CN108770593A - A kind of Lepista mucla (Bull.:Fr.) Cooke bacterial strain and its sporocarp culture method - Google Patents
A kind of Lepista mucla (Bull.:Fr.) Cooke bacterial strain and its sporocarp culture method Download PDFInfo
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- CN108770593A CN108770593A CN201810468261.4A CN201810468261A CN108770593A CN 108770593 A CN108770593 A CN 108770593A CN 201810468261 A CN201810468261 A CN 201810468261A CN 108770593 A CN108770593 A CN 108770593A
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- 235000013192 Lepista nuda Nutrition 0.000 claims abstract description 9
- 239000001963 growth media Substances 0.000 claims description 60
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- 239000000463 material Substances 0.000 claims description 23
- 210000003608 Feces Anatomy 0.000 claims description 20
- 241000283690 Bos taurus Species 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 238000002360 preparation method Methods 0.000 claims description 15
- 238000011081 inoculation Methods 0.000 claims description 12
- 239000010440 gypsum Substances 0.000 claims description 11
- 229910052602 gypsum Inorganic materials 0.000 claims description 11
- 230000035784 germination Effects 0.000 claims description 9
- 235000021028 berry Nutrition 0.000 claims description 8
- CURLTUGMZLYLDI-UHFFFAOYSA-N carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 7
- 239000001569 carbon dioxide Substances 0.000 claims description 7
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 7
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- 239000010902 straw Substances 0.000 abstract description 15
- 241000894007 species Species 0.000 abstract description 10
- 241000233866 Fungi Species 0.000 abstract description 9
- 241000446313 Lamella Species 0.000 abstract description 4
- 239000003205 fragrance Substances 0.000 abstract description 3
- 235000013311 vegetables Nutrition 0.000 abstract description 2
- 238000000855 fermentation Methods 0.000 description 24
- 230000004151 fermentation Effects 0.000 description 24
- 239000001965 potato dextrose agar Substances 0.000 description 10
- 239000002994 raw material Substances 0.000 description 9
- 240000005158 Phaseolus vulgaris Species 0.000 description 6
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 6
- ODINCKMPIJJUCX-UHFFFAOYSA-N calcium monoxide Chemical compound [Ca]=O ODINCKMPIJJUCX-UHFFFAOYSA-N 0.000 description 6
- PASHVRUKOFIRIK-UHFFFAOYSA-L calcium sulfate dihydrate Chemical compound O.O.[Ca+2].[O-]S([O-])(=O)=O PASHVRUKOFIRIK-UHFFFAOYSA-L 0.000 description 5
- 238000003306 harvesting Methods 0.000 description 5
- 239000002609 media Substances 0.000 description 5
- 235000015099 wheat brans Nutrition 0.000 description 5
- 239000011888 foil Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000011068 load Methods 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L mgso4 Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
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- CZMRCDWAGMRECN-UGDNZRGBSA-N D-sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- CZMRCDWAGMRECN-GDQSFJPYSA-N Sucrose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)[C@@]1(CO)[C@H](O)[C@@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-GDQSFJPYSA-N 0.000 description 3
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- 241000287828 Gallus gallus Species 0.000 description 2
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- 240000002809 Lavandula angustifolia Species 0.000 description 2
- 235000003515 Lavandula officinalis Nutrition 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M Monopotassium phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 240000001016 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 244000269722 Thea sinensis Species 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000009264 composting Methods 0.000 description 2
- 230000000875 corresponding Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
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- 239000001102 lavandula vera Substances 0.000 description 2
- 235000018219 lavender Nutrition 0.000 description 2
- 239000010871 livestock manure Substances 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
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- 238000004901 spalling Methods 0.000 description 2
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- GWEVSGVZZGPLCZ-UHFFFAOYSA-N titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N β-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 241000222485 Agaricales Species 0.000 description 1
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- 210000003705 Ribosomes Anatomy 0.000 description 1
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- 235000004338 Syringa vulgaris Nutrition 0.000 description 1
- 235000016639 Syzygium aromaticum Nutrition 0.000 description 1
- 240000005147 Syzygium aromaticum Species 0.000 description 1
- 241000383675 Trama Species 0.000 description 1
- 241000609666 Tuber aestivum Species 0.000 description 1
- 239000010975 amethyst Substances 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
Abstract
The invention discloses a kind of Lepista mucla (Bull.:Fr.) Cooke bacterial strain and its sporocarp culture methods.The present invention provides Lepista mucla (Bull.:Fr.) Cooke Lepista nuda DCH560, preserving number is CGMCC No.15376.Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) provided by the present invention is the isolated bacterial strain of sample for picking up from LiaoNing, China Dandong Manchu Autonomous County of Kuandian, which can obtain fructification after domestication.The fructification cap that the bacterial strain is cultivated is purple, and bacterial context is thicker, lamella purple, has strong fragrance, consistent with wild fructification.The present invention tames to have obtained Lepista mucla (Bull.:Fr.) Cooke fructification, develops Rare edible fungus new varieties, for protecting the germ plasm resource of the species; abundant edible fungus variety resource, abundant vegetable basket etc. are of great significance, while the bacterium is typical straw rotting fungus; agricultural crop straw can be utilized, there is ecological significance.
Description
Technical field
The invention belongs to microorganisms technical field, it is related to a kind of Lepista mucla (Bull.:Fr.) Cooke bacterial strain and its sporocarp culture method.
Background technology
Lepista mucla (Bull.:Fr.) Cooke Lepista nuda (Bull.) Cooke is also known as naked dried mushroom, and amethyst mushroom is under the jurisdiction of Agaricales, dried mushroom
Section, Lepista lentinus because gill fungus handle is in lilac and fragrant with very strong unique mushroom, therefore claim Lepista mucla (Bull.:Fr.) Cooke.Lepista mucla (Bull.:Fr.) Cooke color
Damp pleasant, aromatic flavour, fresh and tender sweet, excellent in taste are known as the good reputation of " a Shiqi taste, ten are heard its perfume ", Europe by
Ratings and truffle and bolete are eponymous, are considered as first-class food materials in France.
Lepista mucla (Bull.:Fr.) Cooke has higher research and development value as rare edible and medical fungi, but main so far next
Source is field acquisition, and natural resources are limited, and quality cannot be guaranteed.Although the domestication that some units have carried out Lepista mucla (Bull.:Fr.) Cooke is planted
Training, but there are no fructification cultivation successfully report.Patent " a kind of Lepista mucla (Bull.:Fr.) Cooke culture medium based on yellow wine lees and purple fourth
Fragrant mushroom liquid spawn preparation method (CN201610552378.1) " provide a kind of Lepista mucla (Bull.:Fr.) Cooke culture medium based on yellow wine lees with
And Lepista mucla (Bull.:Fr.) Cooke liquid spawn preparation method, but do not obtain fructification；" a kind of Lepista mucla (Bull.:Fr.) Cooke bacterial strain and its liquid spawn with
Preparation method CN201510877093.0 " discloses a kind of Lepista mucla (Bull.:Fr.) Cooke bacterial strain and its liquid spawn and preparation method, does not have equally
Obtain fructification.Yanbian University's master thesis《Lepista mucla (Bull.:Fr.) Cooke Mycelium culture characteristic and artificial domesticating cultivation research》
(Zhao Ting, 2010) has studied Lepista mucla (Bull.:Fr.) Cooke Mycelium culture characteristic, and domestication research has been carried out to it, but result there is no
Fructification.Yao Zhiwei etc. studies the induction synthesis of Lepista mucla (Bull.:Fr.) Cooke former base, does not as a result also obtain fructification (Yao Zhiwei；Yuan
Positive wave；Gao Junwei, Lepista mucla (Bull.:Fr.) Cooke former base induce synthetic media pre-test chemistry and bioengineering, 2010,27 (1):59-62.).
1994 once had been reported that the domesticating and cultivating of the spontaneous edible mushroom Lepista mucla (Bull.:Fr.) Cooke in tea place (wore bright cloud and Cui Xiaoming, 1994 Tea Leaves of Guizhou 1：
24) it is reported in tea place outdoor cropping, by 7 months time fruitings, this report method therefor was outdoor cropping, uncontrollable factor
It is more, and fruiting time is very long, so far there are no promoting.
Invention content
It is an object of the present invention to provide Lepista mucla (Bull.:Fr.) Cooke (Lepista nuda) DCH560 bacterial strains.
Lepista mucla (Bull.:Fr.) Cooke (Lepista nuda) DCH560 bacterial strains provided by the invention, preserving number CGMCC
No.15376。
Application of the above-mentioned Lepista mucla (Bull.:Fr.) Cooke in being cross-breeding as parent is also the scope of protection of the invention.
It is also the scope of protection of the invention to cultivate the fructification that above-mentioned Lepista mucla (Bull.:Fr.) Cooke obtains.
It is also the scope of protection of the invention to cultivate the mycelium that above-mentioned Lepista mucla (Bull.:Fr.) Cooke obtains.
Another object of the present invention is to provide the cultural method of the fructification.
Method provided by the invention, includes the following steps：The mycelium of above-mentioned Lepista mucla (Bull.:Fr.) Cooke is subjected to original seed system successively
Standby, cultigen preparation, inoculation and bacterium germination, earthing, management of producing mushroom and fruiting, obtain fructification.
In the above method, method prepared by the original seed includes the following steps：For by Lepista mucla (Bull.:Fr.) Cooke described in claim 1
Mycelium be inoculated into pedigree seed culture medium, 15-25 DEG C is protected from light culture, and relative air humidity, which is 60%-70%, to be covered with to mycelia
Culture medium obtains original seed；
Or, method prepared by the cultigen includes the following steps：The original seed is inoculated into culture medium for cultivating, 15-
25 DEG C are protected from light culture, and relative air humidity is that 60%-70% covers with culture medium to mycelia, obtains cultigen；
Or, the method for the inoculation and bacterium germination includes the following steps：The cultigen is accessed into culture material, 15-25 DEG C is kept away
Optical culture, relative air humidity cover with culture material for 65%-75% to mycelia；
Or, the method for the earthing includes the following steps：In the inoculation and bacterium germination step after mycelia covers with charge level
The earthing on the charge level of the culture material, 15-25 DEG C is protected from light culture, relative air humidity 70%-75%, titanium dioxide in air
Carbon relative concentration 0.1%-0.5%, until mycelia grows into soil layer surface；
Or, the method for the management of producing mushroom includes the following steps：Wait for that the mycelia that the earthing step obtains grows into soil layer
When surface, one layer of fine earth is mended, maintains 15-23 DEG C of temperature, intensity of illumination 500-800Lux, relative air humidity 85%-
95%, Carbon Dioxide in Air relative concentration 0.05%-0.15%, culture obtain mushroom former base in 5-10 days；
Or, the method for the fruiting includes the following steps：The mushroom former base that the management of producing mushroom step obtains is transferred to
Environment temperature is 18-20 DEG C, relative air humidity 75%-90%, and carbon dioxide relative concentration is the item of 0.05%-0.15%
It is cultivated under part, obtains fructification.
The method further includes harvesting fructification step, specially waits for that fructification is grown to medium well, cap is not yet opened up completely
It opens, you can harvesting mushroom.
In the above method, the pedigree seed culture medium and the Cultivar culture medium are wheat culture medium or the training of wheat cow dung
Support base；The pedigree seed culture medium is identical with the Cultivar culture medium.
The wheat culture medium is the starting material with water tune being made of 98% wheat berry of mass fraction, 1% gypsum and 1% lime
Biodiversity number processed is 65% obtained culture medium；
The wheat cow dung culture medium is by wheat berry 93%, cow dung 5%, and the raw material that gypsum 1% and lime 1% form is used
It is 65% obtained culture medium that water, which modulates biodiversity number,.
In the above method, in method prepared by the original seed, described 15-25 DEG C is protected from light culture and is protected from light culture for 22 DEG C.On
It is excrement grass fermentation material to state culture material, is fermented by agricultural crop straw, cow dung, dregs of beans and gypsum.
Above-mentioned culture material can be obtained by following culturing raw material after secondary fermentation：The culturing raw material is by agricultural crop straw
(straw, wheat straw), animal wastes (such as dry cow dung and/or chicken manure), beans foil and land plaster mix.Agricultural crop straw, animal
Quality (dry weight) proportioning of excrement, beans foil and land plaster is 55：45：1.2：4.
In the present invention, the secondary fermentation is divided into primary fermentation and rear fermentation.The primary fermentation is：It 3-4 days before composting will
Agricultural crop straw and cow dung are prewetted, moisture 65%-70%, south-north direction fermentation reactor system, heap temperature are monitored, as long as heap
A turning is carried out when body internal temperature is begun to decline after reaching 70 DEG C or more, after 3-4 turning, completes the preceding hair
Ferment.It ferments after described and is：It will again be fermented by the culture material of the primary fermentation, heap body internal temperature risen into 58-62
DEG C and keep 12-24h, then divulge information and heap temperature be down to 50-52 DEG C of holding 4-6d, then heap temperature is down to room temperature (25-
28℃).The rear fermentation is completed, the secondary fermentation comes to an end.By the secondary fermentation culturing raw material should without ammonia taste,
Without excrement stink, it can be seen that the apparent actinomyces albus layer of charge level, by the water content by the culturing raw material of the secondary fermentation
It is adjusted to 65% (mass fraction), pH value is adjusted to 7.0-7.5 to get to the culture material.
In the method, planting type concretely cultivate by layer frame.
The layer frame is cultivated：Layer frame specification is 3-5 layers, and floor height 0.6m, wide 1.5m, bottom is apart from ground 0.3m (length
Depending on needing)；The culture material is laid in every layer of bedstead and carries out cultivation fruiting.
Application of the above-mentioned fructification in food processing is also the scope of protection of the invention.
Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) provided by the present invention is to pick up from LiaoNing, China Dandong Kuan Dian to expire
The isolated bacterial strain of the sample of autonomous county of race, the bacterial strain can obtain fructification after domestication.The bacterial strain is cultivated
The fructification cap arrived is purple, and bacterial context is thicker, lamella purple, has strong fragrance, consistent with wild fructification.The present invention
Domestication has obtained Lepista mucla (Bull.:Fr.) Cooke fructification, develops Rare edible fungus new varieties, for protecting the germ plasm resource of the species, enriches
Edible fungus variety resource, abundant vegetable basket etc. is of great significance, while the bacterium is typical straw rotting fungus, can utilize crops
Stalk has ecological significance.
Preservation explanation
It is recommended that Classification And Nomenclature：Lepista mucla (Bull.:Fr.) Cooke
Latin name：Lepista nuda
Join the biomaterial of evidence：DCH560
Preservation mechanism：China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is referred to as：CGMCC
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date：On April 17th, 2018
Collection is registered on the books number：CGMCC No.15376
Description of the drawings
Fig. 1 is the Lepista mucla (Bull.:Fr.) Cooke sample of field acquisition.
Fig. 2 Lepista mucla (Bull.:Fr.) Cookes DCH560 (CGMCC No.15376) strain tube strain (parent species).
Fig. 3 is the growth shape of Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) mycelia after PDA plate culture 20d days
Condition.
Fig. 4 is pedigree seed culture medium of Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) inoculations in 3 kinds of different formulations
In upgrowth situation.
Fig. 5 is the situation that full bacterium is sent out after Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) bacterial strain is sowed 30 days.
Fig. 6 is that Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) bacterial strain former base breaks up situation.
Fig. 7 is Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) bacterial strain fructification fruiting situation.
Fig. 8 is the fructification load and basidiospore that Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) bacterial strain is cultivated.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The acquisition and identification of embodiment 1, Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376)
Bacterial strain DCH560 of the present invention is isolated from natural Lepista mucla (Bull.:Fr.) Cooke sample, and (Lepista mucla (Bull.:Fr.) Cooke sample is certainly Chinese in acquisition in 2016
Dandong Manchu Autonomous County of Kuandian), by the observation to its bacterium colony, Mycelial morphological characteristic, combination cell core rDNA-ITS is surveyed
Sequence carries out taxonomic identification to the bacterial strain.
One, morphological feature
Bacterial strain DCH560 is cultivated 2 weeks, colony diameter 5cm or so for 20 DEG C in potato dextrose agar (PDA),
Bacterium colony front lavender flocculence, mycelia is loose, and back side color is slightly deep, and the older mycelia color of the tender mycelia of children is deep (see Fig. 1 and figure
2).Mycelia has every transparent, tool clamp connection, 1.2-5 μm of diameter does not observe that asexual spore generates.
Two, molecular biological characteristics
RDNA-ITS sequences are as bar code (Schoch, C.L.et al.Nuclear ribosomal internal
transcribed spacer(ITS)region as a universal DNA barcode marker for
Fungi.Proceedings of the National Academy of Sciences,2012.109:6241-6246), extensively
It is general to be identified for fungal species.The present invention carries out DNA extractions and rDNA-ITS amplifications sequencing to bacterial strain DCH560, and (the primer is
The primer pair of ITS4 and ITS5 compositions), in sequencing result such as sequence table shown in sequence 1.By the sequence in ncbi database
It carries out BLAST to compare online, as a result shows the sequence and Lepista mucla (Bull.:Fr.) Cooke (Lepista nuda) corresponding sequence (GenBank：
KJ577474 it) compares, homologous similitude is 99%.
ITS4：5'-TCCTCCGCTTATTGATATGC-3'；
ITS5：5'-GGAAGTAAAAGTCGTAACAAGG-3'.
It is studied in view of the taxonomic identification of above-mentioned morphological feature and rDNA-ITS sequences, determines that bacterial strain DCH560 of the present invention is purple
Cloves mushroom (Lepista nuda).The bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center
(abbreviation CGMCC, address are：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is CGMCC No.15376.
Embodiment 2, the method for Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) fructification cultivation
One, flow is cultivated
The cultivation flow of Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) fructification is specific as follows：Parent species preparation → original seed
Preparation → cultigen preparation → inoculation and bacterium germination → earthing → management of producing mushroom → fruiting → harvesting.
(1) preparation of parent species：The mycelia of Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) after tissue separation is tamed,
It being inoculated on mother culture media (test tube slant), 15-25 DEG C (such as 20-23 DEG C) is protected from light culture, and culture medium is covered in culture to mycelia,
Obtained mycelium is as parent species (Fig. 3).
(2) preparation of original seed：Parent species obtained by step (1) are transferred on pedigree seed culture medium, inoculum concentration is per 750mL
The parent species strain block of 6-10 a diameter of 1.0cm sizes is uniformly accessed in the pedigree seed culture medium of volume, relative air humidity is
60%-70%, 15-25 DEG C (such as 20-23 DEG C) are protected from light culture and cover with culture medium to mycelia, obtain original seed.
(3) preparation of cultigen：Original seed obtained by step (2) is transferred in Cultivar culture medium, per 750mL original seeds
The cultigen of 30 bottles of 750mL of bottle inoculation, 15-25 DEG C (such as 20-23 DEG C) are protected from light culture, relative air humidity 60%-70%, training
It supports to mycelia and covers with culture medium, obtain cultigen.
(4) inoculation and bacterium germination：Cultigen obtained by step (3) is accessed into culture material, access ratio is to be cultivated per square meter
The culture material of area is inoculated with 2 bottles of cultigens (750mL), and inoculation method is broadcast for layer, and cultigen is equably sprinkling upon charge level, repaves one
Layered material is covered with thin film moisturizing, and 15-25 DEG C (such as 20-23 DEG C) is protected from light culture, and relative air humidity is 65%-75% (Fig. 5).
(5) earthing：Step (4) mycelia covers with after charge level earthing, 15-25 DEG C of (such as 20-23 on the charge level of above-mentioned culture material
DEG C) it is protected from light culture, relative air humidity 70%-75%, 1 maintenance Carbon Dioxide in Air relative concentration of ventilation daily
0.1%-0.5% (volume fraction)；
The soil of earthing is turfy soil, it is desirable that good permeability, retention ability are strong.
(6) management of producing mushroom：When the mycelia of step (5) culture grows into soil layer surface, one layer of fine earth is mended, starts to cool down, with
And the dim light that intensity of illumination is 500-800Lux stimulates, relative air humidity 85%-95%, ventilation 2-3 times, makes dioxy daily
Change carbon relative concentration and drop to 0.05%-0.15% (volume fraction), cultivates 5-10 days.
(7) fruiting：It was found that after a large amount of mushroom former bases (Fig. 6), it is 18-20 DEG C to be transferred to environment temperature, relative air humidity
For 75%-90%, carbon dioxide relative concentration cultivates (Fig. 7) under conditions of being 0.05%-0.15% (volume fraction).
(8) it harvests：Wait for that fructification is grown to medium well, cap is not yet fully deployed, you can the first damp mushroom of harvesting.It picks laggard
Row cleaning residual mushroom root mends native mushroom cave, and aforesaid operations carry out fruiting again after mycelia restores, and harvest next damp mushroom.
Above-mentioned mother culture media can be that PDA culture medium plus rich PDA culture medium or wheat bran soak juice culture medium；
Every liter of PDA culture medium is prepared obtain as follows：Potato 200g is put into 900mL water and boils 20min,
It takes filtrate that agar 20g heating is added to be allowed to dissolve completely, adds water and complement to 1000mL, 121 DEG C of sterilizing 30min obtain 1L
PDA culture medium.
Every liter plus rich PDA culture medium are prepared obtain as follows：Potato 200g is put into 900mL water and is boiled
20min, takes filtrate that agar 20g, sucrose or glucose 20g, potassium dihydrogen phosphate 3g, magnesium sulfate 1.5g is added, and heating is allowed to complete
It dissolves, adds water and complement to 1000mL, 121 DEG C of sterilizing 30min obtain 1L and add rich PDA culture medium.
Every liter of wheat bran leaching juice culture medium is prepared obtain as follows：Wheat bran 100g is put into 900mL water and is boiled
20min, takes filtrate that agar 20g, sucrose or glucose 20g, potassium dihydrogen phosphate 3g, magnesium sulfate 1.5g is added, and heating is allowed to complete
It dissolves, adds water and complement to 1000mL, obtain wheat bran described in 1L and soak juice culture medium；
Above-mentioned pedigree seed culture medium and above-mentioned Cultivar culture medium are wheat culture medium or wheat cow dung culture medium.
Wheat culture medium prescription：Wheat berry 98%, gypsum 1%, lime 1% (mass fraction).Preparation method is wheat berry
It impregnates 12-24 hours, boils 20min or so, pay attention to preventing wheat spalling.It is pulled out after cooked, clear water rinses, and drains away the water, adds
Enter remaining ingredient, dissolving stirs evenly.
Wheat cow dung culture medium is wheat berry 93%, cow dung 5%, gypsum 1%, lime 1% (mass fraction).Preparation method
It is impregnated 12-24 hours for wheat berry, boils 20min or so, pay attention to preventing wheat spalling.It is pulled out after cooked, clear water rinses, and drains
Remaining ingredient is added in moisture, stir evenly.
Above-mentioned culture material is excrement grass fermentation material, is fermented by agricultural crop straw, cow dung, dregs of beans and gypsum, specifically by such as
Lower culturing raw material obtains after secondary fermentation：
Above-mentioned culturing raw material is by agricultural crop straw (straw, wheat straw), animal wastes (such as dry cow dung and/or chicken manure), beans foil
Mixed with land plaster, agricultural crop straw, animal wastes, beans foil and land plaster quality (dry weight) proportioning be 55：45：1.2：
4。
Above-mentioned secondary fermentation is divided into primary fermentation and rear fermentation, and primary fermentation is：Before composting 3-4 days by agricultural crop straw and cow dung
It prewets, moisture 65%-70%, south-north direction fermentation reactor system monitors heap temperature, as long as heap body internal temperature reaches 70
DEG C or more after a turning is carried out when beginning to decline, after 3-4 turning, completion primary fermentation.
It ferments after above-mentioned and is：It will again be fermented by the culture material of primary fermentation, heap body internal temperature risen into 58-62
DEG C and keep 12-24h, then divulge information and heap temperature be down to 50-52 DEG C of holding 4-6d, then heap temperature is down to room temperature (25-
28℃).It ferments after the completion, secondary fermentation comes to an end.
It should be without ammonia taste, no excrement stink by the culturing raw material of secondary fermentation, it can be seen that the apparent actinomyces albus of charge level
The water content of culturing raw material by secondary fermentation will be adjusted to 65% (mass fraction) by layer, and pH value is adjusted to 7.0-7.5,
Obtain culture material.
Above-mentioned steps (4) planting type concretely cultivate by layer frame.
Above-mentioned layer frame is cultivated：Layer frame specification is 3-5 layers, and floor height 0.6m, wide 1.5m, bottom is apart from ground 0.3m (length
Depending on needing)；Above-mentioned culture material is laid in every layer of bedstead and carries out cultivation fruiting.
Two, condition optimizing
The following factor in cultivation is optimized respectively：Primary spawn temperature is groped and Primary spawn basigamy
Side.
1, the determination of Primary spawn temperature
It will organize the mycelia of isolated Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) by way of punching, press
It according to the inoculation block amount of a diameter of 1.0cm, is inoculated in PDA culture medium, is placed at different temperature (15,20,22,25,30 DEG C)
It is protected from light culture, measures the speed of growth of mycelia in incubation, and observe the growing state of mycelia.
In triplicate, results are averaged for quantitative data for experiment.
The results are shown in Table 1, it is seen that Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) is under the conditions of 22 DEG C, mycelia growth
Speed is significantly higher than other temperature conditions.
Table 1 is the measurement result (unit of mycelial growth rate under different temperatures：mm/d)
Note：Identical letter indicates that multiple check does not have significant difference (P=0.05) in same column.
2, the determination of pedigree seed culture medium
By the parent species for the same a batch of Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) cultivated under the same terms, inoculation
In different pedigree seed culture mediums (formula sees below) 750mL culture bottles, it is placed in 22 DEG C of constant incubators and is protected from light continuous culture, it is empty
Gas relative humidity is 60%-70%, and observation statistics mycelium germination time and mycelia cover with a bottle time.
Pedigree seed culture medium for examination is following three kinds (Fig. 4, A wheat cow dung culture medium, B wheat culture mediums, C sawdust cultures
Base)：
Formula 1：Cotton seed hulls sawdust medium
Cotton seed hulls 40%, sawdust 50%, wheat bran 8%, gypsum 1%, sucrose 1%；
Formula 2：Wheat culture medium
Wheat 98%, gypsum 1%, quick lime 1%；
Formula 3：Wheat cow dung culture medium
Wheat 93%, cow dung 5%, gypsum 1%, quick lime 1%；
The number of each substance is parts by weight (dry weight) in the above formula.Add water damping on the basis of above each formula
It is 65% to get to three kinds of pedigree seed culture mediums for examination to biodiversity number.
Quick lime and land plaster produce for Tianjin Zhi Yuan chemical reagent Co., Ltd.
As a result (Fig. 4) is shown, Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) is in 3 corresponding original seeds of formula 2 and formula
In culture medium (being named as wheat culture medium and wheat excrement grass culture medium), sprout time is relatively early, mycelial growth rate is very fast, mycelia
It is sturdy, upgrowth situation is preferable.And mycelial growth rate is relatively slow on being formulated 1 sawdust medium and tapers off growth.
Three, Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376) cultivates obtained fructification feature
According to one method, fructification is obtained.
Not only successfully cultivation has obtained fructification to Lepista mucla (Bull.:Fr.) Cooke DCH560 (CGMCC No.15376), but also observed load
Son and basidiospore (Fig. 8, A B：Basidiospore, basidiospore of the C long in load).Cultivate obtained fructification bacteria cover diameter 3-
12cm, cap purple, edge is involute, and bacterial context is thicker, lavender, and discoloration is light after maturation, lamella purple, has strong fragrance.
Mycelia thin-walled is to thick wall, and moderate branch, bending, the expansion of some mycelia, interleaved arrangement, diameter is usually 4-10 μm, expands bacterium
Silk diameter is up to 15 μm.Trama hyphae colorless in lamella, thin-walled, few branch frequently separate, slightly curved song is regularly arranged, a diameter of
2.5-6μm；Without cystidium in hymenium；The nearly club-like of load, has 2-4 stigma, and size is (24.0-26.0) × (5.0-
7.0)μm.Basidiospore oblong, colourless, thin-walled to thick wall, size are (6.7-9.0) × (4.0-5.5) μm.
Sequence table
<110>Institute of Microorganism, Academia Sinica
<120>A kind of Lepista mucla (Bull.:Fr.) Cooke bacterial strain and its sporocarp culture method
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 649
<212> DNA
<213>Lepista mucla (Bull.:Fr.) Cooke Lepista nuda (Bull.)
<400> 1
atcattattg aataaacttg gttgggttgt gctggctttt cggagcatgt gcacgcctag 60
cgccattttt accacctgtg cacattttgt agatttggaa caattctcga gtttactcgg 120
tttgaggaat gctatgccaa aagcttagct ttccttgtgt ttcaagtcta tgtttttata 180
taccccatac gaatgtaata gaatgtcatt aatgggctta tagcctttaa attaatacaa 240
ctttcaacaa cggatctctt ggctctcgca tcgatgaaga acgcagcgaa atgcgataag 300
taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacctt gcgctccttg 360
gtattccgag gagcatgcct gtttgagtgt cattaaattc tcaacctttt cagcttttac 420
aagttgtatt ggcttggatg tggaggtttt gcaggcttct taagaagtca gctcctctta 480
aatgcattag cggaaccttt gtggaccagc tttggtgtga taattatcta cgccatggtt 540
gtaaagcagc tttatatggg gttcagcttc taacagtcca tttacttgga caatttctga 600
catcttgacc tcaaatcagg taggactacc cgctgaactt aagcattca 649
Claims (9)
- Lepista mucla (Bull.:Fr.) Cooke 1. (Lepista nuda) DCH560, preserving number is CGMCC No.15376.
- 2. application of the Lepista mucla (Bull.:Fr.) Cooke described in claim 1 in being cross-breeding as parent.
- 3. the fructification that cultivation Lepista mucla (Bull.:Fr.) Cooke described in claim 1 obtains.
- 4. the mycelium that culture Lepista mucla (Bull.:Fr.) Cooke described in claim 1 obtains.
- 5. the cultural method of fructification described in claim 3, includes the following steps：By Lepista mucla (Bull.:Fr.) Cooke described in claim 1 Mycelium carries out original seed preparation, cultigen preparation, inoculation and bacterium germination, earthing, management of producing mushroom and fruiting successively, obtains fructification.
- 6. according to the method described in claim 5, it is characterized in that：Method prepared by the original seed includes the following steps：For the mycelium of Lepista mucla (Bull.:Fr.) Cooke described in claim 1 is inoculated into In pedigree seed culture medium, 15-25 DEG C is protected from light culture, and relative air humidity is that 60%-70% covers with culture medium to mycelia, obtains original Kind；Or, method prepared by the cultigen includes the following steps：The original seed is inoculated into culture medium for cultivating, 15-25 DEG C It is protected from light culture, relative air humidity is that 60%-70% covers with culture medium to mycelia, obtains cultigen；Or, the method for the inoculation and bacterium germination includes the following steps：The cultigen is accessed into culture material, 15-25 DEG C is protected from light training It supports, relative air humidity covers with culture material for 65%-75% to mycelia；Or, the method for the earthing includes the following steps：In institute after mycelia covers with charge level in the inoculation and bacterium germination step Earthing on the charge level of culture material is stated, 15-25 DEG C is protected from light culture, relative air humidity 70%-75%, Carbon Dioxide in Air phase To concentration 0.1%-0.5%, until mycelia grows into soil layer surface；Or, the method for the management of producing mushroom includes the following steps：Wait for that the mycelia that the earthing step obtains grows into soil layer surface When, one layer of fine earth is mended, maintains 15-23 DEG C of temperature, intensity of illumination 500-800Lux, relative air humidity 85%-95% are empty Carbon dioxide relative concentration 0.05%-0.15% in gas, culture obtain mushroom former base in 5-10 days；Or, the method for the fruiting includes the following steps：The mushroom former base that the management of producing mushroom step obtains is transferred to environment Temperature is 18-20 DEG C, relative air humidity 75%-90%, under conditions of carbon dioxide relative concentration is 0.05%-0.15% Culture, obtains fructification.
- 7. according to the method described in claim 6, it is characterized in that：The pedigree seed culture medium and the Cultivar culture medium are wheat culture medium or wheat cow dung culture medium；The wheat culture medium is to modulate water by the starting material with water that 98% wheat berry of mass fraction, 1% gypsum and 1% lime form It is 65% obtained culture medium to divide mass fraction；The wheat cow dung culture medium is the starting material with water tune being made of wheat berry 93%, cow dung 5%, gypsum 1% and lime 1% Biodiversity number processed is 65% obtained culture medium.
- 8. the method described according to claim 6 or 7, it is characterised in that：In method prepared by the original seed, described 15-25 DEG C is protected from light culture and is protected from light culture for 22 DEG C.
- 9. application of the fructification described in claim 3 in food processing.
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| CN110679389A (en) * | 2019-11-18 | 2020-01-14 | 仁怀酱香白酒科研所 | Lepista nuda planting method |
| CN112725192A (en) * | 2021-01-05 | 2021-04-30 | 中华全国供销合作总社昆明食用菌研究所 | Lepista nuda strain and cultivation method thereof |
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| CN106047725A (en) * | 2016-07-14 | 2016-10-26 | 安徽天明生态林农科技开发有限公司 | Lepista nuda culture medium based on yellow wine lees and preparation method of lepista nuda liquid strain |
| CN106479901A (en) * | 2016-10-11 | 2017-03-08 | 山东惠民春生食用菌科技开发有限公司 | A kind of Xianggu mushroom strain and its application in method for cultivating mushroom |
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| CN106604650A (en) * | 2014-08-26 | 2017-04-26 | 麦可科技有限公司 | Methods for the production and use of mycelial liquid tissue culture |
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| CN106047725A (en) * | 2016-07-14 | 2016-10-26 | 安徽天明生态林农科技开发有限公司 | Lepista nuda culture medium based on yellow wine lees and preparation method of lepista nuda liquid strain |
| CN106479901A (en) * | 2016-10-11 | 2017-03-08 | 山东惠民春生食用菌科技开发有限公司 | A kind of Xianggu mushroom strain and its application in method for cultivating mushroom |
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| CN110679389A (en) * | 2019-11-18 | 2020-01-14 | 仁怀酱香白酒科研所 | Lepista nuda planting method |
| CN110679389B (en) * | 2019-11-18 | 2021-10-26 | 仁怀酱香白酒科研所 | Lepista nuda planting method |
| CN112725192A (en) * | 2021-01-05 | 2021-04-30 | 中华全国供销合作总社昆明食用菌研究所 | Lepista nuda strain and cultivation method thereof |
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