CN103283478B - Cordyceps militaris producing method - Google Patents
Cordyceps militaris producing method Download PDFInfo
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- CN103283478B CN103283478B CN201210044959.6A CN201210044959A CN103283478B CN 103283478 B CN103283478 B CN 103283478B CN 201210044959 A CN201210044959 A CN 201210044959A CN 103283478 B CN103283478 B CN 103283478B
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Abstract
The invention discloses a cordyceps militaris producing method which comprises the following steps: (1) a cordyceps militaris strain is cultivated in a compost until a growth stage when aerial hyphae are generated while fruiting body primordial are not generated; and (2) the aerial hyphae are subjected to mechanical stimulation and then cultivated until a growth stage when a cordyceps militaris fruiting body is generated, wherein the operation of the mechanical stimulation comprises that the surfaces of the aerial hyphae are cut open, the aerial hyphae with cut-open surfaces are lodging on the surface of the compost, and the cordyceps militaris fruiting body comes from a cordyceps militaris bacterial strain whose preservation number is CGMCC (China general microbiological culture collection center) NO. : 5606. With the adoption of the technical scheme, the cordyceps militaris producing method provided by the invention can achieve the effect that the forming time of the cordyceps militaris fruiting body primordia is shortened and the differentiation of the cordyceps militaris primordial is uniform, so that the cultivation period of the cordyceps militaris is shortened, and the yield and quality of the cordyceps militaris are improved.
Description
Technical field
The present invention relates to biological technical field, particularly, relate to a kind of method of producing Cordyceps militaris.
Background technology
Cordyceps militaris (Cordyceps militsris) is a kind of entomogenous fungi, is under the jurisdiction of Ascomycota, gang pyrenomycetes, and Sphaerials, Clavicipitaceae, Cordyceps, have another name called northern Chinese caterpillar Fungus, northern Chinese caterpillar fungus or pupa grass, belongs to together not of the same race with Cordyceps sinensis.Cordyceps militaris has higher medical value, sees the earliest Compendium of Material Medica: " cicada fungus can cure mainly children's sky and hang frightened epilepsy, the morbid night crying of babies, palpitaition " in addition, also has record in " Xinhua's Compendium of Materia Medica ": " taste is sweet, and property is flat, and beneficial lung is mended marrow, hemostasis and phlegm ".Modern study shows, contains the various bioactivators such as cordycepin, Cordyceps sinensis polysaccharide in Cordyceps militaris, anti-inflammatory, anticancer, antibacterial, strengthen immunity etc. aspect play an important role, similar with Cordyceps sinensis, be one of desirable substitute of Cordyceps sinensis.2009, Ministry of Health of the People's Republic of China was according to the regulation of " Food Hygiene Law of the People's Republic of China " and " new resource food management method ", and approval Cordyceps militaris is new resource food, and this is that Cordyceps militaris and converted products thereof are opened up wide market and laid a good foundation.Because Cordyceps militaris has high nutritive value and health-care efficacy, Cordyceps militaris and products thereof is accepted by increasing consumer, is particularly subject to liking of the national consumers such as China, Japan and Korea S.
Tissue Culture of Cordyceps militaris is to solve the not enough good approach in wild Chinese caterpillar fungus source, and relevant research is a lot.At present, the planting type of adopt the most extensively, technology is the most ripe is substituting stuff cultivation, and main flow process comprises inoculation, sends out bacterium, urges flower bud, cultivates and gather; Wherein, inoculation refers to Cordyceps militaris spawn is placed in to medium; Sending out bacterium is to instigate Cordyceps militaris spawn to be grown in medium to produce mycelia and the aerial hyphae in composts or fertilisers of cultivating; Urge flower bud to refer to the formation of induction fruit body primordium; Cultivation is to instigate fruit body primordium to grow to meet harvesting standard; Gather and refer to and will meet the fruiting bodies of cordyceps militaris results of harvesting standard.This planting type raw material sources are extensive, easy and simple to handle, are applicable to scale and standardized production.
In existing Tissue Culture of Cordyceps militaris, the operation of the formation of induction fruit body primordium, urges the step of flower bud mainly to utilize thermal stimulation and increase light application time and intensity to induce the generation of fruit body primordium.But facts have proved that the method for existing production Cordyceps militaris, due to the limitation of the operation of the formation of above-mentioned induction fruit body primordium, exists the defect that the time that forms cordycep militaris fruiting body entity primodium grows.
And in existing Tissue Culture of Cordyceps militaris, the hereditary capacity of cordyceps militaris link bacterial strain is poor, be a large bottleneck of restriction industrialization development thereby cause the decline on the yield and quality of Cordyceps militaris.The poor form performance of the hereditary capacity of cordyceps militaris link bacterial strain is generally: angle cash resemble serious, the generation weak, fruit body primordium of mycelia growing way is less, cultivation cycle is long, mycelia more aging, mycelium color and luster is dark, fruiting body yield is less and fruit body abnormal rate is more high.
Summary of the invention
The object of the invention is to overcome the defect that time of the formation cordycep militaris fruiting body entity primodium that the method for existing production Cordyceps militaris exists grows, the method for the production Cordyceps militaris of shorter and former base differentiation neat and consistent of a kind of time that forms cordycep militaris fruiting body entity primodium is provided.
To achieve these goals, the present inventor finds unexpectedly, if the aerial hyphae of Cordyceps militaris is carried out to mechanical stimulus, just can promote significantly the formation of fruit body primordium, has obtained thus the present invention.
The invention provides a kind of method of producing Cordyceps militaris, the method comprises: (1) is cultured to Cordyceps militaris spawn the vegetative stage that produces aerial hyphae annesl but do not produce fruit body primordium in composts or fertilisers of cultivating; (2) described aerial hyphae is carried out to mechanical stimulus, be then cultured to the vegetative stage that produces fruiting bodies of cordyceps militaris; The operation of described mechanical stimulus comprises: the surface of described aerial hyphae is cut, and the lip-deep aerial hyphae of composts or fertilisers of cultivating is lodged in the surface of described composts or fertilisers of cultivating; Wherein, described Cordyceps militaris spawn is from a kind of cordyceps militaris link bacterial strain, this cordyceps militaris link bacterial strain is a bacterial strain for Cordyceps militaris (Cordyceps militaris), (address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City to be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 23rd, 2011, Institute of Microorganism, Academia Sinica), preserving number is CGMCC NO:5606.This bacterial strain is that the present inventor obtains after complicated screening.
Pass through technique scheme, the method of production Cordyceps militaris provided by the invention can reach and shorten the cordycep militaris fruiting body entity primodium time forming and the effect that makes the former base differentiation of Cordyceps militaris neat and consistent, thereby shorten the cordyceps militaris plantation cycle, improved production efficiency and the quality of Cordyceps militaris.
Other features and advantages of the present invention are described in detail the embodiment part subsequently.
Embodiment
Below the specific embodiment of the present invention is elaborated.Should be understood that, embodiment described herein only, for description and interpretation the present invention, is not limited to the present invention.
In the present invention, do not do in the situation of contrary explanation, as known in the art, for fear of living contaminants, the various operations of carrying out are all preferably carried out under substantially aseptic condition, various tool used and material are had sterilized, for example, under 110-130 DEG C and 0.12-0.18MPa, carry out after steam sterilizing 20-80min.
The invention provides a kind of method of producing Cordyceps militaris, the method comprises: (1) is cultured to Cordyceps militaris spawn the vegetative stage that produces aerial hyphae annesl but do not produce fruit body primordium in composts or fertilisers of cultivating; (2) described aerial hyphae is carried out to mechanical stimulus, be then cultured to the vegetative stage that produces fruiting bodies of cordyceps militaris; The operation of described mechanical stimulus comprises: the surface of described aerial hyphae is cut, and the lip-deep aerial hyphae of composts or fertilisers of cultivating is lodged in the surface of described composts or fertilisers of cultivating; Wherein, described Cordyceps militaris spawn is from a kind of cordyceps militaris link bacterial strain, this cordyceps militaris link bacterial strain is a bacterial strain for Cordyceps militaris (Cordyceps militaris), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 23rd, 2011, preserving number is CGMCC NO:5606.This bacterial strain is that the present inventor obtains after complicated screening.
Wherein, this cordyceps militaris link bacterial strain can use the vegetative propagating method of conventional Cordyceps militaris to go down to posterity, also can carry out preservation with conventional Cordyceps militaris spawn method for preserving, can also use the method for conventional cordyceps militaris strain rejuvenation to carry out rejuvenation and expand cultivating.
Wherein, described Cordyceps militaris spawn can, before being inoculated in composts or fertilisers of cultivating, according to the conventional method of this area, be cultivated the form for solid spawn or liquid spawn in advance, is preferably the form of liquid spawn.Wherein, described Cordyceps militaris spawn can be inoculated in composts or fertilisers of cultivating according to conventional method, and the present invention does not have special requirement.
Wherein, the history of life and grown form under Cordyceps militaris carded sliver part out of office and under various culture conditions have been conventionally known to one of skill in the art, and (publishing house of the Central China University of Science and Technology, publishes for example document for 2010 for cordyceps militaris cultivation new technology, Xu Xiuhua; New cultivation method Cordyceps militaris, Zhang Shengyou, publishing house of the Central China University of Science and Technology, 2010) in record content.Due in the history of life of Cordyceps militaris, the mycelia of Cordyceps militaris can be all saffron mycelia by the mycelia annesl of white (or transparent) occurring before fruit body primordium, and, because the fruit body primordium of Cordyceps militaris is the mycelium of the specialization of grain of rice shape, also very easily identification.Therefore can whether come to determine that Cordyceps militaris is whether in producing aerial hyphae annesl but do not produce the vegetative stage of fruit body primordium by the appearance of fruit body primordium.Wherein, in step (1), annesl can refer to that part annesl is crocus, also can refer to that substantially all annesl is crocus.
The method according to this invention, wherein, under preferable case, in step (1), described cultivation comprises the dark cultivation stage and the light stimulation cultivation stage that successively carry out; In the time that the mycelia in composts or fertilisers of cultivating is had thorough grasp composts or fertilisers of cultivating, described dark cultivation stage finishes; In the time that aerial hyphae annesl is crocus, described light stimulation cultivation stage finishes.
Wherein, normally, when Cordyceps militaris spawn is inoculated in composts or fertilisers of cultivating, Cordyceps militaris spawn is sprouted, and grows mycelia, and a part stretches into (i.e. mycelia in composts or fertilisers of cultivating) in composts or fertilisers of cultivating, a part is to composts or fertilisers of cultivating Growth In Space in addition, form aerial hyphae (being stretched over the mycelia in the space outside composts or fertilisers of cultivating), the continued growth simultaneously of the mycelia in composts or fertilisers of cultivating and aerial hyphae, until reach the state of the saturating composts or fertilisers of cultivating of mycelia of Cordyceps militaris.The state of the saturating composts or fertilisers of cultivating of mycelia of Cordyceps militaris is a kind of growth conditions well known in the art, and for example, when the mycelia in composts or fertilisers of cultivating is dispersed throughout composts or fertilisers of cultivating substantially, when aerial hyphae covers composts or fertilisers of cultivating surface substantially, the mycelia that reaches Cordyceps militaris is had thorough grasp the state of composts or fertilisers of cultivating.The state of having thorough grasp composts or fertilisers of cultivating in order to reach as soon as possible mycelia in composts or fertilisers of cultivating, preferably carries out the cultivation of dark cultivation stage.The condition of described dark cultivation stage can be the selection in conventional Tissue Culture of Cordyceps militaris method, as long as without under optical condition, the condition of for example described dark cultivation stage comprises: temperature is 21-27 DEG C, is preferably 23-25 DEG C; Relative air humidity can be 50-70%, is preferably 60%.Typically, in step (1), the time of conventional dark cultivation stage is generally 8-10 days.
Wherein, preferably, the mechanical stimulus in method of the present invention has been experienced the cultivation of light stimulation cultivation stage and annesl carries out after being crocus mycelia.
The method according to this invention, wherein, be crocus in order to make quickly aerial hyphae annesl, under preferable case, in step (1), the condition of described light stimulation cultivation stage comprises: the light application time of every day is 15-20 hour, and intensity of illumination is 100-150 lux, temperature between illumination period is 18-25 DEG C, is preferably 20-23 DEG C; Temperature during unglazed photograph is 18-25 DEG C, is preferably 20-23 DEG C.Wherein, relative air humidity can be 50-70%, is preferably 60%.Typically, in step (1), the time of conventional light stimulation cultivation stage is generally 5-7 days.
The method according to this invention, wherein, although the mechanical stimulus in method of the present invention can be after Cordyceps militaris mycelia becomes crocus carries out to random time before there is fruit body primordium, but to form the time of cordycep militaris fruiting body entity primodium and improve output in order further to shorten, under preferable case, in step (2), described mechanical stimulus is crocus and carries out immediately while just having there is mound shape protuberance at Cordyceps militaris mycelia annesl.Wherein, described mound shape protuberance refer to the mound shape that forms on the surface of Cordyceps militaris mycelia and area be 0.7-1cm
2, be highly the mycelium of the protuberance of 0.7-1mm.
The method according to this invention, wherein, in step (2), as long as reaching, the operation that the surface of described aerial hyphae is cut scratches described aerial hyphae, to form the time of cordycep militaris fruiting body entity primodium and make the former base of Cordyceps militaris break up to obtain neat and consistent more in order further to shorten, under preferable case, in step (2), the operation of described mechanical stimulus comprises: with the spaced and parallel of 0.5-1.0cm the surface of described aerial hyphae is cut.Wherein, the degree of depth of incision can reach the deposition surface of composts or fertilisers of cultivating deeply, for example, can be 2-3mm.
The method according to this invention, wherein, instrument used is cut in the surface of described aerial hyphae can be for making mycelia rupture and the instrument of the cutter shape that lodges; Under preferable case, in step (2), it is at least one in wood chip, bamboo chip, plastic sheet and sheet metal that instrument used is cut in the surface of described aerial hyphae.
The method according to this invention, wherein, in step (2), described cultivation is cultivated and is the cultivation that usually said Cordyceps militaris goes out the grass stage and cultivates, the present invention is requirement especially not, under preferable case, described cultivation is cultivated and is comprised former base induction period and the sporophore growth stage of successively carrying out; In the time there is fruit body primordium on described aerial hyphae, described former base induction period finishes; In the time of fruit body growth and maturity, the described sporophore growth stage finishes.
Wherein, because the fruit body primordium of Cordyceps militaris is the aerial mycelium of the specialization of grain of rice shape, very easily whether identification, therefore can come to determine whether former base induction period finishes by the appearance of fruit body primordium.
Wherein, sporophore growth maturation refers to that sporophore growth arrives Cordyceps militaris nutritive value the highest state relatively.Typically, when the size of the fruit body of Cordyceps militaris is substantially no longer grown up, and while there is spinule in top, can think that sporophore growth has arrived Cordyceps militaris nutritive value the highest state relatively.
The method according to this invention, wherein, in step (2), the condition of described former base induction period can be conventional condition, the present invention does not have special requirement, in order further to shorten the time that forms cordycep militaris fruiting body entity primodium, under preferable case, in step (2), the condition of described former base induction period comprises: the light application time of every day is 15-18 hour, intensity of illumination is 100-150 lux, cultivation temperature between illumination period is 19-24 DEG C, be preferably 20-22 DEG C, temperature during unglazed photograph is 13-18.5 DEG C, be preferably 15-18 DEG C, relative air humidity is 60-70%, be preferably 70%.Typically, owing to having carried out mechanical stimulus, in step (2), the time of conventional former base induction period is generally 4-10 days, is preferably 4-6 days.
The method according to this invention, wherein, in step (2), the condition in described sporophore growth stage can be conventional condition, the present invention does not have special requirement, in order further to improve the yield and quality of Cordyceps militaris, under preferable case, the condition in described sporophore growth stage comprises: the light application time of every day is 12-15 hour, and intensity of illumination is 100-150 lux, and the cultivation temperature between illumination period is 16-22 DEG C, be preferably 18-20 DEG C, temperature during unglazed photograph is 16-22 DEG C, is preferably 18-20 DEG C, and relative air humidity is 80-90%.Typically, in step (2), the time in conventional sporophore growth stage is generally 15-20 days.
The method according to this invention, wherein, described composts or fertilisers of cultivating can be conventional can be for the various composts or fertilisers of cultivating of cultivating for material of Cordyceps militaris, the present invention does not have special requirement, under preferable case, contains base-material and basic nutrient solution.Wherein, the weight ratio of described base-material and described basic nutrient solution is 1: 1.1 to 1: 1.3.Wherein, described basic culture solution contains: pupa extract, 0.6-1g/L magnesium sulfate, the potassium dihydrogen phosphate of 0.8-1.2g/L and the egg white of 50-70g/L of 15-25g/L.Wherein, described base-material contains rice and analysis for soybean powder, and the weight ratio of rice and analysis for soybean powder be 3: 1 to 4: 1 wherein, described pupa extract can dissolve for conventional water the DDGS that (can for boil dissolving) goes out from silkworm chrysalis.Described egg white can be generally the egg white of raw egg.
Below will exemplarily describe in detail the present invention by embodiment.
Preparation Example 1
Cordyceps militaris spawn is cultivated the form for liquid spawn by this Preparation Example.
200g potato is boiled to 30min in 1L boiling water, filter and obtain potato leachate, then in whole potato leachate obtained above, add the MgSO of 20g glucose, 3g peptone, 1.5g
47H
2the KH of O and 3g
2pO
4, and be settled to 1L, under 121 DEG C and 0.14Mpa, after steam sterilizing 30min, be cooled to room temperature, obtain potato liquid nutrient medium for subsequent use.
Get Cordyceps militaris spawn (bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC NO:5606, is the Cordyceps militaris mycelia tissue of preserving under 4 DEG C of conditions) the about 3mm of volume
33 of bacterial classification pieces be inoculated in 150ml potato liquid nutrient medium obtained above, turn per minute 120, shaken cultivation 7 days at 25 DEG C of temperature, obtain Cordyceps militaris bacterial classification.
Preparation Example 2
This Preparation Example is prepared composts or fertilisers of cultivating.
20g silkworm chrysalis is filtered and obtains filtrate and be cooled to room temperature after digestion 30min in the water of 800ml, in whole filtrate obtained above, add the MgSO of 0.8g
47H
2the KH of O, 1g
2pO
4, the peptone of 6g and the egg of 60g, be settled to 1L, obtain basic nutrient solution.
After being mixed by the weight ratio of 3: 1, rice and analysis for soybean powder obtain base-material.Base-material is mixed by the weight ratio of 1: 1.2 with basic nutrient solution, under 121 DEG C and 0.14Mpa, after steam sterilizing 60min, be cooled to room temperature, obtain composts or fertilisers of cultivating for subsequent use.
Embodiment 1
In the blake bottle of the composts or fertilisers of cultivating obtaining in the Preparation Example 2 that contains 88g, the Cordyceps militaris spawn of the liquid spawn form that the Preparation Example 1 of inoculation 3ml obtains, then carries out the cultivation of dark cultivation stage.
Wherein, the cultivation of dark cultivation stage is carried out in lucifuge environment, and actual conditions comprises: temperature is 23-25 DEG C, and relative air humidity can be 60%.Dark cultivation stage carried out after 9 days, and the mycelia in visible composts or fertilisers of cultivating is dispersed throughout composts or fertilisers of cultivating substantially, aerial hyphae covers composts or fertilisers of cultivating surface substantially, had reached the mycelia in composts or fertilisers of cultivating and have thorough grasp the state of composts or fertilisers of cultivating.Now, finish the cultivation of dark cultivation stage, then carry out the cultivation of light stimulus cultivation stage.
Wherein, the condition of light stimulation cultivation stage comprises: the light application time of every day is 18 hours, and intensity of illumination is 150 luxs, and the temperature between illumination period is 22 DEG C, and the temperature during unglazed photograph is 22 DEG C, and relative air humidity is 60%.Light stimulation cultivation stage carried out after 6 days, visible aerial hyphae substantially all annesls be crocus and just having started form mound shape protuberance (the surperficial mound shape of Cordyceps militaris mycelia and area be 0.7-1mm
2, be highly the mycelium of the protuberance of 0.7-1mm).Now, finish light stimulation cultivation stage, and carry out mechanical stimulus.
Wherein, the operation of mechanical stimulus comprises: use 0.5mm thick, 2cm is wide, bamboo chip after the sterilization of 20cm length, with the interval of 1cm, the surface of aerial hyphae is cut, the degree of depth of incision is 2.5mm left and right, and aerial hyphaes all surface is smoothed with this bamboo chip, the lip-deep aerial hyphae of composts or fertilisers of cultivating is all lodged in the surface of described composts or fertilisers of cultivating, then carry out the cultivation of former base induction period and cultivate.
Wherein, the condition of former base induction period comprises: the light application time of every day is 15 hours, and intensity of illumination is 100 luxs, and the temperature between illumination period is 22 DEG C, and the temperature during unglazed photograph is 16 DEG C, and relative air humidity is 70%.Former base induction period carried out after 4 days, on visible aerial hyphae, started to form fruit body primordium (projection of grain of rice shape), now, finished the cultivation of former base induction period and cultivated, and then carried out the cultivation in sporophore growth stage and cultivated.
Wherein, the condition in sporophore growth stage comprises that the light application time of every day is 12 hours, and intensity of illumination is 150 luxs, and the temperature between illumination period is 20 DEG C, and the temperature during unglazed photograph is 20 DEG C, and relative air humidity is 80-90%.The sporophore growth stage carried out after 18 days, and the size of the fruit body of visible Cordyceps militaris is substantially no longer grown up, and spinule appears in top, and now, Cordyceps militaris nutritive value is relatively the highest, finishes immediately the cultivation in sporophore growth stage and cultivates, and gathers.
In the present embodiment, being inoculated into composts or fertilisers of cultivating from the Cordyceps militaris spawn of liquid spawn form starts, through 37 days, the fruiting bodies of cordyceps militaris (fresh weight) of average results 28g in each blake bottle, after dry, the shape of fruit body meets " bar-shaped, long 4-7cm, diameter 0.1-0.5cm, surface light brown yellow is to khaki, there is thin vertical wrinkle, quality is pliable and tough " standard be normal-sub entity, incongruent is lopsided fruit body, the fruit body abnormal rate that statistics obtains the present embodiment production is 8%, the coefficient of variation (CV value) of the harvest yield (fresh weight) of the fruit body of single blake bottle is 4%.
Embodiment 2
Carry out the cultivation of Cordyceps militaris according to the method for embodiment 1, difference is: light stimulation cultivation stage carries out finishing for 4 days, now aerial hyphae part annesl be crocus, but also do not start to form mound shape protuberance.
In the present embodiment, former base induction period carried out after 9 days, just on visible aerial hyphae, started to form fruit body primordium.
In the present embodiment, being inoculated into composts or fertilisers of cultivating from the Cordyceps militaris spawn of liquid spawn form starts, through 40 days, the fruiting bodies of cordyceps militaris (fresh weight) of average results 26g in each blake bottle, after dry, according to the method identical with embodiment 1, the fruit body abnormal rate that statistics obtains the present embodiment production is 10%, and the coefficient of variation (CV value) of the harvest yield (fresh weight) of the fruit body of single blake bottle is 6%.
Embodiment 3
Carry out the cultivation of Cordyceps militaris according to the method for embodiment 1, difference is: the operation of mechanical stimulus comprises: use 0.5mm thick, 2cm is wide, bamboo chip after the sterilization of 20cm length, the surface of aerial hyphae is cut at interval with 0.3cm, and the degree of depth of incision is 2.5mm left and right, and aerial hyphaes all surface is smoothed, the lip-deep aerial hyphae of composts or fertilisers of cultivating is all lodged in the surface of described composts or fertilisers of cultivating, then carry out the cultivation of former base induction period and cultivate.
In the present embodiment, former base induction period carried out after 7 days, just on visible aerial hyphae, started to form fruit body primordium.
In the present embodiment, being inoculated into composts or fertilisers of cultivating from the Cordyceps militaris spawn of liquid spawn form starts, through 40 days, the fruiting bodies of cordyceps militaris (fresh weight) of average results 25g in each blake bottle, after dry, according to the method identical with embodiment 1, the fruit body abnormal rate that statistics obtains the present embodiment production is 10%, and the coefficient of variation (CV value) of the harvest yield (fresh weight) of the fruit body of single blake bottle is 8%.
Embodiment 4
Carry out the cultivation of Cordyceps militaris according to the method for embodiment 1, difference is: the operation of mechanical stimulus comprises: use 0.5mm thick, 2cm is wide, bamboo chip after the sterilization of 20cm length, the surface of aerial hyphae is cut at interval with 1.5cm, and the degree of depth of incision is 1mm left and right, and aerial hyphaes all surface is smoothed, the lip-deep aerial hyphae of composts or fertilisers of cultivating is all lodged in the surface of described composts or fertilisers of cultivating, then carry out the cultivation of former base induction period and cultivate.
In the present embodiment, former base induction period carried out after 5 days, on visible aerial hyphae, started to form fruit body primordium.
In the present embodiment, being inoculated into composts or fertilisers of cultivating from the Cordyceps militaris spawn of liquid spawn form starts, through 38 days, the fruiting bodies of cordyceps militaris (fresh weight) of average results 24g in each blake bottle, after dry, according to the method identical with embodiment 1, the fruit body abnormal rate that statistics obtains the present embodiment production is 10%, and the coefficient of variation (CV value) of the harvest yield (fresh weight) of the fruit body of single blake bottle is 10%.
Comparative example 1
In the blake bottle of the composts or fertilisers of cultivating obtaining in the Preparation Example 2 that contains 40g, the Cordyceps militaris spawn of the liquid spawn form that the Preparation Example 1 of inoculation 3ml obtains, carries out the cultivation of dark cultivation stage.
Wherein, the cultivation of dark cultivation stage is carried out in lucifuge environment, and actual conditions comprises: temperature is 23-25 DEG C, and relative air humidity can be 60%.Dark cultivation stage carried out after 9 days, and the mycelia in visible composts or fertilisers of cultivating is dispersed throughout composts or fertilisers of cultivating substantially, aerial hyphae covers composts or fertilisers of cultivating surface substantially, had reached the mycelia in composts or fertilisers of cultivating and have thorough grasp the state of composts or fertilisers of cultivating.Now, finish the cultivation of dark cultivation stage, then carry out the cultivation of light stimulus cultivation stage.
Wherein, the condition of light stimulation cultivation stage comprises: the light application time of every day is 18 hours, and intensity of illumination is 150 luxs, and the temperature between illumination period is 22 DEG C, and the temperature during unglazed photograph is 22 DEG C, and relative air humidity is 60%.Light stimulation cultivation stage carried out after 6 days, and visible aerial hyphae substantially whole annesls is crocus and has just started to form mound shape protuberance.Now, finish light stimulation cultivation stage, and directly carry out the cultivation of former base induction period.
Wherein, the condition of former base induction period comprises: the light application time of every day is 15 hours, and intensity of illumination is 100 luxs, and the temperature between illumination period is 22 DEG C, and the temperature during unglazed photograph is 16 DEG C, and relative air humidity is 70%.Former base induction period carried out after 12 days, on visible aerial hyphae, started to form fruit body primordium, now, finished the cultivation of former base induction period and cultivated, and then carried out the cultivation in sporophore growth stage and cultivated.
Wherein, the condition in sporophore growth stage comprises that the light application time of every day is 12 hours, and intensity of illumination is 150 luxs, and the temperature between illumination period is 20 DEG C, and the temperature during unglazed photograph is 20 DEG C, and relative air humidity is 80-90%.The sporophore growth stage carried out after 18 days, and the size of the fruit body of visible Cordyceps militaris is substantially no longer grown up, and spinule appears in top, and now, Cordyceps militaris nutritive value is relatively the highest, finishes immediately the cultivation in sporophore growth stage and cultivates, and gathers.
In this comparative example, being inoculated into composts or fertilisers of cultivating from the Cordyceps militaris spawn of liquid spawn form starts, through 45 days, the fruiting bodies of cordyceps militaris (fresh weight) of average results 22g in each blake bottle, after dry, according to the method identical with embodiment 1, the fruit body abnormal rate that statistics obtains the production of this comparative example is 15%, and the coefficient of variation (CV value) of the harvest yield (fresh weight) of the fruit body of single blake bottle is 25%.
Preparation Example 3
A kind of cordyceps militaris link bacterial strain being commercially available is cultivated the form for liquid spawn by this Preparation Example.
Get Cordyceps militaris spawn (purchased from Beijing agricultural and forest science institute, being the Cordyceps militaris mycelia tissue of preserving under 4 DEG C of conditions) the about 3mm of volume
33 of bacterial classification pieces be inoculated in the potato liquid nutrient medium that 150ml is identical with Preparation Example 1, turn per minute 120, shaken cultivation 7 days at 25 DEG C of temperature, obtain the Cordyceps militaris bacterial classification of this Preparation Example.
Comparative example 2
In the blake bottle of the composts or fertilisers of cultivating obtaining in the Preparation Example 2 that contains 88g, the Cordyceps militaris spawn of the liquid spawn form that the Preparation Example 1 of inoculation 3ml obtains, then carries out the cultivation of dark cultivation stage.
Wherein, the cultivation of dark cultivation stage is carried out in lucifuge environment, and actual conditions comprises: temperature is 23-25 DEG C, and relative air humidity can be 60%.Dark cultivation stage carried out after 9 days, and the mycelia in visible composts or fertilisers of cultivating is dispersed throughout composts or fertilisers of cultivating substantially, aerial hyphae covers composts or fertilisers of cultivating surface substantially, had reached the mycelia in composts or fertilisers of cultivating and have thorough grasp the state of composts or fertilisers of cultivating.Now, finish the cultivation of dark cultivation stage, then carry out the cultivation of light stimulus cultivation stage.
Wherein, the condition of light stimulation cultivation stage comprises: the light application time of every day is 18 hours, and intensity of illumination is 150 luxs, and the temperature between illumination period is 22 DEG C, and the temperature during unglazed photograph is 22 DEG C, and relative air humidity is 60%.Light stimulation cultivation stage carried out after 6 days, visible aerial hyphae substantially all annesls be crocus and just having started form mound shape protuberance (the surperficial mound shape of Cordyceps militaris mycelia and area be 0.7-1mm
2, be highly the mycelium of the protuberance of 0.7-1mm).Now, finish light stimulation cultivation stage, and carry out mechanical stimulus.
Wherein, the operation of mechanical stimulus comprises: use 0.5mm thick, 2cm is wide, bamboo chip after the sterilization of 20cm length, with the interval of 1cm, the surface of aerial hyphae is cut, the degree of depth of incision is 2.5mm left and right, and aerial hyphaes all surface is smoothed with this bamboo chip, the lip-deep aerial hyphae of composts or fertilisers of cultivating is all lodged in the surface of described composts or fertilisers of cultivating, then carry out the cultivation of former base induction period and cultivate.
Wherein, the condition of former base induction period comprises: the light application time of every day is 15 hours, and intensity of illumination is 100 luxs, and the temperature between illumination period is 22 DEG C, and the temperature during unglazed photograph is 16 DEG C, and relative air humidity is 70%.Former base induction period carried out after 4 days, on visible aerial hyphae, started to form fruit body primordium (projection of grain of rice shape), now, finished the cultivation of former base induction period and cultivated, and then carried out the cultivation in sporophore growth stage and cultivated.
Wherein, the condition in sporophore growth stage comprises that the light application time of every day is 12 hours, and intensity of illumination is 150 luxs, and the temperature between illumination period is 20 DEG C, and the temperature during unglazed photograph is 20 DEG C, and relative air humidity is 80-90%.The sporophore growth stage carried out after 18 days, and the size of the fruit body of visible Cordyceps militaris is substantially no longer grown up, and spinule appears in top, and now, Cordyceps militaris nutritive value is relatively the highest, finishes immediately the cultivation in sporophore growth stage and cultivates, and gathers.
In this comparative example, being inoculated into composts or fertilisers of cultivating from the Cordyceps militaris spawn of liquid spawn form starts, through 37 days, the fruiting bodies of cordyceps militaris (fresh weight) of average results 25g in each blake bottle, after dry, according to the method identical with embodiment 1, the fruit body abnormal rate that statistics obtains this comparative example production is 28%, and the coefficient of variation (CV value) of the harvest yield (fresh weight) of the fruit body of single blake bottle is 5%.
By the contrast of embodiment 1-4 and comparative example 1 and 2, can find that the production method of Cordyceps militaris provided by the invention, by mechanical stimulus, can reach the effect of the former base differentiation of the time chien shih Cordyceps militaris neat and consistent that shortens cordycep militaris fruiting body entity primodium; And, by having selected specific bacterial strain, improved the yield and quality of Tissue Culture of Cordyceps militaris.
And, be crocus and carry out immediately while just having there is mound shape protuberance in the situation that in preferred described mechanical stimulus at Cordyceps militaris mycelia annesl, can reach better shorten cordycep militaris fruiting body entity primodium time chien shih Cordyceps militaris former base differentiation neat and consistent effect.
And, in the case of the operation of preferred described mechanical stimulus comprise with the spaced and parallel of 0.5-1.0cm the surface of described aerial hyphae is cut, can reach better shorten cordycep militaris fruiting body entity primodium time chien shih Cordyceps militaris former base differentiation neat and consistent effect.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characterictic described in above-mentioned embodiment, in reconcilable situation, can combine by any suitable mode, for fear of unnecessary repetition, the present invention is to the explanation no longer separately of various possible combinations.
In addition, between various embodiment of the present invention, also can be combined, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1. a method of producing Cordyceps militaris, the method comprises:
(1) Cordyceps militaris spawn is cultured in composts or fertilisers of cultivating to the vegetative stage that produces aerial hyphae annesl but do not produce fruit body primordium;
(2) described aerial hyphae is carried out to mechanical stimulus, be then cultured to the vegetative stage that produces fruiting bodies of cordyceps militaris; The operation of described mechanical stimulus comprises: the surface of described aerial hyphae is cut, and the lip-deep aerial hyphae of composts or fertilisers of cultivating is lodged in the surface of described composts or fertilisers of cultivating;
Wherein, described Cordyceps militaris spawn is from a kind of cordyceps militaris link bacterial strain, this cordyceps militaris link bacterial strain is a bacterial strain for Cordyceps militaris (Cordyceps militaris), is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC NO:5606.
2. method according to claim 1, wherein, in step (1), described cultivation comprises the dark cultivation stage and the light stimulation cultivation stage that successively carry out; In the time that the mycelia of Cordyceps militaris is had thorough grasp composts or fertilisers of cultivating, described dark cultivation stage finishes; In the time that aerial hyphae annesl is crocus, described light stimulation cultivation stage finishes.
3. method according to claim 2, wherein, in step (1), the condition of described light stimulation cultivation stage comprises: the light application time of every day is 15-20 hour, intensity of illumination is 100-150 lux, temperature between illumination period is 18-25 DEG C, and the temperature during unglazed photograph is 18-25 DEG C.
4. according to the method described in any one in claim 1-3, wherein, in step (2), described mechanical stimulus is crocus and carries out immediately while just having there is mound shape protuberance at Cordyceps militaris mycelia annesl.
5. method according to claim 4, wherein, in step (2), the operation of described mechanical stimulus comprises: with the spaced and parallel of 0.5-1.0cm the surface of described aerial hyphae is cut.
6. method according to claim 1 or 5, wherein, in step (2), it is at least one in wood chip, bamboo chip, plastic sheet and sheet metal that instrument used is cut in the surface of described aerial hyphae.
7. method according to claim 1 or 5, wherein, in step (2), described cultivation is cultivated and is comprised former base induction period and the sporophore growth stage of successively carrying out; In the time there is fruit body primordium on described aerial hyphae, described former base induction period finishes; In the time of fruit body growth and maturity, the described sporophore growth stage finishes.
8. method according to claim 7, wherein, in step (2), the condition of described former base induction period comprises: the light application time of every day is 15-18 hour, intensity of illumination is 100-150 lux, cultivation temperature between illumination period is 18-24 DEG C, and the temperature during unglazed photograph is 13-18.5 DEG C, and relative air humidity is 60-70%.
9. method according to claim 7, wherein, in step (2), the condition in described sporophore growth stage comprises: the light application time of every day is 12-15 hour, intensity of illumination is 100-150 lux, cultivation temperature between illumination period is 16-22 DEG C, and the temperature during unglazed photograph is 16-22 DEG C, and relative air humidity is 80-90%.
10. method according to claim 1, wherein, described composts or fertilisers of cultivating contains base-material and basic nutrient solution; The weight ratio of described base-material and described basic nutrient solution is that 1:1.1 is to 1:1.3; Described basic culture solution contains: pupa extract, 0.6-1g/L magnesium sulfate and the potassium dihydrogen phosphate of 0.8-1.2g/L and the egg white of 50-70g/L of 15-25g/L; Described base-material contains rice and analysis for soybean powder, and the weight ratio of rice and analysis for soybean powder is that 3:1 is to 4:1.
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