CN103404368B - Rejuvenation culture method for artificial breeding of cordyceps militaris strain - Google Patents
Rejuvenation culture method for artificial breeding of cordyceps militaris strain Download PDFInfo
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- CN103404368B CN103404368B CN201310293611.5A CN201310293611A CN103404368B CN 103404368 B CN103404368 B CN 103404368B CN 201310293611 A CN201310293611 A CN 201310293611A CN 103404368 B CN103404368 B CN 103404368B
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- A01G18/00—Cultivation of mushrooms
Abstract
The invention provides a strain character rejuvenation culture method for artificial breeding of strain. The method is mainly characterized in that cordyceps militaris strain which is continuously used for many generations is placed in the wild environment for being trained and managed, and reproduction materials undergoing rejuvenation are recovered. The method not only effectively solves the strain character degeneration problem of artificial breeding of the strain and more importantly, achieves sustainable production of artificial breeding of the strain in the practical production process of cordyceps militaris, and a strain guarantee is provided for large-scale sustainable production of the cordyceps militaris.
Description
Technical field
The present invention relates to a kind of strain improvement technology, relate to a kind of rejuvenation cultural method of artificial breeding cordyceps species in particular.
Technical background
Chinese caterpillar fungus mainly refers to Cordyceps sinensis, is also the bacterium worm combination that other are formed with the fungi of Clavicipitaceae (Cordyceps Militaris) and Hepialidae larva under specific condition simultaneously, comprises cordyceps hawkesii gary, liangshan cordyceps herb and Cordyceps militaris etc.; Cordyceps sinensis is the peculiar rare rare traditional Chinese medicine of China.Have invigorate the lung and the kidney, relieving cough and reducing sputum, cure mainly the diseases such as cough, hemoptysis, impotence and seminal emission, after being ill body void, senile chronic cough.Cordyceps sinensis main product the grassy marshland area, high and cold mountain area, provinces and regions height above sea level 3500-5200m local such as in China Tibet, Qinghai, Yunnan, Sichuan and Gansu.Natural resources are very limited, excavate the deterioration excessively causing ecotope in recent years, and natural resources are increasingly exhausted.Therefore, artificial cultivation Cordyceps sinensis is imperative.The key of artificial cultivation is that can aweto infect host insect, enters in the body cavity of host, and in body cavity flourish, finally produce stroma, form Cordyceps sinensis medicinal material.Wild cordyceps host bat moth larvae route of infection be, bat Genus Hepialus insect larvae in wild environment by body surface or Esophageal infections China by hair spore bacterium, but said method, microbiological contamination rate is low, and the cycle is long, and can not carry out large-scale production.
Current host artificial infection method being applied in host's body surface for being adopted by Hirsutella sinensis pityrosporion ovale liquid, being expelled in pin main body, pouring into or being entered in pin main body from host's esophagus by feed." Genus Hepialus larva infects the experimental study of Cordyceps sinensis mattress " that Tu Yongqin etc. deliver in periodical " edible mushroom " 2010 (3) discloses the multiple strain material infected as host of Cordyceps sinensis by hair spore pityrosporion ovale liquid using China.But it is large by hair spore strain separating purifying difficulty to there is China in said method, and the problem that after larva microbiological contamination rate and microbiological contamination, survival rate is lower.Patent applicant is studied the strain material that host of Cordyceps sinensis infects under the subsidy of state natural sciences fund general project " mechanism of host of Cordyceps sinensis larval adaptation feed and ecological fitness evaluation thereof " (being numbered 81072997), " molecule epidemic disease-ology research of Paecilomyces Muscardine of Cordyceps Sinensis Host Insect " (being numbered 81173479) and state natural sciences fund youth fund project " Study on influencing factors of artificial breeding host of Cordyceps sinensis larvae development variation " (project approval number is 30901964).Applicant finds artificial cultivation in the process of Chinese caterpillar fungus research, ascospore, conidium and mycoderma is gathered as propagating materials from the Cordyceps sinensis after several generations's work is cultivated, the Ascospore development of the Cordyceps sinensis of turning out is imperfect, and the propagating materials that the propagating materials simultaneously gathered obtains compared with wild resource the infection rate of host of Cordyceps sinensis larva is low.
Cordyceps species is bred 3-5 and is just started to occur that offspring's ascus development complete or agensis and the reduction of bacterial classification infecting potential etc. comparatively significantly plant sexual involution phenomenon for rear under artificial condition.And for the problem of all sexual involutions of Cordyceps Militaris in producing, traditional solution gathers bacterial classification exactly every year continuously from Proterozoic environment.Due to wild-type strain resource-constrained, xcessive digging not only makes microorganism resource endangered without limit, also by heavy damage natural ecological environment, so the forever continuous from numerous and Chinese caterpillar fungus industry sustainable development of bacterial classification will be realized, the genus rejuvenation technology of artificial breeding bacterial classification must be taked.
Summary of the invention
For solving the kind sexual involution problem of artificial breeding cordyceps species, the invention provides a kind of genus rejuvenation method of artificial breeding cordyceps species, comprising the following steps:
1) with the second generation of artificial breeding (or the third generation, forth generation, the 5th generation is with first-class) cordyceps species, microbiological contamination is carried out to Chinese caterpillar fungus host larva (artificial or wild larva can), screen the sense bacterium larva rejuvenated at first, after continuing to cultivate 1-2 age, render to rejuvenation base;
2) the transitional period management of bacterium larval adaptation wild environment is felt;
3) the hardening period management of bombys batryticatus kind;
4) rejuvenation of sexual spore and mycoderma kind;
5) gather the Cordyceps sinensis of sexual spore and mycoderma maturation, after cleaning entomogenous fungi complex, render to and continue to cultivate in the culture matrix of sterilization;
6) after-ripening of Chinese caterpillar fungus;
7) to gather cordyceps species.
Screen the sense bacterium larva rejuvenated at first.The standard rejuvenated starts feed and starts to attack other companions, and the larva rejuvenated using Article 1 starts the Chinese caterpillar fungus host larva cultivated as rejuvenation to the amount of account for whole vigor host larva to be restored 40%.
Step 2) described in transitional period management for the sense bacterium larva rejuvenated being rendered to the initial 1-2 after primitive environment month; Should control at 5-25 DEG C by temperature in this period, soil moisture controls at mass water content 50-80%, avoids rodent, birds catch and kill Chinese caterpillar fungus host larva, avoid herding to trample Chinese caterpillar fungus host larva.
Step 3) the hardening period management of described bombys batryticatus kind is: throw in sense bacterium larva that rodent will be avoided in region, birds are catched and killed Chinese caterpillar fungus host larva, but region should be thrown in sense bacterium larva and extrude the soil matrix feeling the life of bacterium larva in the mode of herding, thus completely cut off the invasion of companion or natural enemy with the soil matrix of consolidation; Keep vegetation base portion exposed, increase oxygen-supplying amount and the illumination of soil.
Step 4) described in sexual spore and mycoderma kind rejuvenation for control soil moisture be mass water content 60-80%; Avoid rodent, birds catch and kill Chinese caterpillar fungus host larva, avoid herding to trample, until develop into the Chinese caterpillar fungus of sexual spore and mycoderma maturation.
Step 6) after-ripening of described Chinese caterpillar fungus be will the Chinese caterpillar fungus of sexual spore maturation clean after be transplanted in the matrix after indoor 60-100 DEG C high-temperature disinfection treatment, indoor temperature controls at 8-25 DEG C, adopt the cold water moisturizing after 60-100 DEG C of high-temperature disinfection treatment, adopt natural daylight cycle and natural daylight intensity to cultivate 7-15 days, collect strain material.
The stroma that is masked as of sexual spore maturation starts to occur white filiform.
Advantageous Effects of the present invention is: the genus rejuvenation method of artificial breeding bacterial classification provided by the invention; not only effectively solve the kind sexual involution problem of artificial breeding bacterial classification; the more important thing is the problem of the sustainable production solving artificial breeding bacterial classification in Chinese caterpillar fungus actual production process, the scale sustainability for Chinese caterpillar fungus is produced and is provided bacterial classification guarantee.
Embodiment
The screening of embodiment 1 microbiological contamination host larva is investigated
Collect bat moth artificial feeding each 1500 of the larva six groups of 1-2 month respectively, give group I, experimental group II, experimental group III, experimental group IV and experimental group V by name respectively.The method that the patent application " a kind of Cordyceps sinensis low altitude area artificial culture method " (application number: CN201210292550.6) that collection is submitted to through applicant is recorded cultivates Cordyceps sinensis, the ascospore of Cordyceps sinensis cultivated from the method, conidium and mycoderma, as strain material, repeat to cultivate Cordyceps sinensis by the method; Abundant aforesaid operations more than 3 times, gather obtain ascospore, conidium and mycoderma as propagating materials, using the mixing of these propagating materialss as mass rearing propagating materials.Mass rearing propagating materials is proceeded under air purity requirement is not less than the environmental condition of 100,000 grades and inoculate; Staff requirement dresses work clothes, mouth mask and cap.
Respectively mass rearing propagating materials is carried out to group I, experimental group II, experimental group III, experimental group IV and experimental group V and infect process.A kind of method that the method infected is recorded with reference to " strain material for host of Cordyceps sinensis infection and host infect method " (application number: CN201210338583.X).Constantly microbiological contamination larva is collected after infecting; The standard of screening is: 1) host larva liveness reduces, 2) do not take food.Often group selects 500.By the larva of hair spore bacterium, raising is concentrated to above-mentioned infection China, between concentrated feeding period except the feed consumption of routine is provided, needs to provide enough moisture.That inoculated by spray pattern and increase follow-up output by filling with needing of inoculating of the mode of feeding; By admixing feed or being mixed into the humidity needing to increase feed or matrix that matrix mode inoculates.Intensive farm completes under the temperature condition of 8-18 DEG C.Between concentrated feeding period, constantly select the larva that liveness recovers, until screen specified quantity, the standard of judgement is: 1) start to recover feed; 2) the host larva attacking other is started.The amount of group I screening is 100, and the amount that experimental group II is screened is 150, and the amount that experimental group III is screened is 200, and the amount that experimental group IV is screened is 250, and the amount that experimental group V is screened is 300.
Respectively group I, experimental group II, experimental group III, experimental group IV, experimental group V are rendered to region, Cordyceps sinensis producing region and cultivated, for preventing the larger animals such as cattle and sheep from trampleing at any time, cultivating ground, region surrounding wire netting and surrounding.The rejuvenation base gradient is advisable between 10-30 degree, and draining is good; There are other shrubs except lawn plant in interior growth to avoid sunshine of high strength and birds to the harm of adult.Cultivate the survival rate of adding up each group of host larva for 50 days afterwards.Specifically as shown in table 1.
The different screening ratio of table 1 is on the impact of host's survival rate of larvae
The content recorded in table 1 proves, to the selection of host larva higher than after 40%, the survival rate of host larva just significantly declines, the ratio of therefore screening strong larva with 40% for best.
The larva that the host larva of group I, experimental group II, experimental group III, experimental group IV, experimental group V is thrown in is adapted to gradually the living environment in field, and start the primary humus soil life crawling into periphery from input region.The larva of this one-phase will accept natural temperature, naturally the taking exercise of humidity and illumination, and produces the resistance to all kinds of disease, insect pest and the plague of rats, until larvae development is bombys batryticatus.When base green grass starts growth and covers earth's surface gradually, can appropriately allow the herbivore such as ox, sheep to enter base and gnaw green grass, to make turf base portion exposed outside for degree, the sense bacterium larva grown for underground is provided sufficient Oxygen Condition by this, and illumination simultaneously also will be grown larval growth and be produced active influence.The genus rejuvenation phase will forbid the larger animal such as ox, sheep to enter base trample; Physics or chemical measure is taked to prevent the harm of muroid and birds sky hostile sense bacterium larva.Bombys batryticatus is formed into stroma, and to start the soil humidity that duration of germination will prevent it from growing not enough, and the morning and evening of fine day will water, and how many standards is advisable with the soil layer at drenched bombys batryticatus place.Sufficient moisture condition is not only conducive to the growth of stroma, is also conducive to generating more solid mycoderma.The stroma of this one-phase is grown accepting natural temperature, the free period of humidity and illumination takes exercise, and produces the resistance to all kinds of disease together with mycoderma.The genus rejuvenation phase will forbid the larger animal such as ox, sheep to enter base trample; Physics or chemical measure is taked to prevent the harm of muroid and birds sky hostile sense bacterium larva, until grow for the ripe Cordyceps sinensis of sexual spore (comprising ascospore and conidium).When the sexual spore of Cordyceps sinensis and mycoderma are after reaching maturity in genus rejuvenation base, need to adapt to get back to indoor process from field through a latter stage of ripening.During operation, with instruments such as spades Cordyceps sinensis is transplanted in strain material catch tray and carries out After-mature cultivation.The matrix or other impurity of carrying is removed during transplanting.With the cold water moisturizing through 60-100 DEG C of high-temperature sterilization between latter stage of ripening, and under being placed in 5-25 DEG C of condition, natural daylight cycle and natural daylight intensity is adopted to cultivate.In about 1 week of after-ripening, the sexual spore (comprising ascospore and conidium) of rejuvenation Cordyceps sinensis and mycoderma have adapted to indoor environment gradually.Collect the pycnospore through the after-ripening cycle and mycoderma mixing, as rejuvenation culture propagation material.
The activity of embodiment 2 rejuvenation culture propagation material is investigated
The method that the patent application " a kind of Cordyceps sinensis low altitude area artificial culture method " (application number: CN201210292550.6) that application reference people submits to is recorded cultivates Cordyceps sinensis; Collect little Jin bat Hepialus xiaojinensisTuetMa larva, 4000 cestode six deciles are put in 8 1m
2in the clean enamel tray through sterilizing, often organize 500 cestodes, upper dried leaves through sterilization, movable and hidden in order to insect, spray some distilled water and keep humidity.First to feed 7d with aseptic serpentgrass, carrot, sweet potato, dish leaf, make polypide restoration ecosystem.Then residue is removed all edible.Stop feeding after 2 ~ 3d, get serpentgrass that above-mentioned any 3 groups of mass rearing propagating materials suspensions of feeding sprayed, carrot, sweet potato, dish leaf, as a control group; Get serpentgrass that rejuvenation culture propagation material suspension that any 4 groups of embodiments 1 of feeding obtain sprayed, carrot, sweet potato, dish leaf, as experimental group; Every day removes residue, and 2d same method is fed, and connects and feeds 30d, observes worm growth and active situation, at this moment rejects those robust growth.Movable quick, sensitive worm, then the alpine meadow soil and turf adopted back in 1Ocm larval growth district is covered on, control temperature is at 1 ~ 5 DEG C, continue to feed, respectively after 1 month, 3 months, 6 months and 1 year, respectively experimental group and control group a group is dug out polypide, calculate that infect surviving of ossifing with polypide number that is death; Obtain mixed feed feeding method group data.
Investigate by randomly drawing 20 larvas in the time phase periodic collection larva colony of 1 month, 3 months, 6 months and 1 year.After being sterilized larva surface, adopt special instrument aseptically to extract the hemolymph film-making of trace, microscopy is observed, the form of thalline in record hemolymph.Tracing study larva is ossify situation.While thalline is observed, extract the trace hemolymph that carries disease germs and spread upon on sterilizing PDA culture medium flat plate, be placed in 18 DEG C of insulating boxs and cultivate.Take out the state that culture dish microscopy observes growth and sporulation once a day, or picking thalline and culture film-making thereof are observed.Thalline place to be coated forms bacterium colony, and the mycelium film-making microscopy on picking colony surface is observed; Concrete outcome is as shown in table 2.
The infection conditions of table 2 different strain material larva
Result shows, host larva infection aweto is a complicated process, needs the regular hour, just can complete whole infection processs.As shown in Table 1, adopt the rejuvenation culture propagation material described in embodiment 1, bacterial classification is the strongest to the infection ability of host larva, can infect aweto after host larva inoculates 1 month.Inoculate 6 months postoperative infections and reach peak, infection rate is 100%.
Raise about 1 year, larva can fall ill rigid, then germinates and young shoot, grow into fruit body.The Chinese caterpillar fungus that the Chinese caterpillar fungus of being unearthed 20 days of bat moth larvae bacterial classification microbiological contamination provided by the invention cultivated and wild sense bacterium larva perithecium fully-developed worm and mass rearing propagating materials sense bacterium larva are unearthed 20 days compares; Find the ascocarp of the Chinese caterpillar fungus that the rejuvenation culture propagation material microbiological contamination that obtains of the present invention obtains and wild cordyceps, ascospore is considered to the ascocarp natural with Cordyceps sinensis, ascospore morphological feature is just the same; The larva perithecium fully-developed worm infected with mass rearing propagating materials compares, find there is larger difference in thecasporous form He on polypide, the Cordyceps sinensis polypide produced with mass rearing propagating materials is less, there is not full phenomenon in ascospore simultaneously in fruit body, and the output of each group Cordyceps sinensis is as shown in table 3.
The different impact on cordyceps yield and form of table 3 strain material
Research proves, there will be the phenomenon of kind of sexual involution through 3-5 for the cordyceps species of artificial breeding; Presentation is that offspring's ascus development complete or agensis and the reduction of bacterial classification infecting potential etc. comparatively significantly plant sexual involution phenomenon.Adopt method rejuvenation bacterial classification provided by the invention, the Chinese caterpillar fungus of production, in Chinese caterpillar fungus rank and the full rate of ascus all relatively.
Claims (7)
1. the genus rejuvenation method of artificial breeding cordyceps species, is characterized in that: comprise the following steps:
1) with cordyceps species more than 2 generations of artificial breeding, microbiological contamination is carried out to Chinese caterpillar fungus host larva, screen the sense bacterium larva that rejuvenates at first, after continuing to cultivate 1-2 age, render to rejuvenation base;
2) the transitional period management of bacterium larval adaptation wild environment is felt;
3) the hardening period management of bombys batryticatus kind;
4) rejuvenation of sexual spore and mycoderma kind;
5) gather the Chinese caterpillar fungus of sexual spore maturation, after cleaning entomogenous fungi complex, be transplanted to and continue to cultivate in the culture matrix of sterilization;
6) after-ripening of Chinese caterpillar fungus;
7) to gather cordyceps species material.
2. the genus rejuvenation method of artificial breeding cordyceps species according to claim 1, is characterized in that: the standard rejuvenated starts feed and starts to attack other companions; The sense bacterium larva rejuvenated with the 1st article starts to collecting the larva of 40% quantity accounting for whole sense bacterium larva, as the sense bacterium larva that rejuvenation is cultivated.
3. the genus rejuvenation method of artificial breeding cordyceps species according to claim 1, is characterized in that:
Step 2) described in transitional period management for the sense bacterium larva rejuvenated being rendered to the initial 1-2 after primitive environment month; Should control at 8-25 DEG C by temperature in this period, soil moisture controls at mass water content 50-80%, avoids rodent, birds catch and kill Chinese caterpillar fungus host larva, avoid herding to trample Chinese caterpillar fungus host larva.
4. the genus rejuvenation method of artificial breeding cordyceps species according to claim 1, it is characterized in that: the management of the hardening period of bombys batryticatus kind described in step 3) is: throw in sense bacterium larva that rodent will be avoided in region, birds are catched and killed Chinese caterpillar fungus host larva, but region should be thrown in sense bacterium larva and extrude the soil matrix feeling the life of bacterium larva in the mode of herding, thus completely cut off the invasion of companion or natural enemy with the soil matrix of consolidation; Keep vegetation base portion exposed, increase oxygen-supplying amount and the illumination of soil.
5. the genus rejuvenation method of artificial breeding cordyceps species according to claim 1, is characterized in that: the sexual spore described in step 4) and the genus rejuvenation of mycoderma are mass water content 60-80% for controlling soil moisture; Avoid rodent, birds trampling and taking food Chinese caterpillar fungus, until develop into the Chinese caterpillar fungus of sexual spore and mycoderma maturation.
6. the genus rejuvenation method of artificial breeding cordyceps species according to claim 1, it is characterized in that: the after-ripening of Chinese caterpillar fungus described in step 6) is be transplanted in the matrix after indoor 60-100 DEG C high-temperature disinfection treatment after the Chinese caterpillar fungus cleaning by sexual spore and mycoderma maturation, indoor temperature controls at 8-25 DEG C, adopt the cold water moisturizing after 60-100 DEG C of high-temperature disinfection treatment, adopt natural daylight cycle and natural daylight intensity to cultivate 7-15 days, collect bacterial classification.
7. the genus rejuvenation method of artificial breeding cordyceps species according to claim 5, is characterized in that: the stroma that is masked as of sexual spore and mycoderma maturation starts to occur white filiform.
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| PCT/CN2014/080068 WO2015003547A1 (en) | 2013-07-12 | 2014-06-17 | Culture method for natural species rejuvenation of artificially bred cordyceps strains |
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| CN103404368B (en) * | 2013-07-12 | 2015-04-15 | 重庆市中药研究院 | Rejuvenation culture method for artificial breeding of cordyceps militaris strain |
| CN104145719B (en) * | 2014-09-04 | 2016-06-08 | 重庆市中药研究院 | A kind of Cordyceps fungus mycelium fermentation production method |
| CN105176834A (en) * | 2015-08-24 | 2015-12-23 | 赵彦龙 | Efficient insecticidal fungus strain screening technology for preventing and controlling forest insects including yponomeuta evonymellus, naxa seriaria motschulsky and malacosoma neustria testacea |
| CN108018251A (en) * | 2018-01-31 | 2018-05-11 | 广西师范大学 | One species impoverishment strain returns the rejuvenation cultural method of natural environment |
| CN112673890B (en) * | 2019-10-19 | 2024-01-16 | 东莞市东阳光冬虫夏草研发有限公司 | Method for reducing arsenic content and heavy metal content of ecological breeding cordyceps sinensis |
| CN112342145A (en) * | 2020-11-04 | 2021-02-09 | 青海久实虫草生物科技有限公司 | Rejuvenation method of hirsutella hepiali Chen et Shen fungi |
| CN115399295A (en) * | 2022-08-31 | 2022-11-29 | 力源新资源开发(广东)有限公司 | Method for improving survival rate of hirsutella sinensis infected bat moth larvae |
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| CN1094091A (en) * | 1994-02-05 | 1994-10-26 | 李春燕 | The seed selection of cordyceps militaris excellent species and high yield cultivating method |
| CN1156569C (en) * | 2001-09-04 | 2004-07-07 | 上海大中生物种苗有限公司 | Artificial culture method of north cordyceps sporophores and its living product |
| CN101215527A (en) * | 2008-01-11 | 2008-07-09 | 丁啸南 | Method for cultivating silkworm chrysalis Cordyceps sinensis |
| CN102726216B (en) * | 2012-07-17 | 2013-07-10 | 杨毅 | Method for culturing cordyceps militaris anti-degradation strain |
| CN103404368B (en) * | 2013-07-12 | 2015-04-15 | 重庆市中药研究院 | Rejuvenation culture method for artificial breeding of cordyceps militaris strain |
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Effective date of registration: 20220809 Address after: Huang Jueya South Road 400065 Chongqing Nan'an District No. 34 Patentee after: CHONGQING ACADEMY OF CHINESE MATERIA MEDICA Address before: 400065 Nanan District, Chongqing City, No. 34 (2) Building, Nanshan Road, Huangshuangya Town, 2 floors Patentee before: Chongqing Pharmaceutical Research Institute Technology Development Co.,Ltd. |
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