CN102986537A - Tricholoma lobayense strain KJH-3 and preparation method thereof - Google Patents
Tricholoma lobayense strain KJH-3 and preparation method thereof Download PDFInfo
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- CN102986537A CN102986537A CN201210503842XA CN201210503842A CN102986537A CN 102986537 A CN102986537 A CN 102986537A CN 201210503842X A CN201210503842X A CN 201210503842XA CN 201210503842 A CN201210503842 A CN 201210503842A CN 102986537 A CN102986537 A CN 102986537A
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Abstract
The invention relates to a selection and preparation method of tricholoma lobayense strains, belonging to the technical field of microorganisms. A strain tricholoma lobayense KJH-3 provided by the invention is preserved in the China general microbiological culture collection center with a preservation number of CGMCCNO.6703 on October 11, 2012. Excellent liquid or excellent solid cultures prepared from the strain are applied to the reproduction improvement of tricholoma lobayense so that obvious social and economic effects and a wide application prospect are realized.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to golden good fortune mushroom bacterial strain KJH-3 and preparation method.
Background technology
Jin Fugu
Tricholoma lobayenscBeing called again Tricholoma Giganteum Massee, large spoken parts in traditional operas mushroom, Tricholoma lobayense, is rare edible mushroom new varieties, at south China and Taiwan Province the small size cultivation is arranged all
In golden good fortune mushroom culture technique, the acquisition of excellent species is key technique.The acquisition of gold good fortune mushroom bacterial strain utilizes normally that fruit body or spore separate, purifying, directly applies to cultivation or the mycelium production of Jin Fugu.The deficiencies in the prior art are that its biological property still is in the experimental study stage at present, and biological transformation ratio is lower, but also will cultivate with grog, and production technology and technology essential factor are had relatively high expectations, thereby have restricted the development of golden good fortune mushroom commodity economy.Can only tentatively determine whether to be golden good fortune mushroom pure culture according to mycelia form and separator in addition; The bacterial classification of producing is generally used solid spawn.Adopting at home at present liquid spawn to carry out golden good fortune mushroom protection of resources uses not extensive.
Summary of the invention
Main purpose of the present invention overcomes the prior art deficiency exactly; utilize original producton location, Yunnan gold good fortune massee fruiting bodies to filter out strain excellent; adopt biotechnology to carry out bacterial classification and identify, prepare golden good fortune mushroom liquid and solid excellent species, for the protection of resources of golden good fortune mushroom provides excellent species
The fungi that the present invention adopts is Jin Fugu
Tricholoma lobayenscKJH-3; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: Da Tun road, Chaoyang District, BeiJing, China city, Institute of Microorganism, Academia Sinica; Preservation date: on October 11st, 2012; The numbering CGMCC No.6703 that preservation is registered on the books
Gold good fortune massee fruiting bodies is grown thickly or is clustered, and shape is large, white or milky, and cap is open and flat smooth, is hemispherical.Stem is up-small and down-big, is long bar-shaped, and mushroom shape is beautiful, attitude is graceful.Gold good fortune mushroom bacterial context is plump tender white, and mouthfeel is unique, and it is little sweet, tender and crisp and delicious, nutritious to distinguish the flavor of.According to one's analysis, its gross protein value 27%, crude fat 9.5%, total reducing sugar 38.4%, and contain multivitamin.Tasty mouthfeel, beautiful, the commodity valency extra-high-speed of shape.Anti-assorted power is strong, Functionality, quality and appealing design.22-36 ℃ of fruiting temperature adapts to most with 24-33 ℃, the spring and summer autumn produces
Technical scheme of the present invention:
1. gather wild golden good fortune massee fruiting bodies;
2. tissue separates or spore separation;
3. purifying bacterial strain and identification of strains;
4. biological property relatively reaches the production traits relatively;
5. determine strain excellent;
6. bacterial classification production and bacterial classification are used;
Through repeated screening, obtain mycelium production height, resistant to pollution golden good fortune mushroom liquid strain special strain therefore KJH-3
Fungi of the present invention
Tricholoma lobayenscCultural method (below be weight percentage):
Strain separating pure medium: 2% gold medal good fortune mushroom offcuts, 0.001%V
B1, 2% glucose, 2% agar;
Gold good fortune mushroom bacterial strain Isolation and Identification method:
1, golden good fortune massee fruiting bodies tissue block or spore liquid are inoculated on the above-mentioned test tube agar medium inclined-plane, in 18-24 ℃ of lower the cultivation 5-10 days, to separating test tube observed and recorded every day of cultivating, the test tube that in time rejecting has been polluted, pick out bacterial strain pollution-free, that grow fine, obtain golden good fortune mushroom separation test tube kind
2, separation test tube kind inoculated by hypha block is carried out purifying agaric to the dull and stereotyped culture dish of above-mentioned agar medium, get most advanced and sophisticated inoculated by hypha block after 2 days on medium slant, in 18-24 ℃ of lower the cultivation 5-7 days, obtain golden good fortune mushroom purifying test tube kind
3, adopt for examination fruit body and corresponding hypha separation thing, extract total DNA by the SDS method respectively, carry out pcr amplification and ITS sequencing, the ITS sequence by the Blast comparative sample and the Jin Fugu among the GENBANK (
Tricholoma lobayensc) Identities=542/559 (97%), Gaps=7/559 (1%), it is golden good fortune mycelium that Analysis deterrmination separates the pure culture that obtains
The preparation method of the present invention's gold good fortune mushroom strains is divided into: two kinds of liquid culture and solid culture
The liquid spawn preparation method:
The liquid culture based formulas is: 10 ~ 20% wheat brans, 10 ~ 20% potatoes, 0.5 ~ 1% sucrose, 10% corn flour, remainder is water, the pH nature
1, the mycelium with Jin Fugu is inoculated on the test tube agar medium inclined-plane, and culture medium prescription is the PDA medium, in 18-24 ℃ of lower the cultivation 5-7 days, obtains test tube strains
2, the test tube kind is inoculated in 500ml triangular flask (the every bottled 200ml) liquid nutrient medium, culture medium prescription is the aforementioned liquids culture medium prescription, cultivates in 20-26 ℃ of lower shaking table, and rotating speed is 120-160r/min, incubation time 5-7 days
3, with the shaking flask bacterial classification inoculation in the fermentation tank of the automatic fermenting and producing line of 25L, throughput is 1:0.8v/ (vmin); Speed of agitator is 120-160r/min; Inoculum concentration is 5-10%; Tank pressure: 0.04Mpa; PH is 6.0; Temperature is 20-26 ℃; Ventilation was cultivated 72-96 hour, obtained golden good fortune mushroom liquid strain
The solid spawn preparation method:
The solid culture based formulas is: cotton seed hulls 90%, wheat bran 9%, calcium carbonate 1%, pH nature
1, the mycelium with Jin Fugu is inoculated on the test tube agar medium inclined-plane, and culture medium prescription is the PDA medium, in 18-24 ℃ of lower the cultivation 5-7 days, obtains test tube strains
2, the test tube kind is inoculated in 500ml triangular flask (the every bottled 200ml) liquid nutrient medium, culture medium prescription is the aforementioned liquids culture medium prescription, cultivates in 20-26 ℃ of lower shaking table, and rotating speed is 120-160 r/min, incubation time 5-7 days
2, adopt aforementioned solid culture based formulas.After composts or fertilisers of cultivating mixed, after adding water and mixing evenly, in the jar of the 750ml that packs into, compress after the sealing 120 ℃ of sterilizations 60 minutes, the access liquid spawn was cultivated 15-20 days in 20-26 ℃, until mycelia is covered with
Embodiment:
Embodiment one:
1, golden good fortune massee fruiting bodies tissue block is inoculated on the above-mentioned strain separating pure medium inclined-plane, in 18 ℃ of lower cultivations 10 days, to separating test tube observed and recorded every day of cultivating, the test tube that in time rejecting has been polluted, pick out bacterial strain pollution-free, that grow fine, obtain golden good fortune mushroom separation test tube kind
2, separation test tube kind inoculated by hypha block is carried out purifying agaric to the dull and stereotyped culture dish of above-mentioned agar medium, get inoculated by hypha block on medium slant after 2 days, in 18 ℃ of lower cultivations 7 days, obtain golden good fortune mushroom purifying test tube kind
3, adopt for examination fruit body and corresponding hypha separation thing, extract total DNA by the SDS method respectively, carry out pcr amplification and ITS sequencing, the ITS sequence by the Blast comparative sample and the Jin Fugu among the GENBANK (
Tricholoma lobayensc) Identities=547/551 (99%), Gaps=1/551 (0%), it is golden good fortune mycelium that Analysis deterrmination separates the pure culture that obtains
4, will
Tricholoma lobayenscThe mycelium of KJH-3 is inoculated on the test tube agar medium inclined-plane, and culture medium prescription is the PDA medium, and 18 ℃ of lower cultivations 7 days obtain the test tube kind
5, the test tube kind is inoculated in the 500ml triangular flask in (every bottled 200ml) liquid nutrient medium, culture medium prescription is 10% wheat bran, 10% potato, 0.5% sucrose, 10% corn flour, remainder is water, the pH nature, cultivate in 20 ℃ of lower shaking tables, rotating speed is 120rpm, and incubation time 7 days obtains the level liquid bacterial classification
In the fermentation tank of the automatic fermenting and producing line of 25L, throughput is 1:0.8v/ (vmin) with the level liquid bacterial classification inoculation; Speed of agitator is 120r/min; Inoculum concentration is 5%; Tank pressure: 0.04Mpa; PH is 6.0; Temperature is 22 ℃; Ventilation was cultivated 96 hours, obtained golden good fortune mushroom liquid strain
Embodiment two:
The step of bacterial classification preparation is identical with embodiment one
1, golden good fortune massee fruiting bodies tissue block is inoculated on the above-mentioned strain separating pure medium inclined-plane, in 22 ℃ of lower cultivations 6 days, to separating test tube observed and recorded every day of cultivating, the test tube that in time rejecting has been polluted, pick out bacterial strain pollution-free, that grow fine, obtain golden good fortune mushroom separation test tube kind
2, separation test tube kind inoculated by hypha block is carried out purifying agaric to the dull and stereotyped culture dish of above-mentioned agar medium, get most advanced and sophisticated inoculated by hypha block after 2 days on medium slant, in 22 ℃ of lower cultivations 6 days, obtain golden good fortune mushroom purifying test tube kind
3, adopt for examination fruit body and corresponding hypha separation thing, extract total DNA by the SDS method respectively, carry out pcr amplification and ITS sequencing, the ITS sequence by the Blast comparative sample and the Jin Fugu among the GENBANK (
Tricholoma lobayensc) Identities=547/551 (99%), Gaps=1/551 (0%), it is golden good fortune mycelium that Analysis deterrmination separates the pure culture that obtains
4, will
Tricholoma lobayenscThe mycelium of KJH-3 is inoculated on the test tube agar medium inclined-plane, and culture medium prescription is the PDA medium, and 22 ℃ of lower cultivations 6 days obtain the test tube kind
5, the test tube kind is inoculated in the 500ml triangular flask in (every bottled 200ml) liquid nutrient medium, culture medium prescription is 15% wheat bran, 15% potato, 0.6% sucrose, 10% corn flour, and remainder is water, the pH nature.Cultivate in 22 ℃ of lower shaking tables, rotating speed is 140rpm, and incubation time 6 days obtains the level liquid bacterial classification
In the fermentation tank of the automatic fermenting and producing line of 25L, throughput is 1:0.8v/ (vmin) with the level liquid bacterial classification inoculation; Speed of agitator is 140r/min; Inoculum concentration is 8%; Tank pressure: 0.04Mpa; PH is 6.0; Temperature is 24 ℃; Ventilation was cultivated 84 hours, obtained golden good fortune mushroom liquid strain
Embodiment three:
1, golden good fortune massee fruiting bodies tissue block is inoculated on the above-mentioned strain separating pure medium inclined-plane, in 24 ℃ of lower cultivations 5 days, to separating test tube observed and recorded every day of cultivating, the test tube that in time rejecting has been polluted, pick out bacterial strain pollution-free, that grow fine, obtain golden good fortune mushroom separation test tube kind
2, separation test tube kind inoculated by hypha block is carried out purifying agaric to the dull and stereotyped culture dish of above-mentioned agar medium, get most advanced and sophisticated inoculated by hypha block after 2 days on medium slant, in 24 ℃ of lower cultivations 5 days, obtain golden good fortune mushroom purifying test tube kind
3, adopt for examination fruit body and corresponding hypha separation thing, extract total DNA by the SDS method respectively, carry out pcr amplification and ITS sequencing, the ITS sequence by the Blast comparative sample and the Jin Fugu among the GENBANK (
Tricholoma lobayensc) Identities=547/551 (99%), Gaps=1/551 (0%), it is golden good fortune mycelium that Analysis deterrmination separates the pure culture that obtains
4, will
Tricholoma lobayenscThe mycelium of KJH-3 is inoculated on the test tube agar medium inclined-plane, and culture medium prescription is the PDA medium, and 24 ℃ of lower cultivations 5 days obtain the test tube kind
5, the test tube kind is inoculated in the 500ml triangular flask in (every bottled 200ml) liquid nutrient medium, culture medium prescription is 20% wheat bran, 20% potato, 0.5% sucrose, 10% corn flour, and remainder is water, the pH nature.Cultivate in 26 ℃ of lower shaking tables, rotating speed is 160rpm, and incubation time 5 days obtains the level liquid bacterial classification
To solid culture medium, culture medium prescription is cotton seed hulls 90%, wheat bran 9%, calcium carbonate 1%, pH nature with the level liquid bacterial classification inoculation.Each raw material is mixed after the weighing in proportion, in the seed bottle of the 750ml that packs into behind the poach, every bottled 500ml, 120 ℃ of sterilizations are after 60 minutes, and inoculation is placed on 26 ℃ and cultivated 15 days, obtains golden good fortune mushroom solid spawn
Embodiment four:
1, golden good fortune massee fruiting bodies tissue block is inoculated on the above-mentioned strain separating pure medium inclined-plane, in 24 ℃ of lower cultivations 5 days, to separating test tube observed and recorded every day of cultivating, the test tube that in time rejecting has been polluted, pick out bacterial strain pollution-free, that grow fine, obtain golden good fortune mushroom separation test tube kind
2, separation test tube kind inoculated by hypha block is carried out purifying agaric to the dull and stereotyped culture dish of above-mentioned agar medium, get inoculated by hypha block on medium slant after 2 days, in 24 ℃ of lower cultivations 5 days, obtain golden good fortune mushroom purifying test tube kind
3, adopt for examination fruit body and corresponding hypha separation thing, extract total DNA by the SDS method respectively, carry out pcr amplification and ITS sequencing, the ITS sequence by the Blast comparative sample and the Jin Fugu among the GENBANK (
Tricholoma lobayensc) Identities=547/551 (99%), Gaps=1/551 (0%), it is golden good fortune mycelium that Analysis deterrmination separates the pure culture that obtains
4, will
Tricholoma lobayenscThe mycelium of KJH-3 is inoculated on the test tube agar medium inclined-plane, and culture medium prescription is the PDA medium, and 24 ℃ of lower cultivations 5 days obtain the test tube kind
5, the test tube kind is inoculated in the 500ml triangular flask in (every bottled 200ml) liquid nutrient medium, culture medium prescription is 18% wheat bran, 10% potato, 1% sucrose, 10% corn flour, and remainder is water, the pH nature.Cultivate in 26 ℃ of lower shaking tables, rotating speed is 160rpm, and incubation time 5 days obtains the level liquid bacterial classification
To solid culture medium, culture medium prescription is cotton seed hulls 90%, wheat bran 9%, calcium carbonate 1%, pH nature with the level liquid bacterial classification inoculation.Each raw material is mixed after the weighing in proportion, in the seed bottle of the 750ml that packs into behind the poach, every bottled 500ml, 120 ℃ of sterilizations are after 60 minutes, and inoculation is placed on 20 ℃ and cultivated 20 days, obtains golden good fortune mushroom solid spawn.
Claims (2)
1. golden good fortune mushroom bacterial strain, it is characterized in that described bacterial strain for (
Tricholoma lobayensc)KJH-3, the preserving number of this bacterial strain are CGMCC NO.6703.
2. the preparation method of golden good fortune mushroom bacterial strain according to claim 1, described bacterial strain is to cultivate through the liquid of routine and solid fermentation to obtain, and it is characterized in that described liquid and solid fermentation culture medium prescription are as follows:
The liquid culture based formulas is: 5-10% wheat bran, 0.5-1% analysis for soybean powder, 0.5-1% maltose, 1-3% corn flour, and remainder is water, the pH nature;
The solid culture based formulas is: wood chip 75-90%, wheat bran 5-20%, phosphate fertilizer 1%, gypsum 1%, hickory chick be humus soil 3% vegetatively, water content 60%, pH nature.
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Cited By (4)
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| CN103858668A (en) * | 2013-11-29 | 2014-06-18 | 昆明菌苑食品有限公司 | Agaricus vaporarius strain and preparing method of agaricus vaporarius strain |
| CN105274010A (en) * | 2015-12-03 | 2016-01-27 | 中华全国供销合作总社昆明食用菌研究所 | Huge tricholomagambosum strain and test tube culture thereof and preparation method |
| CN108293599A (en) * | 2018-02-28 | 2018-07-20 | 中华全国供销合作总社昆明食用菌研究所 | A kind of Lepista sordida and its separation expanding propagation method and soil covering culture method |
| CN112438158A (en) * | 2019-09-02 | 2021-03-05 | 北京市房山区种植业技术推广站 | Ramaria strain and application thereof, and mother culture medium for artificially culturing Ramaria and application thereof |
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| CN101663960A (en) * | 2009-08-07 | 2010-03-10 | 江苏江南生物科技有限公司 | Method for cultivating tricholoma lobayense by utilizing fungus dregs |
| CN102728610A (en) * | 2012-06-20 | 2012-10-17 | 四川大学 | Method for reinforcing soil heavy metal enrichment with mushroom by using serratia marcescens |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101663960A (en) * | 2009-08-07 | 2010-03-10 | 江苏江南生物科技有限公司 | Method for cultivating tricholoma lobayense by utilizing fungus dregs |
| CN102728610A (en) * | 2012-06-20 | 2012-10-17 | 四川大学 | Method for reinforcing soil heavy metal enrichment with mushroom by using serratia marcescens |
Non-Patent Citations (1)
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| 邓优锦等: "野生金福菇的鉴定及其生物学特性研究", 《浙江食用菌》, vol. 16, no. 6, 31 December 2008 (2008-12-31) * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103858668A (en) * | 2013-11-29 | 2014-06-18 | 昆明菌苑食品有限公司 | Agaricus vaporarius strain and preparing method of agaricus vaporarius strain |
| CN103858668B (en) * | 2013-11-29 | 2016-02-17 | 中华全国供销合作总社昆明食用菌研究所 | A kind of wool mushroom mycopremna and process for preparing strain thereof |
| CN105274010A (en) * | 2015-12-03 | 2016-01-27 | 中华全国供销合作总社昆明食用菌研究所 | Huge tricholomagambosum strain and test tube culture thereof and preparation method |
| CN108293599A (en) * | 2018-02-28 | 2018-07-20 | 中华全国供销合作总社昆明食用菌研究所 | A kind of Lepista sordida and its separation expanding propagation method and soil covering culture method |
| CN108293599B (en) * | 2018-02-28 | 2020-05-19 | 中华全国供销合作总社昆明食用菌研究所 | Lepista sordida, and separation propagation method and soil-covering cultivation method thereof |
| CN112438158A (en) * | 2019-09-02 | 2021-03-05 | 北京市房山区种植业技术推广站 | Ramaria strain and application thereof, and mother culture medium for artificially culturing Ramaria and application thereof |
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Application publication date: 20130327 |