CN101735952B - Novel strain of pholiota adiosapose - Google Patents
Novel strain of pholiota adiosapose Download PDFInfo
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- CN101735952B CN101735952B CN2008102260578A CN200810226057A CN101735952B CN 101735952 B CN101735952 B CN 101735952B CN 2008102260578 A CN2008102260578 A CN 2008102260578A CN 200810226057 A CN200810226057 A CN 200810226057A CN 101735952 B CN101735952 B CN 101735952B
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Abstract
The invention provides a novel pholiota adiosapose strain caas-II with a preservation number of CGMCC NO. 1840. The strain is suitable for artificial cultivation and has good mouthfeel, rich nutrients and high pholiota adiosapose polysaccharide content, wherein the pholiota adiosapose polysaccharide has the functions of improving body immunity, resisting tumor and removing oxygen radical. Therefore, the strain of the invention or an extract thereof can be used as a raw material to prepare various health-care foods and drinks.
Description
Technical field
The present invention relates to the new bacterial strain of a kind of edible mushrooms, the new bacterial strain of the yellow umbrella of a strain specifically (Pholiotaadiposa (Fr.) Qu é l.).
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.Yellow destroying angel lovely luster is golden yellow, is covered with the tawny scale on the cap stem.This mushroom rich in proteins, carbohydrate, VITAMIN and multi mineral prime element, the glutinous smooth mouth of food, delicious flavour, unique flavor.A kind of special mucus is arranged on this mushroom cap, show according to biochemical analysis, this material is a kind of nucleic acid, and human body energy, mental recovery are had good result.Yellow umbrella is a kind of middle low temperature modification edible mushrooms, and production technique is simple, and output is higher, and the market price is better, is one of big rising edible mushrooms new variety.
" Chinese macro fungi " (fourth of the twelve Earthly Branches morning mist, 2000)
(1)Put down in writing yellow destroying angel form: " sporophore is general medium big, bacteria cover diameter 3～12cm, first flat hemisphere is capable, the edge is often involute, after gradually open and flat, paddy yellow, dirty yellow to tawny, very sticking has the scale of the nearly calm shape of brown, central authorities are closeer.Bacterial context white or faint yellow.The lamella yellow is directly given birth to or nearly curved giving birth to the brown of becoming rusty, and is close slightly, not isometric.Thick 0.5～the 3cm of the long 5～15cm. of stem, cylindrical, homochromy with lid, the scale of brown warp is arranged, sticking or sticking slightly, the bottom is often curved, and cellulosic is interior tangible.Collarium is faint yellow, and is membranous, gives birth on the stem, easily comes off.The spore rust, level and smooth, ellipse or oblong, (7.5～9.5) μ m * (5～6.3) μ m.Pleat side utricule is colourless or filbert, bar-shaped, not protruding thalamium more ".
" Chinese macro fungi "
(1)Also 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)
Summary of the invention
The object of the present invention is to provide a kind of novel strain of pholiota adiosapose.
Bacterial strain of the present invention is to gather wild bacterium when investigating in the Xinglong County, Hebei province, obtain original strain through separation and Culture, after cultivate through laboratory domestication, not only mouthfeel is good to find this bacterial classification, it is suitable for artificial culture, and have the effect that improves body immunity, with yellow umbrella (Pholiota adiposa) caas-11 of this bacterial strain called after.This bacterial strain on October 18th, 2006 in (address: Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, the yellow umbrella (Pholiota adiosapose) of classification called after, preserving number is CGMCC No.1840.
One, strain culturing of the present invention and suitable growth conditions
1, bacterial classification mother culture media prescription and growth conditions
Mother culture media: potato 200g, glucose 20g, malt extract 2g, MgSO
40.2g, KH
2PO
40.25g, K
2HPO
40.25g, VB
10.5mg, agar powder 15g, water 1000ml, pH nature.
This bacterial strain mycelial growth temperature scope is 8～30 ℃, and optimal temperature is 23～26 ℃, covers with the test tube slant about 10d.
2, pedigree seed culture medium prescription and growth conditions
Cotton seed hull substratum: cotton seed hull 78%, wheat bran 20%, gypsum 1%, lime 1% or cotton seed hull 45%, wood chip 30%, wheat bran 20%, lime 1%, gypsum 1%, sucrose 2%, urea 1%.Optimal temperature is 23～25 ℃, and after mycelia was covered with, general room temperature staging life was no more than 7d, and low-temp storage is no more than 15d, and expired bacterial classification can not work as the cultivar use.
3, cultivating bag composition of raw materials and mushroom producing culture condition
Prescription:
1. cotton seed hulls 88%, wheat bran 10%, gypsum 1%, calcium superphosphate 1%.
2. cotton seed hulls 48%, weed tree sawdust 30%, wheat bran 10%, gypsum 1%, calcium superphosphate 1%.
3. cotton seed hulls 78%, corn cob meal 10%, wheat bran 10%, gypsum 1%, calcium superphosphate 1%.
12～25 ℃ of sporophore growth temperature ranges, optimal temperature are 15～20 ℃.Optimum humidity is 85%～90%.pH5.5～6.5。
Two, morphological specificity
1. sporophore feature (Fig. 1):
The sporophore list is given birth to or is grown thickly; Cap 3～10cm, the initial stage semisphere, the edge curls inward has diaphragm, after gradually open and flat one-tenth umbrella; Color is sorrel when original hase forms, last tool white scale, after thin out gradually to beige, aging for sorrel to Vandyke brown.How upright scale is at the beginning on the cap, its edge Bai Maozhuan, after calm gradually, or mark only, color is a tawny, central authorities are not obvious, the edge is more; Just there is tool mucus behind the moist feeling on the surface, and bacterial context white is to faint yellow.Bacterial context white or faint yellow.Stem is cylindrical, and is sturdy, and cellulosic has the warp ramentum, and long 5～12cm is real among diameter 1～3cm..The collarium yellow, less, easily come off, only there is vestige in the later stage; Lamella is just milky white, and milk yellow when gathering discharges a large amount of rust brown spores when ripe, and the spore ellipse is smooth, diameter 7.5 μ m～9.5 μ m * 5 μ m～6.2 μ m.Under opticmicroscope, mycelia has clamp connexion.
2. cultural characteristic:
This bacterial strain is grown on the PDA substratum normally, and mycelia is sturdy, and bacterium colony suede glues shape, densification, and mycelia initial stage white, the later stage is light yellow to safran.Test tube is female plant in the shelf-time long, see that yellow destroying angel forms more.
Three, cultivation feature
In bag was planted, former Quito easily was formed at old bacterial classification piece place, but after the yellow agaric silk purseful, needed the certain hour after-ripening, saw the light cultivation, and the mycelia variable color just is beneficial to original hase and forms.With most of edible fungis seemingly, annesl after the mycelia maturation (yellow) is told yellow water, kink then forms original hase.
The present invention also provides a kind of cultivating method of above-mentioned bacterial strains:
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.Culture medium prescription:
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 or wood chip will add water-water reactor by 1:1 before use and put 8～12h, help the decomposition of suction of cotton seed hulls or wood chip and part nutrition like this, thereby guarantee 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 5～10d.
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 10h, and the stewing 5h～6h that ceases fire takes out cooling inoculation with bag then.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 30～35d 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 6～10d after the mushroom producing room, 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, about each 1～2h, carry out in conjunction with weather condition, morning in summer, evening ventilate, and autumn and winter, cold weather was taked to ventilate when noon, temperature was high, with constantly additional fresh air, got rid of 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 7～10d 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%.
The present invention also provides the mycelium cultural method of above-mentioned bacterial strains:
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, the pH nature, autoclaving is standby;
Substratum packing and sterilization can be carried out as follows: dress liquid nutrient medium 80mL in the 250mL triangular flask, and through high pressure (0.14～0.15Mpa) sterilization 0.5h.
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 10～14d;
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 7～10d.
Wherein step 1. the preferred liquid culture medium prescription be: 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, pH5.5～7, surplus is a water.
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 12d.
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), 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 1.5 (V/Vmin).
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 for 3:1.5=1:0.5, being called for short 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 ℃.
Three, esterase isozyme feature
Press agricultural industry criteria " edible fungus species authenticity identification esterase isozyme electrophoretic method " (NY/T1097-2006), this bacterial strain is carried out esterase isozyme measure, measure The esterase isozyme and see Fig. 2.Fig. 2 shows that this bacterial strain repeats the esterase isozyme electrophoretogram of point sample for 9 times.
Esterase isozyme electrophoretic method principle: in biology, the expression of enzyme is subjected to genetic regulation and control.Esterase is the enzyme of multiple alleles regulation and control, in gel electrophoresis through the concentrating of polyacrylamide gel, under the electrocharge effect effect of molecular sieve effect and electric field and separate.The crude enzyme liquid that extracts from edible fungus mycelium carries out the biological chemistry dyeing of esterase behind electrophoresis, different esterase components is presented at the different positions of gel and forms esterase isozyme zymogram.Identical bacterial classification genetic background is identical, and its esterase isozyme zymogram is also identical.
Laboratory apparatus: electrophoresis apparatus vertical slab electrophoresis groove high speed freezing centrifuge (more than the 10000r/min) mortar analytical balance microsyringe.
Culture medium prescription: potato 200g (using diffusion juice), glucose 20g, agar 20g, water 1000ml, PH nature.
Mycelium is cultivated: diameter 75mm or 90mm culture dish, 24 ℃ of lucifuges are cultivated 9d.
Specimen preparation: take by weighing mycelium 0.5g, adding liquid nitrogen grinds, with sample collection in the 1.5mL centrifuge tube, add the 1mL sample extracting solution, in 4 ℃ of centrifugal 10min of following 10000r/min, get its supernatant, with the mixed that supernatant liquor, sucrose solution (400g/L), tetrabromophenol sulfonphthalein indicating liquid are pressed 5+1+1, mixed solution is the electrophoresis sample.
Reagent preparation:
1. sample extracting solution: take by weighing 6.02g Na
2HPO
4.12H
2O, 0.5gNaH
2PO
4.2H
2O, soluble in water, and be diluted to 100mL;
2. separation gel damping fluid
Take by weighing the amino alkane (C of 36.3g trishydroxymethyl
4H
11NO
3, Tris),, add N then, N, N ', N '-methyl ethylenediamine (C with 48Ml 1mol/L dissolving with hydrochloric acid
6H
16N
2, TEMED) 0.23M1, water is settled to 100M1,4 ℃ of storages.
3. concentrate the glue damping fluid
5.98g the amino alkane (C of trishydroxymethyl
4H
11NO
3, Tris),, add N then, N, N ', N '-methyl ethylenediamine (C with 48Ml 1mol/L dissolving with hydrochloric acid
6H
16N
2, TEMED) 0.46Ml, water is settled to 100Ml, 4 ℃ of storages.
4. separation gel mother liquor
Take by weighing 28g acrylamide (C
3H
5NO, Acr), 0.735g methylene diacrylamide (C
7H
10N
2O
2, B is) and soluble in water, and be diluted to 100mL, with 4 ℃ of storages of brown bottle
5. concentrate the glue mother liquor
Take by weighing 10.0g acrylamide (C
3H
5NO, Acr), 2.5g methylene diacrylamide (C
7H
10N
2O
2, B is) and soluble in water, and be diluted to 100mL, with 4 ℃ of storages of brown bottle
6. electrode buffer
Amino alkane (the C of 6g trishydroxymethyl
4H
11NO
3, Tris), glycine (C
2H
2NO
2) 28.8g, soluble in water, and be diluted to 1L, dilution is 10 times during use.
7. phosphoric acid buffer
Take by weighing 2.26g Na
2HPO
4.12H
2O, 2.14gNaH
2PO
4.2H
2O, soluble in water, and be diluted to 100mL;
8. staining fluid
Take by weighing how ester (C of 50mg acetate-1-
12H
10O
2), 50mg acetate-2-is ester (C how
12H
10O
2), the solid blue RR salt (CHClNO.1/2ZnCl2) of 100mg is used the 5mL acetone solution, adds phosphoric acid buffer 150mL again, filters.
9. tetrabromophenol sulfonphthalein indicating liquid: tetrabromophenol sulfonphthalein 0.1g adds 0.05mol/L sodium hydroxide solution 3.0mL and makes it dissolving, is diluted with water to 100MmL.
Preparing gel and electrophoresis:
Separation gel is with the separation gel damping fluid, and the ammonium persulfate solution of separation gel mother liquor, water and 1.4g/L is pressed the 1+2+1+4 mixed, stirs, be separation gel, separation gel is poured into the glue chamber to short sheet glass upper edge 2.5cm～3.0cm, add suitable quantity of water and seal the glue face, treat after the gel polymerisation the water sucking-off.To concentrate the glue damping fluid, concentrate glue mother liquor, riboflavin solution and sucrose solution, stir, pour into the glue chamber, insert good sample comb, polymerization under daylight or the light immediately by the 1+2+1+4 mixed.After concentrating the glue polymerization, carefully extract sample comb out,, the injecting electrode damping fluid is drawn an amount of sample with microsyringe and is added in the sample well.With after electrophoresis apparatus is connected, opening power adopts the current stabilization electric current with electrophoresis chamber, and electric current is 1mA/cm～2mA/cm, treat that indicator enters separation gel after, electrophoresis chamber is put into refrigerator carries out the low temperature electrophoresis, and electric current is risen to 2～3mA/cm.When treating that the tetrabromophenol sulfonphthalein indicator is displaced downwardly to apart from the about 1cm in glue bottom, stop electrophoresis.Pour out electrode buffer, unload adhesive tape, breakdown sheet glass in the water takes out gel film, immerses in the staining fluid, and the 15～30min that dyes under the room temperature is with fixing in the rearmounted acetic acid solution of distilled water flushing (70mL/L).
Bacterial strain nutritive ingredient of the present invention is abundant, except that containing gal4 amino acid, also contains abundant vitamin-E, and content is up to 665mg/kg (dry product) (being measured by spectrum Buddhist nun test center); Pholiota adiosapose polysaccharide content height adopts the conventional water extraction of polysaccharide, and the mycelium polysaccharides yield is up to 23.41%, polysaccharide content 55.69%; Fruitbody polysaccharide yield 9.39%, polysaccharide content 41.89%, purified post analysis, a kind of mixed polysaccharide that pholiota adiosapose polysaccharide is made up of glucose, fructose, seminose and four kinds of polysaccharide of semi-lactosi.Prove that through experimentation on animals pholiota adiosapose polysaccharide has enhance immunity power, antitumor and removing oxyradical effect.Therefore, bacterial strain of the present invention or its extract can be made various protective foodss or beverage as raw material.
Description of drawings
Fig. 1 is the sporophore of the yellow agaric strain of the present invention;
Fig. 2 is an esterase isozyme picture, and T1～T9 is 9 repetitions.
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.
Embodiment 1
1, the female kind of yellow agaric strain cultivated
Mother culture media: potato 200g, glucose 20g, malt extract 2g, MgSO
40.2g, KH
2PO
40.25g, K
2HPO
40.25g, VB
10.5mg, agar powder 15g, water 1000ml, pH nature.
Culture temperature is 23～26 ℃, covers with the test tube slant about 10d.
2, original seed is cultivated
Cotton seed hull substratum: cotton seed hull 78%, wheat bran 20%, gypsum 1%, lime 1% or cotton seed hull 75%, lime 1%, gypsum 1%, sucrose 2%, urea 1%.
Mother's kind is transferred to the cotton seed hull substratum, and culture temperature is 23～25 ℃, relative air humidity 50～60%, and the well-ventilated, lucifuge is cultured to mycelia and covers with container.The general room temperature of original seed staging life, be no more than 15d, and low-temp storage is no more than 20d, and expired bacterial classification can not be worked as cultivar and be used.
3, cultivating bag composition of raw materials and mushroom producing culture
Prescription:
1. cotton seed hulls 88%, wheat bran 10%, gypsum 1%, calcium superphosphate 1%.
2. cotton seed hulls 68%, weed tree sawdust 20%, wheat bran 10%, gypsum 1%, calcium superphosphate 1%.
3. cotton seed hulls 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 5～10d.
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 10h, and the stewing 5h～6h that ceases fire takes out cooling inoculation with bag then.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 30～35d 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 6～10d after the mushroom producing room, 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, about each 1～2h, carry out in conjunction with weather condition, temperature is high early, ventilate to evening; Temperature is low, takes to ventilate when noon, temperature was high, constantly to replenish fresh air, gets rid of 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 7～10d 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%.
Embodiment 2 pholiota adiposa mycelium liquid fermentings
1. the mycelium liquid cultural method of the described bacterial strain of claim 1 is characterized in that: 1. liquid culture based formulas: promptly glucose 20g, yeast soak powder 8g, VITMAIN B1 300 μ g, Mg
2SO
40.4g, KH
2PO
41g, K
2HPO
41g, water 1000mL, pH nature.
The substratum packing: dress liquid nutrient medium 80mL in the 250mL triangular flask, through high pressure (0.14～0.15Mpa) sterilization 0.5h.
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 with diameter 0.5cm punch tool.Culture temperature: at 25 ℃; Under shaking speed 120r/min～140r/min condition, incubation time 12d.
3. fermentation culture: with the seed culture bacterial classification, the aseptic technique method is seeded in the fermentor tank, routinely fermentation culture.Fermentation condition: the substratum compositing formula is: glucose 3%, corn steep liquor 2%, potassium primary phosphate 0.2%, pH value 6.5～7.0.When agitator speed is 150～180r/min, air flow is 1.5 (V/Vmin).
The extraction of embodiment 3 pholiota adiosapose polysaccharides
1. pholiota adiosapose polysaccharide extracts
1. material pre-treatment mycelium freeze-drying that embodiment 1 is made, or sporophore is air-dry, is crushed to 140 orders; Material-water ratio 1:30, ultrasonic power 600W, treatment time 10min.
2. lixiviate is put into Backflow bottle and is extracted, and extraction time 3h, extracts 75 ℃ of temperature, the polysaccharide vat liquor;
3. alcohol is analysed the dehydrated alcohol that adds 3.5 times of amounts in the polysaccharide vat liquor, and alcohol is analysed 12h;
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%.
According to above-mentioned identical method dry sporophore is carried out polysaccharide and extract, the result shows, yellow umbrella Crude polysaccharides extraction rate reached to 9.39%, polysaccharide content 41.89%.
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.
Claims (5)
1. yellow umbrella (Pholiota adiosapose) the bacterial strain caas-11 of a strain is characterized in that its preserving number is CGMCC No.1840.
2. the mycelium liquid cultural method of the described bacterial strain of claim 1 is characterized in that:
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, surplus is a water, and autoclaving is standby;
2. seed culture: by the aseptic technique method, get female mycelium of planting, by inoculum size 1～5%V/V, in the liquid nutrient medium after the switching sterilization, culture temperature is 24～26 ℃; Under shaking speed 120r/min～140r/min condition, incubation time 10～14d;
3. fermentation culture: with the seed culture bacterial classification, adopt the aseptic technique method, be seeded in the fermentor tank by inoculum size 5～10%V/V, fermentation culture routinely, 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 0.8～2.0V/Vmin, fermentation culture 7～10d.
3. method as claimed in claim 2 is characterized in that, step 1. liquid culture based formulas is: 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 5.5～7, and surplus is a water.
4. method as claimed in claim 2 is characterized in that step is fermentation culture 3.: with the seed culture bacterial classification, the aseptic technique method is seeded in the fermentor tank, inoculum size is 8%V/V, and controlled temperature is 25 ℃, and agitator speed is 150～180r/min, air flow is 1.5V/Vmin, fermentation culture 8d, fermention medium is: glucose 30g/L, corn steep liquor 20g/L, potassium primary phosphate 2g/L, pH value 6.5～7.0.
5. the application of the described bacterial strain of claim 1 in the preparation protective foods.
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| CN102668885B (en) * | 2012-05-30 | 2013-06-05 | 北京市农林科学院 | Pholiota adipose new strain and method for cultivating fruiting body of pholiota adiposa new strain |
| CN103733873A (en) * | 2013-06-03 | 2014-04-23 | 鲁东大学 | Study on liquid pholiota adiposa strains and polysaccharide |
| CN103918481B (en) * | 2014-04-08 | 2016-04-06 | 德州学院 | Yellow umbrella liquid spawn mixes bacteria cultivation technique |
| CN105950479B (en) * | 2016-05-13 | 2019-05-03 | 广东省微生物研究所 | The novel bacterial and its cultural method of a kind of high polysaccharide and application |
| CN106613359B (en) * | 2016-12-30 | 2020-01-21 | 临沂大学 | Bottle cultivation fruiting culture method of cordyceps militaris liquid medium |
| CN109644775B (en) * | 2018-10-10 | 2021-03-19 | 黑龙江黑臻生物科技有限公司 | Morchella strain YD99 and cultivation method thereof |
| CN113637594B (en) * | 2021-10-18 | 2022-01-07 | 山东蓬勃生物科技有限公司 | Pholiota adiposa strain YX1, culture method and application thereof |
| CN115349401B (en) * | 2022-09-16 | 2023-10-20 | 绵阳市农业科学研究院 | Domestication cultivation method for wild Pingwu mushrooms |
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