CN101790937A - Screening and culture preparing method of Russula.alutacea strain - Google Patents
Screening and culture preparing method of Russula.alutacea strain Download PDFInfo
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Abstract
The invention relates to a screening and culture preparing method of a Russula.alutacea strain, belonging to the microbiological technical field. The fungus used in the invention is Russula.alutacea which was collected by China General Microbiological Culture Collection Center on December 15th, 2009 with the collection and register number of CGMCC.NO.3525. If applied to the propagation of Russula.alutacea, the good liquid and solid culture prepared from the strain has remarkable social and economic benefits and wide application prospects.
Description
Technical field:
The invention belongs to microbial technology field, be specifically related to the bacterial strain screening and the process for preparing strain thereof of DAHONGGU.
Background technology:
DAHONGGU (Russula alutacea) is world-renowned famous and precious wild edible fungus.DAHONGGU can't artificial cultivation, and the DAHONGGU on the market is all from wild.Wild in recent years DAHONGGU causes natural production to decline to a great extent owing to excessively gather and acquisition mode science and do not have corresponding technical measures not.Studies show that, under the natural environmental condition of producing region, adopt artificial bacterial classification and supporting technical measures, is one of most economical valid approach that solves the DAHONGGU imbalance between supply and demand.
In DAHONGGU protection multiplication technique, the acquisition of excellent species is a key technique.The acquisition of DAHONGGU bacterial strain utilizes normally that fruit body or spore separate, purifying, directly applies to the mycelium production of DAHONGGU.The deficiencies in the prior art are that the DAHONGGU bacterial classification can not carry out bacterial classification with the method for fruiting and identify, can only tentatively determine whether to be the DAHONGGU pure culture according to mycelia form and separator; The bacterial classification of producing is generally used solid spawn.It is not extensive that numerous application is expanded in the protection of at present adopting liquid spawn to carry out the DAHONGGU resource at home.
Summary of the invention:
Main purpose of the present invention overcomes the prior art deficiency exactly; utilize original producton location, Yunnan DAHONGGU fruit body to filter out strain excellent; adopt biotechnology to carry out bacterial classification and identify, preparation DAHONGGU liquid and solid excellent species are for numerous excellent species that provides is provided the protection of DAHONGGU wild resource.
The fungi that the present invention adopts is DAHONGGU (Russula alutacea); Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: China. Beijing. the Zhong Guan-cun; Preservation date: on December 15th, 2009; The numbering CGMCC NO.3525 that preservation is registered on the books.
The DAHONGGU fruit body is medium big, cap 6～16cm, and flat hemispherical, the back is open and flat, and the middle part is recessed, and is sticking when wet, dry soon, peony, bright or dark violet redness, edge-smoothing or not obvious striped is arranged.Bacterial context white.Lamella is isometric or almost isometric, and transverse vein is arranged between pleat, and directly give birth to or closely prolong life, first milky, the light ochre yellow in back, the pleat leading edge is usually redly.The stem subcircular, long 3～12cm, thick 1～3.5cm, white, top or side often redly, or whole pink and gradually light downwards.The spore print yellow.
Technical scheme of the present invention:
1. gather wild DAHONGGU fruit body;
2. separate tissue 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 screening, obtain mycelium production height, resistant to pollution DAHONGGU liquid spawn special strain therefore bacterium garden-8.
Fungi of the present invention (Russula alutacea) cultural method (below be weight percentage):
Strain separating purifying medium: 2% DAHONGGU offcuts, 0.001%V
B1, 2% glucose, 2% agar;
The DAHONGGU bacterial strain separates and authentication method:
1, DAHONGGU fruit body piece of tissue or spore liquid are inoculated on the above-mentioned test tube agar medium inclined-plane, cultivated 10-15 days down in 18-24 ℃, test tube observed and recorded every day to separation and Culture, the test tube that rejecting has in time been polluted, pick out bacterial strain pollution-free, that grow fine, obtain DAHONGGU and separate the test tube kind.
2, will separate test tube kind inoculated by hypha block and to the dull and stereotyped culture dish of above-mentioned agar medium, carry out the bacterial classification purifying, get most advanced and sophisticated inoculated by hypha block after 2 days on medium slant, cultivate 10-15 days down, obtain DAHONGGU purifying test tube kind in 18-24 ℃.
3, adopt for examination fruit body and corresponding mycelia separator, extract total DNA by the SDS method respectively, carry out pcr amplification and ITS sequencing, ITS sequence and (Russula alutacea) Identities=499/510 of the DAHONGGU among the GENBANK (97%) by the Blast comparative sample, Gaps=2/510 (0%) analyzes and determines that separating the pure culture that obtains is the DAHONGGU mycelium.
The preparation method of DAHONGGU bacterial classification of the present invention is divided into: two kinds of liquid culture and solid culture.
The liquid spawn preparation method:
The liquid culture based formulas is: 5-10% wheat bran, 0.5-1% maltose, 1-2% soluble starch, remainder is a water, the pH nature.
1, the mycelium with DAHONGGU is inoculated on the test tube agar medium inclined-plane, and culture medium prescription is the PDA medium, cultivates 10-15 days down in 18-24 ℃, 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 following shaking table, and rotating speed is 120-160r/min, incubation time 5-7 days.
3, will shake bottle 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 22-26 ℃; Aerobic culture 72-96 hour, obtain the DAHONGGU liquid spawn.
The solid spawn preparation method:
The solid culture based formulas is: wood chip 55-70%, cotton seed hulls 20%, wheat bran 5-20%, phosphate fertilizer 1%, gypsum 1%, DAHONGGU be humus soil 3% vegetatively, water content 60%, pH nature.
1, the mycelium with DAHONGGU is inoculated on the test tube agar medium inclined-plane, and culture medium prescription is the PDA medium, cultivates 10-15 days down in 18-24 ℃, 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 following shaking table, and rotating speed is 120-160r/min, incubation time 5-7 days.
3, 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 and seal the back, insert liquid spawn, cultivated 30-40 days in 22-26 ℃, cover with up to mycelia 120 ℃ of sterilizations 60 minutes.
Embodiment:
Embodiment one:
1, DAHONGGU fruit body piece of tissue is inoculated on the above-mentioned strain separating purifying medium slant, cultivated 15 days down in 22 ℃, to test tube observed and recorded every day of separation and Culture, the test tube that rejecting has in time been polluted, pick out bacterial strain pollution-free, that grow fine, obtain DAHONGGU and separate the test tube kind.
2, will separate test tube kind inoculated by hypha block and to the dull and stereotyped culture dish of above-mentioned agar medium, carry out the bacterial classification purifying, get most advanced and sophisticated inoculated by hypha block after 2 days on medium slant, cultivate 15 days down, obtain DAHONGGU purifying test tube kind in 22 ℃.
3, adopt for examination fruit body and corresponding mycelia separator, extract total DNA by the SDS method respectively, carry out pcr amplification and ITS sequencing, ITS sequence and (Russula alutacea) Identities=499/510 of the DAHONGGU among the GENBANK (97%) by the Blast comparative sample, Gaps=2/510 (0%) analyzes and determines that separating the pure culture that obtains is the DAHONGGU mycelium.
4, the mycelium with DAHONGGU (Russula alutacea) bacterium garden-8 is inoculated on the test tube agar medium inclined-plane, and culture medium prescription is the PDA medium, cultivates 15 days down, obtains the test tube kind for 22 ℃.
5, the test tube kind is inoculated in the 500ml triangular flask in (every bottled 200ml) liquid nutrient medium, culture medium prescription is potato 20%, glucose 2%, peptone 0.2%, potassium dihydrogen phosphate 0.05%, magnesium sulfate 0.05%, and remainder is a water, the PH nature.Cultivate in 20 ℃ of following 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 with the level liquid bacterial classification inoculation: 0.8v/ (vmin); Speed of agitator is 120r/min; Inoculum concentration is 5%; Tank pressure: 0.04Mpa; PH is 6.0; Temperature is 22 ℃; Aerobic culture 96 hours obtains the DAHONGGU liquid spawn.
Embodiment two:
The step of strain preparation is identical with embodiment one.
1, DAHONGGU fruit body piece of tissue is inoculated on the above-mentioned strain separating purifying medium slant, cultivated 10 days down in 25 ℃, to test tube observed and recorded every day of separation and Culture, the test tube that rejecting has in time been polluted, pick out bacterial strain pollution-free, that grow fine, obtain DAHONGGU and separate the test tube kind.
2, will separate test tube kind inoculated by hypha block and to the dull and stereotyped culture dish of above-mentioned agar medium, carry out the bacterial classification purifying, get most advanced and sophisticated inoculated by hypha block after 2 days on medium slant, cultivate 10 days down, obtain DAHONGGU purifying test tube kind in 25 ℃.
3, adopt for examination fruit body and corresponding mycelia separator, extract total DNA by the SDS method respectively, carry out pcr amplification and ITS sequencing, ITS sequence and (Russula alutacea) Identities=499/510 of the DAHONGGU among the GENBANK (97%) by the Blast comparative sample, Gaps=2/510 (0%) analyzes and determines that separating the pure culture that obtains is the DAHONGGU mycelium.
4, the DAHONGGU mycelium is inoculated on the test tube agar medium inclined-plane, culture medium prescription is the PDA medium, cultivates 6 days down, obtains the test tube kind for 22 ℃.
5, the test tube kind is inoculated in the 500ml triangular flask in (every bottled 200ml) liquid nutrient medium, culture medium prescription is 6% wheat bran, 0.6% maltose, 1.2% soluble starch, and remainder is a water, the pH nature.Cultivate in 22 ℃ of following shaking tables, rotating speed is 140rpm, 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 with the level liquid bacterial classification inoculation: 0.8v/ (vmin); Speed of agitator is 140r/min; Inoculum concentration is 8%; Tank pressure: 0.04Mpa; PH is 6.0; Temperature is 24 ℃; Aerobic culture 84 hours obtains the DAHONGGU liquid spawn.
Embodiment three:
1, DAHONGGU fruit body piece of tissue is inoculated on the above-mentioned strain separating purifying medium slant, cultivated 6 days down in 25 ℃, to test tube observed and recorded every day of separation and Culture, the test tube that rejecting has in time been polluted, pick out bacterial strain pollution-free, that grow fine, obtain DAHONGGU and separate the test tube kind.
2, will separate test tube kind inoculated by hypha block and to the dull and stereotyped culture dish of above-mentioned agar medium, carry out the bacterial classification purifying, get most advanced and sophisticated inoculated by hypha block after 2 days on medium slant, cultivate 6 days down, obtain DAHONGGU purifying test tube kind in 25 ℃.
3, adopt for examination fruit body and corresponding mycelia separator, extract total DNA by the SDS method respectively, carry out pcr amplification and ITS sequencing, ITS sequence and (Russula alutacea) Identities=499/510 of the DAHONGGU among the GENBANK (97%) by the Blast comparative sample, Gaps=2/510 (0%) analyzes and determines that separating the pure culture that obtains is the DAHONGGU mycelium.
4, the DAHONGGU mycelium is inoculated on the test tube agar medium inclined-plane, culture medium prescription is the PDA medium, cultivates 5 days down, obtains the test tube kind for 25 ℃.
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, 0.5% maltose, 1% soluble starch, and remainder is a water, the pH nature.Cultivate in 26 ℃ of following shaking tables, rotating speed is 160rpm, and incubation time 6 days obtains the level liquid bacterial classification.
To solid culture medium, culture medium prescription is weed tree sawdust 78%, wheat bran 20%, sucrose 1%, gypsum 1%, water content 60%, PH nature with the level liquid bacterial classification inoculation.Wood chip, wheat bran, phosphate fertilizer, gypsum, humus soil are 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 30 days, obtains the DAHONGGU solid spawn.
Embodiment four:
1, DAHONGGU fruit body piece of tissue is inoculated on the above-mentioned strain separating purifying medium slant, cultivated 5 days down in 24 ℃, to test tube observed and recorded every day of separation and Culture, the test tube that rejecting has in time been polluted, pick out bacterial strain pollution-free, that grow fine, obtain DAHONGGU and separate the test tube kind.
2, will separate test tube kind inoculated by hypha block and to the dull and stereotyped culture dish of above-mentioned agar medium, carry out the bacterial classification purifying, get most advanced and sophisticated inoculated by hypha block after 2 days on medium slant, cultivate 5 days down, obtain DAHONGGU purifying test tube kind in 24 ℃.
3, adopt for examination fruit body and corresponding mycelia separator, extract total DNA by the SDS method respectively, carry out pcr amplification and ITS sequencing, ITS sequence and (Russula alutacea) Identities=499/510 of the DAHONGGU among the GENBANK (97%) by the Blast comparative sample, Gaps=2/510 (0%) analyzes and determines that separating the pure culture that obtains is the DAHONGGU mycelium.
4, the DAHONGGU mycelium is inoculated on the test tube agar medium inclined-plane, culture medium prescription is the PDA medium, cultivates 5 days down, obtains the test tube kind for 24 ℃.
5, the test tube kind is inoculated in the 500ml triangular flask in (every bottled 200ml) liquid nutrient medium, culture medium prescription is 8% wheat bran, 1% maltose, 1% soluble starch, and remainder is a water, the pH nature.Cultivate in 26 ℃ of following 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 35%, wood chip 45%, wheat bran 18%, sucrose 1%, gypsum 1% with the level liquid bacterial classification inoculation, water content 60%, PH nature.Wood chip, wheat bran, phosphate fertilizer, gypsum, humus soil are 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 40 days, obtains the DAHONGGU solid spawn.
Claims (2)
1. DAHONGGU bacterial strain is characterized in that described bacterial strain is that the preserving number of this bacterial strain of DAHONGGU (Russula alutacea) bacterium garden-8 is CGMCC NO.3525.
2. DAHONGGU according to claim 1 (Russula alutacea) bacterium garden-8 and preparation method, described bacterial strain obtains through solid or liquid culture, it is characterized in that described solid culture based formulas and liquid culture based formulas are as follows:
(1) the solid culture based formulas is: 1. weed tree sawdust 78%, wheat bran 20%, sucrose 1%, gypsum 1%, water content 60%, PH are naturally; 2. cotton seed hulls 35%, wood chip 45%, wheat bran 18%, sucrose 1%, gypsum 1%, water content 60%, PH nature;
(2) the liquid culture based formulas is: potato 20%, glucose 2%, peptone 0.2%, potassium dihydrogen phosphate 0.05%, magnesium sulfate 0.05%, remainder is a water, the PH nature.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103004465A (en) * | 2012-12-02 | 2013-04-03 | 中华全国供销合作总社昆明食用菌研究所 | Coprinus comatus strain and preparation method |
| CN103535183A (en) * | 2013-08-14 | 2014-01-29 | 上海雪榕生物科技股份有限公司 | Simple inoculation method for improving liquid spawn running points |
| 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 |
| CN105359831A (en) * | 2015-11-30 | 2016-03-02 | 江苏康盛农业发展有限公司 | Inoculation method for transforming solid culture to liquid culture of golden mushroom |
| CN107711290A (en) * | 2017-09-28 | 2018-02-23 | 山西省农业科学院食用菌研究所 | The culture medium and its synchronous cultural method of mycorhiza edible mushroom symbiosis seedling |
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2010
- 2010-03-02 CN CN2010101158758A patent/CN101790937B/en active Active
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| 《湖北农业科学》 19941231 彭浩然等 湖北大红菇的采集鉴定及驯化培养 第47-48页 1-2 , 第3期 * |
| 《食用菌》 19941231 黄世典等 大红菇及其分离驯化栽培初探 第11页 1-2 , 第S1期 * |
| 《食用菌》 20051231 杜顺刚等 西峡县野生大红菇资源及生态调查初报 第7-8页 1-2 , 第3期 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103004465A (en) * | 2012-12-02 | 2013-04-03 | 中华全国供销合作总社昆明食用菌研究所 | Coprinus comatus strain and preparation method |
| CN103535183A (en) * | 2013-08-14 | 2014-01-29 | 上海雪榕生物科技股份有限公司 | Simple inoculation method for improving liquid spawn running points |
| 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 |
| CN105359831A (en) * | 2015-11-30 | 2016-03-02 | 江苏康盛农业发展有限公司 | Inoculation method for transforming solid culture to liquid culture of golden mushroom |
| CN107711290A (en) * | 2017-09-28 | 2018-02-23 | 山西省农业科学院食用菌研究所 | The culture medium and its synchronous cultural method of mycorhiza edible mushroom symbiosis seedling |
| CN107711290B (en) * | 2017-09-28 | 2020-07-24 | 山西省农业科学院食用菌研究所 | Culture medium for mycorrhizal edible fungus symbiotic seedling and synchronous culture method thereof |
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