CN104620855A - Pleurotus spodotecus strain, and culture medium, culture method and application of pleurotus spodotecus strain and a culture medium - Google Patents

Pleurotus spodotecus strain, and culture medium, culture method and application of pleurotus spodotecus strain and a culture medium Download PDF

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CN104620855A
CN104620855A CN201510106164.7A CN201510106164A CN104620855A CN 104620855 A CN104620855 A CN 104620855A CN 201510106164 A CN201510106164 A CN 201510106164A CN 104620855 A CN104620855 A CN 104620855A
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pleurotus
medium
long handle
parts
cgmcc
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CN104620855B (en
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王淑敏
宫诗林
齐放
谢兆丽
邓西贝
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Changchun University of Chinese Medicine
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Changchun University of Chinese Medicine
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor

Abstract

The invention relates to the field of fungus cultivation and the field of fungus liquid fermentation, and particularly relates to a pleurotus spodotecus strain, and a culture medium, a culture method and an application pleurotus spodotecus strain and a culture medium. The invention provides a pleurotus spodotecus strain of which the preservation number is CGMCC No.7359, provides a seed culture medium, a seed culture method and a cultivation method of the strain, and a culture medium, a liquid fermentation method and a culture medium, and further provides a hypha bacterium liquid (fermentative hyphae and a fermentation liquid) after liquid fermentation of the strain, and an application of metabolites of the hypha bacterium liquid. The strain provided by the invention has the advantages of good stability and high metabolic capability, and does not age or degenerate after 25-generation passage; the strain is cultivated by the cultivation method and a solid culture medium provided by the invention; and the obtained pleurotus spodotecus is high in yield, good in quality and high in biotransformation efficiency. The fermentative hyphae is prepared by the liquid culture medium and the fermentation method provided by the invention, and can grow rapidly, branches are uniform and few, and the metabolites are abundant.

Description

Long handle oyster mushroom strain and medium, cultural method and application
Technical field
The present invention relates to fungi cultivation field and fungi liquid fermentation arts, particularly relate to long handle oyster mushroom strain and medium, cultural method and application.
Background technology
Long handle is picked up the ears as the fruit body of Basidiomycetes, Agaricales, Pleurotaceae, Pleurotus Pleurtus spodotecus Fr..Another name is grey foliosous parnassia herb, edible.This bacterium encroaches on white poplar, white birch trees at occurring in nature, makes down wood, lumbering etc. form the rotten wood of white.Its fruit body meat, median size, scattered or grow thickly.The flat hemispherical of cap, gradually open and flat is fan-shaped, wide 3-10cm; Bacterium face is smooth, white, and central authorities are faint yellow, yellowish-brown after dry.Bacterial context is thick, soft, white, and stem is wilfully raw, closely cylindrical to side, middle reality, white, long 4-12cm, thick 0.5-1.5cm.Lamella white, prolongs life, slightly rare.Spore is colourless, smooth, cylindrical, 8-10.5 μm × 3-4 μm.Autumn grows thickly on broad-leaved tree is dried-up.Distribution Area in Jilin, Yunnan, Tibet etc.Long handle is picked up the ears meat exquisiteness, agreeable to taste, nutritious, fruit body dry product contains rich in protein, Thick many candies, raw fiber etc., also containing several amino acids, vitamin and mineral element, its Thick many candies have antitumor, improve effect of immunity, the pick up the ears Aqueous extracts of fruit body of long handle is 72% to the tumour inhibiting rate of small white mouse according to the literature.Long handle also contains the secondary metabolites such as ergosterol, Lovastatin, mannitol in picking up the ears.Ergosterol has anti-inflammatory and antitumaous effect; Lovastatin has the effect of reducing blood lipid and prevention tissue fibrosis; Mannitol has dehydration, diuresis.Mannitol can reduce intracranial pressure, intraocular pressure, prevent renal tubular necrosis etc.; Long handle polysaccharide of picking up the ears is immunopotentiator and immunoactivator, improves the immunity of human body, strengthens disease-resistant ability.
Pick up the ears to be a kind of cuisines due to long handle, have again certain drug action, the demand that existing market is picked up the ears to long handle is increasing, but long handle is picked up the ears, wild resource is less, and a large amount of harvestings have in recent years caused its germplasm resource to be on the verge of exhaustion simultaneously.Therefore; in order to develop new resources; maintaining ecological balance; accomplish both reserved resources; utilize resource again; utilize the biological property of fungi, absorb the feature of nourishing and generating by mycelium in growth and development process of picking up the ears in conjunction with long handle, carry out being separated, purifying and select excellent species and use it for cultivation that long handle picks up the ears or fermentation has important economic implications and social effect.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of long handle oyster mushroom strain and medium, cultural method and application.Long handle oyster mushroom strain provided by the invention grows soon, cultivates the fruiting body yield obtained high, and the mycelia metabolite obtained that ferments enriches.
The invention provides a kind of long handle Pleurotus sp2, its deposit number is CGMCC No.7359.
Separation, purifying, screening are passed through from the long handle that Zhu Cheng township of Dehui City of Jilin Province willow withered tree grows picks up the ears fruit body by bacterial strain system provided by the invention, and the bacterial strain of the stable performance obtained by systematic breeding.Long handle oyster mushroom strain bacterium colony provided by the invention is in white, and mycelia is that white cotton is cotton-shaped, dense pure white.Mycelial growth rate of development is fast, and suitable condition can cover with full inclined-plane in lower 4 days.After Shaking culture or seed tank culture, bacterium liquid is clear and bright, and yellowish-brown, mycelia is spheroid radially, has denseer fruit aroma.By 16 × 100 times of biology microscope spectroscopy, mycelia is faint yellow, sturdy, and evenly, branch is many, and clamp connection is obviously visible, without living contaminants.Long handle oyster mushroom strain provided by the invention has good stability, and metabolic capability is high, and safety is high, in its 25 generations of going down to posterity, have no aging or degenerate, and fermented incubation time is short, and cultivating and growing speed is fast, its secondary metabolites content is high, has good edible and medical value.Experiment proves, after the fermented hypha drying that fermented and cultured obtains, the pick up the ears mass fraction of polysaccharide of long handle is no less than 3%; Lovastatin content is no less than 188.00 μ g/g; Quantitative Determination of Ergosterol is no less than 70.00 μ g/g.
Deposit number provided by the invention be the fungi preservation of the long handle Pleurotus sp2 of CGMCC No.7359 in 2 DEG C ~ 4 DEG C, the long-term bacterial classification preserved, must at about 180 days tubes once.As thin in found the bacterium colony of bacterial classification, white flock mycelia is very thin, darken, and illustrates that bacterial classification is aging, then needs to carry out rejuvenation of spawn.
The method of rejuvenation of spawn is that bacterial classification substrate mycelium is separated rejuvenation, bridge cutoff type detoxification technology or changes the method rejuvenation bacterial classification of bacterium culture medium.
Wherein, bacterial classification substrate mycelium is separated rejuvenation: select pollution-free, eugonic bacterial classification, with dissecting needle picking tip mycelia, be seeded on PDA medium, and in 25 DEG C of incubators, lucifuge is cultivated.
Bridge cutoff type detoxification technology: prepare PDA medium with conventional method, after culture medium solidifying, aseptically, with inoculating rake, medium thinner foremost for medium slant being disconnected about 1 centimetre, mother waiting being planted and is connected on the middle medium of test tube, when mycelia grows to gap, then can continue to extend, pass through gap, stretch on the fritter medium of front end, extend again through fracture, mycelia seems especially sturdy, strong.During switching, picking fritter medium one piece of tissue foremost, inoculates routinely, cultivates like this, just can get rid of virus through 3-4 most advanced and sophisticated picking.
Change the method rejuvenation bacterial classification of bacterium culture medium: bacterial classification PDA medium is changed into potato synthetic medium (glucose 2%, potato 20%, agar 2%, KH 2sO 40.3%, MgSO 40.15%, VB10.001%, pH nature) or change perfect medium (glucose 2%, peptone 0.2%, agar 1.5%, KH into 2sO 40.046%, MgSO 40.05%, pH nature) cultivate.Select pollution-free, eugonic bacterial classification, with dissecting needle picking tip mycelia, be seeded on PDA medium, in 25 DEG C of incubators, lucifuge is cultivated.
Deposit number provided by the invention is the medium in the fungi preservation process of the long handle Pleurotus sp2 of CGMCC No.7359 is PDA medium.
In PDA medium, the mass fraction of each component is: glucose 2%, potato 20%, agar 2%, pH nature.
Deposit number provided by the invention is that the bacterial classification of the long handle Pleurotus sp2 of CGMCC No.7359 is for cultivating or before fermentation tank culture, first need preparing seed.
The invention provides the seed culture medium that deposit number is the long handle Pleurotus sp2 of CGMCC No.7359, comprise slant medium, Shake flask medium, seed tank culture base;
Wherein, slant medium is PDA medium;
Shake flask medium comprises the component of following mass fraction: 2.5% ~ 3.5% carbon source, 1% ~ 2% nitrogenous source, 0.1% ~ 0.15%KH 2sO 4, 0.1% ~ 0.15%MgSO 4, natural ph;
Seed tank culture base comprises the component of following mass fraction: 1% ~ 4% carbon source, 0.2% ~ 4% nitrogenous source and 0.1% ~ 0.15%KH 2pO 4, natural ph;
In Shake flask medium, carbon source is sucrose or glucose; Nitrogenous source is peptone or dusty yeast;
In seed tank culture base, carbon source is a kind of in sucrose, glucose or corn flour or both above mixtures; Nitrogenous source is a kind of in wheat bran juice, peptone, dusty yeast or beancake powder or both above mixtures.
In certain embodiments, Shake flask medium comprises the component of following mass fraction: sucrose 2% ~ 3%, peptone 1% ~ 2%, KH 2sO 40.1% ~ 0.15%, 0.1% ~ 0.15%MgSO 4, natural ph.
In certain embodiments, Shake flask medium comprises the component of following mass fraction: glucose 2% ~ 3%, dusty yeast 1% ~ 2%, KH 2sO 40.1% ~ 0.15%, 0.1% ~ 0.15%MgSO 4, natural ph.
In certain embodiments, Shake flask medium comprises the component of following mass fraction: sucrose 3%, peptone 2%, KH 2sO 40.1%, MgSO 40.1%, natural ph.
In certain embodiments, seed tank culture base comprises the component of following mass fraction: sucrose 1% ~ 3%, wheat bran juice 1% ~ 3%, KH 2pO 40.1% ~ 0.15%, natural ph.
In certain embodiments, seed tank culture base comprises the component of following mass fraction: glucose 1% ~ 3%, peptone 0.2% ~ 2%, KH 2pO 40.1% ~ 0.15%, natural ph.
In certain embodiments, seed tank culture base comprises the component of following mass fraction: corn flour 2% ~ 4%, dusty yeast 0.2% ~ 2%, KH 2pO 40.1% ~ 0.15%, natural ph.
In certain embodiments, seed tank culture base comprises the component of following mass fraction: corn flour 2% ~ 4%, beancake powder 2% ~ 4%, KH 2pO 40.1% ~ 0.15%, natural ph.
In certain embodiments, seed tank culture base comprises the component of following mass fraction: sucrose 3%, wheat bran juice 2%, KH 2pO 40.1%, natural ph.
Present invention also offers the seed culture method that deposit number is the long handle Pleurotus sp2 of CGMCC No.7359, comprise the following steps:
Step 1: by deposit number be the strain inoculation of CGMCC No.7359 in slant medium provided by the invention, cultivate 90h ~ 96h for 25 DEG C ± 3 DEG C, obtain inclined-plane mycelia;
Step 2: by inclined-plane mycelium inoculation in Shake flask medium provided by the invention, 25 DEG C ± 3 DEG C, rotating speed 160r/min ~ 220r/min, cultivates 140h ~ 150h, obtains shaking flask bacterium liquid;
Step 3: shaking flask bacterium liquid is inoculated in seed tank culture base provided by the invention, cultivates 100h ~ 150h for 25 DEG C ± 3 DEG C, obtains the long handle Pleurotus sp2 seed that deposit number is CGMCC No.7359.
As preferably, the temperature of cultivating in step 1 is 25 DEG C ± 1 DEG C.
In certain embodiments, inclined-plane mycelia can in 2 DEG C ~ 4 DEG C preservations before being inoculated in Shake flask medium.
In certain embodiments, the holding time of inclined-plane mycelia is not more than 30d.
As preferably, in step 2, the ratio of inclined-plane mycelia and Shake flask medium is 1:6.
Concrete, in step 2, each slant medium inoculates 6 shaking flasks.
As preferably, the temperature of cultivating in step 2 is 25 DEG C ± 1 DEG C, and the time is 144h.
As preferably, cultivate through step 2, in often liter of zymotic fluid, dry mycelial weight is not less than 12g.
Cultivate through step 2, bacterium liquid is clear and bright, yellowish-brown, and mycelia is spheroid radially, has denseer fruit aroma, without living contaminants.
As preferably, in step 3, the mass ratio of shaking flask bacterium liquid and seed tank culture base is 1:10.
As preferably, the condition of cultivating in step 3 is for passing into filtrated air, and throughput is 1:1v/vmin.
As preferably, the temperature of cultivating in step 2 is 25 DEG C ± 1 DEG C, and the time is 120h.
As preferably, cultivate through step 3, in often liter of zymotic fluid, dry mycelial weight is not less than 15g, and pH value is 5.5 ~ 6, and the mass fraction of reducing sugar is 3% ~ 5%, and the mass fraction of amino nitrogen is 10% ~ 20%.
Cultivate through step 3, microscopy mycelia is even, and sturdy, branch is many, and clamp connection is obviously visible; Zymotic fluid is yellowish-brown, clear and bright, has light fruit aroma; Without living contaminants.
Adopt the present invention to carry common method activation culture seed, the Seed Vitality obtained is strong, and active high, growing way is good, and metabolite is many.
Deposit number is a solid culture medium for the handle Pleurotus sp2 of CGMCC No.7359, comprises the component of following mass parts: carbon source 60 parts ~ 90 parts, 5 parts ~ 38 parts, nitrogenous source, 0 part ~ 2 parts, gypsum, sucrose 0 part ~ 2 parts, 0 part ~ 2 parts, lime;
Carbon source is a kind of in rice straw powder, corn stalk powder, wheat straw powder, rice straw powder, maize cob meal, wood sawdust or cotton seed hulls or both above combinations;
Nitrogenous source is a kind of in wheat bran, rice bran or beancake powder or both above combinations.
In certain embodiments, solid culture medium comprises the component of following mass parts: 80 parts ~ 90 parts, rice straw powder, wheat bran 8 parts ~ 16 parts, 1 part ~ 2 parts, gypsum, sucrose 1 part ~ 2 parts.
In certain embodiments, solid culture medium comprises the component of following mass parts: corn stalk powder 80 parts ~ 90 parts, 8 parts ~ 16 parts, rice bran, 1 part ~ 2 parts, gypsum, sucrose 1 part ~ 2 parts.
In certain embodiments, solid culture medium comprises the component of following mass parts: 80 parts ~ 90 parts, wheat straw powder, 8 parts ~ 16 parts, wheat bran, 1 part ~ 2 parts, gypsum, sucrose 1 part ~ 2 parts.
In certain embodiments, solid culture medium comprises the component of following mass parts: rice straw powder 80 parts ~ 90 parts, 10 parts ~ 20 parts, wheat bran.
In certain embodiments, solid culture medium comprises the component of following mass parts: rice straw powder 80 parts ~ 90 parts, 10 parts ~ 20 parts, rice bran.
In certain embodiments, solid culture medium comprises the component of following mass parts: rice straw powder 90 parts ~ 95 parts, beancake powder 5 parts ~ 10 parts.
In certain embodiments, solid culture medium comprises the component of following mass parts: maize cob meal 60 parts ~ 70 parts, 30 parts ~ 38 parts, wheat bran, 1 part ~ 2 parts, gypsum.
In certain embodiments, solid culture medium comprises the component of following mass parts: maize cob meal 60 parts ~ 70 parts, 30 parts ~ 38 parts, rice bran, 1 part ~ 2 parts, gypsum.
In certain embodiments, solid culture medium comprises the component of following mass parts: wood sawdust 75 parts ~ 80 parts, 16 parts ~ 21 parts, wheat bran, 1 part ~ 2 parts, gypsum, sucrose 1 part ~ 2 parts.
In certain embodiments, solid culture medium comprises the component of following mass parts: wood sawdust 75 parts ~ 80 parts, 16 parts ~ 21 parts, rice bran, 1 part ~ 2 parts, gypsum, sucrose 1 part ~ 2 parts.
In certain embodiments, solid culture medium comprises the component of following mass parts: cotton seed hulls 80 parts ~ 90 parts, 8 parts ~ 16 parts, wheat bran, 1 part ~ 2 parts, gypsum, sucrose 1 part ~ 2 parts.
In certain embodiments, solid culture medium comprises the component of following mass parts: cotton seed hulls 80 parts ~ 90 parts, 8 parts ~ 16 parts, rice bran, 1 part ~ 2 parts, gypsum, sucrose 1 part ~ 2 parts.
In certain embodiments, solid culture medium comprises the component of following mass parts: rice straw powder 87 parts, 10 parts, wheat bran, 2 parts, gypsum, sucrose 2 parts.
Present invention also offers the cultivation method that deposit number is the long handle Pleurotus sp2 of CGMCC No.7359, for cultivating the seed that deposit number is the long handle Pleurotus sp2 of CGMCC No.7359, be inoculated in solid culture medium provided by the invention, after sending out bacterium, sporophore growth, gathers and obtains long handle Pleurotus sp2;
The condition sending out bacterium is: medium humidity 55% ~ 65%, relative air humidity 60% ~ 70%, temperature 20 DEG C ~ 30 DEG C;
The condition of sporophore growth is: medium humidity 55% ~ 65%, relative air humidity 85% ~ 95%, temperature 7 DEG C ~ 20 DEG C.
In certain embodiments, the condition sending out bacterium is: medium humidity 55% ~ 65%, relative air humidity 60% ~ 70%, temperature 24 DEG C ~ 26 DEG C.
In certain embodiments, the condition of sporophore growth is: medium humidity 55% ~ 65%, relative air humidity 85% ~ 95%, temperature 10 DEG C ~ 20 DEG C.
In certain embodiments, deposit number is the seed of the long handle Pleurotus sp2 of CGMCC No.7359 and the volume-mass ratio of solid culture medium is 1:10.
That is, every 100g solid culture medium inoculation is through the invention provides seed (culture fluid) 10mL that the deposit number of method activation is the long handle Pleurotus sp2 of CGMCC No.7359.
In certain embodiments, the time sending out bacterium is 19 ~ 21 days.
In certain embodiments, the time of sporophore growth is 10 ~ 20 days.
In certain embodiments, in the condition of sporophore growth, temperature is: daytime 20 DEG C ~ 22 DEG C, night 10 DEG C ~ 12 DEG C.The fruit body cap produced is pure white, and quality is tender and crisp and plump, and can receive 4-5 tide mushroom, total biological transformation ratio can reach 150%.
When mushroom lid expansion degree reaches eighty per cant, cap edge do not have complete open and flat time, gather in time.
Practice shows, adopts solid culture medium provided by the invention and adopts cultivation method provided by the invention, and gained long handle is picked up the ears, and quality is good, output is high, biological transformation ratio is higher, and cap white is to canescence, and quality is tender and crisp and plump.
The invention provides the liquid nutrient medium that deposit number is the long handle Pleurotus sp2 of CGMCC No.7359, it is characterized in that, comprising: carbon source 1% ~ 4%, nitrogenous source 0.2% ~ 4%, KH 2pO 40.1% ~ 0.15%, natural ph;
Carbon source is a kind of in sucrose, glucose or corn flour or both above combinations;
Nitrogenous source is a kind of in wheat bran juice, peptone, dusty yeast or beancake powder or both above combinations.
In certain embodiments, liquid nutrient medium comprises the component of following mass fraction: sucrose 1% ~ 3%, wheat bran juice 1% ~ 3%, KH 2pO 40.1% ~ 0.15%, natural ph.
In certain embodiments, liquid nutrient medium comprises the component of following mass fraction: glucose 1% ~ 3%, peptone 0.2% ~ 2%, KH 2pO 40.1% ~ 0.15%, natural ph.
In certain embodiments, liquid nutrient medium comprises the component of following mass fraction: corn flour 2% ~ 4%, dusty yeast 0.2% ~ 2%, KH 2pO 40.1% ~ 0.15%, natural ph.
In certain embodiments, liquid nutrient medium comprises the component of following mass fraction: corn flour 2% ~ 4%, beancake powder 2% ~ 4%, KH 2pO 40.1% ~ 0.15%, natural ph.
In certain embodiments, liquid nutrient medium comprises the component of following mass fraction: sucrose 3%, wheat bran juice 2%, KH 2pO 40.1%, natural ph.
Present invention also offers the liquid fermentation method that deposit number is the long handle Pleurotus sp2 of CGMCC No.7359: cultivate the seed that deposit number is the long handle Pleurotus sp2 of CGMCC No.7359, be inoculated in liquid nutrient medium provided by the invention, after sterilizing, 100h ~ 150h is cultivated with 25 DEG C ± 3 DEG C, put tank, obtain zymocyte liquid;
The condition of cultivating is: pass into filtrated air, throughput is 1:1v/vmin, tank pressure 0.5kg/cm 2.In certain embodiments, deposit number is the seed of the long handle Pleurotus sp2 of CGMCC No.7359 and the mass ratio of liquid nutrient medium is 1:(8 ~ 12).
That is, every 100mL liquid nutrient medium inoculation is through the invention provides seed (culture fluid) 8 ~ 12mL that the deposit number of method activation is the long handle Pleurotus sp2 of CGMCC No.7359.
As preferably, liquid nutrient medium is aseptic liquid nutrient medium.
In certain embodiments, the temperature of cultivation is 24 DEG C ± 1 DEG C, and the time is 120h.
In certain embodiments, sterilizing adopts autoclaving.
As preferably, the condition of sterilizing is: pressure 1.3kg/cm 2~ 1.5kg/cm 2, time 1h.
Deposit number provided by the invention be the long handle Pleurotus sp2 of CGMCC No.7359 after liquid fermentation, the zymocyte liquid of generation comprises fermented hypha and zymotic fluid, and wherein mycelia becomes emitting shape spherical, and bacterium liquid is in yellow clear and bright.
In certain embodiments, the pH value of inoculation wild Oryza species is natural ph, and through cultivating 100h ~ 150h, the pH value of zymocyte liquid is 5.5 ~ 6, and wherein the mass fraction of reducing sugar is 2% ~ 3%, and the mass fraction of amino nitrogen is not more than 20%.
Adopt liquid nutrient medium provided by the invention and fermentation process to prepare fermented hypha, gained mycelial growth is rapid, and biomass is large, and metabolite enriches.After fermentation ends, dry mycelial weight is not less than 12g/L; By 16 × 100 times of biology microscope spectroscopy, mycelia is even, and more carefully, branch reduces, and clamp connection is rare, accidental cavity; Bacterium liquid is clear and bright, and yellow, mycelium is radially spherical, has light fruit aroma.After fermented hypha drying, " long handle pick up the ears polysaccharide " content is no less than 3%; Lovastatin content is no less than 188.00 μ g/g; Quantitative Determination of Ergosterol is no less than 70.00 μ g/g.
Zymocyte liquid is preparing the application in food, drink, medicine or health products.
The pick up the ears preparation method of beverage of long handle is: the long handle Pleurotus sp2 fermented hypha being CGMCC No.7359 by the deposit number that obtains the invention provides method, water extraction is utilized to get, extract and zymotic fluid merge, and mix with water, citric acid, sucrose, obtain long handle to pick up the ears beverage through homogeneous.
The pick up the ears preparation method of health products of long handle is: the long handle Pleurotus sp2 fermented hypha being CGMCC No.7359 by the deposit number that obtains the invention provides method and zymotic fluid dry, after pulverizing, mix with auxiliary material acceptable on health products, compressing tablet obtains long handle and to pick up the ears health products.
The pick up the ears preparation method of food of long handle is: after the long handle Pleurotus sp2 fermented hypha being CGMCC No.7359 by the deposit number obtained the invention provides method and zymotic fluid dry, pulverize, mix with honey, and obtained long handle is picked up the ears food.
The pick up the ears preparation method of medicine of long handle is: the long handle Pleurotus sp2 fermented hypha being CGMCC No.7359 by the deposit number that obtains the invention provides method is dry, extracts ergosterol, mixes with pharmaceutically acceptable auxiliary material, and obtained long handle is picked up the ears medicine.
The pick up the ears preparation method of medicine of long handle is: the long handle Pleurotus sp2 fermented hypha being CGMCC No.7359 by the deposit number obtained the invention provides method drying mixes with pharmaceutically acceptable auxiliary material, and obtained long handle is picked up the ears medicine.
The invention provides the long handle oyster mushroom strain that deposit number is CGMCC No.7359, and provide the seed culture medium of this bacterial strain, seed culture method, cultivation method and medium, liquid fermentation method and medium, and the purposes of mycelia and zymotic fluid after providing the fermentation of this strain liquid.Bacterial classification provided by the invention has good stability, and metabolic capability is strong, and 25 generations of going down to posterity have no aging or degenerate.Adopt cultivation method provided by the invention and solid culture medium to cultivate, gained long handle output of picking up the ears is high, and quality better, biological transformation ratio is high.Adopt liquid nutrient medium provided by the invention and fermentation process to prepare fermented hypha, mycelial growth is rapid, and the even branch of mycelia is few, and metabolite enriches.
Biological deposits explanation
Long handle Pleurotus sp2 Pspo-1, Classification And Nomenclature: Pleurtus spodotecus, be deposited on 03 20th, 2013 and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.Deposit number is CGMCC No.7359.
Embodiment
The invention provides long handle oyster mushroom strain and medium, cultural method and application, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
The raw material that the present invention adopts is all common commercially available product, all can be buied by market.
Below in conjunction with embodiment, set forth the present invention further:
The production of embodiment 1 seed
(1) slant strains:
Medium: PDA medium glucose 2%, potato 20%, agar 2%, pH nature.
Be that the long handle of CGMCC No.7359 bacterial classification of picking up the ears is inoculated in PDA medium, cultivation temperature 23 DEG C ~ 25 DEG C by deposit number, within 90 ~ 96 hours, mycelia covers with inclined-plane, puts refrigerator (2 ~ 4 DEG C) and saves backup.
(2) second-level shake flask bacterial classification
Medium: sucrose 3%, peptone 2%, KH 2sO 40.1%, MgSO 40.1%, natural ph.Sterilizing is for subsequent use.
Get that cell age is short, the sturdy dense slant strains of mycelia, aseptically, access sterilized Shake flask medium (in 1:6 ratio, namely 1 inclined-plane connects 6 shaking flasks), 23 DEG C ~ 25 DEG C temperature controls are cultivated, shaking flask rotating speed is 160 ~ 220r/min (rotary), when the every 100ml of zymotic fluid obtains more than dry mycelial weight 1.2g; Microscopy reaches standard under bacterial classification item, and sense of taste has light fruit aroma, and without living contaminants, incubation time is 144 hours, can proceed to seeding tank fermented and cultured.
(3) three grades of seeding tank bacterial classifications
Medium: sucrose 3%, wheat bran juice 2%, KH 2pO 40.1%, natural ph.Sterilizing is for subsequent use.
Get fresh shaking flask bacterial classification, in 10% ratio, aseptically access in sterilized seed tank culture base, 23 DEG C ~ 25 DEG C temperature controls are cultivated, and pass into filtrated air and stir, throughput is 1:1v/vmin.Fermentation time is 120 hours.
The production of embodiment 2 seed
(1) slant strains:
Medium: PDA medium glucose 2%, potato 20%, agar 2%, pH nature.
Be that the long handle of CGMCC No.7359 bacterial classification of picking up the ears is inoculated in PDA medium, cultivation temperature 23 DEG C ~ 28 DEG C by deposit number, within 90 ~ 96 hours, mycelia covers with inclined-plane, puts refrigerator (2 ~ 4 DEG C) and saves backup.
(2) second-level shake flask bacterial classification
Medium: sucrose 2% ~ 3%, peptone 1% ~ 2%, KH 2sO 40.1% ~ 0.15%, 0.1% ~ 0.15%MgSO 4, natural ph.Sterilizing is for subsequent use.
Get that cell age is short, the sturdy dense slant strains of mycelia, aseptically, access sterilized Shake flask medium (in 1:6 ratio, namely 1 inclined-plane connects 6 shaking flasks), 23 DEG C ~ 28 DEG C temperature controls are cultivated, shaking flask rotating speed is 160 ~ 220r/min (rotary), when the every 100ml of zymotic fluid obtains more than dry mycelial weight 1.2g; Microscopy reaches standard under bacterial classification item, and sense of taste has light fruit aroma, and without living contaminants, incubation time is 140 hours, can proceed to seeding tank fermented and cultured.
(3) three grades of seeding tank bacterial classifications
Medium: sucrose 1% ~ 3%, wheat bran juice 1% ~ 3%, KH 2pO 40.1% ~ 0.15%, natural ph.Sterilizing is for subsequent use.
Get fresh shaking flask bacterial classification, in 10% ratio, aseptically access in sterilized seed tank culture base, 23 DEG C ~ 28 DEG C temperature controls are cultivated, and pass into filtrated air and stir, throughput is 1:1v/vmin.Fermentation time is 150 hours.
The production of embodiment 3 seed
(1) slant strains:
Medium: PDA medium glucose 2%, potato 20%, agar 2%, pH nature.
Be that the long handle of CGMCC No.7359 bacterial classification of picking up the ears is inoculated in PDA medium, cultivation temperature 22 DEG C ~ 25 DEG C by deposit number, within 90 ~ 96 hours, mycelia covers with inclined-plane, puts refrigerator (2 ~ 4 DEG C) and saves backup.
(2) second-level shake flask bacterial classification
Medium: glucose 2% ~ 3%, dusty yeast 1% ~ 2%, KH 2sO 40.1% ~ 0.15%, 0.1% ~ 0.15%MgSO 4, natural ph.Sterilizing is for subsequent use.
Get that cell age is short, the sturdy dense slant strains of mycelia, aseptically, access sterilized Shake flask medium (in 1:6 ratio, namely 1 inclined-plane connects 6 shaking flasks), 22 DEG C ~ 25 DEG C temperature controls are cultivated, shaking flask rotating speed is 160 ~ 220r/min (rotary), when the every 100ml of zymotic fluid obtains more than dry mycelial weight 1.2g; Microscopy reaches standard under bacterial classification item, and sense of taste has light fruit aroma, and without living contaminants, incubation time is 150 hours, can proceed to seeding tank fermented and cultured.
(3) three grades of seeding tank bacterial classifications
Medium: corn flour 2% ~ 4%, beancake powder 2% ~ 4%, KH 2pO 40.1% ~ 0.15%, natural ph.Sterilizing is for subsequent use.
Get fresh shaking flask bacterial classification, in 10% ratio, aseptically access in sterilized seed tank culture base, 22 DEG C ~ 25 DEG C temperature controls are cultivated, and pass into filtrated air and stir, throughput is 1:1v/vmin.Fermentation time is 100 hours.
Embodiment 4 seed quality is identified
Seed liquor prepared by Example 1 ~ 3, by 16 × 100 times of biology microscope spectroscopy, observes zymotic fluid situation, weighs dry mycelial weight, and detects the quality of reducing sugar in zymotic fluid, amino nitrogen content qualification gained seed.Result is as shown in table 1:
Table 1 seed quality qualification result
Visible, the seed that the embodiment of the present invention 1 provides is the most healthy and the strongest, active high.
The cultivation that embodiment 5 ~ 9 long handle is picked up the ears
Example 1 obtains seed, picks up the ears to cultivate to long handle with solid culture medium shown in table 2.Inoculum concentration is: 10ml seed liquor is inoculated in 100g medium.Send out bacterium condition of culture: humidity 55%-65%, cultivation temperature 20 DEG C ~ 30 DEG C.Relative air humidity 60% ~ 70%.
Sporophore growth condition: suitably strengthen day and night temperature, cultivation temperature 7 DEG C ~ 20 DEG C.Relative air humidity 85% ~ 95%.Gather: when mushroom lid expansion degree reaches eighty per cant, cap edge is completely not open and flat, gathers in time.
Table 2 embodiment 5 ~ 9 medium
Culture medium prescription
Embodiment 5 Rice straw powder 870g, wheat bran 100g, gypsum 20g, sucrose 20g
Embodiment 6 Rice straw powder 850g, wheat bran 120g, gypsum 15g, sucrose 15g
Embodiment 7 Rice straw powder 850g, wheat bran 150g
Embodiment 8 Maize cob meal 650g, rice bran 340g, gypsum 15g
After gathering, investigate quality, output and biological transformation ratio that embodiment 5 ~ 8 gained long handle is picked up the ears.Result is as shown in table 3:
Biological transformation ratio (%)=fruit body fresh weight (g)/composts or fertilisers of cultivating dry weight (g) × 100%
Table 3 embodiment 5 ~ 8 gained long handle is picked up the ears quality (1000g composts or fertilisers of cultivating)
Quality Fruit body fresh weight (g) Biological transformation ratio (%)
Embodiment 5 Cap white, quality is tender and crisp and plump 1400～1500 140～150
Embodiment 6 Cap white, quality is tender and crisp and plump 1400～1500 140～150
Embodiment 7 Cap white, quality is tender and crisp and plump 1300～1400 130～140
Embodiment 8 Cap white, quality is tender and crisp and plump 1300～1500 130～150
As shown in Table 3, embodiment 5 gained long handle is picked up the ears best in quality, and output is the highest.
The liquid fermentation that embodiment 10 ~ 12 long handle is picked up the ears
Seed prepared by Example 1, in 10% ratio, aseptically access in sterilized liquid nutrient medium, liquid nutrient medium is as shown in table 4.25 DEG C ± 3 DEG C temperature controls are cultivated, and pass into filtrated air and stir, throughput is 1:1v/vmin, tank pressure: 0.5kg/cm 2.Fermentation 100 ~ 150h, puts tank.Detect long handle in the dry weight of mycelia in often liter of zymotic fluid, dry mycelia to pick up the ears the mass fraction of polysaccharide, in dry mycelia, the content of Lovastatin and ergosterol, the results are shown in Table 5.
Table 4 embodiment 10 ~ 12 medium
culture medium prescription
embodiment 10 sucrose 3%, wheat bran juice 2%, KH 2pO 40.1%, natural ph
embodiment 11 corn flour 3%, beancake powder 3%, KH 2pO 40.1%, natural ph
embodiment 12 glucose 2%, peptone 1%, KH 2pO 40.1%, natural ph.
Table 5 Quality Detection result
The liquid fermentation that comparative example 1 ~ 2 long handle is picked up the ears
Seed prepared by Example 1, in 10% ratio, aseptically access in sterilized liquid nutrient medium, liquid nutrient medium is as shown in table 5.25 DEG C ± 3 DEG C temperature controls are cultivated, and pass into filtrated air and stir, throughput is 1:1v/vmin, tank pressure: 0.5kg/cm 2.Fermentation 100 ~ 150h, puts tank.Detect long handle in the dry weight of mycelia in often liter of zymotic fluid, dry mycelia to pick up the ears the mass fraction of polysaccharide, in dry mycelia, the content of Lovastatin and ergosterol, the results are shown in Table 6.
Table 6 comparative example 1 ~ 2 medium
Culture medium prescription
Comparative example 1 Lactose 2%, urea 2%, NaH 2PO 40.1％、MgSO 40.15%, natural ph
Comparative example 2 Wood sugar 2%, ammonium nitrate 1%, NaH 2PO 40.15％，MgSO 40.15%, natural ph
Table 7 Quality Detection result
Embodiment 13 long handle is picked up the ears the preparation of beverage
The fermented hypha that Example 10 is obtained, utilizes water extraction to get, and extract and zymotic fluid merge, and mix with water, citric acid, sucrose, obtains long handle to pick up the ears beverage through homogeneous.
Embodiment 14 long handle is picked up the ears the preparation of health products
After the obtained zymocyte liquid (fermented hypha and zymotic fluid) of Example 12 dry, pulverize, mix with auxiliary material acceptable on health products, compressing tablet obtains long handle and to pick up the ears health products.
Embodiment 15 long handle is picked up the ears the preparation of food
After the obtained zymocyte liquid (fermented hypha and zymotic fluid) of Example 13 dry, pulverize, mix with honey, obtained long handle is picked up the ears food.
Embodiment 16 long handle is picked up the ears the preparation of medicine
The obtained fermented hypha of Example 13 is dry, extracts ergosterol (or Lovastatin, mannitol etc.), mixes with pharmaceutically acceptable auxiliary material, and obtained long handle is picked up the ears medicine.
Embodiment 17 long handle is picked up the ears the preparation of medicine
The zymocyte liquid (fermented hypha and zymotic fluid) that Example 13 is obtained, dry, mix with pharmaceutically acceptable auxiliary material, obtained long handle is picked up the ears medicine.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (10)

1. a long handle Pleurotus sp2, is characterized in that, its deposit number is CGMCC No.7359.
2. deposit number is a seed culture medium for the long handle Pleurotus sp2 of CGMCC No.7359, it is characterized in that, comprises slant medium, Shake flask medium, seed tank culture base;
Wherein, described slant medium is PDA medium;
Described Shake flask medium comprises the component of following mass fraction: 2% ~ 3% carbon source, 1% ~ 2% nitrogenous source, 0.1% ~ 0.15%KH 2sO 4, 0.1% ~ 0.15%MgSO 4, natural ph;
Described seed tank culture base comprises the component of following mass fraction: 1% ~ 4% carbon source, 0.2% ~ 4% nitrogenous source and 0.1% ~ 0.15%KH 2pO 4, natural ph;
In described Shake flask medium, described carbon source is sucrose or glucose; Described nitrogenous source is peptone or dusty yeast;
In described seed tank culture base, described carbon source is a kind of in sucrose, glucose or corn flour or both above mixtures; Described nitrogenous source is a kind of in wheat bran juice, peptone, dusty yeast or beancake powder or both above mixtures.
3. deposit number is a seed culture method for the long handle Pleurotus sp2 of CGMCC No.7359, it is characterized in that, comprises the following steps:
Step 1: by deposit number be the strain inoculation of CGMCC No.7359 in slant medium as claimed in claim 2, cultivate 90h ~ 96h for 25 DEG C ± 3 DEG C, obtain inclined-plane mycelia;
Step 2: by described inclined-plane mycelium inoculation in Shake flask medium as claimed in claim 2,25 DEG C ± 3 DEG C, rotating speed 160r/min ~ 220r/min, cultivates 140h ~ 150h, obtains shaking flask bacterium liquid;
Step 3: described shaking flask bacterium liquid is inoculated in seed tank culture base as claimed in claim 2, cultivates 100h ~ 150h for 25 DEG C ± 3 DEG C, obtains the long handle Pleurotus sp2 seed that deposit number is CGMCC No.7359.
4. a deposit number is the solid culture medium of the long handle Pleurotus sp2 of CGMCC No.7359, it is characterized in that, comprise the component of following mass parts: carbon source 60 parts ~ 90 parts, 5 parts ~ 38 parts, nitrogenous source, 0 part ~ 2 parts, gypsum, sucrose 0 part ~ 2 parts, 0 part ~ 2 parts, lime;
Described carbon source is a kind of in rice straw powder, corn stalk powder, wheat straw powder, rice straw powder, maize cob meal, wood sawdust or cotton seed hulls or both above combinations;
Described nitrogenous source is a kind of in wheat bran, rice bran or beancake powder or both above combinations.
5. a deposit number is the cultivation method of the long handle Pleurotus sp2 of CGMCC No.7359, it is characterized in that, cultivate the seed that deposit number is the long handle Pleurotus sp2 of CGMCC No.7359, be inoculated in solid culture medium as claimed in claim 4, after sending out bacterium, sporophore growth, gathers and obtains long handle Pleurotus sp2;
The condition of described bacterium is: medium humidity 55% ~ 65%, relative air humidity 60% ~ 70%, temperature 20 DEG C ~ 30 DEG C;
The condition of described sporophore growth is: medium humidity 55% ~ 65%, relative air humidity 85% ~ 95%, temperature 7 DEG C ~ 24 DEG C.
6. cultivation method according to claim 5, is characterized in that, in the condition of described sporophore growth, temperature is: daytime 20 DEG C ~ 22 DEG C, night 10 DEG C ~ 12 DEG C.
7. deposit number is a liquid nutrient medium for the long handle Pleurotus sp2 of CGMCC No.7359, it is characterized in that, comprises the component of following mass fraction: carbon source 1% ~ 4%, nitrogenous source 0.2% ~ 4%, KH 2pO 40.1% ~ 0.15%, natural ph;
Described carbon source is a kind of in sucrose, glucose or corn flour or both above combinations;
Described nitrogenous source is a kind of in wheat bran juice, peptone, dusty yeast or beancake powder or both above combinations.
8. a deposit number is the liquid fermentation method of the long handle Pleurotus sp2 of CGMCC No.7359, it is characterized in that, comprise: cultivate the seed that deposit number is the long handle Pleurotus sp2 of CGMCC No.7359, be inoculated in liquid nutrient medium as claimed in claim 7, after sterilizing, cultivate 100h ~ 150h in 25 DEG C ± 3 DEG C, put tank, obtain zymocyte liquid;
The condition of described cultivation is: pass into filtrated air, and throughput is 1:1v/vmin, tank pressure 0.5kg/cm 2.
9. liquid fermentation method according to claim 8, is characterized in that, described deposit number is the seed of the long handle Pleurotus sp2 of CGMCC No.7359 and the mass ratio of liquid nutrient medium as claimed in claim 7 is 1:(8 ~ 12).
10. the zymocyte liquid that as described in claim 8 or 9 prepared by method is preparing the application in food, drink, medicine or health products.
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CN105454957A (en) * 2015-11-19 2016-04-06 河北大学 A pleurotus citrinopileatus-common cattail solid fermentation functional drink and a preparing method
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