CN1752109A - Method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis - Google Patents
Method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis Download PDFInfo
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Abstract
A process for preparing the Huishuhua polyose by enzymolyzing includes such steps as slant culture and gradually enlarge culture to obtain the fermented liquid of fungus Huishuhua, regulating its pH value and temp, enzymolyzing by the enzyme line contained by itself, adding cellulase, beta-glucanase, pectinase and neutral proteinase, enzymolyzing, deactivating enzyme, filtering, concentrating, depositing in alcohol and separating.
Description
Technical field
The invention belongs to the edible fungi polysaccharide preparation field, particularly relate to and utilize enzymatic treatment Grifola frondosa submerged fermentation liquid, to improve the production method of active grifolan content.
Technical background
Grifola frondosa (Polyporus frondosus) is under the jurisdiction of Mycophyta, Basidiomycotina, Aphyllophorales, polyporaceae, Polyporus.Grifola frondosa is famous edible, medicinal fungi, and is nutritious, tasty, is the macro fungi of " but edible, can mend medicine, the whole body is precious ".Grifola frondosa is flat, flavor is sweet, can be used for treating diseases such as dysuria, oedema, beriberi, hepatic ascites, diabetes, hypertension and obesity.
The functional component grifolan that contains in the Grifola frondosa has tangible antitumous effect.In recent years, people have carried out big quantity research to its physico-chemical property and pharmacological action.That grifolan has is significantly antitumor, AIDS virus resisting, improve function of immune system, blood sugar regulation, blood fat and cholesterol levels, effect such as hypotensive grade to generation and the transfer that prevents cancer cells, has certain curative effect to hypertension, obesity, diabetes, anemia etc.The grifolan product that has gone on the market at present has the MAIEXT nutritional supplements of Japan, the Grifron series product of the U.S., guarantor's Lik-Sang capsule of China etc.Grifolan has the characteristics of antitumor action safely and effectively, and it has broad application prospects in medicine, food.
Grifolan mainly is present in Grifola frondosa fungus sporophore, sclerotium, mycelium or the fermented liquid.The grifolan of molecular weight between 6000-50000, its biological activity is higher, and the grifolan molecule is a branched structure, and glycosidic link is main with β-1,4 key, and β-1,6 key is auxilliary.Macromolecular grifolan in the maitake mushroom mycelia (polymerization degree is nearly millions of) is cross-linked with each other compact structure with protein, lipid and pectin substance.Therefore, how to excise part or all protein and the pectin composition crosslinked with grifolan, and the grifolan macromole degraded effectively, thereby improve the content of the active grifolan of molecular weight between 6000-50000, be a problem that needs to be resolved hurrily.
At present, correlative study both at home and abroad mainly concentrates on the purifying extraction and the functional study aspect of grifolan.And particularly handle fermented liquid (containing mycelium) by mycelium self enzyme system and additional enzyme system with enzyme process, still there is not report in order to the technology that improves active grifolan content.
Summary of the invention:
The method that the purpose of this invention is to provide a kind of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis.By to the Grifola frondosa fungal fermented filtrate enzymatic treatment of (containing mycelium), improve the content of active grifolan effectively.
The method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis of the present invention is summarized as follows:
(1) acquisition of fermented liquid: adopt slant strains to spread cultivation step by step and obtain Grifola frondosa fungi liquid submerged fermentation liquid;
(2) mycelium self enzyme is an enzymolysis: regulating step (1) obtains contains mycelial fermented liquid, making its pH is 5～7, at 30～70 ℃, by self enzyme system that exists in the maitake mushroom mycelia maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis time 30～100 minutes, 10～100 rev/mins of stirring velocitys;
(3) additional enzyme enzymolysis: the fermented liquid that obtains in the step (2) is adjusted temperature and pH respectively, making temperature is 30～65 ℃, pH is 5～7, add mixed enzyme in the fermented liquid, the weight percent of mixed enzyme and fermented liquid is 0.03～0.05%, and mixed enzyme consists of by weight percentage: cellulase 5～95%, beta-glucanase 5～80%, polygalacturonase 0～70%, neutral protease 0～70%, enzymolysis time 0.3～8 hour, open in the enzymolysis process and stir 10～100 rev/mins of stirring velocitys;
(4) enzyme that goes out: after enzyme control reaction finishes, fermented liquid is warmed up to 90～100 ℃, kept 10～30 minutes, enzyme goes out;
(5) filter: enzyme treatment process carries out solid-liquid separation to fermented liquid after finishing, and available equipment has plate and frame(type)filter press or whizzer etc.;
(6) concentrate: filtrate is concentrated into 1/5～1/10 of original volume, and concentrated method therefor can be single-action, multiple-effect vacuum concentration and freeze concentration;
(7) alcohol precipitation: add 70～100% ethanol and carry out alcohol precipitation in concentrated filtrate, its volume ratio is 1: 3～4;
(8) centrifugation: throw out is a grifolan.
At mycelium self enzyme is to be advisable with pH 5.5～6 in the enzymolysis step, and hydrolysis temperature preferably selects 45～50 ℃, and enzymolysis time preferably selects 60～70 minutes, and stirring velocity is preferably selected 20～60 rev/mins.
The temperature that adds mixed enzyme in the additional enzyme enzymolysis step is advisable with 50～55 ℃, and pH preferably selects 5.5～6, and enzymolysis time preferably selects 2～2.5 hours, and stirring velocity is preferably selected 20～50 rev/mins.
Xu Jin hall volume " Chinese medicinal fungi " is seen in the cultivation of Grifola frondosa fungi liquid submerged fermentation liquid among the present invention.
Remarkable advantage of the present invention is:
The present invention is on the basis of the Grifola frondosa fungal fermented filtrate that cultivation obtains, the mixed enzyme of utilizing mycelium self enzyme system dexterously and adding is carried out enzymolysis to fungal fermented filtrate under suitable condition, behind the enzymolysis in the thick product grifolan grifolan content be 35-60%.The active grifolan of molecular weight between 6000-50000 accounts for 70-90% in the grifolan.Thick Grifola frondosa polysaccharide yield can reach 10-17.5 kilogram/ton fermented liquid.
Description of drawings:
Fig. 1 is the blue dextran elution curve in the sephadex chromatography measuring method;
Fig. 2 is the wash-out characteristic standard curve in the sephadex chromatography measuring method.
Embodiment:
The following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.
10 tons of fermented liquid grifolan method of enzymatically treating
(1) adopts slant strains to spread cultivation step by step and obtain Grifola frondosa liquid submerged fermentation liquid: the Grifola frondosa slant strains is inserted carry out the first order seed cultivation in 500 ml shake flasks that 100 milliliters of substratum are housed, culture condition: 150 rev/mins of rotary shaking tables, cultivated 72 hours for 28 ℃.Then the one-level shake-flask seed is inserted with 10% inoculum size and carry out the secondary seed cultivation in 500 ml shake flasks that 100 milliliters of substratum are housed, cultivate according to shaking the identical condition of bottle with one-level.And then with 10% inoculum size secondary seed is transferred and to carry out three grades of seed culture, culture condition in 5000 ml shake flasks that 1000 milliliters of substratum are housed: 180 rev/mins of rotary shaking tables, cultivated 60 hours for 28 ℃.And then with 10% inoculum size three grades of seeds to be inserted the cubic capacity that 0.1 ton of fermention medium is housed be 0.15 ton first class seed pot.Culture condition is: 28 ℃ of culture temperature, and 150 rev/mins of stirring velocitys, ventilation (V/V) 1: 0.5, tank pressure 0.05Mpa cultivated 48 hours.Be in 1.5 tons of secondary seed jars with the bacterium liquid of first class seed pot with 10% the inoculum size cubic capacity that 1 ton of fermention medium is housed of transferring equally, the secondary seed jar is identical with the culture condition of first class seed pot.At last with 10% inoculum size secondary seed jar seed being inserted the cubic capacity that 10 tons of fermention mediums are housed is in 15 tons the fermentor tank.28 ℃ of fermentor cultivation temperature, 100 rev/mins of stirring velocitys, ventilation (V/V) 1: 0.5, tank pressure 0.05Mpa cultivated 120 hours.
(2) mycelium self enzyme is an enzymolysis: fermented liquid is warmed up to 50 ℃ with steam with the fermented liquid in the fermentor tank after reaching jar standard of putting, regulate PH to 6.0 with 10% NaOH, system carries out enzymolysis to the maitake mushroom mycelia in the fermented liquid by maitake mushroom mycelia self enzyme, enzymolysis time 60 minutes, 30 rev/mins of stirring velocitys;
(3) additional enzyme enzymolysis: at 55 ℃, pH is under 6 the condition, to in 10 tons of fermented liquids that step (2) obtains, adding 2.5 kilograms of cellulases, 1 kilogram of beta-glucanase, 1 kilogram of polygalacturonase, 0.5 kilogram of neutral protease, enzymolysis time 3 hours, open in the enzymolysis process and stir 30 rev/mins of stirring velocitys;
(4) enzyme that goes out: the additional enzyme enzymolysis is warmed up to 95 ℃ with fermented liquid after finishing, and 30 minutes, enzyme went out;
(5) filter: fermented liquid is carried out filter press, collect filtrate;
(6) concentrate: concentrating under reduced pressure filtrate volume to 1 ton;
(7) alcohol precipitation: add 3.8 ton 95% ethanol, alcohol was analysed 12 hours;
(8) centrifugation: adopted horizontal centrifuge 3500rpm centrifugal 60 minutes, with the dissolved in distilled water precipitation, after the vacuum drying process preparation obtains 110 kilograms in thick grifolan dry powder.Wherein grifolan content is 45%, and active grifolan (molecular weight is at 6000-50000) accounts for 80% of grifolan, and ratio sees Table 1.
The Grifola frondosa bacterial classification is purchased in Chinese industrial microbial strains preservation administrative center among the present invention, bacterium number: CICC 14098.
Slant medium is formed in this example: potato (liquor) 20%, glucose 2%, potassium primary phosphate 0.1%, sal epsom 0.15%, agar 2.0%.
The used substratum of seeds at different levels and fermentative production consists of in this example: W-Gum 5%, yeast extract paste 0.5%, sucrose 1%, dipotassium hydrogen phosphate 0.2%, sal epsom 0.1%, VITMAIN B1 2PPm, PH6.0.
Measure grifolan content with the phenol sulfuric acid process in this example, measuring method is as follows:
Accurately measure sample 1 gram, put in 100 milliliters of volumetric flasks and be settled to scale, and then get 1 milliliter and add 100 milliliters of volumetric flasks and be settled to scale with distilled water with distilled water.Draw that 1 milliliter of diluent is put in 10 milliliters of measuring bottles and mend and add water to 2 milliliters, the accurate 1 milliliter of mixing of 5% phenol test solution that adds, add 5.0 milliliters in sulfuric acid rapidly, jolting 5 minutes is put and is placed in 100 ℃ of the water-baths after 15 minutes, and cooling is 30 minutes in cold water, add water to scale, shake up, measure optical density at 490 nano wave length places, do blank simultaneously.
Calculate polysaccharide content according to formula.
P＝(0.289x-0.0131)*d*f/(1000*W)*100。
X is the dilution factor of light absorption value d for test agent solution, and f is a reduction factor 0.9, and W is an example weight, and P is a polysaccharide content in the sample.
Measure the grifolan molecular weight with the sephadex chromatography method in this example, measuring method is as follows:
Gel chromatography column when treating that elutriant flow to the following 1-2 millimeter of bed surface, is closed outlet after balance, draw a certain amount of (about 0.3 milliliter) dextran standardized solution with sample injector, add to the bed surface, open outlet from the application of sample mouth, receiver pope is installed, liquid to be marked penetrates in the bed, carefully adds an amount of elutriant, connects constant voltage wash-out bottle, the beginning chromatography, and fraction collection, every pipe is collected 3 milliliters, flow velocity 0.5 ml/min.From every pipe, draw 0.5 milliliter, measure with the phenol sulfuric acid process.With light absorption value to the collection tube number map dextran standard elution curve (see figure 1), thereby obtain elution volume Ve.Survey void volume Vo with blue dextran equally, with Ve/Vo the logarithm of molecular weight is drawn characteristic standard curve (see figure 2) at last.Typical wash-out characteristic standard curve is Y=-0.95X+5.84.
Take by weighing polyoses extract sample 1g, with hot elutriant dissolving, be incubated 0.5 hour, cooling constant volume to 50 milliliter (certain volume).Get that chromatography column carries out chromatographic separation on the supernatant liquor 0.3-0.5 milliliter, try to achieve elution volume Ve, look into wash-out characteristic standard curve with the Ve/Vo value then, obtain molecular weight.Polysaccharide to different molecular weight ranges adopts the phenolsulfuric acid method to measure its polysaccharide content.
Active grifolan molecular weight distribution and ratio in table 1 grifolan
| Molecular weight | ≥50000 | 50000- 30000 | 30000- 10000 | 10000- 6000 | ≤6000 |
| Proportion % | 9 | 10 | 44 | 26 | 11 |
Embodiment 2 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 5.5, at 45 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 70 minutes, 40 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 5.5 the condition, adds mixed enzyme: 0.15 kilogram of cellulase, 1.2 kilograms of beta-glucanases, 0.9 kilogram of polygalacturonase, 0.75 kilogram of neutral protease in fermented liquid, enzymolysis time 2 hours, 40 rev/mins of stirring velocitys;
In the enzyme process that goes out, fermented liquid is warmed up to 90 ℃, 10 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 2 tons;
In the alcohol precipitation process, the ethanol that adds 6 ton 70% carries out alcohol precipitation.
Embodiment 3 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 5, at 30 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 30 minutes, 60 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 30 ℃, pH under 6.0 the condition, adds mixed enzyme: 0.2 kilogram of cellulase, 1.6 kilograms of beta-glucanases, 1.2 kilograms of polygalacturonases, 1 kilogram of neutral protease in fermented liquid, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys;
In the enzyme process that goes out, fermented liquid is warmed up to 100 ℃, 30 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1 ton;
In the alcohol precipitation process, the ethanol that adds 4 ton 100% carries out alcohol precipitation.
Embodiment 4 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 7, at 70 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 100 minutes, 100 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 65 ℃, pH under 5 the condition, adds mixed enzyme: 2.0 kilograms of cellulases, 1.0 kilograms of beta-glucanases, 1.5 kilograms of polygalacturonases, 0.5 kilogram of neutral protease in fermented liquid, enzymolysis time 0.3 hour, 100 rev/mins of stirring velocitys;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Embodiment 5 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 6, at 50 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 65 minutes, 10 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 7 the condition, adds mixed enzyme: 0.25 kilogram of cellulase, 2.0 kilograms of beta-glucanases, 1.5 kilograms of polygalacturonases, 1.25 kilograms of neutral proteases in fermented liquid, enzymolysis time 8 hours, 10 rev/mins of stirring velocitys;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.2 tons;
In the alcohol precipitation process, the ethanol that adds 4 ton 78% carries out alcohol precipitation.
Embodiment 6 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 6, at 50 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 65 minutes, 50 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 6 the condition, adds mixed enzyme: 2.85 kilograms of cellulases, 0.15 kilogram of beta-glucanase, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys in fermented liquid;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Embodiment 7 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 6, at 50 ℃, by self the enzyme system that exists in the maitake mushroom mycelia maitake mushroom mycelia in the fermented liquid is carried out enzymolysis; Enzymolysis 65 minutes, 50 rev/mins of stirring velocitys;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 6 the condition, adds mixed enzyme: 3.8 kilograms of cellulases, 0.2 kilogram of beta-glucanase, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys in fermented liquid;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Embodiment 8 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 6, at 50 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 65 minutes, 50 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 6 the condition, adds mixed enzyme: 4.75 kilograms of cellulases, 0.25 kilogram of beta-glucanase, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys in fermented liquid;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Embodiment 9 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 6, at 50 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 65 minutes, 50 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 6 the condition, adds mixed enzyme: 2.4 kilograms of cellulases, 0.3 kilogram of beta-glucanase, 0.3 kilogram of neutral protease, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys in fermented liquid;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Embodiment 10 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 6, at 50 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 65 minutes, 50 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 6 the condition, adds mixed enzyme: 3.2 kilograms of cellulases, 0.4 kilogram of beta-glucanase, 0.4 kilogram of neutral protease, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys in fermented liquid;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Embodiment 11 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 6, at 50 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 65 minutes, 50 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 6 the condition, adds mixed enzyme: 4 kilograms of cellulases, 0.5 kilogram of beta-glucanase, 0.5 kilogram of neutral protease, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys in fermented liquid;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Embodiment 12 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 6, at 50 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 65 minutes, 50 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 6 the condition, adds mixed enzyme: 0.3 kilogram of cellulase, 2.4 kilograms of beta-glucanases, 0.3 kilogram of polygalacturonase, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys in fermented liquid;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Embodiment 13 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 6, at 50 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 65 minutes, 50 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 6 the condition, adds mixed enzyme: 0.4 kilogram of cellulase, 3.2 kilograms of beta-glucanases, 0.4 kilogram of polygalacturonase, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys in fermented liquid;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Embodiment 14 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 6, at 50 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 65 minutes, 50 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 6 the condition, adds mixed enzyme: 0.5 kilogram of cellulase, 4 kilograms of beta-glucanases, 0.5 kilogram of polygalacturonase, 0.32 kilogram of neutral protease in fermented liquid, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Embodiment 15 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 6, at 50 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 65 minutes, 50 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 6 the condition, adds mixed enzyme: 0.3 kilogram of cellulase, 0.3 kilogram of beta-glucanase, 2.1 kilograms of polygalacturonases, 0.3 kilogram of neutral protease in fermented liquid, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Embodiment 16 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 7, at 60 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 55 minutes, 10 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 6 the condition, adds mixed enzyme: 0.4 kilogram of cellulase, 0.4 kilogram of beta-glucanase, 2.8 kilograms of polygalacturonases, 0.4 kilogram of neutral protease in fermented liquid, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Embodiment 17 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 6, at 50 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 65 minutes, 50 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 6 the condition, adds mixed enzyme: 0.5 kilogram of cellulase, 0.5 kilogram of beta-glucanase, 3.5 kilograms of polygalacturonases, 0.5 kilogram of neutral protease in fermented liquid, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Embodiment 18 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 6, at 50 ℃, by self the enzyme system that exists in the maitake mushroom mycelia maitake mushroom mycelia in the fermented liquid is carried out enzymolysis; Enzymolysis 65 minutes, 50 rev/mins of stirring velocitys;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 6 the condition, adds mixed enzyme: 0.3 kilogram of cellulase, 0.3 kilogram of beta-glucanase, 0.3 kilogram of polygalacturonase, 2.1 kilograms of neutral proteases in fermented liquid, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Embodiment 19 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 7, at 50 ℃, by self the enzyme system that exists in the maitake mushroom mycelia maitake mushroom mycelia in the fermented liquid is carried out enzymolysis: enzymolysis 60 minutes, 100 rev/mins of stirring velocitys;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 6 the condition, adds mixed enzyme: 0.4 kilogram of cellulase, 0.4 kilogram of beta-glucanase, 0.4 kilogram of polygalacturonase, 2.8 kilograms of neutral proteases in fermented liquid, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Embodiment 20 (10 tons of fermented liquids)
A kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis, its step are with embodiment 1:
At mycelium self enzyme is in the enzymolysis process, regulates fermented liquid, and making its pH is 6, at 50 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis 65 minutes, 50 rev/mins of stirring velocitys by self the enzyme system that exists in the maitake mushroom mycelia;
In the additional enzyme enzymolysis process, at 50 ℃, pH under 6 the condition, adds mixed enzyme: 0.5 kilogram of cellulase, 0.5 kilogram of beta-glucanase, 0.5 kilogram of polygalacturonase, 2.5 kilograms of neutral proteases in fermented liquid, enzymolysis time 2.5 hours, 50 rev/mins of stirring velocitys;
In the enzyme process that goes out, fermented liquid is warmed up to 95 ℃, 20 minutes, enzyme went out;
In concentration process, filtrate is concentrated into 1.5 tons;
In the alcohol precipitation process, the ethanol that adds 4.5 ton 75% carries out alcohol precipitation.
Claims (4)
1, a kind of method of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis is characterized in that it comprises the steps:
(1) acquisition of fermented liquid: adopt slant strains to spread cultivation step by step and obtain Grifola frondosa liquid submerged fermentation liquid;
(2) mycelium self enzyme is an enzymolysis: regulating step (1) obtains contains mycelial fermented liquid, making its pH is 5～7, at 30～70 ℃, the maitake mushroom mycelia in the fermented liquid is carried out enzymolysis, enzymolysis time 30～100 minutes by self enzyme system that exists in the maitake mushroom mycelia;
(3) additional enzyme enzymolysis: at 30～65 ℃, pH is under 5～7 the condition, in the fermented liquid that obtains through step (2), add mixed enzyme, the weight percent of described mixed enzyme and fermented liquid is 0.03～0.05%, described mixed enzyme comprises by weight percentage: cellulase 5～95%, beta-glucanase 5～80%, polygalacturonase 0～70%, neutral protease 0～70%, enzymolysis time 0.3～8 hour;
(4) enzyme that goes out: after enzyme control reaction finishes, fermented liquid is warmed up to 90～100 ℃, 10～30 minutes, enzyme went out;
(5) filter;
(6) concentrate: filtrate is concentrated into 1/5～1/10 of original volume;
(7) alcohol precipitation: add 70～100% ethanol and carry out alcohol precipitation in concentrated filtrate, its volume ratio is 1: 3～4;
(8) centrifugation: throw out is a grifolan.
2, the method for a kind of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis according to claim 1, it is characterized in that described mycelium self enzyme is that pH is 5.5～6 in the enzymolysis step, hydrolysis temperature is 45～50 ℃, and enzymolysis time is 60～70 minutes, 20～60 rev/mins of stirring velocitys.
3, the method for a kind of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis according to claim 1, the temperature that it is characterized in that adding mixed enzyme in the described additional enzyme enzymolysis step is 50～55 ℃, pH is 5.5～6, described enzymolysis time 2～2.5 hours, 20～50 rev/mins of stirring velocitys.
4, the method for a kind of preparing huishuhua (grey tree flower) polysaccharide by enzymolysis according to claim 1 is characterized in that the temperature of adding mixed enzyme in the described additional enzyme enzymolysis step is 50 ℃, and pH is 6, described enzymolysis time 2.5 hours, 50 rev/mins of described stirring velocitys.
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| CN101914131A (en) * | 2010-08-27 | 2010-12-15 | 山东京博控股发展有限公司 | Method for preparing Griflola frondosa proteoglycan |
| CN101396378B (en) * | 2007-09-24 | 2011-06-22 | 钱卫群 | Nano gene peptides products and preparation method and use thereof |
| CN101348817B (en) * | 2008-08-25 | 2012-07-04 | 浙江省林业科学研究院 | Method for extracting skunk bush polysaccharide by zymohydrolysis |
| CN105754005A (en) * | 2016-03-04 | 2016-07-13 | 江苏江大源生态生物科技股份有限公司 | Method for extracting grifola frondosa polysaccharase with high yield |
| CN106008027A (en) * | 2016-06-30 | 2016-10-12 | 江苏挚信花卉发展有限公司 | Green environment-friendly nutrient solution beneficial to plant growth |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2859843B2 (en) * | 1996-03-08 | 1999-02-24 | 株式会社雪国まいたけ | Antitumor substance extracted from Maitake |
| CN1110566C (en) * | 1999-11-18 | 2003-06-04 | 莱阳农学院 | Process for extracting polyose from polyporus frondosus fermenting liquor |
| CN1157473C (en) * | 2001-09-29 | 2004-07-14 | 李宝健 | Large-scale deep liquid fermentation process for gray tree flower |
| JP2003119149A (en) * | 2001-10-12 | 2003-04-23 | Miwa Seiyaku Co Ltd | Antitumor macromolecular polysaccharide, method of producing the same and antitumor agent |
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| CN101396378B (en) * | 2007-09-24 | 2011-06-22 | 钱卫群 | Nano gene peptides products and preparation method and use thereof |
| CN101348817B (en) * | 2008-08-25 | 2012-07-04 | 浙江省林业科学研究院 | Method for extracting skunk bush polysaccharide by zymohydrolysis |
| CN101914131A (en) * | 2010-08-27 | 2010-12-15 | 山东京博控股发展有限公司 | Method for preparing Griflola frondosa proteoglycan |
| CN101914131B (en) * | 2010-08-27 | 2013-02-27 | 山东京博控股发展有限公司 | Method for preparing Griflola frondosa proteoglycan |
| CN105754005A (en) * | 2016-03-04 | 2016-07-13 | 江苏江大源生态生物科技股份有限公司 | Method for extracting grifola frondosa polysaccharase with high yield |
| CN106008027A (en) * | 2016-06-30 | 2016-10-12 | 江苏挚信花卉发展有限公司 | Green environment-friendly nutrient solution beneficial to plant growth |
| CN112370487A (en) * | 2020-11-11 | 2021-02-19 | 黄河三角洲京博化工研究院有限公司 | Raspberry and grifola frondosa fermented product and fermentation method thereof |
| CN113355378A (en) * | 2021-06-08 | 2021-09-07 | 绥化市蓝源生物工程有限公司 | R-polysaccharide fermentation broth, fermentation method and bacteriostatic application thereof, and food preservative containing R-polysaccharide fermentation broth |
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