CN1323157C - Chinese caterpillar fungus bionic culturing method - Google Patents
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Abstract
The present invention relates to a bionic method for culturing Chinese caterpillar fungus mycelium. The culturing process comprises the separation and the pure culture of monospore, the configuration of a liquid culture medium, the inoculation of pure culture strains of mycelium, shallow liquid static culture and the harvest of mycelium after six months. The mycelium is broken into unicellular hyphal fragments or multicellular hyphal fragments by using a mechanical method in each month in the culturing process. The pure culture obtained by the single spore separating method of the present invention is proven to be unique sterile type-Hirsutella sinensis. The culturing method of the present invention does not need the investment of expensive equipment, such as fermentation tanks, rocking beds, etc., and the cost is greatly reduced. Compared with submerged liquid fermentation culture, the production cost of 1g of dry mycelium is reduced by more than 50%.
Description
Technical field
The present invention relates to a kind of bionic culturing method of Cordyceps fungus, belong to the microbial culture method of biological field.
Background technology
Cordyceps sinensis Cordyceps sinensis (Berk.) Sacc., belong to Ascomycetes, Hypocreales, Clavicipitaceae, Cordyceps, the another name Chinese caterpillar fungus is a kind of entomogenous fungi that parasitizes phosphorus wing order bat moth Hepialus spp. larva, also is a kind of famous and precious medicinal fungi.Ancient Times in China is among the people just to have certain understanding to the pharmaceutical use of Cordyceps sinensis.The Chinese caterpillar fungus sweet property of distinguishing the flavor of is flat, the lung kidney be can protect, marrow, preventing phlegm from forming and stopping coughing mended, treatment pulmonary tuberculosis, old man's chilly, cough, weak, puerpera and old man's anaemia weakness, seminal emission, impotence, spontaneous sweating, soreness of waist and knee joint, neurasthenia, weak for a long time, spitting of blood haematemesis after being ill, chronic nephritis etc. there is good effect, normal edible regulating immunological function strengthens body immunity.Just because of Cordyceps sinensis the so many medicinal functions and the medicinal history of origin are arranged, this wild resource is on the verge of exhaustion in recent years, makes scientific research person and exploitation commercial city pay special attention to the research of the replacement resources of Cordyceps sinensis.
Partly head it off of three kinds of approach is arranged: the one, the artificial culture sporophore, China has spent very big manpower and materials, though grasped tame basic fundamental, still can't commercially produce so far; The 2nd, utilize the gene of these function things of molecular biotechnology clones coding, at these function things of other biological expression in vivo, but at present the basic substance of most of function of Cordyceps sinensis not to be studied as yet clearly, gene clone is not just known where to begin yet; The 3rd, utilize the anamorph bacterial strain to carry out mycelium fermentation and cultivate, abstraction function component from mycelium again, perhaps this be comparatively reality and a simple and direct development approach (Mo Ming and etc., fungus system 20 (4): 482-485,2001) at present.
Very for active, kind surplus worldwide Bao Dao Cordyceps sinensis anamorph has 10 belongs to 10 genus in the research of Cordyceps sinensis anamorph.Can imagine that Cordyceps sinensis can not have so many anamorph, and belong to different genus.(CHINA JOURNAL OF CHINESE MATERIA MEDICA, 1995,20 (12): think that 707-710) imperfect stage of Cordyceps sinensis is a novel species, name and be China pilose spore H.sinensis such as Yin Dinghua.Liu makes easy (Guizhou agricultural sciences such as grade, 2003,31 (1): 3-5) proved that also China pilose spore is the real anamorph of Cordyceps sinensis, other isolating and Cordyceps sinensis from the Cordyceps sinensis have the bacterial strain of same or similar chemical ingredients, pharmacological action and clinical efficacy, can be used as the surrogate of Cordyceps sinensis.Zhao Jin etc. adopt molecular biology method, from gene level the imperfect stage of Cordyceps sinensis is carried out Molecular Identification, further its imperfect stage of proof is a China pilose spore, and think why can repeated isolation go out some assorted bacterium, may be because on the alpine meadow of C.sinensis height above sea level 3000-5000m occurring in nature mainly is distributed in the Himalaya mountain range near or comparatively cold northern area, the segregator cultivates the place that isolate is placed on higher temperatures by general custom, cause the death of C.sinensis mycelia, and turn out those often and the fungal species that grows together of C.sinensis, and to be mistaken as be the anamorph of C.sinensis.
Therefore, obtaining the real anamorph of Cordyceps fungus---it is the first step of it being carried out artificial culture that the sterile pure of China pilose spore is cultivated.Three kinds of separating pathways are arranged usually, promptly separate, wherein separate the most reliable with monospore by fresh Chinese caterpillar fungus bombys batryticatus tissue, sporophore and thecaspore.Because the influence of Cordyceps sinensis growing environment, the integral body sterilization of collect specimen is difficult to obtain its sterile culture thing, and thecasporous separation to just launching out in the sporophore, its success ratio is far above with the separation after the sample surface sterilization.(Mo Ming and etc., fungus system 20 (4): 482-485).Proving institute's isolate really in the separation method of China pilose spore, (Mo Ming and etc., fungus system 20 (4): 482-485) (Liu's tin Jin etc., fungi journal, 8 (1): 35-40,1989), only the method for Liu's tin Jin etc. is to carry out isolating with monospore.Its method is to get to contain thecaspore stroma to be put and sterilize, under aseptic condition, the two ends of whole stroma are made and be suspended from culture dish and cover, then this lid is placed on the culture dish that fills substratum, or stroma is suspended in the peptone PDA slant tube with its shank of fine rule bolt.After thecaspore launched, through microscopy, the thecaspore that picking is sprouted obtained pure culture.But its separable programming exists loaded down with trivial details shortcoming.
The China pilose spore mycelium is carried out artificial fermentation's cultivation and adopts its fermented product to replace wild cordyceps, and domestic have the scientific worker of more than 30 unit studying, and obtained comparatively ideal results.Method therefor is deep fermentation and cultivates, generally the mode that stirs oxygen supply and shaking table oxygen supply with fermentor tank cultivate improve output (Guo Hongchun etc., JOURNAL OF MICROBIOLOGY, 2003,1,23 (1), 50-54).Open apparent shame, He Daozhen explores zymotechnique and is: original seed--three grades of cultivation-level Four cultivation-bacterium liquid separation-dryings of shaking table cultivations-one-level cultivation-secondary cultivation.(open apparent shame etc., pharmaceutical analysis magazine, 1999,7 (5): 5-6).Because China is by hair spore growing environment height above sea level, temperature is low, have and have a liking for low temperature, the extremely slow characteristics of growth, and process of growth needs oxygen.Long with equipment culture cycle such as fermentor tank, shaking tables, it is very big to consume energy, and the cost that equipment itself such as fermentor tank, shaking table drop into is also very high, and therefore, with the valuable product that this method is produced, market is difficult to accept.Be difficult to industrialized scale operation.
Summary of the invention
The objective of the invention is to cultivate Cordyceps mycelium, make the product price of production cheap, can be accepted by market with the method for low input, high production.
Cultural method of the present invention is that hyphal fragment cell or tissue with Cordyceps fungus carries out liquid and leaves standstill cultivation.
The hyphal fragment cell or tissue of the present invention by Cordyceps fungus carries out liquid and leaves standstill cultivation, improved the rate of growth of hyphal fragment biomass greatly, reduced production cost significantly, makes it be more conducive to big rule and touches production.
Culturing process of the present invention may further comprise the steps: the substratum configuration, and the inoculation of hyphal fragment (inoculum size is the 5%-30% of culture volume per-cent), the results of hyphal fragment, temperature is 10-20 ℃ in the culturing process, and pressure is normal pressure, and light intensity is an indoor natural light.
The used hyphal fragment cell or tissue of the present invention is to sprout formed by aseptic single thecaspore.
Monospore separating step of the present invention is:
(1) collection of cordyceps species
Excavate sporophore sturdy full, can pregnant grow the complete bacterium worm of tangible Cordyceps sinensis complex body, put into 13-16 ℃ of incubator maintenance, keep suitable humidity, pinch not lump behind the earth with have gentle hands and be advisable.
(2) thecasporous collection and sprouting
Remove the sporophore surface impurity, wash with sterile distilled water, alcohol with 75% carries out surface sterilization, put in the 13-16 ℃ of incubator and cultivate, when treating that ascus begins to launch thecaspore, the sporophore that begins to launch spore is stretched in the physiological saline of sterilization, fully the wash-out thecaspore, the thecaspore of wash-out is placed in the 13-16 ℃ of incubator 3-5 days, so that sprout.
(3) preparation of inoculating needle
With the most small size sewing-needle needle point blunt, the wear down of being tried one's best in the head both sides again forms the scuppit shape, is inserted in the common loop-carrier fixing then.
(4) monospore separates and pure culture
Under aseptic technique, to contain thecasporous liquid shakes up, on a small quantity in the isolation medium flat board of sterilization, leave standstill 5-10min, after treating that water is inhaled into agar, the thecaspore of the single germination of inoculating needle picking that under low-power microscope, prepares with (three), insert in the isolation medium flat board of sterilization, 6-8 of each dull and stereotyped evenly access, inoculation position is performed mark, putting into 13-16 ℃ of incubator cultivated 2-4 days, take with the sterile surgical cutter and untaintedly to have thecasporous substratum and put into new isolation medium flat board, every dull and stereotyped strain, sealing with adhesive tape prevents moisture loss, put into 13-16 ℃ of incubator and cultivate, form macroscopic bacterium colony about 45-55 days.
Used isolation medium was when monospore of the present invention separated: 8-12g/L peptone, 14-23g/L glucose, 13-16g/L agar, 180-230g peeled potatoes are fried in shallow oil water juice/L, pH6.
With reference to the authentication method of reports such as Chen Yueqin, (Biochemical Systematics and Ecology, 2001,29,597-607), prove that the Cordyceps strain that separation method separation thus obtains is a China pilose spore.
The liquid of Hirsutella sinensis mycelium of the present invention leaves standstill cultural method and is meant that liquid depth is that the shallow-layer liquid of 1-5cm leaves standstill cultivation.
The substratum that the shallow-layer liquid that the present invention adopts leaves standstill cultivation is the substratum that is fit to the China pilose spore growth.
The substratum of the suitable China pilose spore growth that the present invention adopts is soyflour 4-7g/L, peptone 2-5g/L, glucose 90-120g/L, yeast extract paste 0.2-0.6g/L, K
2HPO
40.7-1.1g/L, MgSO
40.3-0.8g/L, pH6, culture temperature is 10-20 ℃, and pressure is normal pressure, and light intensity is an indoor natural light.
Every cultivation for some time of hyphal fragment cell or tissue of the present invention smashs to pieces once with the method for machinery under aseptic condition.
For some time of the present invention is meant one month.
The degree that hyphal fragment cell or tissue machinery of the present invention is smashed to pieces is unicellular or the many cells hyphal fragment.
Cultural method of the present invention may further comprise the steps: monospore separates and pure culture, the substratum configuration, the inoculation of mycelium pure culture bacterial classification, every cultivation was broken into mycelium unicellular or the many cells hyphal fragment with mechanical method under aseptic condition in one month, cultivate results after six months, temperature is 15-18 ℃ in the culturing process, and pressure is normal pressure, and light intensity is an indoor natural light.
The invention has the advantages that:
1. Cordyceps fungus has the amphitypy phenomenon, is the yeast type when colonizing in the larva body, and hyphal body is to breed in the mode of sprouting or rupture.(Li Quansen etc., the heterogamous preliminary study of Cordyceps fungus, CHINA JOURNAL OF CHINESE MATERIA MEDICA 1998,23 (4), 210-212).Each cell of its filamentous fungus all has the biology characteristics that are divided into same bacterial strain.The host is the mycelia type when after death saprophytic, with normal mycelia form growth.Because the growth of filamentous fungus can only be undertaken by the mode that the top prolongs, can not resemble it is to form by merismatic division the intravital hyphal fragment of larva, and the speed of growth is relatively slow.If the intravital propagation mode of the parasitic larva of imitation hyphal body in mycelial vitro culture, manually impel China pilose spore mycelia type to change to the yeast type, to increase bacterial strain number and vegetative point, will increase substantially the speed of growth of the extremely slow China pilose spore of growth.The present invention is left standstill in the cultivation at hyphal fragment liquid, adopts bionic method, and every cultivation for some time is broken into small segment by the artificial method with mycelia, even it is unicellular, with abundant increase bacterial strain number and vegetative point, improved the speed of growth of hyphal fragment, mycelial yield is improved greatly.This technology is to common fungi--and the cultivation of the fungi of fast growth is worth little, but concerning the extremely slow fungi of this class growth of Cordyceps sinensis, effect is very remarkable.From turnout, can get 2 gram left and right trunk mycelium in one month with the cultivation of shaking table 100ml liquid nutrient medium merely, by the inventive method, the 100ml liquid nutrient medium can get the dried mycelium of 5.7 grams.
2. the present invention separates it by the monospore of Cordyceps fungus, and resulting pure culture is the most reliable.The step of separation method is simple, has broken away from the pollution rate height, the predicament that screening process is loaded down with trivial details, the cycle is long.Separate the Cordyceps strain that obtains by separation method of the present invention and proved its unique anamorph--China pilose spore.
3. the liquid depth that liquid lamella of the present invention is cultivated is no more than 5cm, therefore can natural oxygen supply, and it is sufficient to obtain oxygen, does not need power to stir, and does not need the input of expensive device such as fermentor tank, shaking table.The consumption of substratum is less relatively simultaneously.In six middle of the month of cultivating, only inoculation has once been saved the manpower of inoculation, amplification culture repeatedly of fermentor cultivation, has reduced the chance of polluting.Greatly reduce cost.Only need surplus nontoxic, resistant to elevated temperatures thin layer plastic containers customized or Glass Containers and autoclaving equipment commonly used, the investment 10 ten thousand yuan can produce, economic benefit is very remarkable.
Embodiment
Further illustrate the present invention in the following embodiments, this does not limit the scope of the invention.
Embodiment one
Aseptic Cordyceps mycelium is inoculated in soyflour 6g/L, peptone 4g/L, glucose 100g/L, yeast extract paste 0.4g/L, K by 15% volume percent
2HPO
40.8g/L, MgSO
40.4g/L, in the aseptic liquid nutrient medium of pH6, being statically placed on the culturing rack, culture temperature is 18 ℃, and pressure is normal pressure, and light intensity is an indoor natural light.
Gather in the crops mycelium after six months.
Embodiment two
The cultivation program is all undertaken by embodiment one, and institute's difference is that be placed on magnetic stirring apparatus on stirring 0.5 hour (Celsius 18 ℃) with culture vessel every month between incubation period, and mycelium is smashed.
Embodiment three
1. the separation of Cordyceps fungus China pilose spore
(1) gathers cordyceps species
In July, 2002 in the kangding by local medicinal herb grower help to excavate from the Ya Jia ridge many strains sporophore sturdy full, can pregnant grow the complete bacterium worm of tangible Cordyceps sinensis complex body, put into 15 ℃ of incubator maintenances, pay special attention to the humidity that keeps suitable, do not lump after earth is pinched with have gentle hands and be advisable.
(2) thecasporous collection and sprouting
Remove the sporophore surface impurity, with the sterile distilled water flushing, the alcohol with 75% carries out surface sterilization, puts in 15 ℃ of incubators and cultivates, and every day, record was observed.When treating that ascus begins to launch thecaspore, the sporophore that begins to launch spore is stretched in the penicillin bottle that fills sterile saline, abundant wash-out thecaspore is placed in 15 ℃ of incubators 4 days with the thecaspore of wash-out, so that sprout.
(3) preparation of inoculating needle
With the most small size sewing-needle needle point blunt, the wear down of being tried one's best in the head both sides again forms the scuppit shape with whetstone, is inserted in the common inoculating needle fixing then.
(4) monospore separates and pure culture
Low-power microscope was put into the Bechtop uv irradiating 1 hour, aseptic technique shakes up the penicillin bottle that (two) handle, on a small quantity in isolation medium (the 11g/L peptone of sterilization, 22g/L glucose, 14g/L agar, the 190g peeled potatoes is fried in shallow oil water juice/L, pH6) in the flat board, leave standstill 8min, after treating that water is inhaled into agar, the thecaspore of the single germination of inoculating needle picking that under low-power microscope, prepares with (three), insert in the isolation medium flat board of sterilization, 6 of each dull and stereotyped evenly accesses, inoculation position is performed mark, putting into 15 ℃ of incubators cultivated 3 days, take with the sterile surgical cutter and untaintedly to have thecasporous substratum and put into new isolation medium flat board, every dull and stereotyped strain, sealing with adhesive tape prevents moisture loss, put into 15 ℃ of incubators and cultivate, mirror is observed upgrowth situation down.Formed macroscopic bacterium colony in 50 days, the colony diameter that forms about 90 days can reach 1.5-2.5cm, and bacterium colony is made up of the mycelium of densification, and is hard; The bacterium colony air spots resembles the wormcast shape; Periphery of bacterial colonies has this bacterium excretory brown material of circle, and the back side also is brown.Mycelia is translucent, is difficult for picking.Later stage, mycelia goed deep in the substratum, and with the substratum absorption, the projection of formation hollow was cultivated the aerial hyphae prosperity 5 months.
2. the preparation of the substratum of mycelia large scale culturing
Soyflour 6g/L, peptone 4g/L, glucose 105g/L, yeast extract paste 0.5g/L, K
2HPO
40.9g/L, MgSO
40.7g/L, pH6,115 ℃ of high pressure 30min.
3. the shallow-layer liquid of China pilose spore leaves standstill cultivation
The bacterial classification of above-mentioned purifying is rejected the agar part, under aseptic condition, grind to form unicellular or many cells hyphal fragment (the naked eyes section of being visible as is bar-shaped), add an amount of aqua sterilisa, volume percent by 5% is inoculated in the nutrient solution that is cooled in the culture vessel below 18 ℃, the nutrient solution degree of depth is 4cm, leave standstill cultivation under 18 ℃, light intensity is an indoor natural light, between incubation period, the refiner homogenate two minutes that the mycelium of cultivating in the vessel places sterilization is taken out in the every month aseptic technique, again mixture is relay in the former cultivation vessel of meeting 6 months results.
Embodiment four
The cultivation program is all undertaken by embodiment 1, institute's difference be will be to be inoculated under aseptic technique Cordyceps mycelium and the magnetic bar of sterilization put into culture vessel, after sealing mouth, be placed on the magnetic stirring apparatus, 18 ℃ were stirred 10 hours, stir into mycelia unicellular or many cells hyphal fragment (the naked eyes section of being visible as is bar-shaped), be inoculated in the nutrient solution that is cooled in the culture vessel below 18 ℃, the nutrient solution degree of depth is 4cm, leave standstill cultivation under 18 ℃, light intensity is an indoor natural light, and be placed on culture vessel and stir 0.5-1 hour (18 degree Celsius), six months results mycelium on the magnetic stirring apparatus every month between incubation period.
Embodiment five
The cultivation program is all undertaken by embodiment 1, and institute's difference is
The monospore isolation medium is: 9g/L peptone, 18g/L glucose, 15g/L agar, 230g peeled potatoes are fried in shallow oil water juice/L, pH6.
The liquid nutrient medium that the big rule of mycelium are touched cultivation is: soyflour 4g/L, peptone 3g/L, glucose 90g/L, yeast extract paste 0.2g/L, K
2HPO
40.7g/L, MgSO
40.4g/L, pH6, culture temperature is 17 ℃,
Embodiment six
The cultivation program is all undertaken by embodiment 1, and institute's difference is
The monospore isolation medium is: 10g/L peptone, 14g/L glucose, 16g/L agar, 180g peeled potatoes are fried in shallow oil water juice/L, pH6.
The liquid nutrient medium that the big rule of mycelium are touched cultivation is: soyflour 5g/L, peptone 4g/L, glucose 120g/L, yeast extract paste 0.3g/L, K
2HPO
40.9g/L, MgSO
40.3g/L, pH6, culture temperature is 16 ℃,
Embodiment seven
The cultivation program is all undertaken by embodiment 1, and institute's difference is
The monospore isolation medium is: 11g/L peptone, 16g/L glucose, 15g/L agar, 220g peeled potatoes are fried in shallow oil water juice/L, pH6.
The liquid nutrient medium that the big rule of mycelium are touched cultivation is: soyflour 7g/L, peptone 2g/L, glucose 100g/L, yeast extract paste 0.6g/L, K
2HPO
41.1g/L, MgSO
40.7g/L, pH6, culture temperature is 18 ℃.
Claims (6)
1. the bionic culturing method of a Cordyceps fungus, it is characterized in that, carry out liquid with the hyphal fragment cell or tissue of described bacterium and leave standstill cultivation, culturing process may further comprise the steps: substratum configuration, the inoculation of hyphal fragment, the results of hyphal fragment, temperature is 10-20 ℃ in the culturing process, and pressure is normal pressure, and light intensity is an indoor natural light, described hyphal fragment cell or tissue is formed by aseptic single thecaspore sprouting, and wherein the monospore separating step is:
(1) gathers cordyceps species
Excavate sporophore sturdy full, can pregnant grow the complete bacterium worm of tangible Cordyceps sinensis complex body,
Put into 13-16 ℃ of incubator maintenance, keep suitable humidity,
(2) thecasporous collection and sprouting
Remove the sporophore surface impurity, wash with sterile distilled water, alcohol with 75% carries out surface sterilization, put in the 13-16 ℃ of incubator and cultivate, when treating that ascus begins to launch thecaspore, the sporophore that begins to launch spore is stretched in the physiological saline of sterilization, abundant wash-out thecaspore, the thecaspore of wash-out is placed in the 13-16 ℃ of incubator 3-5 days, so that sprout
(3) preparation of inoculating needle
With the most small size sewing-needle needle point blunt, the wear down of being tried one's best in the head both sides again forms the scuppit shape with whetstone, and be inserted in then in the common loop-carrier and fix,
(4) monospore separates and pure culture
Under aseptic technique, to contain thecasporous liquid shakes up, on a small quantity in the isolation medium flat board of sterilization, leave standstill 5-10min, after treating that water is inhaled into agar, the thecaspore of the single germination of inoculating needle picking that under low-power microscope, prepares with (three), insert in the isolation medium flat board of sterilization, 6-8 of each dull and stereotyped evenly access, inoculation position is performed mark, put into 13-16 ℃ of incubator and cultivated 2-4 days, take with the sterile surgical cutter and untaintedly have thecasporous substratum and put into new isolation medium flat board, every dull and stereotyped strain, seal with adhesive tape and prevent moisture loss, put into 13-16 ℃ of incubator and cultivate, formed macroscopic bacterium colony in 45-55 days
Wherein said liquid leaves standstill to cultivate and is meant that liquid depth is that the shallow-layer liquid of 1-5cm leaves standstill cultivation,
Every cultivation for some time of described hyphal fragment cell or tissue smashs to pieces once with the method for machinery under aseptic condition, described for some time is meant one month, the degree that described hyphal fragment cell or tissue machinery is smashed to pieces is unicellular or the many cells hyphal fragment, cultivates results after six months.
2. according to the described cultural method of claim 1, it is characterized in that isolation medium is: it is 6 that 8-12g/L peptone, 14-23g/L glucose, 13-16g/L agar, 180-230g peeled potatoes are fried in shallow oil water juice/L, pH.
3. according to the described cultural method of claim 1, it is characterized in that described Cordyceps fungus is a China pilose spore.
4. according to the described cultural method of claim 1, it is characterized in that the substratum that described shallow-layer liquid leaves standstill cultivation is the substratum that is fit to the China pilose spore growth.
5. according to the described cultural method of claim 4, it is characterized in that the substratum of described suitable China pilose spore growth is soyflour 4-7g/L, peptone 2-5g/L, glucose 90-120g/L, yeast extract paste 0.2-0.6g/L, K
2HPO
40.7-1.1g/L, MgSO
40.3-0.8g/L pH is 6, culture temperature is 10-20 ℃, and pressure is normal pressure, and light intensity is an indoor natural light.
6. according to the described cultural method of claim 1, it is characterized in that, described cultural method may further comprise the steps: monospore separates and pure culture, the substratum configuration, the inoculation of mycelium pure culture bacterial classification, every cultivation was broken into mycelium unicellular or the many cells hyphal fragment with mechanical method under aseptic condition in one month, cultivate results after six months, temperature is 15-18 ℃ in the culturing process, and pressure is normal pressure, and light intensity is an indoor natural light.
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