CN102726216A - Method for culturing cordyceps militaris anti-degradation strain - Google Patents
Method for culturing cordyceps militaris anti-degradation strain Download PDFInfo
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Abstract
The invention provides a method for culturing cordyceps militaris anti-degradation strain, comprising the steps of: preparing a cordyceps militaris mother seed pipe for the growth of low-temperature companion fungus; placing the cordyceps militaris mother seed pipe in low-temperature, high-temperature, anoxia and mixed bacterium growth environments for strain culture; purifying and rejuvenating the cultured mother seed to obtain the cordyceps militaris anti-degradation strain; and entering the application stage of stock seed and cultured seed. In culture, the anti-degradation strain can be continuously used for subculture for 10 times; the biological transformation rate is about 70%; then cordyceps militaris sporophore is obtained; and the cordyceps militaris sporophore has the characteristics that the color is deep golden yellow, the original characteristics are kept, the indexes are stable in normal culture process range, and for the active ingredients, the content of cordycepin is not less than 0.1%, the content of adenosine is not less than 0.055% and the content of polysaccharide is not less than 2.5% after detection, thereby conforming to the index specified by the Announcement of Ministry of Health of the People's Republic of China (No. 3, 2009) about ratifying cordyceps militaris as a new resource of food.
Description
Technical field
The invention belongs to the production of hybrid seeds field of fungi, be specifically related to the new technology that a kind of Cordyceps militaris spawn is cultivated.By this method, can obtain the good quality strain of degeneration.
Background technology
Fruiting bodies of cordyceps militaris [Cordyceps militaris] claim cordyceps militaris fruit body or stroma again, is called for short Cordyceps militaris, Ascomycotina, Clavicipitaceae, Cordyceps.The artificial culture fruit body spreads all over town and country in recent years, has become a new industry of China.But no matter be professional or specialized company, often occur in cultivation practices that only long mycelia is not grown grass (fruit body), the yielding poorly of not annesl, or grass, problem such as of poor quality.Effective component content is few and unstable, not only is difficult to realize standardized production, even makes numerous producers bankruptcy of having no harvest.Tracing it to its cause, mainly is spawn degeneration, and bacterial classification can not use by subculture.Bacterial classification often can only use once, and subculture uses and also was no more than for two generations reluctantly.Numerous technical staff have used up various traditional production of hybrid seeds ways for preventing spawn degeneration, as separate tissue preferred site, spore separation select excellent, purificate and rejuvenate, introduce excellent kind, hybridization and select excellently, but stoped not the quick degeneration of bacterial classification all the time.Although many experts and scholars have delivered many paper suggestions, the theoretical productivity that will become on the practice significance is still needed and is wanted the very long time.The quick degeneration of Cordyceps militaris spawn has become the bottleneck of this industry development.Concerning the numerous producers, the quick degenerate problem of bacterial classification needs to be resolved hurrily.The present invention sums up the simple and easy to do effective ways of coming out in production practices for many years.
Summary of the invention
The objective of the invention is to overcome the weak point of prior art, a kind of method of cultivating Cordyceps militaris degeneration bacterial classification is provided, resulting Cordyceps militaris degeneration bacterial classification can use by continuous ten subcultures.
Technical scheme of the present invention is: a kind of method of cultivating Cordyceps militaris degeneration bacterial classification comprises the following steps:
(1) cultivates winning kind: the female pipe of planting of the Cordyceps militaris that preparation has concomitance bacterium to grow, PDA medium+chloramphenicol, a first inclined-plane inoculation concomitance bacterium Paecilomyces varioti or a quilt hair spore bacterial classification; Cultivate; Insert Cordyceps militaris spawn again, said Cordyceps militaris spawn (Cordyceps militaris) depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation day: on December 12nd, 2011; Preserving number: CGMCC No.5577; Proceed to cultivate, make the Cordyceps militaris growth vigorous and all swallow up concomitance bacterium, obtain the winning kind of Cordyceps militaris kind;
(2) place 0～-20 ℃ environment to cultivate winning kind pipe and be no less than 30 days, be placed on then and recover growth 3～5 days under 18～25 ℃ of conditions;
(3) the winning kind pipe after will recovering removes pipe close, places 15～25 ℃ assorted collarium border to cultivate 5～7 days; And then cover pipe close and be placed in 28～36 ℃ the assorted collarium border and cultivated 5～7 days; Or
Winning kind pipe after will recovering earlier places 28～36 ℃ assorted collarium border to cultivate 5～7 days; And then remove pipe close and place 15～25 ℃ assorted collarium border to cultivate 5～7 days;
(4) after above-mentioned (1)～(3) step is cultivated; The test tube strains variable color, varied bacteria growing wherein has the Cordyceps militaris mycelia that has vitality on a small quantity; It is located away from the PDA medium; Place cultivate 6～8 days under 18～25 ℃ of conditions after, by the conventional purification and rejuvenation of the production of hybrid seeds, obtain Cordyceps militaris degeneration bacterial classification.
Preferably, the concrete steps of the winning kind of above-mentioned cultivation are: many of preparation association tubes, PDA medium+chloramphenicol; 45ug/1000ml, pipe inclined-plane indirect a 0.8～2mm Paecilomyces varioti or one of a quilt hair spore kind are concomitance bacterium, place under 10～18 ℃ of conditions and cultivate 7～10 days; When treating bacterial classification field planting and long opening, outside inclined-plane two end distance concomitance bacterium 1cm, insert each one of Cordyceps militaris spawn 2～5mm respectively; With tube place cultivate 3～5 days under 18～20 ℃ of conditions after, treat the Cordyceps militaris spawn field planting and scatter the growth in, cultivation temperature is brought up to 24～26 ℃; Cordyceps militaris growth is vigorous after 3～5 days, and all swallowed up concomitance bacterium, obtains the winning kind of Cordyceps militaris kind.
It is the principal character of present technique that adaptability is cultivated; I.e. preparation has the female pipe of planting of the Cordyceps militaris of low temperature concomitance bacterium growth; Place the environment cultured strain of low temperature, high temperature, anoxic and varied bacteria growing; Mother after cultivating plants purification and rejuvenation again, obtains Cordyceps militaris degeneration bacterial classification, and then gets into the application stage of original seed and cultivated species.
Practice confirms that the degeneration bacterial classification is used for cultivation, and continuous ten subcultures use can not degenerate; Biological transformation ratio about 70%, the fruiting bodies of cordyceps militaris that obtains, it is golden yellow deeply to show as color and luster; Keep primary characteristic, each item index is stable in the normal cultured processing range, through detecting its effective ingredient; Cordycepin content is no less than 0.1%; Adenosine content is no less than 0.055%, and polyoses content is no less than 2.5%, and meeting " Ministry of Health of the People's Republic of China's bulletin 2009 No. 3 " is the bulletin set quota of new resource food about the approval Cordyceps militaris.
Embodiment
The present invention needs Cordyceps militaris high-quality head to plant for tube is female, and this mother plants all can through what separate tissue, spore separation obtained or introduced.This bacterial classification also can obtain in Chinese common micro-organisms culture presevation administrative center, and preserving number is CGMCC No.5577.Concomitance bacterium is a Paecilomyces varioti or by a kind of in the spore of hair.Paecilomyces varioti (Paecilomyces hepiali) and quilt hair spore (Hirsutella sinensis) can obtain through the commercial channel, like Miyang County, Henan Province fungal studies institute, the wide edible mushroom in Jining City, Shandong Province research institute etc. sale arranged all.
The breeding method of Cordyceps militaris degeneration bacterial classification of the present invention, concrete steps are following:
1, cultivates winning kind.Preparation has female kind of the Cordyceps militaris of concomitance bacterium growth to manage many, so that the grouping of following step is cultivated, once accomplishes a plurality of embodiment, improves culture efficiency.PDA medium+chloramphenicol (45ug/1000ml), pipe inclined-plane indirect a 0.8～2mm Paecilomyces varioti or one of a quilt hair spore kind are concomitance bacterium, place under 10～18 ℃ of conditions and cultivate 7～10 days; When treating bacterial classification field planting and long opening, outside inclined-plane two end distance concomitance bacterium 1cm, insert each one of Cordyceps militaris spawn 2～5mm respectively; With tube place cultivate 3～5 days under 18～20 ℃ of conditions after; Treat the Cordyceps militaris spawn field planting and the growth of scattering in, cultivation temperature is brought up to 24～26 ℃, Cordyceps militaris growth is vigorous after 3～5 days; And all swallowed up concomitance bacterium, obtain the winning kind of Cordyceps militaris kind.
Above-mentioned each temperature condition is preferred fate with cultivating fate, can suitably adjust according to the growing state of concomitance bacterium and Cordyceps militaris spawn in the practical operation, reach the bacterial classification field planting and grow the state of opening, and then cordyceps swallows up concomitance bacterium, obtains the winning kind of Cordyceps militaris kind.Each following temperature condition also is same reason with cultivating fate.
2, winning kind is divided into two groups of A, B, places 0～-20 ℃ environment cultivation to be no less than 30 days simultaneously, be placed on then and recover growth 3～5 days under 18～25 ℃ of conditions;
3, the tube after will recovering is divided into two groups of A, B, and the A group is removed pipe close, places the assorted collarium border about 20 ℃; Simultaneously the B group is encased the assorted collarium border that places 28～36 ℃ with Polypropylence Sheet; Let A, B group in environment separately, cultivate 5～7 days.Then the A group is covered pipe close, the B group is removed pipe close, and A, B organize switched environment.Cultivated 5～7 days by preceding method is same.
Above-mentioned assorted collarium border can be the various environment that are rich in various assorted bacterium, and like undressed soil environment, natural environment, the artificial subenvironment that increases a certain bacterium or several kinds of bacterium is as placing certain bacterium bag etc. in a casing.
4, after above-mentioned 1～3 step is cultivated, test tube strains variable color, varied bacteria growing; Former Cordyceps militaris kind mycelia is dead basically, but still has a small amount of mycelia to have vitality.These great-hearted Cordyceps militaris mycelia carefulnesses are located away from the PDA medium, place cultivate 6～8 days under 18～25 ℃ of conditions after, by the conventional purification and rejuvenation of the production of hybrid seeds, obtain Cordyceps militaris degeneration bacterial classification.
1～20 summer of embodiment, Paecilomyces hepiali chen (Paecilomyces hepiali) association
1.1 60 of preparation association tubes, with PDA medium+chloramphenicol 45ug/1000ml, every inclined-plane tube indirect peacilomyce hepiahi bacterium kind 2mm1 piece places 10～18 ℃ insulating box to cultivate after 7 days, bacterial classification field planting and long opening; Outside every inclined-plane pipe two end distance concomitance bacterium 1cm, insert each one of 4mm Cordyceps militaris spawn respectively, said Cordyceps militaris spawn preserving number is CGMCC No.5577, places 18～20 ℃ of constant warm tubes to cultivate after 4 days, Cordyceps militaris spawn length is opened; At this moment, temperature is brought up to 24～26 ℃, after 4 days Cordyceps militaris kind growth vigorous, and all swallowed up the concomitance bacterium Paecilomyces varioti, indicate that the competition of Cordyceps militaris kind is winning kind.
1.2 after 60 " winning kind " pipe placed 0 ℃ of refrigerator and cooled to freeze to cultivate 30 days simultaneously, be placed on again in 18～25 ℃ the insulating box and recover growth 3 days.
1.3 the bacterial classification that will recover after the growth is divided into two groups of A, B, is 10 groups with each component again, 3 of every groups.The A group is removed the test tube plug, and 10 groups place different indoor different cartons respectively, puts two useless bacterium bags of flat mushroom of having given birth to mould in the case.Room temperature is 18～24 ℃; 10 groups with the B group encase with Polypropylence Sheet respectively simultaneously, place in the heatable adobe sleeping platform that the 20cm of diverse location is dark in the field, bury with soil respectively, and ground temperature is 28～32 ℃; After two groups of A, B respectively cultivated 5 days, the A group was added a cover pipe close, and the B group is removed pipe close, and switched environment was cultivated 5 days by preceding method is same.
1.4 through all variable colors of bacterial classification after the above-mentioned cultivation, red mould, green mold, assorted flora are given birth to.Former Cordyceps militaris spawn is most dead, but in every test tube some great-hearted Cordyceps militaris mycelia is arranged all; These great-hearted mycelia are separated on the PDA medium carefully, in 18～25 ℃ insulating box, cultivate after 7 days, purificate and rejuvenate.Obtain 55 high-quality Cordyceps militaris degeneration bacterial classification pipes altogether, can get into the making and the application stage of original seed and cultivated species.
Through the bacterial classification that the foregoing description 1～20 obtains, be used for cultivation, continuous ten times subculture uses, and biological transformation ratio is 68～71%.The fruiting bodies of cordyceps militaris that obtains, the performance color depth is golden yellow, 4～6 centimetres of height, 3～4 millimeters of diameters, ascocarp is sturdy, keeps primary characteristic.Through detecting its effective ingredient, cordycepin content is between 0.1～0.15%, and adenosine content is between 0.055～0.1%, and polyoses content meets the standard of 2009 years No. three bulletin of the Ministry of Public Health between 2.5～17.6%.
21～40 winters of embodiment, Hirsutella sinensis (Hirsutella sinensis) association
2.1 60 of preparation association tubes are with PDA medium+chloramphenicol 45ug/1000ml.One of every inclined-plane pipe indirect Hirsutella sinensis bacterial classification 2mm places 10～18 ℃ of insulating boxs to cultivate after 10 days the field planting of Hirsutella sinensis kind and the growth of scattering; At this moment, inclined-plane tube two end distances by hair spore kind 1cm outside, connect each one of the big Cordyceps militaris spawn of 5mm respectively; Said Cordyceps militaris spawn preserving number is CGMCC No.5577, places 10～20 ℃ insulating box to cultivate after 4 days, and Cordyceps militaris kind length is opened; At this moment, cultivation temperature is brought up to 24～26 ℃, the growth of Cordyceps militaris kind is vigorous after 5 days; All swallowed up by the hair spore, indicated that the Cordyceps militaris kind is " winning kind ".
2.2 60 " winning kind " pipe is equally divided into two groups of A, B, be respectively charged in two casees, place under the outdoor sunny side wall below 0 ℃, place after 30 days, place 18～25 ℃ of insulating boxs to recover growth 3 days again for two groups A, B.
2.3 the A group that will recover after the growth is removed the test tube plug, is divided into 10 groups, 3 of every groups; Place respectively in the different cartons; Put 1～3 useless bacterium bag of the flat mushroom of having given birth to mould in each case, place the shady place of the diverse location of green house of vegetables, 18～21 ℃ of temperature to carton; Simultaneously the B component is become 10 groups, 3 of every groups encase with Polypropylence Sheet respectively and place on the indoor heated kang, remain on 28～30 ℃; After two groups of A, B cultivated 7 days separately, the A group was added a cover pipe close, and the B group is removed pipe close, and switched environment was cultivated 7 days by preceding method is same.
2.4 60 bacterial classification pipes through after the above-mentioned cultivation have all become look; Red mould, green mold, assorted flora are given birth to; Former Cordyceps militaris spawn is most dead, but the Cordyceps militaris mycelia that every Guan Zhongjun has some that vitality is arranged is separated to these great-hearted mycelia on the PDA medium carefully; In 18～25 ℃ insulating box, cultivate after 7 days, purificate and rejuvenate.Obtain 50 high-quality Cordyceps militaris degeneration bacterial classification pipes altogether, can get into the making and the application stage of original seed and cultivated species.
Through the bacterial classification that the foregoing description 21～40 obtains, be used for cultivation, continuous ten times subculture uses, and biological transformation ratio is 68～71%.The fruiting bodies of cordyceps militaris that obtains, the performance color depth is golden yellow, 4～6 centimetres of height, 3～4 millimeters of diameters, ascocarp is sturdy, keeps primary characteristic.Through detecting its effective ingredient, cordycepin content is between 0.1～0.15%, and adenosine content is between 0.055～0.1%, and polyoses content meets the standard of 2009 years No. three bulletin of the Ministry of Public Health between 2.5～17.6%.
Claims (2)
1. a method of cultivating Cordyceps militaris degeneration bacterial classification is characterized in that comprising the following steps:
(1) cultivates winning kind: the female pipe of planting of the Cordyceps militaris that preparation has concomitance bacterium to grow, PDA medium+chloramphenicol, a first inclined-plane inoculation concomitance bacterium Paecilomyces varioti or a quilt hair spore bacterial classification; Cultivate; Insert Cordyceps militaris spawn again, said Cordyceps militaris spawn (Cordyceps militaris) depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation day: on December 12nd, 2011; Preserving number: CGMCC No.5577; Proceed to cultivate, make the Cordyceps militaris growth vigorous and all swallow up concomitance bacterium, obtain the winning kind pipe of Cordyceps militaris kind;
(2) place 0～-20 ℃ environment to cultivate winning kind pipe and be no less than 30 days, be placed on then and recover growth 3～5 days under 18～25 ℃ of conditions;
(3) the winning kind pipe after will recovering removes pipe close, places 15～25 ℃ assorted collarium border to cultivate 5～7 days; Covering pipe close then is placed in 28～36 ℃ the assorted collarium border again and cultivated 5～7 days; Or
Winning kind pipe after will recovering earlier places 28～36 ℃ assorted collarium border to cultivate 5～7 days; And then remove in the assorted collarium border that pipe close is placed on 15～25 ℃ and cultivated 5～7 days;
(4) after above-mentioned (1)～(3) step is cultivated; The test tube strains variable color, varied bacteria growing wherein has the Cordyceps militaris mycelia that has vitality on a small quantity; It is located away from the PDA medium; Place cultivate 6～8 days under 18～25 ℃ of conditions after, by the conventional purification and rejuvenation of the production of hybrid seeds, obtain Cordyceps militaris degeneration bacterial classification.
2. method according to claim 1 is characterized in that the concrete steps of cultivating winning kind are: many of preparation association tubes, PDA medium+chloramphenicol; 45ug/1000ml, pipe inclined-plane indirect a 0.8～2mm Paecilomyces varioti or one of a quilt hair spore kind are concomitance bacterium, place under 10～18 ℃ of conditions and cultivate 7～10 days; When treating bacterial classification field planting and long opening, outside inclined-plane two end distance concomitance bacterium 1cm, insert each one of Cordyceps militaris spawn 2～5mm respectively; With tube place cultivate 3～5 days under 18～20 ℃ of conditions after; Treat the Cordyceps militaris spawn field planting and the growth of scattering in, cultivation temperature is brought up to 24～26 ℃, Cordyceps militaris growth is vigorous after 3～5 days; And all swallowed up concomitance bacterium, obtain the winning kind of Cordyceps militaris kind.
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| CN103478080A (en) * | 2013-07-12 | 2014-01-01 | 重庆市中药研究院 | Genus rejuvenation culture method for artificially-cultured cordyceps sinensis host insects |
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| CN114431071A (en) * | 2022-01-23 | 2022-05-06 | 内蒙古嘉致农业科技有限公司 | Preparation method of high-quality cordyceps militaris cultivation mother strain |
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| CN102907258A (en) * | 2012-11-13 | 2013-02-06 | 四川省青川县川珍实业有限公司 | Detoxification method for edible fungus strain |
| CN103404368A (en) * | 2013-07-12 | 2013-11-27 | 重庆市中药研究院 | Rejuvenation culture method for artificial breeding of cordyceps militaris strain |
| CN103478080A (en) * | 2013-07-12 | 2014-01-01 | 重庆市中药研究院 | Genus rejuvenation culture method for artificially-cultured cordyceps sinensis host insects |
| WO2015003547A1 (en) * | 2013-07-12 | 2015-01-15 | 重庆市中药研究院 | Culture method for natural species rejuvenation of artificially bred cordyceps strains |
| CN103908476A (en) * | 2014-04-29 | 2014-07-09 | 洛阳奥达特食用菌技术开发有限公司 | Cordyceps militaris extract as well as preparation method and preparation thereof |
| CN103908476B (en) * | 2014-04-29 | 2017-01-25 | 洛阳奥达特食用菌技术开发有限公司 | Cordyceps militaris extract as well as preparation method and preparation thereof |
| CN110637680A (en) * | 2018-06-27 | 2020-01-03 | 贵州金蟾大山生物科技有限责任公司 | Production method of Dictyophora rubrovalvata mildew-removing high-germination-rate cultivation stock |
| CN110637680B (en) * | 2018-06-27 | 2023-08-18 | 贵州金蟾大山生物科技有限责任公司 | Production method of Dictyophora rubrovalvata mildew-removed and high-germination-rate cultivation stock |
| CN111587735A (en) * | 2020-06-19 | 2020-08-28 | 山西万海澳生物科技有限责任公司 | Production method for synergistically improving and stabilizing multiple active substances of cordyceps militaris |
| CN114431071A (en) * | 2022-01-23 | 2022-05-06 | 内蒙古嘉致农业科技有限公司 | Preparation method of high-quality cordyceps militaris cultivation mother strain |
| CN114431071B (en) * | 2022-01-23 | 2023-09-05 | 内蒙古嘉致农业科技有限公司 | Preparation method of high-quality cordyceps militaris cultivation mother seed |
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