CN105331548A - Lepista nuda strain and liquid culture and preparation method thereof - Google Patents
Lepista nuda strain and liquid culture and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a lepista nuda strain and a liquid culture and preparation method thereof. The lepista nuda strain is a lepista nuda strain M-012<#>, the preservation unit is China General Microbiological Culture Collection Center (CCMCC), Microbiological Culture Collection Committee, the preservation date is the twenty first, August, 2015, and the preservation number is CGMCC No. 11206; the liquid culture of the lepista nuda strain M-012<#> is prepared through the steps that the lepista nuda strain M-012<#> is subjected to slant culture, seed culture and fermentation. A preparation method of the liquid strain of the lepista nuda strain M-012<#> comprises the steps of slant culture, seed culture and fermentation. The lepista nuda strain and the liquid culture and preparation method thereof have the advantages that mycelium biomass is high and can reach 28 g/Kg, and the biomass of solid fungus-package mycelium approximately ranges from 6 g/Kg to 13 g/Kg; the mycelium growth speed is high, and on a slab with the diameter being 10 cm, it only takes seven days for the mycelium to grow all over the slab; the culture contamination rate is low and is less than or equal to 2%. Meanwhile, a liquid fermentation method is adopted, and the time is short and is about 72-96 hours, while it takes over 15 days to culture a common solid package.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of Lepista mucla (Bull.:Fr.) Cooke bacterial strain and liquid spawn thereof and preparation method.
Background technology
Lepista mucla (Bull.:Fr.) Cooke [
lepistanuda(Bull.) Cooke] be a kind of aromatic flavour, beautiful in colour, nutritious edible mushrooms, is acknowledged as first-class food sending out.Moreover, Lepista mucla (Bull.:Fr.) Cooke also shows to the suppression of small white mouse tumour up to 90% in anticancer test, to the inhibiting rate of ehrlich carcinoma especially up to 100%.But bacterial classification is in the market all the bacterial classification of the Lepista mucla (Bull.:Fr.) Cooke of France for monopoly position, and the Lepista mucla (Bull.:Fr.) Cooke bacterial classification of China oneself or rare, this is mainly due to our bacterial classification Character instability, the uniqueness of bacterial classification causes cultural method and French spawn culture method to be not quite similar, therefore we are in the urgent need to working out the bacterial strain of China oneself, break the monopoly position of France at Lepista mucla (Bull.:Fr.) Cooke bacterial classification, and work out corresponding cultural method, carry out supporting production cultivation technique, the effective output etc. improving our Lepista mucla (Bull.:Fr.) Cooke.
Key technique in the acquisition of the excellent species of Lepista mucla (Bull.:Fr.) Cooke.Current domestic most employing solid seeding technique, and seldom use liquid fermenting.For solid seeding technique, liquid fermentation technology has that mycelial growth is good, bacterium ball is even, proterties is stablized, and comparatively less contamination, substratum utilize sufficient advantage.This patent adopts liquid fermentation technology to make liquid spawn best in quality, proterties is stable, provides safeguard to later stage artificial culture and medical research.
Summary of the invention
The present invention first object is to provide a kind of Lepista mucla (Bull.:Fr.) Cooke bacterial strain, and the second object is to provide a kind of Lepista mucla (Bull.:Fr.) Cooke M-012
#liquid spawn, the 3rd object is to provide described Lepista mucla (Bull.:Fr.) Cooke M-012
#the preparation method of liquid spawn.
The present invention first object is achieved in that described Lepista mucla (Bull.:Fr.) Cooke bacterial strain is Lepista mucla (Bull.:Fr.) Cooke M-012
#bacterial strain, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation day: on August 21st, 2015, deposit number: CGMCCNo.11206; Described Lepista mucla (Bull.:Fr.) Cooke M-012
#bacterial strain belongs to mycota, Basidiomycota, agaric guiding principle, Agaricales, Tricholomataceae, Lepista lentinus.
Lepista mucla (Bull.:Fr.) Cooke M-012
#the acquisition of bacterial strain and qualification
1, the Lepista mucla (Bull.:Fr.) Cooke sporophore interior wall tissue block of collection is inoculated on test tube nutrient agar inclined-plane, 5 ~ 10d is cultivated at 18 ~ 24 DEG C, to test tube observed and recorded every day of separation and Culture, the test tube that timely rejecting has been polluted, pick out pollution-free, grow fine and the strong bacterial strain of sclerotium generative capacity, obtain Lepista mucla (Bull.:Fr.) Cooke separation test tube kind, the formula of described test tube nutrient agar: potato 16g, glucose 1.6g, KH
2pO
40.03g, agar 1.6g, MgSO
40.02g, distilled water 100ml, pH7.0; Culture temperature 20 ~ 28 DEG C.
2, be inoculated on slat chain conveyor ware by Lepista mucla (Bull.:Fr.) Cooke separation test tube kind mycelium and carry out purifying agaric, culture condition is lucifuge at 18 ~ 24 DEG C, the formula of described nutrient agar: potato 16g, glucose 1.6g, KH
2pO
40.03g, agar 1.6g, MgSO
40.02g, distilled water 100ml, pH7.0; Culture temperature 20 ~ 28 DEG C; Getting Tip Splitting block after 2d is seeded in medium slant, described culture medium prescription: potato 16g, glucose 1.6g, KH
2pO
40.03g, agar 1.6g, MgSO
40.02g, distilled water 100ml, pH7.0; At 18 ~ 24 DEG C, cultivate 5 ~ 7d, obtain Lepista mucla (Bull.:Fr.) Cooke purifying test tube kind.
3, adopt for swab entity and corresponding hypha separation thing, press CTAB method respectively and extract Lepista mucla (Bull.:Fr.) Cooke STb gene, carry out pcr amplification and ITS sequence to measure, submit to ncbi database to carry out BLAST sequence alignment analysis the ITS sequence of measured test sample, with in NCBI Lepista mucla (Bull.:Fr.) Cooke (
l.nuda) sequence Identities=606/606 (100%), Gaps=0/606 (0%), analyze determine to be separated the pure growth obtained be Lepista mucla (Bull.:Fr.) Cooke of the present invention (
l.nuda) mycelium.
Compared with prior art, the invention has the beneficial effects as follows: one is that mycelial biomass is high, can reach 28g/Kg, and solid bacterium bag mycelial biomass is greatly about 6 ~ 13g/Kg; Two is that mycelial growth rate is fast, as long as mycelia 7d just can cover with flat board on the flat board of diameter 10cm; Three is that cultivation pollution rate is low ,≤2%.Adopt the solution fermentation time short, about 72 ~ 96h, and common solid bag cultivation need more than 15d simultaneously.
The present invention utilizes country of origin, Yunnan Lepista mucla (Bull.:Fr.) Cooke sporophore to filter out strain excellent, for Lepista mucla (Bull.:Fr.) Cooke artificial culture provides excellent species.
Lepista mucla (Bull.:Fr.) Cooke M-012 provided by the present invention
#bacterial strain depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Preservation date: on 08 21st, 2015; The numbering CGMCCNO.11206 that preservation is registered on the books.
The present invention second object is achieved in that with described Lepista mucla (Bull.:Fr.) Cooke bacterial strain M-012
#the liquid spawn of making after slant culture, seed culture, fermentation.
The third object of the present invention is achieved in that and comprises slant culture, seed culture, fermentation step, specifically comprises:
A, slant culture: by Lepista mucla (Bull.:Fr.) Cooke bacterial strain M-012
#mycelium be inoculated on test tube nutrient agar inclined-plane, at temperature is 18 ~ 24 DEG C cultivate 5 ~ 7d, obtain test tube strains;
B, seed culture: test tube strains is inoculated in liquid nutrient medium, at temperature is 20 ~ 26 DEG C, 5 ~ 11d cultivated by shaking table, obtains liquid fermenting seed;
C, fermentation: liquid fermenting seed is inoculated into fermentor tank by the amount of fermention medium massfraction 5 ~ 9%, at tank pressure 0.03 ~ 0.05MPa, temperature 21 ~ 23 DEG C, pH value 7.0 ~ 8.0, stirring velocity 130 ~ 150r/min, air flow cultivates 80 ~ 90h 70 ~ 90% times, obtains Lepista mucla (Bull.:Fr.) Cooke M-012
#liquid spawn.
The present invention solve technical problem be: one be from Lepista mucla (Bull.:Fr.) Cooke (
l.nuda) spot---the sporophore of this bacterium grown near Shuan Long township, Kunming, Yunnan Province Yeyahu is wild is carried out separate tissue cultivation and obtained, and has obvious regional Characteristics; Two is overcome that prior art mycelial growth is uneven, pollution rate is high, substratum utilizes the deficiencies such as insufficient.Its objective is provide a kind of and have that output is high, mycelial growth is quick, resistant to pollution new excellent Lepista mucla (Bull.:Fr.) Cooke bacterial strain and cultural method thereof.
The invention has the beneficial effects as follows: one is that mycelial biomass is high, can reach 28g/Kg, and solid bacterium bag mycelial biomass is greatly about 6-13g/Kg; Two is that mycelial growth rate is fast, as long as mycelia 7d just can cover with flat board on the flat board of diameter 10cm; Three is that cultivation pollution rate is low ,≤2%.Adopt the solution fermentation time short, about 72 ~ 96h, and common solid bag cultivation need more than 15d simultaneously.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, but limited the present invention never in any form, and any conversion done based on training centre of the present invention or improvement, all fall into protection scope of the present invention.
Lepista mucla (Bull.:Fr.) Cooke bacterial strain of the present invention is Lepista mucla (Bull.:Fr.) Cooke M-012
#bacterial strain, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation day: on August 21st, 2015, deposit number: CGMCCNo.11206; Described Lepista mucla (Bull.:Fr.) Cooke M-012
#bacterial strain belongs to mycota, Basidiomycota, agaric guiding principle, Agaricales, Tricholomataceae, Lepista lentinus.
Lepista mucla (Bull.:Fr.) Cooke M-012 of the present invention
#liquid spawn, be with described Lepista mucla (Bull.:Fr.) Cooke bacterial strain M-012
#the liquid spawn of making after slant culture, seed culture, fermentation.
Lepista mucla (Bull.:Fr.) Cooke M-012 of the present invention
#the preparation method of liquid spawn, comprise slant culture, seed culture, fermentation step, specifically comprise:
A, slant culture: by Lepista mucla (Bull.:Fr.) Cooke bacterial strain M-012
#mycelium be inoculated on test tube nutrient agar inclined-plane, at temperature is 18 ~ 24 DEG C cultivate 5 ~ 7d, obtain test tube strains;
B, seed culture: test tube strains is inoculated in liquid nutrient medium, at temperature is 20 ~ 26 DEG C, 5 ~ 11d cultivated by shaking table, obtains liquid fermenting seed;
C, fermentation: liquid fermenting seed is inoculated into fermentor tank by the amount of fermention medium massfraction 5 ~ 9%, at tank pressure 0.03 ~ 0.05MPa, temperature 21 ~ 23 DEG C, pH value 7.0 ~ 8.0, stirring velocity 130 ~ 150r/min, air flow cultivates 80 ~ 90h 70 ~ 90% times, obtains Lepista mucla (Bull.:Fr.) Cooke M-012
#liquid spawn.
Test tube nutrient agar described in step A is improvement PDA substratum, and its composition is: potato 15 ~ 17g, glucose 1.5 ~ 1.7g, KH
2pO
40.02 ~ 0.04g, agar 1.5 ~ 1.7g, MgSO
40.01 ~ 0.03g, all the other are water, and pH value is 7.0.
The composition of the liquid nutrient medium described in step B is: wheat bran 5 ~ 10g, analysis for soybean powder 0.5 ~ 1g, maltose 0.5 ~ 1g, Zulkovsky starch 1 ~ 2g, and all the other are water, and pH value is 7.0.
The composition of the fermention medium described in step C is: wheat bran 5 ~ 10g, analysis for soybean powder 0.5 ~ 1g, maltose 0.5 ~ 1g, Zulkovsky starch 1 ~ 2g, and all the other are water, and pH value is 7.0 ~ 8.0.
Be illustrated below by embodiment:
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The wild Lepista mucla (Bull.:Fr.) Cooke of following examples gathers in Shuan Long township, Kunming, Yunnan Province wild duck lakeside; Acquisition method is free-hand collection, notices that maintenance sporophore is complete, nothing is gone mouldy, without disease and pest when collection.
Embodiment 1
1.1 Lepista mucla (Bull.:Fr.) Cooke M-012
#acquisition
(1) the wild Lepista mucla (Bull.:Fr.) Cooke sporophore gathered is cut into block and tissue block, tissue block is inoculated on strain separating pure medium inclined-plane, 10d is cultivated at 18 DEG C, to test tube observed and recorded every day of separation and Culture, the test tube that timely rejecting has been polluted, pick out bacterial strain that is pollution-free, that grow fine, obtain Lepista mucla (Bull.:Fr.) Cooke separation test tube kind; The formula of described strain separating pure medium is:
(2) Lepista mucla (Bull.:Fr.) Cooke separation test tube kind inoculated by hypha block is carried out purifying agaric to the slat chain conveyor ware of nutrient agar, culture condition is 18 ~ 24 DEG C of lucifuges, the formula of described nutrient agar: potato 30g, glucose 2g, K
2hPO
40.5g, agar 2g, distilled water 100ml, pH7.0, culture temperature 20 ~ 25 DEG C; Getting Tip Splitting block after 2d is seeded in medium slant, at 18 DEG C, cultivate 7d, obtains the new Lepista mucla (Bull.:Fr.) Cooke bacterial strain that Lepista mucla (Bull.:Fr.) Cooke purifying test tube kind is acquisition, described culture medium prescription method: potato 30g, glucose 2g, K
2hPO
40.5g, agar 2g, distilled water 100ml, pH7.0, culture temperature 20 ~ 25 DEG C.
By proterties comparisons such as mycelium production, sclerotium generative capacity, antipollution power, pick out that mycelium production is high, sclerotium generative capacity strong, resistant to pollution Lepista mucla (Bull.:Fr.) Cooke liquid spawn special strain therefore M-012
#.
1.2 qualification and feature, name, preservation
(1) sporophore feature
M-012
#
Separation sporophore is characterized as: bacteria cover diameter 3.5 ~ 10cm, and semisphere is to open and flat, and middle part is recessed sometimes, and brilliant violet look or lilac fade to brown purple, smooth, moistening, and edge is involute, without striped.Bacterial context lavender is thicker.Lamella purple, close, straight raw to slightly prolonging life, Length discrepancy, be spiculation toward raw edge.Stem long 4 ~ 9cm, thick 0.5 ~ 2cm, cylindrical, with cap look, cotton-shaped powder is arranged at initial stage top, and the smooth or tool vertical stripe in bottom, interior reality, base portion slightly expands.Spore print digested tankage look.Spore is colourless, oval, and dipped beam is sliding, has age spot, 5.0 ~ 7.5 μm × 3.0 ~ 5.0 μm.Identification of morphology reference " Macrofungi From China " (fourth of the twelve Earthly Branches morning mist. Zhengzhou: Henan science tech publishing house, 2000.).
(2) Lepista mucla (Bull.:Fr.) Cooke M-012
#bacterial strain purifying test tube kind feature
After inoculation, be under the condition of 22 DEG C in culture temperature, 15h just starts to sprout, and early growth period mycelia is close to substratum, colourless, glossy, is generally the less mycelia of thicker not branch or branch; The long speed of first day is 1.1cm the soonest, the most slowly be 0.3cm, then with the increase of bacterium colony, there is forked or dendritic branch, generally comparatively thin, a few days ago produce without sclerotium and pigment, growth in 3rd day is the fastest, average long speed is 0.70cm/d, and starts have pigment to produce, and within the 9th day, covers with whole culture dish by the tenth day.
(3) ITS sequence analysis
Adopt the corresponding Lepista mucla (Bull.:Fr.) Cooke purifying test tube kind mycelia obtained with embodiment 1 for the wild Lepista mucla (Bull.:Fr.) Cooke sporophore of examination, press CTAB method respectively and extract STb gene, carry out pcr amplification and ITS sequence to measure, by Lepista mucla (Bull.:Fr.) Cooke in the ITS sequence of Blast comparative sample and NCBI (
l.nuda) sequence Identities=606/606 (100%), Gaps=0/606 (0%), analyze and determine that embodiment 1 is separated the Lepista mucla (Bull.:Fr.) Cooke purifying test tube kind that obtains and new Lepista mucla (Bull.:Fr.) Cooke bacterial strain is Lepista mucla (Bull.:Fr.) Cooke mycelium.Its concrete grammar is as follows:
Modified CTAB method flow process
I takes the dry sample of about 0.12g, adds liquid nitrogen and after being ground to powdery rapidly, adding rapidly-CTAB500 μ l, beta-mercaptoethanol 20 μ l, then add+CTAB(65 DEG C of preheating) 700 μ l, mix rearmounted 65 DEG C of water-baths vibration extracting 60min;
II cools, and the centrifugal 10min of 12000r, gets supernatant liquor, adds the isopyknic phenol/chloroform of 700 μ l (1:1), the centrifugal 10min of 12000r after mixing;
III gets supernatant, adds 700 μ l chloroform/primary isoamyl alcohol, mixing, the centrifugal 10min of 12000r;
IV gets supernatant, first adds 80 μ l10%CTAB+4%NaCl solution (65 DEG C of preheatings), then adds 700 μ l chloroform/primary isoamyl alcohol, mixing, the centrifugal 10min of 12000r;
V gets supernatant, adds 600 μ l Virahols ,-18 DEG C of left overnight;
VI is taken out, and the centrifugal 10min of 12000r, abandons supernatant liquor, and precipitation is washed with 600 μ l76% ethanol, 0.2mol/L sodium acetate and 300 μ l70% ethanol successively;
The centrifugal 5min of VII 8000r, is filtered dry centrifuge tube with filter paper, vacuum-drying 5min at 45 DEG C, adds 100 μ lTE buffer solution, 4 DEG C of Refrigerator stores.
DNA purity and Concentration Testing
Get 2 μ lDNA samples, be settled to 50 μ l with TE damping fluid, measure the OD value of wavelength at 230nm, 260nm, 280nm place with ultraviolet spectrophotometer, the OD value according to 260nm place calculates DNA concentration, determines the purity of DNA according to 260/280,260/230 value.
DNA molecular amount detects
Get DNA sample 5 μ l, sample-loading buffer is (containing 0.25% tetrabromophenol sulfonphthalein, 40% sucrose) 2 μ l, mixing, click and enter in 0.8% sepharose containing 0.75 μ l nucleic acid dye, click and enter λ-Lambdamixmarker as mark simultaneously, with 1 × TAE damping fluid, electrophoresis 80min under 80V voltage, finally observes and takes a picture under gel imaging instrument.
ITS-PCR increases
ITS sequence amplimer
The ITS universal primer ITS4/ITS5 of White design is adopted to carry out pcr amplification to the rDNAITS sequence for examination material.Its ITS primer is as follows:
ITS4:5'—TCCTCCGCTTATTGATATGC—3'
ITS5:5'—GGAAGTAAAAGTCGTAACAAGG—3'
ITS-PCR amplification system
ITS-PCR amplification system is 50 μ L, containing rTaq (5U/ μ L) 0.5 μ L, 10 × PCRBuffer6.25 μ L, Mgcl
2(25mmol/L) 5.0 μ L, dNTP mixture (each 2.5mmol/L) 0.75 μ L, ITS4/ITS5 primer (10 μm of ol/L) each 2.5 μ L, template DNA (50ng/ μ L) 2.5 μ L, ddH
2o complements to cumulative volume 50 μ L.
Pcr amplification carries out on GE9700PCR instrument.Response procedures is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 40s; 52 DEG C of annealing 45s; 72 DEG C extend 1min; Totally 40 circulations; Finally fill 10min in 72 DEG C, final temperature is 4 DEG C.The agarose gel electrophoresis of 1.8% is adopted to detect ITS-PCR amplified production, with gel imaging system Taking Pictures recording electrophoretic band.
The sequencing of ITS amplified production
Pcr amplification product is after electrophoresis detection, and reclaim test kit with glue and carry out reclaiming and purifying, sample send order-checking company to check order.
ITS sequence is analyzed
By the ITS sequence of each test sample obtained that increases, adopt ContigExpress software to carry out positive and negative chain-ordering splicing, the sequence of having spliced is saved as FASTA form, then at NCBI website enterprising line order row tetraploid rice.ClustalX1.83 software and BioEdit software is adopted to carry out sequence alignment the ITS sequence of having spliced.Comparison result adopts Mega3.0 software to carry out sequence compositional analysis, adopts non-weighting pairing arithmetic mean method (UPGMA) constructing system tree, and checks the degree of confidence of each branch of Molecular Phylogenetic tree with Bootstrap1000 time.
(4) preservation
Preservation: this Lepista mucla (Bull.:Fr.) Cooke (
lepistanuda) M-012
#depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Preservation date: on 08 21st, 2015; The numbering CGMCCNO.11206 that preservation is registered on the books.
1.3 cultural method
(1) by Lepista mucla (Bull.:Fr.) Cooke M-012
#mycelium be inoculated on test tube nutrient agar inclined-plane, at 18 ~ 24 DEG C, cultivate 5 ~ 7d, obtain test tube strains, the formula of described test tube nutrient agar is improvement PDA substratum, fills a prescription as potato 16g, glucose 1.6g, KH
2pO
40.03g, agar 1.6g, MgSO
40.02g, distilled water 100ml, pH7.0.
(2) test tube strains is inoculated into fills in the 500mL triangular flask of 200mL liquid nutrient medium, at 20 ~ 26 DEG C, shaking table is cultivated, rotating speed is 120 ~ 160r/min, incubation time 5 ~ 7d, obtain level liquid bacterial classification, described liquid culture based formulas is: 5 ~ 10g wheat bran, 0.5 ~ 1g analysis for soybean powder, 0.5 ~ 1g maltose, 1 ~ 2g Zulkovsky starch, distilled water 100ml, pH7.0.
(3) level liquid strain inoculation will be obtained in the fermentor tank of the automatic fermentative production line of 25L, ventilation ratio is 1:0.8, mixing speed is 120r/min, inoculum size is 5%(massfraction), tank pressure: 0.04Mpa, pH are 7.2, temperature is 22 DEG C, aerated culture 72h, namely obtains described Lepista mucla (Bull.:Fr.) Cooke M-012
#liquid spawn.
Embodiment 2
Bacterial strain acquisition, qualification, method for preserving are equal to embodiment 1.
2.1 cultural method
(1) by Lepista mucla (Bull.:Fr.) Cooke M-012
#mycelium be inoculated on test tube nutrient agar inclined-plane, at 18 ~ 24 DEG C, cultivate 5 ~ 7d, obtain test tube strains, the formula of described test tube nutrient agar is improvement PDA substratum, fills a prescription as potato 16g, glucose 1.6g, KH
2pO
40.03g, agar 1.6g, MgSO
40.02g, distilled water 100ml, pH7.0.
(2) test tube strains is inoculated into fills in the 500mL triangular flask of 200mL liquid nutrient medium, at 20 DEG C, shaking table is cultivated, rotating speed is 130r/min, incubation time 9d, obtain level liquid bacterial classification, described liquid culture based formulas is: 7g wheat bran, 0.5g analysis for soybean powder, 0.5g maltose, 1g Zulkovsky starch, distilled water 100ml, pH7.0.
(3) level liquid strain inoculation will be obtained in the fermentor tank of the automatic fermentative production line of 25L, ventilation ratio is 1:0.8, mixing speed is 140r/min, inoculum size is 7%(massfraction), tank pressure: 0.04Mpa, pH are 7.4, temperature is 22 DEG C, aerated culture 84h, namely obtains described Lepista mucla (Bull.:Fr.) Cooke M-012
#liquid spawn.
Embodiment 3
Bacterial strain acquisition, qualification, method for preserving are equal to embodiment 1.
3.1 cultural method
(1) by Lepista mucla (Bull.:Fr.) Cooke M-012
#mycelium be inoculated on test tube nutrient agar inclined-plane, at 18 ~ 24 DEG C, cultivate 5 ~ 7d, obtain test tube strains, the formula of described test tube nutrient agar is improvement PDA substratum, fills a prescription as potato 16g, glucose 1.6g, KH
2pO
40.03g, agar 1.6g, MgSO
40.02g, distilled water 100ml, pH7.0.
(2) test tube strains is inoculated into fills in the 500mL triangular flask of 200mL liquid nutrient medium, at 22 DEG C, shaking table is cultivated, rotating speed is 140r/min, incubation time 11d, obtain level liquid bacterial classification, described liquid culture based formulas is: 5g wheat bran, 0.5g analysis for soybean powder, 0.5g maltose, 1g Zulkovsky starch, distilled water 100ml, pH7.0.
(3) level liquid strain inoculation will be obtained in the fermentor tank of the automatic fermentative production line of 25L, ventilation ratio is 1:0.8, mixing speed is 160r/min, inoculum size is 9%(massfraction), tank pressure: 0.04Mpa, pH are 7.6, temperature is 22 DEG C, aerated culture 96h, namely obtains described Lepista mucla (Bull.:Fr.) Cooke M-012# liquid spawn.
Embodiment 4---shape simultaneous test
Use M-012
#with original 3 bacterial strain M-221
#, M1740
#, M-184
#, all strains tested kinds be Lepista mucla (Bull.:Fr.) Cooke (
l.nuda).
(1) test method
By 4 Lepista mucla (Bull.:Fr.) Cooke inoculation on 18mm × 18mm flat board, each bacterial strain repeats 10 times, and plate culture medium is improvement PDA: potato 16%, glucose 1.6%, KH
2pO
40.03%, agar 1.6%, MgSO
40.02%, described percentage ratio is massfraction.Be placed on lucifuge in the incubator of 20 DEG C after inoculation to cultivate.
Every 8h carries out observed and recorded.
(2) interpretation of result
The growing state of each bacterial strain of Lepista mucla (Bull.:Fr.) Cooke on improvement PDA substratum
note :+represent that mycelium growth vigor is poor; ++ represent that mycelium growth vigor is general; +++ represent that mycelium growth vigor is denseer; ++++represent, mycelium growth vigor was dense; +++ ++ represent that mycelium growth vigor is dense close.
As can be seen from the above table, under identical culture condition, the growing state difference to some extent of each bacterial strain.From growing way and full plate time, M-012
#strain growth speed is fast, grows fine, and the time that mycelia is paved with flat board shortens 1 ~ 3d compared with other three bacterial strains; From pollution condition, M-012
#bacterial strain does not pollute, and antipollution rate is obviously better than other three bacterial strains.
SEQUENCELISTING
<110> KUNMING EODIBLE FUNGUS RES INS
<120> Lepista mucla (Bull.:Fr.) Cooke bacterial strain and liquid spawn thereof and preparation method
<130>2015
<160>2
<170>PatentInversion3.3
<210>1
<211>20
<212>DNA
<213>ITS4
<400>1
tcctccgcttattgatatgc20
<210>2
<211>22
<212>DNA
<213>ITS5
<400>2
ggaagtaaaagtcgtaacaagg22
Claims (6)
1. a Lepista mucla (Bull.:Fr.) Cooke bacterial strain, is characterized in that described Lepista mucla (Bull.:Fr.) Cooke bacterial strain is Lepista mucla (Bull.:Fr.) Cooke M-012
#bacterial strain, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation day: on August 21st, 2015, deposit number: CGMCCNo.11206; Described Lepista mucla (Bull.:Fr.) Cooke M-012
#bacterial strain belongs to mycota, Basidiomycota, agaric guiding principle, Agaricales, Tricholomataceae, Lepista lentinus.
2. a Lepista mucla (Bull.:Fr.) Cooke M-012 according to claim 1
#liquid spawn, it is characterized in that with described Lepista mucla (Bull.:Fr.) Cooke bacterial strain M-012
#the liquid spawn of making after slant culture, seed culture, fermentation.
3. a Lepista mucla (Bull.:Fr.) Cooke M-012 according to claim 2
#the preparation method of liquid spawn, it is characterized in that comprising slant culture, seed culture, fermentation step, specifically comprise:
A, slant culture: by Lepista mucla (Bull.:Fr.) Cooke bacterial strain M-012
#mycelium be inoculated on test tube nutrient agar inclined-plane, at temperature is 18 ~ 24 DEG C cultivate 5 ~ 7d, obtain test tube strains;
B, seed culture: test tube strains is inoculated in liquid nutrient medium, at temperature is 20 ~ 26 DEG C, 5 ~ 11d cultivated by shaking table, obtains liquid fermenting seed;
C, fermentation: liquid fermenting seed is inoculated into fermentor tank by the amount of fermention medium massfraction 5 ~ 9%, at tank pressure 0.03 ~ 0.05MPa, temperature 21 ~ 23 DEG C, pH value 7.0 ~ 8.0, stirring velocity 130 ~ 150r/min, air flow cultivates 80 ~ 90h 70 ~ 90% times, obtains Lepista mucla (Bull.:Fr.) Cooke M-012
#liquid spawn.
4. Lepista mucla (Bull.:Fr.) Cooke M-012 according to claim 3
#the preparation method of liquid spawn, it is characterized in that the test tube nutrient agar described in step A is for improvement PDA substratum, its composition is: potato 15 ~ 17g, glucose 1.5 ~ 1.7g, KH
2pO
40.02 ~ 0.04g, agar 1.5 ~ 1.7g, MgSO
40.01 ~ 0.03g, all the other are water, and pH value is 7.0.
5. Lepista mucla (Bull.:Fr.) Cooke M-012 according to claim 3
#the preparation method of liquid spawn, it is characterized in that the composition of the liquid nutrient medium described in step B is: wheat bran 5 ~ 10g, analysis for soybean powder 0.5 ~ 1g, maltose 0.5 ~ 1g, Zulkovsky starch 1 ~ 2g, all the other are water, and pH value is 7.0.
6. Lepista mucla (Bull.:Fr.) Cooke M-012 according to claim 3
#the preparation method of liquid spawn, it is characterized in that the composition of the fermention medium described in step C is wheat bran 5 ~ 10g, analysis for soybean powder 0.5 ~ 1g, maltose 0.5 ~ 1g, Zulkovsky starch 1 ~ 2g, all the other are water, and pH value is 7.0 ~ 8.0.
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