min～140r/min condition, incubation time 12 days.
The 3. preferred fermentation culture of step wherein: with the seed culture bacterial classification, the aseptic technique method is seeded in the fermentor tank, inoculum size is 8% (V/V), and fermention medium is: glucose 30g/L, corn steep liquor 20g/L, potassium primary phosphate 2g/L, pH value 6.5～7.0, controlled temperature are 25 ℃, and agitator speed is 150～180r/min, air flow is 0.8～2 (V/Vmin), fermentation culture 7～10 days.
In this application, term " air flow " is meant that the volume of air of passing through the unit volume nutrient solution in the per minute is than (V/Vmin).As: interior dress 3m
3The fermentor tank of nutrient solution is if per minute feeds 1.5m
3Sterile air, then ventilation is than being 3: 1.5=1: 0.5, the abbreviation air flow is 0.5 (V/Vmin).
The percentage sign that relates among the application " % " if do not specify, is meant mass percent; But the per-cent of solution except as otherwise herein provided, is meant and contains the some grams of solute among the solution 100ml; Per-cent between the liquid means the ratio of capacity in the time of 20 ℃.
The present invention provides a kind of novel healthy food--pholiota adiosapose polysaccharide for the mankind, and it can improve body immunity, has anti-cancer, anticancer, the anti-ageing effect of waiting for a long time.Therefore, pholiota adiosapose polysaccharide of the present invention can be able to be sent out the medicine that becomes to have therapeutic action, functional food perhaps with health role.In addition, the yellow umbrella of the present invention also can be used with ganoderan, krestin etc.It is low that the present invention has overcome the pholiota adiosapose polysaccharide extraction yield, content low (general Crude polysaccharides content is below 50%), difficult problems such as polysaccharide component complexity, a kind of extracting and purifying method of pholiota adiosapose polysaccharide is provided, polysaccharide yield reaches more than 60%, the purity of polysaccharide is up to 93.67%, ligative hyphae body fluid body culture technique in addition, can improve the mycelial growth amount more than 10%, thereby reduce production costs, enhance productivity, particularly improved the biological action of polysaccharide product greatly, for example anticancer, do tumour, remove interior free yl etc.
Description of drawings
Fig. 1 is the high pressure liquid chromatography figure of polysaccharide PP-HMW;
Fig. 2 is the ultraviolet scintigram of pholiota adiosapose polysaccharide PP-HMW.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
1. liquid culture based formulas: glucose 20g/L, yeast soak powder 8g/L, vitamins B
1300 μ g/L, Mg
2SO
40.4g/L, KH
2PO
41g/L, K
2HPO
41g/L, pH 6, and high-temperature sterilization is standby.
2. seed culture: by the aseptic technique method, get female the kind, get inoculum size and be 3 solid spawn, in the liquid nutrient medium after switching is sterilized (inoculum size about 3%) with diameter 0.5cm punch tool.Culture temperature: at 25 ℃; Under shaking speed 120r/min～140r/min condition, incubation time 12 days.
3. fermentation culture: with the seed culture bacterial classification, the aseptic technique method is seeded in the fermentor tank, inoculum size is 8% (V/V), fermention medium is: glucose 30g/L, corn steep liquor 20g/L, potassium primary phosphate 2g/L, pH value 6.5～7.0, controlled temperature is 25 ℃, and agitator speed is 150～180r/min, and air flow is 0.8 (V/Vmin).After the fermentation ends, the pholiota adiposa mycelium dry weight in every liter of solution reaches 13.6g.
1. liquid culture based formulas: glucose 15g/L, yeast soak powder 10g/L, vitamins B
1200 μ g/L, Mg
2SO
40.5g/L, KH
2PO
40.8g/L, K
2HPO
41.2g/L pH 6, high-temperature sterilization is standby.
2. seed culture: by the aseptic technique method, get female the kind, get inoculum size and be 5 solid spawn, in the liquid nutrient medium after switching is sterilized (inoculum size about 6%) with diameter 0.5cm punch tool.Culture temperature: at 25 ℃; Under shaking speed 120r/min～140r/min condition, incubation time 10 days.
3. fermentation culture: with the seed culture bacterial classification, the aseptic technique method is seeded in the fermentor tank, inoculum size is 10% (V/V), fermention medium is: glucose 40g/L, corn steep liquor 25g/L, potassium primary phosphate 2.5g/L, pH value 6.5, controlled temperature is 25 ℃, and agitator speed is 150～180r/min, and air flow is 1.5 (V/Vmin).After the fermentation ends, the pholiota adiposa mycelium dry weight in every liter of solution reaches 14.5g.
Embodiment 3 pholiota adiposa mycelium liquid culture
1. liquid culture based formulas: glucose 25g/L, yeast soak powder 6g/L, vitamins B
1400 μ g/L, Mg
2SO
40.3g/L, KH
2PO
40.1.2g/L, K
2HPO
40.8g/L pH 6, high-temperature sterilization is standby.
2. seed culture: by the aseptic technique method, get female the kind, get inoculum size and be 2 solid spawn, in the liquid nutrient medium after switching is sterilized (inoculum size about 2%) with diameter 0.5cm punch tool.Culture temperature: at 25 ℃; Under shaking speed 120r/min～140r/min condition, incubation time 16 days.
3. fermentation culture: with the seed culture bacterial classification, the aseptic technique method is seeded in the fermentor tank, inoculum size is 5% (V/V), fermention medium is: glucose 30g/L, corn steep liquor 15g/L, potassium primary phosphate 1.5g/L, pH value 6.5, controlled temperature is 25 ℃, and agitator speed is 150～180r/min, and air flow is 2 (V/Vmin).After the fermentation ends, the pholiota adiposa mycelium dry weight in every liter of solution reaches 13.1g.
Adopt generation material slaking cultivation planting type, autumn culture should be at 1～February system cultivating bag in spring, 3～May fruiting.Autumn is at 8～September system cultivating bag, 10～December fruiting.Autumn culture is few than the spring cultivation disease and pest, and the fruiting quality is higher.Yellow umbrella cultured mushroom room can be selected plastic greenhouse or indoor layer frame fruiting mode for use; Also can the factory culture pattern, can under the temperature control condition, carry out multiple batches of artificial culture of anniversary.
The cultivating in bag of yellow umbrella has 6 schedule of operation: a culture medium spice → pack sterilization → aseptic inoculation → bacterium cultivation → management of producing mushroom → processing of gathering.
Culturing raw material: major ingredient wood chip, cotton seed hulls, auxiliary material wheat bran, terra alba.The artificial culture culture medium prescription is as follows:
1. cotton seed hull 88%, wheat bran 10%, gypsum 1%, calcium superphosphate 1%.
2. cotton seed hull 68%, weed tree sawdust 20%, wheat bran 10%, gypsum 1%, calcium superphosphate 1%.
3. cotton seed hull 78%, corn cob meal 10%, wheat bran 10%, gypsum 1%, calcium superphosphate 1%.
Substratum preparation cotton seed hull will add water-water reactor by 1: 1 before use and put 8～12h, helps the decomposition of cotton seed hulls suction and part nutrition like this, thereby guarantees the high yield of yellow umbrella.The above-mentioned cotton seed hulls of banking up is added auxiliary material by each prescription and mix thoroughly, add suitable quantity of water again and will expect that the culture material water content transfers to 60%～65%, pH naturally.
Pack: adopt polyethylene or the polypropylene plastics pocket of 17cm * 33cm * 0.04cm, the heavily about 0.45kg～0.55kg of every packed siccative.Material wants appropriateness to compress, and then sack is sealed with the collar, and the collar is ventilated, and can make to send out bacterium time shortening 5～10 days.
Sterilization: high pressure or normal-pressure sterilization.Normal-pressure sterilization requires to cultivate pocket and is positioned in the Autoclave, is heated to 100 ℃, keeps 10 hours, ceases fire stewing 5 hours～6 hours, then bag is taken out cooling inoculation.Autoclaving is used the large vol high-pressure sterilizing pot, and pocket also layering is placed, and 1.2kg/cm keep-ups pressure
2Sterilization 2h, when naturally cooling to pressure then and be zero, the lid that boils takes out cooling with pocket.
Inoculation: undertaken by the aseptic technique program.
Mycelium culture: after the inoculation pocket direct code is put in the culturing rack of clean culturing room, culturing room's temperature should remain on 23 ℃～25 ℃.Send out about about 30～35 days of bacterium time.When the bacterium bag becomes tawny fully, can move into mushroom producing room, carry out management of producing mushroom.
Management of producing mushroom: the bacterium bag is taken off the collar after moving into mushroom producing room, and sack is screwed two circles, and the bacterium bag is reduced and the air contact area as far as possible, helps the bacterium bag and keeps moisture, prevents that surperficial mycoderma drying from hardening.Enter after the mushroom producing room 6～10 days, original hase begins differentiation, and can open the bacterium bag fully this moment.Management of producing mushroom mainly is the control of humidity, concrete measure: increase relative air humidity: according to the needs of bacterium bag different times to moisture, mainly can be divided into three different stepss and manage 1..Before the fs original hase differentiation, adjust between the fruiting indoor air relative humidity 80%～90%, break up to stimulate original hase, if but humidity is excessive, and cause green mold to infect easily; Subordinate phase small mushroom bud vegetative period, this moment, relative air humidity was difficult for excessively, otherwise can cause little mushroom to be rotted, and reduced output, humidity should be controlled between 70%～85%; Yellow umbrella fast growing period of phase III is adjusted relative air humidity between 85%～95%, grows to the needs of moisture fast to satisfy yellow umbrella, helps improving the yield and quality of yellow umbrella simultaneously.The mode that humidification can adopt humidifier atomizing and ground to water and combine.2. temperature: yellow umbrella be a kind of in the low temperature modification edible mushrooms, be suitable for the fruiting in spring and autumn, temperature should maintain 15～20 ℃ in the yellow destroying angel growth and development stage mushroom room, therefore to calculate the time in difference cultivation area, guarantee the fruiting temperature, as have ready conditions also and can increase cooling intensification equipment, but can increase cost in mushroom producing room.3. suitably ventilation: ventilate every day 1～2 time, each about 1～2 hour, carry out in conjunction with weather condition, it is warm early, ventilate to evening, and sky cold is taked to ventilate when noon, temperature was high, constantly to replenish fresh air, eliminating CO
24. certain illuminance in the maintenance mushroom producing room, yellow umbrella can not normal developments under dark fully condition, and mushroom producing room should have the illumination about 300～800 luxs, and the small character that can see clearly on the newspaper with normal people's eyesight is passable at ordinary times.5. keep the fruiting indoor cleaning, regularly clean health in the mushroom producing room, to reduce bacterium rod pollution rate.
Gather: yellow umbrella occurs beginning can plucking through 7～10 days approximately from original hase under the management of producing mushroom of strictness.Standard of plucking is: not regrowth of stem, cap have open and flat trend, and should in time pluck this moment, otherwise parachute-opening reduces commodity value.Pluck the back mycelium stimulation, sack is tightened, enter mycelia decubation, reduce the mushroom producing room relative air humidity, be controlled at 60%～80%.Treat that mycelia recovers back repetition aforesaid operations and carries out fruiting, generally can adopt 3～4 damp mushrooms, the biology transformation efficiency reaches 100%～120%.
1. pholiota adiosapose polysaccharide extracts
1. the material pre-treatment is crushed to 140 orders with the mycelium freeze-drying that embodiment 1 makes, and material-water ratio 1: 30, ultrasonic power 600W handle 10min;
2. lixiviate is put into Backflow bottle with pretreated mycelium, and 3 hours extraction times, 75 ℃ of extraction temperature get the polysaccharide vat liquor during lixiviate;
3. alcohol is analysed the dehydrated alcohol that adds 3.5 times of amounts in the polysaccharide vat liquor, and alcohol was analysed 12 hours;
4. concentrated alcohol is analysed the centrifugal 10min of back 4500rpm and is abandoned supernatant liquor, gets throw out, uses dehydrated alcohol (1 time), acetone (2 times) washing precipitation successively.
5. vacuum lyophilization gets the mycelium Crude polysaccharides.
By said extracted method, yellow umbrella Crude polysaccharides extraction rate reached to 23.41%, polysaccharide content reaches 55.69%.
2. pholiota adiosapose polysaccharide purifying
The above-mentioned polysaccharide that makes is prepared into 3% polysaccharide soln with distilled water, drips an amount of strong aqua and make its pH value reach 10, leave standstill behind the 10min that centrifugal 10min removes albumen precipitation under 4500rpm.With one times of polysaccharide soln dilution, (propyl carbinol: chloroform=1: 4) behind the thermal agitation 30min, centrifugal 10min under the 4500rpm gets the supernatant liquor repetitive operation once to add isopyknic seveg reagent more then.Polysaccharide soln is concentrated postlyophilization.
Get that polysaccharide is dissolved in an amount of distilled water after 20～28 ℃ of following purifying of the further room temperature of ion exchange column of forming via D152 and D301T series connection, elutriant is a distilled water, behind the purifying, again through Sephadex G-100 gel filtration chromatography purifying (20～28 ℃ of room temperatures), as elutriant, can obtain the high purity polysaccharide product with distilled water.Polysaccharide content reaches 93.67% after testing.
3, purity of polysaccharide is identified:
(1) adopt high pressure lipuid chromatography (HPLC) (HPLC) that the holosaccharide sample that aforesaid method obtains is identified that the result is as Fig. 1. shown in.High pressure liquid chromatography figure is single symmetrical peak, illustrates that gained pholiota adiosapose polysaccharide component is the homogeneous component.
(2) UV scanning result: as shown in Figure 2, do not find the nucleic acid absorption peak (260nm～290nm) and protein absorption peak (and 250～300nm), prove free nucleic acid and albumen in the pholiota adiosapose polysaccharide solution.
4. packing is preserved: after dried holosaccharide is ground into fine powder, seals preservation in the Aluminium Foil Package of packing into the pack or carry out product processing.
1. pholiota adiosapose polysaccharide extracts
1. the material pre-treatment is crushed to 120 orders with the mycelium freeze-drying that embodiment 1 makes, and material-water ratio 1: 30, ultrasonic power 1000W handle 15min;
2. lixiviate is put into the Backflow bottle hot water extraction with pretreated mycelium, and 2.5 hours extraction times, 90 ℃ of extraction temperature get the polysaccharide vat liquor;
3. alcohol is analysed the dehydrated alcohol that adds 3 times of amounts in the polysaccharide vat liquor, and alcohol was analysed 12 hours;
4. concentrated alcohol is analysed the centrifugal 25min of back 3500rpm and is abandoned supernatant liquor, gets throw out, uses dehydrated alcohol (1 time), acetone (2 times) washing precipitation successively.
5. vacuum lyophilization gets the mycelium Crude polysaccharides.
By said extracted method, yellow umbrella Crude polysaccharides extraction rate reached to 21.32%, polysaccharide content reaches 53.42%.
2. pholiota adiosapose polysaccharide purifying
The above-mentioned polysaccharide that makes is prepared into 5% polysaccharide soln with distilled water, drips an amount of strong aqua and make its pH value reach 9, leave standstill behind the 15min that centrifugal 15min removes albumen precipitation under 4500rpm.With one times of polysaccharide soln dilution, (propyl carbinol: chloroform=1: 4) behind the thermal agitation 30min, centrifugal 10min under the 4500rpm gets the supernatant liquor repetitive operation once to add isopyknic seveg reagent more then.Polysaccharide soln is concentrated postlyophilization.
Get that polysaccharide is dissolved in an amount of distilled water after 20～28 ℃ of following purifying of the further room temperature of ion exchange column of forming via D152 and D301T series connection, elutriant is a distilled water, behind the purifying, again through Sephadex G-100 gel filtration chromatography purifying (20～28 ℃ of room temperatures), as elutriant, can obtain the high purity polysaccharide product with distilled water.Polysaccharide content reaches 91.82% after testing.
3. packing is preserved: after dried holosaccharide is ground into fine powder, seals preservation in the Aluminium Foil Package of packing into the pack or carry out product processing.
Embodiment 7 pholiota adiosapose polysaccharide extraction and purifications
1. pholiota adiosapose polysaccharide extracts
1. the material pre-treatment is crushed to 160 orders with yellow destroying angel freeze-drying, and material-water ratio 1: 20, ultrasonic power 600W handle 5min;
2. lixiviate is put into the Backflow bottle hot water extraction with pretreated sporophore, and 3 hours extraction times, 80 ℃ of extraction temperature get the polysaccharide vat liquor;
3. alcohol is analysed the dehydrated alcohol that adds 5 times of amounts in the polysaccharide vat liquor, and alcohol was analysed 8 hours;
4. concentrated alcohol is analysed the centrifugal 10min of back 4500rpm and is abandoned supernatant liquor, gets throw out, uses dehydrated alcohol (1 time), acetone (2 times) washing precipitation successively.
5. vacuum lyophilization gets the sporophore Crude polysaccharides.
By said extracted method, yellow umbrella Crude polysaccharides extraction rate reached to 9.39%, polysaccharide content reaches 41.89%.
2. pholiota adiosapose polysaccharide purifying
The above-mentioned polysaccharide that makes is prepared into 2% polysaccharide soln with distilled water, drips an amount of strong aqua and make its pH value reach 10, leave standstill behind the 10min that centrifugal 10min removes albumen precipitation under 4500rpm.With one times of polysaccharide soln dilution, (propyl carbinol: chloroform=1: 4) behind the thermal agitation 30min, centrifugal 10min under the 4500rpm gets the supernatant liquor repetitive operation once to add isopyknic seveg reagent more then.Polysaccharide soln is concentrated postlyophilization.
Get that polysaccharide is dissolved in an amount of distilled water after 20～28 ℃ of following purifying of the further room temperature of ion exchange column of forming by D152 and D301T series connection, elutriant is a distilled water, behind the purifying, again through Sephadex G-100 gel filtration chromatography purifying (20～28 ℃ of room temperatures), as elutriant, can obtain the high purity polysaccharide product with distilled water.Detect polysaccharide content through the phenolsulfuric acid method and reach 89.69%.
3. packing is preserved: after dried holosaccharide is ground into fine powder, seals preservation in the Aluminium Foil Package of packing into the pack or carry out product processing.
The active check of embodiment 8 pholiota adiosapose polysaccharides
(1) pholiota adiosapose polysaccharide promotes the mice spleen lymphocytes proliferation effect
Test materials: a. test sample: pholiota adiposa mycelium polysaccharide (sample A); (sample a) for the Pholiota adiposa fermentation liquid polysaccharide
B. test mice: the C57 mouse inbred lines, body weight 20 grams are purchased in Institute of Experimental Animals, Chinese Academy of Medical Sciences.B. main experiment reagent and instrument: new-born calf serum, GIBCO product, Cat No.16010-159.Lot No.600202; RPMI 1640 substratum, GIBCO product, Cat No.31800-022.LotNo.1165062; 96 well culture plates, Costar product, the place of production U.S..ATP bioluminescence detection kit is Beijing gold amethyst biological medicine technology company limited product, Beijing, the place of production; The microwell plate fluorescence analyser, BMP9504, Binsong Photon Technology Co., Ltd. Beijing's product; CO2gas incubator, Forma 3111, the place of production U.S.; Whizzer, OLYMPUS OPTICAL CO.LTD, place of production Japan.
Test method: the preparation of a. mouse spleen lymphocyte suspension: mouse sacrificed by decapitation, the aseptic spleen of getting.Spleen placed fill an amount of aseptic Hank`s liquid plate, gently spleen is ground, make the individual cells suspension with tweezers.Filter through 200 eye mesh screens, wash 2 times each centrifugal 10min (1000r/min) with Hank`s liquid.Cell suspension in 10% NBS-RPMI-1640, is expected blue dyeing counting with platform, and cytoactive is 98%, and adjusting cell concn is 2 * 106/mL.B. cell cultures and polysaccharide concentration are set: under aseptic condition, polysaccharide is dissolved with complete RPMI1640 nutrient solution, and the degerming of polysaccharide soln filter, redilution is to desired concn.Every kind of polysaccharide is established 6 concentration, and concentration is respectively 25,50,100,200,400 and 600 μ g/mL, and every hole adds polysaccharide soln 100 μ L; Blank group (CK) adds 100 μ L nutrient solutions, all establishes 3 repeating holes.In 96 porocyte culture plates, every hole adds 100 μ l splenocyte suspensions, adds for test agent, in 37 ℃, CO then
2Concentration is 5%, relative air humidity is 95% CO
2Cultivated 4 days in the incubator, take out the every hole of culture plate and add ATP extracting solution 50 μ L, place 5min under the room temperature, 50 μ l are got in every hole, join to measure in the blank, and every then hole adds 50 μ L luciferases/fluorescein and measures liquid, measures absorption value on fluor tester.C. detect and data analysis: thus relatively determine the cultivation effect of sample according to experimental group light absorption value and blank group to splenocyte.SI (stimulation index)=sample well light absorption value/blank well light absorption value.The SI value shows that greater than 1 sample has proliferation function to splenocyte, and SI is big more, and its value-added effect is good more.Experimental result adopts the t check, and when P≤0.05, each group relatively has statistical significance with control group (CK).
Test-results: as can be seen from table 1.: when pholiota adiposa mycelium polysaccharide (sample A) is 25 μ g/mL～400 μ g/mL at dosage, have proliferation function to mouse spleen lymphocyte, and with control group significant difference (P＜0.05).(sample a) each dosage group has proliferation function to mouse spleen lymphocyte to the Pholiota adiposa fermentation liquid polysaccharide, and along with its proliferation function of dosage strengthens, each dosage is compared significant difference with control group.Compare with same dose, when concentration was 50～600 μ g/mL, the proliferation function of Pholiota adiposa fermentation liquid polysaccharide was significantly greater than mycelium polysaccharides.
Table 1 pholiota adiosapose polysaccharide is to the proliferation function of mouse spleen lymphocyte
(2) pholiota adiosapose polysaccharide is to the effect of Lvwis lung cancer solid tumor resistance
Test materials: A. pholiota adiposa mycelium polysaccharide (embodiment 5 makes); B. for the examination mouse: male for the C57 mouse inbred lines, 18～22g, Li Keaida biotechnology consultative centre provides by Beijing, conformity certification scxk2004-0001.C. for the strain of examination knurl: the strain of Lewis lung cancer knurl is available from zooscopy institute of the Chinese Academy of Medical Sciences.Positive control drug: endoxan, SHANXI POWERDONE PHARMACEUTICAL.,LTD produces, lot number: 20061102.
Test method:
A. animal grouping
Laboratory animal is divided into 5 groups at random, blank group CK1, positive controls CK2, the basic, normal, high dosage group of polysaccharide.Every group 10.
CK1---physiological saline control group is with volume physiological saline;
CK2---positive controls, dosage are 30mg/kg;
A1---pholiota adiosapose polysaccharide low dose group, dosage 50mg/kg;
A2---dosage group in the pholiota adiosapose polysaccharide, dosage 100mg/kg;
A3---pholiota adiosapose polysaccharide high dose group, dosage 200mg/kg;
B. inoculation
To inoculate well-grown C57 tumor-bearing mice dislocation 7 day after tomorrow of Lewis lung cancer and put to death, and choose well-grown tumor tissue, and make cell suspension, in the oxter inoculation, 0.2ml/ only (5 * 10
6Individual).
C. dosage regimen: 8 groups of mouse are divided into behind inoculation tumor cell suspension 24h and begin gastric infusion, once a day, successive administration 9 days, each capacity is 0.2ml.The blank group gives equivalent physiological saline every day.
D. observation index: mouse is put to death in cervical vertebra dislocation in the 10th day, strips knurl piece, spleen, thymus gland, uses scales/electronic balance weighing.Carry out evaluating drug effect with following formula calculating tumor control rate, spleen index, thymus index.
Tumour inhibiting rate=(C-T)/C * 100%, C: be the average knurl of blank group heavy (mg), T: be the average knurl of experimental group heavy (mg)
Thymus gland or index and spleen index=thymus gland (mg) or spleen (mg) quality/body weight (g)
Data analysis: data with mean number ± standard deviation (expression of x ± s),, each group compares data with negative control group respectively and analyzes with SPSS12.0 software.P＝0.05。
Test-results:
A. as can be seen from Table 2, low, in, the high density tumour inhibiting rate is respectively 54.7%, 34.7%, and 16.5%.The low dose group tumour inhibiting rate is the highest, and test group and physiological saline control group and the equal difference of positive controls are remarkable.
The influence that table 2 cap mycelium polysaccharides is heavy to the Lewis lung cancer mouse tumor
Annotate: ▲ expression is remarkable with physiological saline control group comparing difference, and △ represents with the positive controls comparing difference remarkable.
B. aspect thymus index, positive control (CK2) is very low, only is 0.39 ± 0.24, and all there were significant differences for positive controls and polysaccharide group and physiological saline group; Each dosage group of polysaccharide is compared with blank, and equal difference not significantly (table 3) between the polysaccharide group.
Table 3 pholiota adiposa mycelium polysaccharide is to the influence of Lewis lung cancer mouse thymus
Annotate: ▲ expression is remarkable with physiological saline control group comparing difference, and △ represents with the positive controls comparing difference remarkable.
C. aspect index and spleen index, (CK2) is lower for positive control, only is 2.06 ± 0.55, and all there were significant differences for positive controls and polysaccharide group and physiological saline group; Each dosage group of polysaccharide is compared with blank, and equal difference not significantly (table 4) between the polysaccharide group.
Table 4 pholiota adiposa mycelium polysaccharide is to the influence of Lewis lung cancer mouse spleen
Annotate: ▲ expression is remarkable with physiological saline control group comparing difference, and △ represents with the positive controls comparing difference remarkable.
(3) immunoregulation effect of pholiota adiosapose polysaccharide
Test materials:
A. laboratory animal: the inbred lines that the Shandong Experimental Animal Center provides, 18-22 gram, male white mouse, test is handled totally 24 groups, 10 of every group of tests.
B. reagent and instrument
Physiological saline, purpurum bromocresolis indicator, methyl alcohol, Giemsa dyestuff, KH
2PO
4, N
AH
2PO
412H
2O, DMPO (5.5-dimethyl-1-pyrroline-1-oxide), xanthine, XOD are U.S. Sigma company product; Diethylenetriamine pentaacetic acid, hydrogen peroxide, ferrous sulfate amine, Beijing chemical reagents corporation chemically pure reagent.SRBC: the sheep jugular vein is got blood, and sheep blood is put into the sterilization Erlenmeyer flask of granulated glass sphere, shakes towards a direction, shakes defiber, puts into 4 ℃ of refrigerators and preserves standby.Giemsa dye liquor: a. gets Giemsa dyestuff 0.5g, neutral glycerine 33mL, methyl alcohol 333mL.Elder generation puts the Giemsa dyestuff in the cleaning mortar body, behind the glycerol adding, grinds a moment, pours in the brown bottle, places 55～60 ℃ of water baths interior 2 hours, constantly shakes up, and shakes up at adding methyl alcohol, preserves standby.B. dilute Ji's nurse Sa Albert'stain Albert liquid---the damping fluid with pH6.8 during use adds 1 part of Ji's nurse Sa Albert'stain Albert stoste for 8 parts, use liquid.
PBS damping fluid: KH
2PO
46.66g, NaH
2PO
412H
2O is with adjust pH to 7.2 in the water-soluble 1000mL distilled water of mentioned reagent.
1% chicken erythrocyte suspension: get chicken vein or arterial blood before the experiment, place the triangular flask that fills granulated glass sphere (about 20), fully shake 5～10min along a direction continuously, remove and defibrinate, 4 ℃ of refrigerators are preserved.Wash 3 times with physiological saline before the experiment, 1500r/min, centrifugal 10min, supernatant discarded is mixed with 1% chicken erythrocyte suspension by hematocrit Hank ' s liquid.
3% agar: get agar 3 grams, add water 100mL, heated and boiled adds 1% purpurum bromocresolis indicator 1～2 to transparent.
The destainer of Giemsa dye liquor: methyl alcohol 20mL, distilled water 80mL.Mix two of back 2N HCl.
DMPO (5.5-dimethyl-1-pyrroline-1-oxide): with preceding through activated carbon treatment.
C. instrument
Counter, instruments, syringe, staining trough are the conventional instrument in laboratory; Microhemagglutination brassboard: Jinan Bo Sai company.
Experimental technique: the A. humoral immune function is measured: serum hemolysin determination experiment (blood clotting method)
[7]B. monokaryon-macrophage function is measured: Turnover of Mouse Peritoneal Macrophages is engulfed chicken red blood cell experiment (dripping the sheet method)
[7]C. given the test agent dosage and time: yellow umbrella sample is established 3 dosage groups, i.e. 1mg/d, 2mg/d, 10mg/d intake; Other establishes negative control group.Test 10 for every group.D. given the test agent gives mode: stomach is irritated in autoclaving pure water dissolving back.Irritate stomach every day once, continue 15 days.E. the statistical method experimental data all adopts the SPSS13.0 statistical software to carry out statistical study.F. the strengthening immunity function is judged according to the decision principle as a result in " protective foods check and evaluation technique standard ", that is: any two positives as a result aspect four of cellular immune functions, humoral immune function, monokaryon-macrophage function, NK cytoactive have the strengthening immunity function with regard to this given the test agent of decidable.
Experimental result:
Irritate respectively every day by mouse and to feed 5 times, 10 times and 50 times of human dose A1, behind A2 and the PA 15d, the mice serum hemolysin with irritate not feed blank and compare, inquire into three kinds of polysaccharide and whether have the human body fluid of raising immunologic function, the result shows: 5 times of human doses of A1 sample, 10 times of human doses, 50 times of human doses, 5 times of human doses of A2 sample, 10 times of human doses, 10 times of human dose experimental results of PA sample are positive, as seen equal in various degree the raising mouse humoral immune function of three kinds of polysaccharide, it is positive that wherein A1 and A2 all have two (comprising) above level experiment, therefore can judge the A1 and the A2 humoral immune function determination experiment positive, promptly have the humoral immunity of raising function.Experimental result shows that also three dosage of A1 all have the raising humoral immune function, and A2 only works when lower concentration.
(4) pholiota adiosapose polysaccharide antioxidation in vitro effect
Test materials: 5 kinds of samples: sporophore extract Polysaccharide A 1, mycelium extract Polysaccharide A 2,, mycelium powder A3, fruit body powder A4.
Above-mentioned sample with the tri-distilled water dissolved dilution to desired concn (1.0,10.0,100.0mg/mL+1.5mL50% ethanol) at the mensuration A2 of 517nm place; The control group distilled water.
Instrument reagent: DMPO (5.5-dimethyl-1-pyrroline-1-oxide), xanthine, XOD are U.S. Sigma company product.DMPO with preceding through activated carbon treatment.Detecting instrument is Japanese REIX type EPR spectrometer (EPR) (test condition: experiment parameter: microwave frequency 9.66GHz, microwave power 20mW, modulating frequency 100kHz, modulation amplitude 0.5mT).
Test method: EPR measures: get respectively a certain amount of sample solution (≤0.1ml), test by working method that test kit is indicated, the cumulative volume of reaction system is 3.4ml.
The generation of oxyradical: producing the oxyradical model is xanthine-XOD system: XH+O
2→ X+H
++ O
- 2The model that hydroxy radical qiao produces is Fenton reaction: H
2O
2+ Fe
2+→ Fe
3++ 2OH.
Spectrum Analysis: the spectrum spectroscopic signal is respectively adducts DMPO-OH and the DMPO-OOH after representing hydroxy radical qiao and ultra-oxygen anion free radical to be captured, and the concentration of oxygen free radicals that produces in its peak value and the system is proportional.
Method of calculation: sample is to the percentage E of free radical scavenging, E=[(h
0-h
x)/h
0] * 100%, h
0Be control group electron spin resonance signal average peak, hx is an experimental group peak height mean value.
Test-results:
Three kinds of pholiota adiosapose polysaccharides have antioxidant radical and hydroxy radical qiao effect preferably.The effect of A2 mycelium extraction polysaccharide in the oxyradical experiment-10mg/mL dosage removing oxyradical is best, reach 72.7%, other two kinds of polysaccharide clearance rate when maximum concentration also reaches 69.1%, all is better than mycelium powder and fruit body powder about 55%, and except that A2; A1-100mg/mL dosage is the highest to clearance rate in the hydroxy radical qiao experiment, reaches 95.8%, also is better than about 70% of A3 and A4, and polysaccharide is to the scavenging(action) and the polysaccharide concentration also linear (table 5, table 6) of hydroxy radical qiao.
Table 5 polysaccharide is to the scavenging(action) of oxyradical
Reference:
1. fourth of the twelve Earthly Branches morning mist. Chinese macro fungi [M]. Henan: Henan science tech publishing house, 2000,248;
2. Su Yan friend, Kang Li, Yang Zhixiao etc. the extraction of pholiota adiosapose polysaccharide and to the activation effect research of Turnover of Mouse Peritoneal Macrophages. Taishan Hospital's journal, 2004,25 (1): 9～11.
3. Wang Qian, Zhang Jungang, Wang Shikui etc. the research of thing regulating blood fat effect is obtained through refining in yellow umbrella fermentation. University Of Hebei's journal (natural science edition), 2006 (1): 101～103.
4. Li Rong spring, Fu Ziyan, Lee's letter. the research of yellow agaric silk nutritive property. edible mushrooms journal, 2001,8 (1): 19～23
5. Hui Fengli, Wei Minghui, Du Min China etc. the culture condition of yellow agaric silk submerged fermentation, Wuxi Light Industry Univ.'s journal 2004,23 (4): 24～27
6. Hu Qingxiu, Gong Chunyu, Yan Meixia. the research of yellow umbrella and the effect of pholiota adiosapose polysaccharide antioxidation in vitro. Sino-South African Forestry University of Science and Technology's journal, 2007 (6): 58～62
7. protective foods check and evaluation technique standard (2003 editions). Ministry of Health of the People's Republic of China, 2003.22～34
Claims (6)
1. pholiota adiosapose polysaccharide, it prepares by the following method:
1) extraction of yellow umbrella Crude polysaccharides
With pholiota adiposa mycelium or sporophore is raw material, warp: 1. material pre-treatment, and 2. lixiviate, 3. alcohol is analysed, and 4. concentrates, and 5. vacuum lyophilization gets yellow umbrella Crude polysaccharides;
2) pholiota adiosapose polysaccharide purifying
Yellow umbrella Crude polysaccharides is made 1～5% the aqueous solution, drip an amount of strong aqua and make its pH value reach 8～10, leave standstill behind 10～15min that centrifugal 10～15min removes albumen precipitation under 4500～5000rpm; Then with 1～3 times of polysaccharide soln dilution, add isopyknic seveg reagent again, behind thermal agitation 25～35min, centrifugal 10～15min under 4500～5000rpm, get supernatant liquor adding equal-volume seveg reagent and repeat to remove the albumen operation once, polysaccharide soln is concentrated postlyophilization; Get that polysaccharide is dissolved in distilled water after 20～28 ℃ of following purifying of the further room temperature of ion exchange column of forming by D152 and D301T series connection, elutriant is a distilled water, behind the purifying, again through Sephadex G-100 gel filtration chromatography purifying, carry out wash-out with distilled water as elutriant, promptly get pholiota adiosapose polysaccharide.
2. method for preparing pholiota adiosapose polysaccharide, it comprises the steps:
1) extraction of yellow umbrella Crude polysaccharides
With pholiota adiposa mycelium or sporophore is raw material, warp: 1. material pre-treatment, and 2. lixiviate, 3. alcohol is analysed, and 4. concentrates, and 5. vacuum lyophilization gets yellow umbrella Crude polysaccharides;
2) yellow umbrella Crude polysaccharides is made 1～5% the aqueous solution, drip an amount of strong aqua and make its pH value reach 8～10, leave standstill behind 10～15min that centrifugal 10～15min removes albumen precipitation under 4500～5000rpm; Then with 1～3 times of polysaccharide soln dilution, after adding isopyknic seveg reagent thermal agitation 25～30min again, centrifugal 10～15min under 4500～5000rpm gets supernatant adding equal-volume seveg reagent and repeats to remove the albumen operation once, and polysaccharide soln is concentrated postlyophilization; Get that polysaccharide is dissolved in distilled water after 20～28 ℃ of following purifying of the further room temperature of ion exchange column of forming by D152 and D301T series connection, elutriant is a distilled water, behind the purifying, again through Sephadex G-100 gel filtration chromatography purifying, carry out wash-out with distilled water as elutriant, promptly get pholiota adiosapose polysaccharide.
3. method as claimed in claim 2 is characterized in that, wherein step 1)
1. the material pre-treatment is crushed to 120～160 orders with pholiota adiposa mycelium or sporophore freeze-drying, adds deionized water 1: 20～30, ultrasonication 5～15min;
2. lixiviate refluxing extraction, 1～4 hour extraction time, 60～90 ℃ of extraction temperature;
3. alcohol is analysed the dehydrated alcohol that adds 3～5 times of amounts in vat liquor, and alcohol was analysed 8～16 hours;
4. concentrated alcohol is analysed the centrifugal 15～25min of back 3500～5000rpm and is abandoned supernatant liquor, gets throw out, with the absolute ethanol washing precipitation once, precipitates twice with washing with acetone more earlier;
5. vacuum lyophilization.
4. method as claimed in claim 3 is characterized in that, wherein step 1)
1. the material pre-treatment is crushed to 140 orders with pholiota adiposa mycelium or sporophore freeze-drying, material-water ratio 1: 30, ultrasonic power 600W, treatment time 10min;
2. lixiviate refluxing extraction, 3 hours extraction times, 75 ℃ of extraction temperature;
3. alcohol is analysed the dehydrated alcohol that adds 4 times of amounts in vat liquor, and alcohol was analysed 12 hours;
4. concentrated alcohol is analysed the centrifugal 20min of back 3500-5000rpm and is abandoned supernatant liquor, gets throw out, with the absolute ethanol washing precipitation once, precipitates twice with washing with acetone more earlier;
5. vacuum lyophilization.
5. as each described method of claim 2～4, wherein said pholiota adiposa mycelium prepares by the following method:
1. liquid culture based formulas: glucose 15～25g/L, yeast soak powder 6～10g/L, vitamins B
1200～400 μ g/L, Mg
2SO
40.3～0.5g/L, KH
2PO
40.8～1.2g/L, K
2HPO
40.8～1.2g/L, water surplus, autoclaving is standby;
2. seed culture: by the aseptic technique method, get female mycelium of planting, by inoculum size 1～10% (V/V), in the liquid nutrient medium after the switching sterilization, culture temperature: at 24～26 ℃; Under shaking speed 120r/min～140r/min condition, incubation time 10～14 days;
3. fermentation culture: with the seed culture bacterial classification, the aseptic technique method, (V/V) is seeded in the fermentor tank by inoculum size 5～10%, fermentation culture routinely, and fermention medium is: glucose 20～40g/L, corn steep liquor 15～25g/L, potassium primary phosphate 1.5～2.5g/L, pH value 6.5～7.0,24～26 ℃ of controlled temperature, mixing speed 150～180r/min, air flow is 0.8～2 (V/Vmin), fermentation culture 7～10 days.
6. the medicine or the protective foods that contain the described pholiota adiosapose polysaccharide of claim 1.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102405764A (en) * | 2011-04-19 | 2012-04-11 | 唐延军 | Method for fermenting piptoporus soloniensis |
| CN103733873A (en) * | 2013-06-03 | 2014-04-23 | 鲁东大学 | Study on liquid pholiota adiposa strains and polysaccharide |
| CN103919224A (en) * | 2014-04-08 | 2014-07-16 | 德州学院 | Pholiota adipose fermentation liquor fermented lactic acid beverage and preparation method thereof |
| CN106852367A (en) * | 2015-12-09 | 2017-06-16 | 深圳先进技术研究院 | A kind of lactic acid drink formula containing algal oil and pholiota adiosapose polysaccharide |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102405764A (en) * | 2011-04-19 | 2012-04-11 | 唐延军 | Method for fermenting piptoporus soloniensis |
| CN102405764B (en) * | 2011-04-19 | 2015-11-18 | 唐延军 | The fermentation process of suolunbguan bacterin |
| CN103733873A (en) * | 2013-06-03 | 2014-04-23 | 鲁东大学 | Study on liquid pholiota adiposa strains and polysaccharide |
| CN103919224A (en) * | 2014-04-08 | 2014-07-16 | 德州学院 | Pholiota adipose fermentation liquor fermented lactic acid beverage and preparation method thereof |
| CN106852367A (en) * | 2015-12-09 | 2017-06-16 | 深圳先进技术研究院 | A kind of lactic acid drink formula containing algal oil and pholiota adiosapose polysaccharide |
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