CN103710271A - Morchella esculenta bacterial strain and culture method thereof - Google Patents

Morchella esculenta bacterial strain and culture method thereof Download PDF

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CN103710271A
CN103710271A CN201310730625.9A CN201310730625A CN103710271A CN 103710271 A CN103710271 A CN 103710271A CN 201310730625 A CN201310730625 A CN 201310730625A CN 103710271 A CN103710271 A CN 103710271A
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bacterial strain
morel
liquid
test tube
strain
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CN103710271B (en
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桂明英
刘蓓
郭相
马明
邰丽梅
吴素蕊
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Scientific Research Institute Of Supply And Marketing Cooperative Society Of Yunnan Province
Yunnan Cloud Technology (group) Co Ltd
Kunming Edible Fungus Institute Of China Federation Of Supply And Marketing Cooperatives
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Scientific Research Institute Of Supply And Marketing Cooperative Society Of Yunnan Province
Yunnan Cloud Technology (group) Co Ltd
Kunming Edible Fungus Institute Of China Federation Of Supply And Marketing Cooperatives
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Abstract

The invention discloses a morchella esculenta bacterial strain and a culture method thereof. The culture method of the morchella esculenta M-02# bacterial strain with a preservation number of CGMCC (China General Microbiological Culture Collection Center ) No.7058 is as follows: inoculating a mycelium of the morchella esculenta bacterial strain into a test tube culture medium inclined surface which contains an improved PDA (Potato Dextrose Agar) culture medium for culturing for 5 days-7 days under 18 DEG C-24 DEG C to obtain a test tube strain; inoculating the test tube strain into a triangular flask which contains a liquid culture medium for culturing on a shaking bed at 20 DEG C-26 DEG C, and culturing for 5 days-7days at rotation speed of 120 r/minute-160r/minute to obtain a primary liquid strain; and inoculating the primary liquid strain into a fermentation tank, ventilating for culturing for 72 hours-96 hours to obtain a liquid strain of the bacterial strain. The bacterial strain disclosed by the invention has mycelium biomass live-weight as high as 25g/kg, strong sclerotium generating capacity, pollution resistance, a low culturing contamination rate not greater than 2%, and short liquid fermentation time of 72 hours-96 hours, so that an excellent strain is provided for morchella esculenta wild resource protection and reproduction promotion.

Description

One strain morel bacterial strain and cultural method thereof
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of new morel bacterial strain and cultural method thereof.
Background technology
Morchella (Morchella) fungi, it is world-renowned famous and precious wild edible fungus, morel (M.esuclenta (L.) Pers.) is one of main species of Yunnan product morchella fungi, distributes comparatively extensive, at Drainage Area of Jinsha River, occurs in a large number.The artificial culture of morchella fungi, because output is unstable, still can not form commercialization and mass-producing at present, and the morel on market is mostly from wild.Climate worsening condition and the excessively impact of the undesirable element such as collection in recent years, wild toadstool natural production declines to a great extent.Research shows, in the former habitat of morel, adopts artificial bacterial classification and supporting technical measures, can effectively improve the output of morel in unit surface.
In the short propagating technology in the former habitat of morel, the acquisition of excellent species is key technique.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 evenly, compared with less contamination, the sufficient advantage of substratum utilization.This patent adopts liquid fermentation technology to make good liquid spawn, to later stage bionics is short numerous, provides safeguard with artificial culture.
Summary of the invention
The technical problem to be solved in the present invention is to overcome that the mycelial biomass of prior art morel is lower, sclerotium generative capacity is weak, mycelial growth is inhomogeneous, pollution rate is high, substratum utilizes the deficiencies such as insufficient.Its objective is provide a kind of and have that output is high, sclerotium generative capacity strong, resistant to pollution new good morel bacterial strain and cultural method thereof, be the short numerous excellent species that provides of morel wild resource protection.
For solving the problems of the technologies described above and reaching the object of the invention, technical scheme of the present invention is as follows:
1. a strain morel (Mochella esculenta) M-02 #bacterial strain CGMCC No.7058.
2. morel (Mochella esculenta) M-02 #the cultural method of bacterial strain CGMCC No.7058 liquid spawn, its step is as follows:
(1) by morel (Mochella esculenta) M-02 #the mycelium of bacterial strain CGMCC No.7058 is inoculated on test tube nutrient agar inclined-plane, at 18-24 ℃, cultivates 5-7d, obtains test tube strains, and described test tube nutrient agar is improvement PDA substratum, and its formula is potato 30%, glucose 2%, K 2hPO 40.5%, agar 2%, surplus is water, described percentage ratio is massfraction;
(2) test tube strains is inoculated in the 500mL triangular flask that fills 200mL liquid nutrient medium, at 20-26 ℃, 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: 5-10% wheat bran, 0.5-1% analysis for soybean powder, 0.5-1% maltose, 1-2% Zulkovsky starch, and surplus is water, described percentage ratio is massfraction;
(3) the level liquid bacterial classification of acquisition is inoculated in the fermentor tank of the automatic fermentative production line of 25L, ventilation ratio is 1:0.8, and mixing speed is 120-160r/min, and inoculum size is 5-10% massfraction, tank pressure: 0.04Mpa; PH is 7.2-7.5; Temperature is 22-26 ℃; Aerated culture 72-96h, obtains morel (Mochella esculenta) M-02 #bacterial strain CGMCC No.7058 liquid spawn.
Compared with prior art, the invention has the beneficial effects as follows: the one, mycelial biomass is high, can reach 25g/kg, and solid bacterium bag mycelial biomass is greatly about 5-12g/kg; The 2nd, sclerotium generative capacity is strong, and on the flat board of diameter 10cm, golden yellow sclerotium particle is many or area coverage is large, and sclerotium particle can reach 50, and single particle diameter can reach 1cm; The 3rd, antipollution, to cultivate pollution rate low, and≤2%.Adopt the solution fermentation time short simultaneously, about 72-96h, and more than common solid bag cultivates and need 15d.
The present invention utilizes country of origin, Yunnan Morchella esculenta (L.) Pers sporophore to filter out strain excellent, is the short numerous excellent species that provides of morel wild resource protection.
Morel provided by the present invention (Mochella esculenta) M-02 #bacterial strain CGMCC No.705 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 01 11st, 2013; Preserving number: CGMCC No.7058.
In sequence table shown in SEQ ID NO:1 is in embodiment 1, (3) bacterial strain to be carried out during ITS sequential analysis the base sequence of ITS sequence amplification ITS4 primer used.
In sequence table shown in SEQ ID NO:2 is in embodiment 1, (3) bacterial strain to be carried out during ITS sequential analysis the base sequence of ITS sequence amplification ITS5 primer used.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., all can obtain from commercial channels.
Following examples wild toadstool gathers (being also primary source ground) in Xin Tang village, Meng Gu township, Zhaotong City Qiaojia County, Yunnan Province sugarcane field, its genetic resources is taken from microorganism; Acquisition method is free-hand collection (voluntarily gather), in the time of collection, note keeping sporophore complete, without going mouldy, without disease and pest, acquisition time: in November, 2012 (being also the acquired original time).
Embodiment 1
Strain morel (Mochellaesculenta) M-02 provided by the present invention #acquisition, evaluation and the cultural method of bacterial strain CGMCC No.7058.
1.1 morels (Mochellaesculenta) M-02 #the acquisition of bacterial strain CGMCC No.7058
(1) the wild toadstool sporophore interior wall tissue piece of collection is inoculated on strain separating pure medium inclined-plane, at 18 ℃, cultivate 10d, test tube observed and recorded every day to separation and Culture, the test tube that rejecting has in time been polluted, referring to the method described in embodiment 4, by proterties such as mycelium production, sclerotium generative capacity, antipollution power, compare the morel separation test tube kind that pick out that mycelium production is high, sclerotium generative capacity is strong, antipollution, the bacterial strain that grows fine are acquisition; The formula of described strain separating pure medium is: potato 30%, glucose 2%, K 2hPO 40.5%, agar 2%, surplus is water, described percentage ratio is massfraction;
(2) morel separation test tube kind inoculated by hypha block is carried out to purifying agaric to the dull and stereotyped culture dish of nutrient agar, culture condition is 18-24 ℃ of lucifuge, after 2d, get most advanced and sophisticated inoculated by hypha block in medium slant, at 18 ℃, cultivate 7d, obtain morel purifying test tube kind and be morel of the present invention (Mochella esculenta) M-02 #bacterial strain CGMCC No.7058; Culture medium prescription method in substratum in described dull and stereotyped culture dish and described medium slant is: potato 30%, glucose 2%, K 2hPO 40.5%, agar 2%, surplus is water, described percentage ratio is massfraction.
1.2 evaluations and feature thereof, name, preservation
(1) sporophore feature
Morel (Mochellaesculenta) M-02 #bacterial strain CGMCC No.7058 separation is characterized as by sporophore: high 6~14.5cm.Long 4~the 6cm of cap, wide 4~6cm, irregular cycle, ellipse, there are many pits on surface, like sheep tripe shape, khaki or light yellow.Long 5~the 7cm of stem, thick 2~2.5cm, white, has shallow groove, and base portion slightly expands.Ascus (200~300) μ m * (18~22) μ m; 8 of thecaspores, single file is arranged, wide ellipse, (20~24) μ m * (12~15) μ m.Expand at lateral filament top, sometimes have every.Identification of morphology with reference to < < Macrofungi From China > > (fourth of the twelve Earthly Branches morning mist. Zhengzhou: Henan science tech publishing house, 2000.) and < < Macrofungi From China primary colors illustrated handbook > > (over yellow year. Beijing: Chinese agriculture press, 1998.).
(2) morel (Mochellaesculenta) M-02 #bacterial strain purifying test tube kind feature
After inoculation, be under the condition of 22 ℃ in culture temperature, 8h 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 1.3cm the soonest, is 0.8cm the most slowly, then with the increase of bacterium colony, there is forked or dendroid branch, generally thinner, a few days ago without sclerotium and pigment formation, growth in the 3rd day is the fastest, average long speed is 1.65cm/d, and starts there is pigment formation, within the 4th day to the 5th day, covers with whole culture dish, within the 6th day, start to have sclerotium to form, along with the generation of pigment, it is aging, weak that mycelia starts, and color starts to deepen.
Sclerotium started at the 5th day to form, and was distributed in inoculation circle around, less, diameter average out to 0.5~1.0mm, and look shallow.Some is planted as strip, and hard, some is smooth, and some is coarse.Picking, is placed on slide glass, knocks crushing, Microscopic observation, and visible sclerotium is intertwined and is formed by many mycelia distortions; To sclerotium on the 8th day whole bacterium colony, be uniformly distributed, between diameter 0.8~3.2mm, color depth.Picking crushes paintedly on slide glass, and Microscopic observation, has been hard to tell interweaving between mycelia, can see outer wall thickening, is one deck shelly material, middle for can be by the spindle cell of the dense nutritive substance of having of engrain.Under lucifuge condition, easily form sclerotium, and density is large.
(3) ITS sequential analysis
Adopt the corresponding morel purifying test tube kind mycelia obtaining with embodiment 1 for examination wild toadstool sporophore, by CTAB method, extract total DNA respectively, carry out pcr amplification and ITS sequencing, by the ITS sequence of Blast comparative sample and morel (M.esculenta) Identities=1114/1138 (97.89%) in NCBI, Gaps=24/1138 (2.10%), the morel purifying test tube kind that Analysis deterrmination embodiment 1 separation obtains is that new morel bacterial strain is Morchella esculenta (L.) Pers mycelium.Its concrete grammar is as follows:
Modified CTAB method flow process
I takes the dry sample of 0.12g left and right, adds liquid nitrogen and is ground to rapidly after powdery, add rapidly-CTAB500 μ l, beta-mercaptoethanol 20 μ l, then add+CTAB(65 ℃ preheating) 700 μ l, mix rearmounted 65 ℃ of water-baths vibration extracting 60min;
II is cooling, and the centrifugal 10min of 12000r, gets supernatant liquor, adds the isopyknic phenol/chloroform of 700 μ l (1:1), mixes the centrifugal 10min of rear 12000r;
III is got supernatant, adds 700 μ l chloroform/primary isoamyl alcohol, mixes the centrifugal 10min of 12000r;
IV is got supernatant, first adds 80 μ l10%CTAB+4%NaCl solution (65 ℃ of preheatings), then adds 700 μ l chloroform/primary isoamyl alcohol, mixes the centrifugal 10min of 12000r;
V is got supernatant, adds 600 μ l Virahols, places at-18 ℃ and spends the night;
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, and vacuum-drying 5min at 45 ℃ adds 100 μ l TE damping fluids to dissolve, 4 ℃ of Refrigerator stores.
DNA purity and concentration detect
Get 2 μ l DNA samples, with TE damping fluid, be settled to 50 μ l, with ultraviolet spectrophotometer, measure wavelength in the OD value at 230nm, 260nm, 280nm place, according to the OD value at 260nm place, calculate DNA concentration, according to 260/280,260/230 value, determine the purity of DNA.
DNA molecular amount detects
Get DNA sample 5 μ l, sample-loading buffer is (containing 0.25% tetrabromophenol sulfonphthalein, 40% sucrose) 2 μ l, mix, click and enter containing in 0.8% sepharose of 0.75 μ l nucleic acid dye, click and enter λ-Lambda mix marker simultaneously and serve as a mark, with 1 * TAE damping fluid, electrophoresis 80min under 80V voltage finally observes and takes a picture under gel imaging instrument.
ITS-PCR amplification
ITS sequence amplification primer
Adopt the ITS universal primer ITS4/ITS5 of White design to carrying out pcr amplification for the rDNA ITS sequence of examination material.Its ITS primer is as follows:
ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ' (being the base sequence shown in SEQ ID NO:1 in sequence table)
ITS5:5 '-GGAAGTAAAAGTCGTAACAAGG-3 ' (being the base sequence shown in SEQ ID NO:2 in sequence table)
ITS-PCR amplification system
ITS-PCR amplification system is 50 μ L, containing rTaq (5U/ μ L) 0.5 μ L, and 10 * PCR Buffer6.25 μ L, Mgcl 2(25mmol/L) 5.0 μ L, dNTP mixture (each 2.5mmol/L) 0.75 μ L, each 2.5 μ L of ITS4/ITS5 primer (10 μ mol/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 ℃ of denaturation 5min; 94 ℃ of sex change 40s; 52 ℃ of annealing 45s; 72 ℃ are extended 1min; Totally 40 circulations; Finally fill 10min in 72 ℃, final temperature is 4 ℃.Adopt 1.8% agarose gel electrophoresis detection ITS-PCR amplified production, with gel imaging system Taking Pictures recording electrophoretic band.
The sequencing of ITS amplified production
Pcr amplification product, after electrophoresis detection, reclaims test kit with glue and reclaims and purifying, and sample send the order-checking of order-checking company.
ITS sequential analysis
The ITS sequence of each test sample that amplification is obtained, adopts ContigExpress software to carry out positive and negative chain-ordering splicing, and the sequence of having spliced is saved as to FASTA form, then on NCBI website, carries out sequence homology comparison.Adopt ClustalX1.83 software and BioEdit software 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 by the degree of confidence of checking molecular system Shu Ge branch for Bootstrap1000 time.
(4) preservation
Preservation: morel of the present invention (Mochella esculenta) M-02 #the depositary institution of bacterial strain CGMCC No.7058: 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 01 11st, 2013; The numbering that preservation is registered on the books (preserving number): CGMCC No.7058.
1.3 cultural method
(1) by morel (Mochella esculenta) M-02 #the mycelium of bacterial strain CGMCC No.7058 is inoculated on test tube nutrient agar inclined-plane, at 18 ℃, cultivates 6d, obtains test tube strains, and the formula of described test tube nutrient agar is improvement PDA substratum: potato 30%, glucose 2%, K 2hPO 40.5%, agar 2%, surplus is water, described percentage ratio is massfraction.
(2) test tube strains is inoculated in the 500mL triangular flask that fills 200mL liquid nutrient medium, at 20 ℃, shaking table is cultivated, rotating speed is 120r/min, incubation time 7d, obtain level liquid bacterial classification, described liquid culture based formulas is: 6% wheat bran, 0.5% analysis for soybean powder, 0.5% maltose, 1% Zulkovsky starch, and surplus is water, described percentage ratio is massfraction.
(3) the level liquid bacterial classification of acquisition is inoculated 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 is 7.2, temperature is 22 ℃, aerated culture 72h, obtains described morel (Mochella esculenta) M-02 #bacterial strain CGMCC No.7058 liquid spawn.
Embodiment 2
Embodiment 2 is to morel (Mochella esculenta) M-02 #the cultural method of bacterial strain CGMCC NO.7058, its cultural method is except following measures difference, and all the other measures are identical with 1.3 cultural methods in embodiment 1, its morel (Mochella esculenta) M-02 #the acquisition of bacterial strain CGMCC No.7058, evaluation, method for preserving and embodiment 1 are equal to.
2.1 cultural method
(1) by morel (Mochella esculenta) M-02 #the mycelium of bacterial strain CGMCC No.7058 is inoculated on test tube nutrient agar inclined-plane, at 20 ℃, cultivates 7d, obtains test tube strains.
(2) test tube strains is inoculated in the 500mL triangular flask that fills 200mL liquid nutrient medium, at 20 ℃, shaking table is cultivated, rotating speed is 130r/min, incubation time 9d, obtain level liquid bacterial classification, described liquid culture based formulas is: 7% wheat bran, 0.5% analysis for soybean powder, 0.5% maltose, 1% Zulkovsky starch, and surplus is water, described percentage ratio is massfraction.
(3) by obtaining level liquid bacterial classification, be inoculated 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 is 7.4, temperature is 24 ℃, aerated culture 84h, obtains described morel (Mochella esculenta) M-02 #bacterial strain CGMCC No.7058 liquid spawn.
Embodiment 3
Embodiment 3 is to morel (Mochella esculenta) M-02 #the cultural method of bacterial strain CGMCC No.7058, its cultural method is except following measures difference, and all the other measures are identical with 1.3 cultural methods in embodiment 1, its morel (Mochella esculenta) M-02 #the acquisition of bacterial strain CGMCC NO.7058, evaluation, method for preserving and embodiment 1 are equal to.
3.1 cultural method
(1) by morel (Mochella esculenta) M-02 #the mycelium of bacterial strain CGMCC No.7058 is inoculated on test tube nutrient agar inclined-plane, at 24 ℃, cultivates 5d, obtains test tube strains.
(2) test tube strains is inoculated in the 500mL triangular flask that fills 200mL liquid nutrient medium, at 22 ℃, shaking table is cultivated, rotating speed is 160r/min, incubation time 7d, obtain level liquid bacterial classification, described liquid culture based formulas is: 10% wheat bran, 1% analysis for soybean powder, 1% maltose, 2% Zulkovsky starch, and surplus is water, described percentage ratio is massfraction.
(3) by obtaining level liquid bacterial classification, be inoculated 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 10%(massfraction), tank pressure: 0.04Mpa, pH is 7.5, temperature is 26 ℃, aerated culture 96h, obtains described morel (Mochella esculenta) M-02 #bacterial strain CGMCC NO.7058 liquid spawn.
Embodiment 4 proterties simultaneous tests
Use morel of the present invention (Mochella esculenta) M-02 #bacterial strain CGMCC No.7058 and original 3 bacterial strain M-16 #, M0710 #, M-501 #, (3 bacterial strain M-16 #, M0710 #, M-501 #can buy by microbial preservation mechanism) all strains tested kinds are morel (M.esculenta).
4.1 mycelial growth situations and pollution condition contrast
(1) test method
By 4 morel inoculation, on 18mm * 18mm flat board, each bacterial strain repeats 10 times, and plate culture medium is improvement PDA: potato 30%, glucose 2%, K 2hPO 40.5%, agar 2%, surplus is water, described percentage ratio is massfraction, is placed on lucifuge in the incubator of 20 ℃ and cultivates after inoculation.
Every 8h carries out observed and recorded.
(2) interpretation of result (referring to table 1)
The growing state of each bacterial strain of table 1 morel on improvement PDA substratum
Note :+represent that mycelia growing way is poor; ++ represent that mycelia growing way is general; +++ represent that mycelia growing way is denseer; ++++represent, mycelia growing way was dense; +++ ++ represent that mycelia growing way is dense close.
As can be seen from Table 1, under identical culture condition, the growing state of each bacterial strain is difference to some extent.From growing way and full plate time, morel of the present invention (Mochella esculenta) M-02 #bacterial strain CGMCC No.7058 fast growth, grows fine, and mycelia is paved with the dull and stereotyped time and has shortened 2-3d compared with other three bacterial strains; From pollution condition, M-02 #bacterial strain does not pollute, and antipollution rate is obviously better than other three bacterial strains.
4.2 sclerotium generative capacity comparisons
(1) test method
By 4 morel inoculation on 18mm * 18mm flat board, 10 repetitions of each bacterial strain, plate culture medium for improvement PDA: potato 30%, glucose 2%, K 2hPO 40.5%, agar 2%, surplus is water, culture temperature 20-25 ℃, described percentage ratio is massfraction.After inoculation, first in the incubator of 20 ℃, lucifuge is cultivated 3d; Then proceed in illumination box, use 250lx continuous illumination to impel mycelium annesl to generate sclerotium.
Every 8h carries out observed and recorded.
(2) interpretation of result (referring to table 2)
Table 2 sclerotium of morchella esculenta formational situation
Note :+the variation of number expression sclerotium.
As can be seen from Table 2, M-02 #bacterial strain sclerotium generative capacity is the strongest, M0710 #, M-501 #secondly, M-16 #the poorest.M-02 #annesl is fast, just can see that the sclerotium of yellow particle shape occurs since the 5th day, and along with the increase of incubation time, sclerotium has pale yellow, the Huang of beginning progressively to change brown Huang, brown into.
Zhaotong Prefecture's Drainage Area of Jinsha River that the present invention occurs from morel (M.esculenta) gathers its wild sporophore isolated strains morel (Mochella esculenta) M-02 #bacterial strain CGMCC No.7058 has obvious regional Characteristics, compared with prior art, has following advantage: the one, and mycelial biomass is high, can reach 25g/kg, and solid bacterium bag mycelial biomass is greatly about 5-12g/kg; The 2nd, sclerotium generative capacity is strong, and on the flat board of diameter 10cm, golden yellow sclerotium particle is many or area coverage is large, and sclerotium particle can reach 50, and single particle diameter can reach 1cm; The 3rd, antipollution, to cultivate pollution rate low, and≤2%.Adopt the solution fermentation time short simultaneously, at 72-96h, and more than common solid bag cultivates and to need 15d.Therefore, morel of the present invention (Mochella esculenta) M-02 #bacterial strain CGMCC No.7058 is the short numerous excellent species that provides of morel wild resource protection.
<110> KUNMING EODIBLE FUNGUS RES INS
Yunnan Supply And Marketing Cooperatives Science Institute
Yunnan cloud Cordycepps skill (group) company limited
<120> mono-strain morel bacterial strain and cultural method thereof
<130> /
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> ITS4 primer
<400> 1
tcctccgctt attgatatgc 20
<210> 2
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> ITS5 primer
<400> 2
ggaagtaaaa gtcgtaacaa gg 22

Claims (2)

  1. One strain morel ( mochella esculenta) M-02 #bacterial strain CGMCC No .7058.
  2. Morel ( mochella esculenta) M-02 #bacterial strain CGMCC No .the cultural method of 7058 liquid spawns, its step is as follows:
    (1) by morel ( mochella esculenta) M-02 #the mycelium of bacterial strain CGMCC NO.7058 is inoculated on test tube nutrient agar inclined-plane, at 18-24 ℃, cultivates 5-7d, obtains test tube strains, and described test tube nutrient agar is improvement PDA substratum, and its formula is potato 30%, glucose 2%, K 2hPO 40.5%, agar 2%, surplus is water, described percentage ratio is massfraction;
    (2) test tube strains is inoculated in the 500mL triangular flask that fills 200mL liquid nutrient medium, at 20-26 ℃, 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: 5-10% wheat bran, 0.5-1% analysis for soybean powder, 0.5-1% maltose, 1-2% Zulkovsky starch, and surplus is water, described percentage ratio is massfraction;
    (3) the level liquid bacterial classification of acquisition is inoculated in the fermentor tank of the automatic fermentative production line of 25L, ventilation ratio is 1:0.8, and mixing speed is 120-160r/min, and inoculum size is 5-10% massfraction, tank pressure: 0.04Mpa; PH is 7.2-7.5; Temperature is 22-26 ℃; Aerated culture 72-96h, obtain morel ( mochella esculenta) M-02 #bacterial strain CGMCC No .7058 liquid spawns.
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* Cited by examiner, † Cited by third party
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CN104756767A (en) * 2015-04-29 2015-07-08 天津农学院 High-density pleurotus eryngii liquid strain and fermentation method thereof
CN105441342A (en) * 2016-01-29 2016-03-30 平武县茂源菌种有限公司 Morchella liquid strain production process
CN105613042A (en) * 2016-01-25 2016-06-01 四川保兴现代农业科技股份有限公司 Breeding method for liquid strain of morchella and industrial cultivation method for morchella
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CN104756767A (en) * 2015-04-29 2015-07-08 天津农学院 High-density pleurotus eryngii liquid strain and fermentation method thereof
CN104756767B (en) * 2015-04-29 2016-11-30 天津农学院 A kind of high density pleurotus eryngii liquid strain and fermentation process thereof
CN106609247A (en) * 2015-10-21 2017-05-03 广东东阳光药业有限公司 Deep fermentation culture method for morel
CN106609247B (en) * 2015-10-21 2019-06-25 广东东阳光药业有限公司 A kind of submerged culturing method for making of hickory chick
CN105613042A (en) * 2016-01-25 2016-06-01 四川保兴现代农业科技股份有限公司 Breeding method for liquid strain of morchella and industrial cultivation method for morchella
CN105613042B (en) * 2016-01-25 2018-12-25 四川保兴现代农业科技股份有限公司 A kind of the liquid spawn mating system and industrial planting method of hickory chick
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CN107513500A (en) * 2016-06-17 2017-12-26 云南清湖山色农业科技有限公司 A kind of preparation method of hickory chick liquid spawn
CN107823225A (en) * 2017-11-14 2018-03-23 上海市农业科学院 The application of preeminent hickory chick
CN109082385A (en) * 2018-08-22 2018-12-25 四川省农业科学院土壤肥料研究所 The preparation method of hickory chick liquid spawn
CN109082385B (en) * 2018-08-22 2021-09-14 四川省农业科学院土壤肥料研究所 Preparation method of morchella liquid strain
CN109136102A (en) * 2018-09-11 2019-01-04 中华全国供销合作总社昆明食用菌研究所 A kind of production method of hickory chick liquid spawn

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