CN1935979A - Coragyceps militaris bacterium culture method - Google Patents
Coragyceps militaris bacterium culture method Download PDFInfo
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Abstract
The invention relates to cordyceps militaris culturing method which can substitute for Cordyceps sinensis to be used as medicine and source. It includes the following steps: preparing mother seed; transforming to original seed and cultivar; preparing suspension fungus seed or liquid fungus seed; manmade culturing for cordyceps militaris.
Description
Technical field
The invention belongs to the Coragyceps militaris bacterium culture method field, especially a kind of Coragyceps militaris bacterium culture method.
Background technology
Cordyceps sinensis is the distinctive class valuable ingredient of traditional Chinese medicine of China, have high value medical health care and add economic worth, at home and abroad enjoy high reputation, have the laudatory title of " the legendary jewellery in east ", be the traditional foreign exchange earning commodity of China, so Cordyceps sinensis is a wonderful work in China's traditional medicine treasure-house.
The value medical health care and the economic worth that have Cordyceps sinensis only are the highest, so at home and abroad mycota, the world of medicine, food circle, organic sphere etc. are devoted to the emphasis researched and developed for many years.Cordyceps sinensis has antifatigue, hypoxia tolerance, anti-ageing, antitumor and improve multiple medical care effect such as immune function of human body.To human body can play nourishing the lung and the kidney, hemostasis and phlegm, relieving asthma, expand tracheae, calm anti-each bacterioid, effect such as hypotensive.Contain cordycepin in the Cordyceps sinensis, towards the multiple essential amino acid of oxalic acid, the VITAMIN of healthy trace elements with household, Cordyceps polysaccharide, superoxide-dismutase multiple nutrients material and anticancer antidotal active substances such as (SOD), one of major ingredient cordycepin is a kind of material that has antibiosis and suppress the cell fission effect, the hyperplasia of energy anticancer.Both at home and abroad the isotope detection technology studies have shown that cordycepin in the Cordyceps sinensis and the effect that not only has kidney invigorating and YANG supporting, beneficial smart Dingchuan towards oxalic acid to Cordyceps sinensis, and can resist cell aging significantly.
Cordyceps sinensis is grown in the extremely frigid zones in China western part and the west and south, growing environment is special, resource-constrained, along with further investigation both domestic and external for many years, further recognize medical treatment and the nourishing research of Cordyceps sinensis to human body, further recognize the important value that Cordyceps sinensis is had the medical treatment of human body and nourishing, demand constantly increases, because the natural cordyceps resource is originally just few, adds that worm grass resources quantity falls sharply, output constantly descends, disparities between supply and demand are increasingly sharpened, cause the market supply and demand to disconnect, cost an arm and a leg, at this situation, input is to the artificial introduction and acclimatization of Cordyceps sinensis, among the research work of aspects such as cultivation heredity and application,, open up medicine source biofermentation technique and have great value and development prospect really as a kind of new technology novel process that development potentiality is arranged.
Summary of the invention
The purpose of this invention is to provide a kind of Coragyceps militaris bacterium culture method that can replace Cordyceps sinensis to be used as medicine, can to replace natural cordyceps medicine source.
Technical scheme of the present invention is: Coragyceps militaris bacterium culture method comprises the following steps:
(1) the female kind prepares; (2) the female kind changeed original seed and cultivar; (3) the suspension bacterial classification or/preparation of liquid spawn; (4) artificial culture of Coragyceps militaris bacterium.
Described female kind prepares and comprises: (1) makes substratum; (2) the female kind of test tube prepares; (3) the female enlarged culturing of planting of test tube; (4) go out the grass test; (5) separate tissue is made female the kind.
Described female kind changes original seed and cultivar comprises: (1) culture medium preparation, (2) female tube of planting enlarge.
Described suspension strain preparation comprises: the preparation of (1) substratum, (2) sterilization, (3) inoculation culture.
Described liquid spawn is made and comprises (1) liquid spawn culture medium prescription, (2) inoculation culture.
Described Coragyceps militaris bacterium artificial culture comprises: (1) is pressed culture medium prescription and is made substratum, and culture medium prescription is one of following five kinds:
1. fry rice 93%, glucose 2%, peptone 2%, citric acid 0.29%, sal epsom 0.2%, dried silkworm chrysalis meal 2.5%, vitamins B
10.01%;
2. sorghum rice 85%, dried silkworm chrysalis meal 10%, sucrose 1.99%, peptone 2%, potassium primary phosphate 0.1%, sal epsom 0.1%, yeast powder 0.8%, vitaminize B in addition
10.01%;
3. millet 95%, glucose 3.49%, yeast extract paste 1.2%, potassium primary phosphate 0.1%, sal epsom 0.2%, vitaminize B in addition
10.01%;
4. sorghum rice 45%, Semen Maydis grit 40%, millet 10%, sucrose 2%, peptone 2%, yeast powder 0.8%, potassium primary phosphate 0.1%, sal epsom 0.1%;
5. rice 50%, wheat bran 25%, Semen Maydis powder 9.99%, glucose (or sucrose) 2%, wood chip 10%, dried silkworm chrysalis meal 2%, urea 0.1%, sal epsom 0.9%, vitaminize B in addition
10.01%;
(2) spice bottling; (3) sterilization; (4) inoculation; (5) culturing room cultivates; (6) final-period management; (7) gather.
Described female the kind prepares, and makes culture medium prescription and be one of two kinds:
(1) glucose 20 grams, agar 20 grams, peeled potatoes 200 gram (liquor), 1000 milligrams in water (PDA substratum); (2) rolled oats 30 gram, agar 20 grams, water are 1000 milliliters;
Test tube is female, and to plant the enlarged culturing based formulas be one of following three kinds:
(1) peptone 10 grams, glucose 10 grams contain fat silkworm chrysalis 10 grams, whole milk powder 10 grams, potassium primary phosphate 15 grams, agar 20 grams, 1000 milliliters in water;
(2) glucose 10 grams, peptone 10 grams, yeast extract paste 1 gram, potassium primary phosphate 1 gram, sal epsom 0.5 gram, agar 20 grams, 5 milliliters in growth hormone, 1000 milliliters in water;
(3) peeled potatoes 200 grams (liquor), glucose 20 grams (or sucrose 40 grams), potassium primary phosphate 1.5 grams, agar 20 grams, vitamins B
110 grams increase spirit 0.5 gram, 1000 milliliters in water.
Female substratum that changes original seed and cultivar of planting is one of following:
(1) sucrose 10 grams, peptone 10 grams, agar 15 grams, sal epsom 0.5 gram, vitamins B
15 1000 milliliters;
(2) glucose 20 grams, agar 20 grams, potato 200 grams (liquor), vitamins B
13, peptone 5 grams, potassium primary phosphate (reagent) 0.5 gram.
Suspension culture of strains base is one of following:
(1) potassium primary phosphate 3 grams, salt 3 grams, vitamins B
15, glucose sugar 15 grams, sal epsom 0.5 gram, monosodium glutamate 0.2 gram, analysis for soybean powder 15 grams, 1000 milliliters in water;
(2) Semen Maydis powder 12 grams, yeast powder 3 grams, potassium primary phosphate 1 gram, glucose 22 grams, analysis for soybean powder 10 grams, vitamins B
1The 180-200 milli, sal epsom 0.5 gram, 1000 milliliters in water;
(3) glucose sugar 35 grams, peptone 25 grams, potassium primary phosphate 1 gram, salt 2.5 grams, vitamins B
13.
The substratum of liquid spawn preparation is one of following:
(1) peptone 12 grams, yeast powder 25 grams, Semen Maydis powder 10 grams, analysis for soybean powder 10 grams, potassium primary phosphate 0.5 gram, sal epsom 0.3 gram, monosodium glutamate 0.1 gram, glucose 20 grams, salt 0.3 gram, vitamins B
11000 milliliters of 100 milligrams, water;
(2) glucose 10 gram, potassium primary phosphate 0.5 gram, sal epsom 1 gram, calcium chloride 1 gram, amino acid composite powder 5 grams, peeled potatoes 250 grams (liquor), cicada pupa 20 grams, water are 1000 milliliters;
(3) potassium primary phosphate 0.05 gram, vitamins B
11000 milliliters of 1150 milligrams, peptone 15 grams, glucose 25 grams, yeast powder 10 grams, sal epsom 0.05 gram, salt 8 grams, water.
Effect of the present invention is: by Coragyceps militaris bacterium culture method of the present invention, can increase the output of Cordyceps sinensis, fundamentally reduce the price of Cordyceps sinensis, the misery that palliates a disease for extensive patients plays a role, for increasing another wonderful work again in China's traditional medicine treasure-house.
The present invention is described further below in conjunction with embodiment.
Embodiment
Coragyceps militaris bacterium culture method comprises the following steps:
One, the female kind prepares
The female kind prepares employing spore separation method, belongs to the sexual propagation method.It is to utilize the mushroom sporophore to launch the principle of spore voluntarily under aseptic condition, and spore is scattered on the artificial medium, by cultivating, obtains original fungus strain body, and then moves the access slant tube and cultivate.Specifically comprise the following steps:
(1) makes substratum
Substratum can select following two kinds of prescriptions to make:
1, glucose 20 grams, agar 20 grams, peeled potatoes 200 gram (liquor), 1000 milligrams in water (PDA substratum)
2, rolled oats 30 gram, agar 20 grams, water are 1000 milliliters
Compound method: rolled oats adds water, and heating 1 hour is supplied amount of preparation with adding water after the filter of two-layer gauze in the boiling water-bath, adds the incubator sterilization of packing into after the agar in-depth.
(2) the female kind of test tube prepares
1, the enamel basin of about 25 centimetres of diameters, 4 layers of gauze of last lining, on gauze, put a medium size incubator (substratum is housed in the device), and in device, put a stainless steel tripod, for inserting Chinese caterpillar fungus stroma usefulness, above incubator, add a cover a bell glass, bind up with gauze together with porcelain dish, sterilization is 1 hour under 147 kpa pressures, and the cooling back is standby.
2, choose fresh, high-quality, maturation, as yet do not distribute the normal Chinese caterpillar fungus stroma of spore clean with flushing with clean water, dry.
3, the complete assembly with the above-mentioned bacterium of going out removes gauze, put into the inoculation tank strict sterilization together with Cordyceps sporophore 0.1% mercuric chloride liquid (or 75% alcohol) sterilized water etc., after the sterilization, under aseptic condition, to the Cordyceps sporophore surface sterilization, and, take bell jar then apart with 0.1% mercuric chloride liquid (or 75% alcohol) with sterile water wash remained on surface soup, Cordyceps sporophore is inserted on tripod, build bell jar again.With gauze that bell jar bottom periphery plug is good, and fall 0.1% mercuric chloride water on the gauze, at last a whole set of spore gathering device is taken out from inoculation tank, put into thermostat container (about 25 degrees centigrade), spore is fallen, treat to have on the substratum Cordyceps to drop out now, the single or multiple bacterium colonies of picking move to be connected on the test tube slant substratum and cultivate after (lucifuge is cultivated under 15 degrees centigrade of-20 degrees centigrade of constant temperatures) cultivate successfully under aseptic condition, can move and connect, expand system.
(3), the female enlarged culturing of planting of test tube
Through make the female kind of test tube with separation method after, also need further tube to transplant enlarged culturing.Female plantation is expanded numerous except increasing greatly the bacterial classification quantity, can also effectively avoid the aging or poor growth of bacterial classification, prevents or eliminate microbial contamination.
The female enlarged culturing based formulas of planting of test tube:
1, peptone 10 grams, glucose 10 grams contain fat silkworm chrysalis 10 grams, whole milk powder 10 grams, potassium primary phosphate 15 grams, agar 20 grams, 1000 milliliters in water.
2, glucose 10 grams, peptone 10 grams, yeast extract paste 1 gram, potassium primary phosphate 1 gram, sal epsom 0.5 gram, agar 20 grams, 5 milliliters in growth hormone, 1000 milliliters in water.
3, peeled potatoes 200 grams (liquor), glucose 20 grams (or sucrose 40 grams), potassium primary phosphate 1.5 grams, agar 20 grams, VB11 0 gram increases spirit 0.5 gram, 1000 milliliters in water.
Sexual Cordyceps mycelium growth is slower, under 20 degrees centigrade constant temperature, generally need cover with the inclined-plane through 3-4 month cultivate one's ability, when seeing the test tube slant substratum 1-2 centimetre of big bacterium colony arranged, bacterium projection and surface are tortuous uneven, and color is that orange illustrates promptly that test tube has been cultivated with mother and finishes.
(4), go out the grass test
The sexual isolating bacterial classification of Chinese caterpillar fungus must be made the grass test before plantation, the new isolating bacterial classification of Chinese caterpillar fungus not necessarily can both go out grass, therefore must test through the strict grass that goes out, can put into production after the syngenesis proterties of mycelia is stable, for go out in the grass test performance position go out grass early, the higher bacterial classification of output will in time expand numerous and preservation.
(annotate: mother's kind of separation and Culture, can not be directly used in production, must make it adapt to cultivation condition, and enlarge quantity through transplanting and habituation, can use.)
(5), separate tissue is made female the kind
Selecting for use good, pure, healthy and strong, of the right age natural cs to make asexual hyphostroma and separate, is to realize high yield, fine key.The asexual cultivation algebraically of Cordyceps militaris (L.) Link. is divided into 3 grades of female kind, original seed and cultivars, the female kind is the mycelium that separate tissue is cultivated, original seed and cultivar are to enlarge to cultivate to form bacterial classification on the female basis of planting of test tube, the female kind should not directly be cultivated usually, tube enlarges and the making original seed and be mainly used in, and as the bacterial classification preservation, original seed then can be used for cultivation and production, also can continue expanding propagation and become cultivar to be mainly used in cultivation and production.
Tissue isolation is vegetative propagation, spore separation sexual propagation owned by France, and the spore separation law technology is strong, complicated operation, difficulty is bigger, be not suitable for common people and adopt, and tissue isolation is simple, the success ratio height.Introduce female separation method of planting below.
1, the notion of aseptic technique, aseptic technique are the keys of success and failure, must perform correct mental preparation, can not ignore.Entire operation is carried out or is operated in sterilisable chamber at inoculation tank.Bacterial classification passes through the space that exposed, must be aseptic area, and the outer spatial channel mouth (mouth of pipe, bottleneck) of bacterial classification and container must be with the sealing of spirit lamp flame sterilization.Various tool all must be with inoculating after spirit lamp flame sterilization, the cooling again with before bacterial classification contacts, and tampon is filled in the test tube notch portion, must not contact with the thing of not sterilizing after extracting, in case infection.
2, production of hybrid seeds instrument:
(1) balance is 1, and weighing is the general utility balance of 500 grams.Mainly be to take by weighing kind of a nutrilite quantity.
(2) test tube: generally select 18 millimeters * 180 millimeters or 20 millimeters * 200 millimeters specification test tubes for use
(3) measuring cup: be mainly used in the volume of metering water, specification is with 500 milliliters or 1000 milliliters.
(4) the little funnel of plastics: be used for the substratum test tube of packing into
(5) inoculation shovel, vaccinating lancet, inoculating needle and long handle tweezers: be used to move and connect bacterial classification.
(6) single-edge blade: be used to cut the female kind of Chinese caterpillar fungus separate tissue.
(7) spirit lamp: a cutting tool carries out flame sterilization usefulness when being used to inoculate.
3, sterilization soup
(1) formalin: containing formaldehyde 37%-40%, to be used for sterilization stifling, every cubic metre of space requirement 2-5 milliliter, or with potassium permanganate (PP powder), restrain with every cubic metre of usefulness 5, add 10 milliliters of formalin solution mixed fumigations.
(2) mercuric chloride solution 0.1%, and mercuric chloride liquid is used for the Chinese caterpillar fungus separate tissue and surface sterilization mercuric chloride solution compound method has two kinds:
Be made into dense stoste 1., earlier, with preceding redilution to desired concn.Original liquid component is as follows: when mercuric chloride 20 grams, 100 milliliters of hydrochloric acid (dense), preparation thimerosal, and 5 milliliters of desirable stostes, 1000 milliliters of dilutions of adding distil water or clean water.
2. directly preparation: mercuric chloride liquid 1 gram, 25 milliliters of hydrochloric acid (dense), 1000 milliliters in water, earlier mercuric chloride liquid is dissolved in the hydrochloric acid, and then thin up.
(3) sterilized water: the clean water (better with distilled water) through high-temperature sterilization is used to clean the cordycep surface.
4, substratum preparation
(1) glucose 20 grams, potassium primary phosphate 1 gram, sal epsom 0.5 gram, agar 20 grams, 1000 milliliters in water
(2) peptone 10 grams, potassium primary phosphate 1 gram, sal epsom 0.5 gram, agar 20 grams, 5 milliliters in growth hormone, glucose 10 grams, yeast extract paste 1 gram, 1000 milliliters in water.
(3) sucrose 30 grams, potassium primary phosphate 1.2 grams, sal epsom 0.3 gram, asparagine 1 gram, SODIUMNITRATE 0.5 gram, Repone K 0.5 gram, agar 20 grams, 1000 milliliters in water.
Collocation method: preparation potato commonly used, glucose, agar CPDA, substratum, fresh potato cleaned go to take by weighing 200 behind the tooth eye and restrain, be cut into small pieces or the bar thin slice all can.Add 1000 milliliters in water and boiled 20 minutes, soft and when mashed, remove slag to potato with soaking the 4-6 layer filtered through gauze of wringing out in advance, get its juice in pot.Thereafter add glucose 20 gram, add agar 20 grams again, sal epsom 0.5 gram adds 1000 milliliters in water, continue to be heated to agar dissolve boil the back culture solution, can divide the test tube of packing into while hot rapidly.
5, autoclaving
The test tube substratum that branch is installed is put into portable pressure kettle and is sterilized, and waits burning to 49 kPa (0.5 kg/cm
2) during pressure, open vent valve and discharge cold air, make pointer return zero; when continuing to be heated to 117.68-147.1 kPa of (1.2-1.5 kg/cm) pressure then, keep after 30-50 minute ceasing fire, treat that pointer gets back to zero after; open pot cover, take out test tube, and while hot test tube is put into the inclined-plane.
6, the selection of starting strain
Starting strain has two kinds: a kind of is the natural cs sporophore, and another kind is tame sporophore, selects growth cycle short, easily grow stroma, the output height, medicinal and nutritive value is big, growth is normal, and no disease and pest does not distribute the healthy and strong stroma of spore and plants the separation starting strain as mother.
Fresh active starting strain after will choosing is earlier cleaned and is dried, together with the instrument of the test tube substratum and the bacterium of going out, and blade, the sterilized water tweezers are together put into inoculation tank, then inoculation tank are sterilized.
Method is by the inoculation volume calculation: every cubic metre of 3 milliliters of formalin solution in space, heating fumigating 30 minutes, or (elder generation poured potassium permanganate in the china bowl in stifling 40-60 minute to add potassium permanganate 5 grams by every cubic metre of space with 10 milliliters of formalin, again formalin is poured into, closed chamber door rapidly.Again with UV-lamp irradiation 20-30 minute, then by aseptic technique, with 0.1% mercuric chloride solution (or 75% alcohol) to the bacterial classification surface sterilization, method is to dip 0.1 mercuric chloride liquid with absorbent cotton to embrocate the Chinese caterpillar fungus tissue surface rapidly, generally need to wipe 3-5 time, be no more than 1 minute at every turn, use sterile water wash 1-3 subsurface residual liquor at last.After surface sterilization, cut extexine with hilted broadsword, cut its interior tissue one fritter (preferably having the top of turtleback decorative pattern to cut tissue block) and can obtain the bacterial strain of good quality and high output from sporophore, move rapidly and be connected on test tube substratum central authorities, and tampon beyond the Great Wall rapidly, the tissue block size that cuts will suit, general 1 millimeter square, excessive easy microbial contamination, too smallly then easily tissue is killed, after all inoculation finishes, test tube is shifted out inoculation tank, in 20 degrees centigrade-28 degrees centigrade thermostat container, cultivate, can obtain female the kind in general 3-4 month.
Two, the female kind changeed original seed and cultivar
Original seed and cultivar derive from female expansion of planting, and original seed is the natural cs bacterial strain, after artificial training, propose the high-quality bacterium colony and change solid medium over to, and through different temperature, humidity of each stage, nutrition obtains the one-level mother and plants.
Female kind can only be made bacterial classification and use, and must not directly cultivate, and cultivates so must make the original seed tube, and general original seed also is difficult for directly cultivation, because the original seed source is few, the cost height needs repeatedly tube to cultivate into cultivar.
(1), culture medium preparation
1, sucrose 10 grams, peptone 10 grams, agar 15 grams, sal epsom 0.5 gram, vitamins B
15 1000 milliliters.
2, glucose 20 grams, agar 20 grams, potato 200 grams (liquor), vitamins B
13, peptone 5 grams, potassium primary phosphate (reagent) 0.5 gram.
Compound method: now various nutrilites are taken by weighing equivalent, close and mix well, agar is melted the heating of 1000 ml waters again, then nutrilite is poured into, boiling time is 5-10 minute, and adjustable value is PH5-6 thereafter, the packing test tube is standby through putting into the inclined-plane behind the autoclaving while hot.
(2) female tube of planting enlarges
Inoculation tank and all apparatus.Will carry out strict sterilization in advance, the inoculation personnel must wash one's hands in case assorted cingula is gone into the lysol solution of soap or 2% before inoculation.
The tube operation
Mother's kind is planted experimentally a mouthful calcination on spirit lamp flame, extract tampon simultaneously, then with all instruments also in the spirit lamp calcination, plant and to cut tube Deng carrying out mother after the cooling, instrument is not run into tube wall during tube, changes test tube central authorities by flame rapidly over to, note substratum not being scratched, bacterial classification is adhered on the test tube wall, extract inoculating tool out, tampon is screwed.
One female plants test tube and once can expand and connect 25-35 test tubes, and inoculation finishes the test tube that connects is labelled, indicates the bacterium name date, the inoculation people, 7-10 props up to a bundle and tightens test tube top with kraft paper, puts into incubator after the end and cultivates, for fear of temperature jump, influencing mycelial growth grows, be preferably in the thermostat container and cultivate, generally under 20 degrees centigrade-28 degrees centigrade, heat insulating culture is about 10 days, secondary is female plants (can cultivate cultivar, also can directly plant).
The method of using is moved and is connect the female kind of the Cordyceps militaris (L.) Link. secondary that expands system, promptly can make pure strain and preserve, and also can directly be used for producing and cultivate, directly plantation.
In general to expand numerous number of times unsuitable too many for 1 former bacterial classification, generally must not surpass 3 times, otherwise easily cause spawn degeneration.
Three, the preparation of suspension bacterial classification
Suspension belongs to the category of liquid spawn, but being suspended in can only be the dilution of female kind and original seed to a great extent, and liquid spawn is only the rejuvenation breeding of Chinese caterpillar fungus, the suspension effect is not as liquid spawn on identical consumption, the suspension bacterial classification is made fairly simple, invest lessly, the cultivation person of lacking experience can use the suspension manufacturing technology earlier, and the bacterial classification consumption is greatly a bit to improve the infection success ratio when just cultivating.
(1), substratum preparation
1, potassium primary phosphate 3 grams, salt 3 grams, vitamins B
15, glucose sugar 15 grams, sal epsom 0.5 gram, monosodium glutamate 0.2 gram, analysis for soybean powder 15 grams, 1000 milliliters in water.
2, Semen Maydis powder 12 grams, yeast powder 3 grams, potassium primary phosphate 1 gram, glucose 22 grams, analysis for soybean powder 10 grams, vitamins B
1The 180-200 milli, sal epsom 0.5 gram, 1000 milliliters in water.
3, glucose sugar 35 grams, peptone 25 grams, potassium primary phosphate 1 gram, salt 2.5 grams, vitamins B
13.
Compound method: take by weighing by above-mentioned equivalent and to put into pot and boiled 10-15 minute, pour in the measuring cup precipitation then into after 15-20 minute, upper strata liquid is packed in the 500 mL of saline bottles, every bottled amount is the 200-250 milliliter.
(2) sterilization
Use portable high-pressure sterilizing pot, will tighten spiral behind the bottled pot of suspension, the intensification firepower should strengthen gradually, and air pressure reaches 49 kPas of (0.5 kg/cm
2) time opens vent valve and discharge cold air.When treating that tensimeter returns zero, shut vent valve again and heat up again, when temperature is raised to 147 (1.5 kg/cm
2) time when keeping cutoff in 40-60 minute cooling to treat that tensimeter returns zero, open vent valve, discharge entrained steam and take the lid of the pot off, it is stand-by to take out sterilized solution cooling back.
(3) inoculation culture
Whole process is all carried out under aseptic condition, and relevant operation measure sees also solid spawn, and every secondary test tube bacterium can connect 4-6 bottle suspension.
The suspension that connects is placed on dark place, under 20-28, cultivated 5-7 days, rock bottle when seeing bottle inner suspension liquid surface adularescent mycelia, make bottle interior mycelia and liquid even, suspension all is under natural temperature mostly, cultivate, not in the thermostat container kind, great majority white hypha occurs and rock not open, this is a normal phenomenon, the conidium of the Cordyceps of surface caking has but entered suspension just as pollen is invisible to the naked eye when rocking, and can cultivate use again after 4-5 days after this kind phenomenon occurs.
Illustrate: suspension, advantage are to produce bacterial classification time weak point, easily learn, and operation is easy, the success ratio height, and the output height, it is prosperous to send out bacterium.
Shortcoming is: after the suspension bacterial classification is made, and the one, the bacterial classification consumption is big, and the 2nd, be difficult for permanent the preservation.
Four, liquid spawn is made
At present, the solid spawn that domestic production bacterial classification great majority all adopt continue the seventies in 20th century to get off, its expense height, labour intensity is big, and the cycle is long, the output instability, we adopt the single-stage culture method to be liquid production of hybrid seeds express method again now, progressively realize multistage cultural method, realize that the stable high yield cycle short, FFI high-efficiency strain.
It is the minimum small-sized production unit of liquid spawn of investment that single-stage is cultivated, a complete set of equipment is also only about hundred yuan, simple installation, equipment is easily purchased, and this bushing device is with short production cycle, can turn out first Chinese caterpillar fungus liquid spawn in 8-10 days, turnout is bigger, can produce 1000-2000 milliliters of liquid bacterial classification as required for 1 time, but and continuous production, cultivation is convenient, can to send out bacterium fast as long as liquid spawn is sprayed onto media surface, be solid send out the bacterium time half, the output height, be 2 times of solid bacterium output, and fundamentally having solved infection time, the success ratio height has been stopped assorted bacterium fully with special strainer, saccharomycetic infection, success ratio needs 6-8 month time up to more than 95% with traditional method plantation Chinese caterpillar fungus, adopts liquid spawn, only need 50-60 days, shortened the Chinese caterpillar fungus production cycle greatly.
(1), liquid spawn culture medium prescription
1, peptone 12 grams, yeast powder 25 grams, Semen Maydis powder 10 grams, analysis for soybean powder 10 grams, potassium primary phosphate 0.5 gram, sal epsom 0.3 gram, monosodium glutamate 0.1 gram, glucose 20 grams, salt 0.3 gram, vitamins B
11000 milliliters of 100 milligrams, water.
2, glucose 10 gram, potassium primary phosphate 0.5 gram, sal epsom 1 gram, calcium chloride 1 gram, amino acid composite powder 5 grams, peeled potatoes 250 grams (liquor), cicada pupa 20 grams, water are 1000 milliliters.
3, potassium primary phosphate 0.05 gram, vitamins B
11000 milliliters of 1150 milligrams, peptone 15 grams, glucose 25 grams, yeast powder 10 grams, sal epsom 0.05 gram, salt 8 grams, water.The bottling sterilization
Select above-mentioned prescription for use, weigh up various raw materials by requirement (this sentences 1000 milliliters of culture materials is example), put into pot, add 1300 milliliters of heated and boiled 5-10 of water minutes, cooling, precipitate 1 hour, get the upper strata stillness of night and pack in the wide-necked bottle, loading amount is 7/10 of a bottle, it is defoamer that adding 3-6 drips edible oil, nutrient solution produces the bubble foam when preventing to ventilate, and bottleneck with replacing the tampon plug good, is placed in the pot, carry out autoclaving, high pressure was sterilized 40-60 minute down for 147 kPas, and sterilization finishes to naturally cool to below 30 ℃, prepares inoculation.
(2), inoculation culture
Pressing does not have the bundle operational requirement, and access secondary kind and cultivar get final product in inoculation tank, and the concrete operations main points are made identical with female kind.
Be placed under 20 ℃-25 ℃ the condition static cultivation after the inoculation 2 days, observing the liquid primary surface does not have heterochromatic, clear can the ventilation, start little air flow earlier, oxygen blast 3 days, restarted the atm number oxygen blast 3-4 days, logical like this oxygen stirs better, and in the culturing process, check at least once every day, whether observe the liquid base becomes turbid, as the explanation of becoming turbid is infected, should destroy, if clear in the liquid base have little mycelia sheet to appear as normally, treat that mycelia sheet diameter reaches 0.3-1 millimeter goods mycelium pellet and is full of the liquid base, illustrate that liquid spawn completes.
Annotate: liquid spawn, can reduce cost effectively, it is fast to send out bacterium speed, it is even that bacterium is sent out in stable high yield, shortcoming is: must in time use after liquid spawn is made, temporarily need not, (below 30 ℃) generally can be placed 2-3 days at normal temperatures, maximum duration must not surpass 7 days, and the time is oversize aging easily and rotten.
Five, Cordyceps militaris (L.) Link. artificial culture
(1), presses culture medium prescription and make substratum
1, fries rice 93%, glucose 2%, peptone 2%, citric acid 0.3%, sal epsom 0.2%, dried silkworm chrysalis meal 2.5%, vitaminize B in addition
10.01%.
2, sorghum rice 85%, dried silkworm chrysalis meal 10%, sucrose 2%, peptone 2%, potassium primary phosphate 0.1%, sal epsom 0.1%, yeast powder 0.8%, vitaminize B10.01% in addition.
3, millet 95%, glucose 3.5%, yeast extract paste 1.2%, potassium primary phosphate 0.1%, sal epsom 0.2%, vitaminize B in addition
10.01%.
4, sorghum rice 45%, Semen Maydis grit 40%, millet 10%, sucrose 2%, peptone 2%, yeast powder 0.8%, potassium primary phosphate 0.1%, sal epsom 0.1%.
5, rice 50%, wheat bran 25%, Semen Maydis powder 10%, glucose (or sucrose) 2%, wood chip (broadleaf tree is advisable) 10%, dried silkworm chrysalis meal (stiff silkworm replaces in available shop of Chinese medicines) 2%, urea 0.1%, sal epsom 0.9%, vitaminize B in addition
10.01%.
(2), spice bottling
Above-mentioned substratum is chosen any one kind of them, and it is even to take by weighing the equivalent spice, and water content must remain on 60%-65%, pH value 5-7, be respectively charged in 500 milliliters of Cans, can adorn about siccative 50 grams for every bottle, nutrilite moisture is meter in addition, available thereafter polyethylene or polypropylene film seal.
(3), sterilization
Sterilization has two kinds of methods: a kind of is autoclaving; A kind of is normal-pressure sterilization.Here only talk normal-pressure sterilization, normal-pressure sterilization helps big row and produces, capacity is big, sterilization time must remain on and get final product in 8-10 hour, and beginning low temperature carries out 3-4 hour (as quick heating, the too high meeting of temperature causes that bottle is broken cruelly) under 50 ℃, thereafter carrying out 100 ℃ of high temperature burnt greatly to 4-6 hour, the cooling that takes the dish out of the pot after having sterilized, inoculation is prepared in the cooling back, and inoculation temp must not be above 20 ℃.
(4), inoculation
Under aseptic condition, operate, it is prosperous burning at first to possess a coal briquette stove, extracts liquid spawn with sterile syringe above flame, injects the culturing bottle kind that bacterium is crossed in death of monks or nuns equably with every bottle of 5-10 milliliter, inoculation finishes, and changes culturing bottle over to the cultivation indoor cultivation.
(5), culturing room cultivates
Before culturing bottle enters the chamber, must carry out disinfection in advance earlier, after the sterilization, can be placed on the bedstead taking over the bottle of planting, each bottle stays point space slightly, be beneficial to ventilation and heat, the simultaneously indoor necessary optimal temperature that keeps, 15-25 days mycelia can be covered with substratum generally speaking, and form fruit body primordium, 45 days-60 days can be ripe, and Chinese caterpillar fungus is cultivated management can be divided into two stages.
1, temperature: the north winter, careless mycelia all can grow at 6 ℃-30 ℃, stopped growing cessation of growth cessation more than 30 ℃ below 6 ℃, very be dead, optimal temperature is 15 ℃-25 ℃, the hyphal development initial stage, room temperature is 15 ℃-20 ℃, and the later stage is 23 ℃-25 ℃, surpasses 25 ℃ and is difficult for forming stroma.
2 light: in the mycelia stage, the dark hyphal development of pipeline is very fast, but light intensity then is difficult for bearing stroma, and poor growth, enters generative growth phase too early, influences output and quality, must wait mycelia to cover with and could see light at the bottom of a bottle face is pricked bottle.
3, humidity: Chinese caterpillar fungus seals growth in bottle, the atmospheric moisture influence is little, but it is long-time dry, also can make water in bottle divide slowly evaporation, influence later stage mycelial growth and sporophore and form, for this reason, will be suitably at flooring water spray humidification, early stage, humidity remained on about 65%, crossed the wet assorted bacterium that easily gives birth to, and was difficult for forming stroma.
4, oxygen: scarlet caterpiller fungus mycelium stage respiratory capacity is less, indoor need not ventilated, need not the special conditions management, can cover with substratum 15-25 days mycelia, the growth of Cordyceps militaris will also soon enter generative growth phase by nourishing and growing, the position that this stage puts bottle can not be touched, because dynamic temperature degree variable effect growth.
5, growing way: after mycelia is covered with bottle, and the bulge projection appears, and beginning to see that the light annesl goes out careless Veraison, 6-8 o'clock every morning ventilates and sees light, after treating that mycelia all transfers yellow to by white, can suitably increase scattered light every day, the annesl initial stage, temperature with about 21 ℃ for well, after annesl is finished, temperature remains on about 23 ℃, and humidity remains on more than 85%, and illumination every day Veraison must not be less than 10 hours.See that Huang promptly ends, light can not shine continuously, and Veraison needs 2-8 days, promptly begins to form on charge level millet shape original hase (being button) behind the annesl.
(6), final-period management
1, when most of charge level forms original hase, temperature can remain on about 23 ℃, humidity is 85-90%, humidity too Da Yi is brought out substratum to produce too much aerial hyphae unfavorable to sporophore growth, the too little substratum that makes easily of humidity loses moisture content too early and influences output, for keeping suitable humidity, sprayable water is in ground, 3-4 days every day.
2, sporophore growth stage management
(1) temperature: the optimum temps that forms Cordyceps sporophore is 20 ℃-25 ℃, and therefore, this stage is the energy temperature control to the greatest extent, is beneficial to sporophore and forms and growth, and temperature control is below 25 ℃, and surpassing 28 ℃ can not the strong point stroma.
(2) light: after mycelia is covered with, can increase natural lighting, mycelia gradually becomes orange or orange by white after seeing light, grain of rice shape orange button promptly can appear in media surface behind the mycelia secretion pigment, be sporophore after the button elongation, can not continuous illumination after turning round, continuous illumination can not generate sporophore.
(3) atmospheric moisture: the sporophore growth stage will be increased to 85%-90% to indoor air humidity, distribute to reduce the water in bottle branch, Cordyceps sporophore forms and will lean on moisture content to absorb nutrition, cell is expanded, total cellular score up to ripe Chinese caterpillar fungus after original hase forms does not increase, and the increase of its whole sporophore is each cell absorption moisture content and nutrition and the result that increases, for this reason, moisture content must be in nutrilite, filled up, just the Chinese caterpillar fungus high yield can be made.
(4) oxygen: even Chinese caterpillar fungus still can normal growth in the bottle of sealing.That is to say that it is less to the demand of oxygen, for this reason, in whole growth process, reduce water in bottle part as far as possible and distribute, need not to remove sealing film, when centimetre high left and right sides of sporophore length to 2 in the bottle, on sealing film, prick several holes with bodkin and get final product.
Again through about 15 days, the Chinese caterpillar fungus stroma can be ripe by above-mentioned condition, and about up to 8 centimetres, the sporophore surface has the yellow powder thing to occur and can gather.
(7), gather
Long to the 5-8 cm long, promptly ripe when the Chinese caterpillar fungus stroma, remove sealing film, Jiang's sporophore is extracted, dry or dry all can, preserve as need, moisture content is lower than 3% to prevent moldy metamorphism, seals with bag film after doing.
Claims (10)
1, Coragyceps militaris bacterium culture method is characterized in that comprising the following steps: that (1) female kind prepares; (2) the female kind changeed original seed and cultivar; (3) the suspension bacterial classification or/preparation of liquid spawn; (4) artificial culture of Coragyceps militaris bacterium.
2, Coragyceps militaris bacterium culture method according to claim 1, it is characterized in that described female plant to prepare comprise: (1) makes substratum; (2) the female kind of test tube prepares; (3) the female enlarged culturing of planting of test tube; (4) go out the grass test; (5) separate tissue is made female the kind.
3, Coragyceps militaris bacterium culture method according to claim 1 and 2 is characterized in that described female kind is changeed original seed and cultivar comprises: (1) culture medium preparation; (2) female tube of planting enlarges.
4, Coragyceps militaris bacterium culture method according to claim 3 is characterized in that described suspension strain preparation comprises: the preparation of (1) substratum, (2) sterilization, (3) inoculation culture.
5, Coragyceps militaris bacterium culture method according to claim 4 is characterized in that described liquid spawn making comprises (1) liquid spawn culture medium prescription, (2) inoculation culture.
6, Coragyceps militaris bacterium culture method according to claim 5 is characterized in that described Coragyceps militaris bacterium artificial culture comprises: (1) is pressed culture medium prescription and is made substratum, and culture medium prescription is one of following five kinds:
1. fry rice 93%, glucose 2%, peptone 2%, citric acid 0.29%, sal epsom 0.2%, dried silkworm chrysalis meal 2.5%, vitamins B
10.01%;
2. sorghum rice 85%, dried silkworm chrysalis meal 10%, sucrose 1.99%, peptone 2%, potassium primary phosphate 0.1%, sal epsom 0.1%, yeast powder 0.8%, vitaminize B in addition
10.01%;
3. millet 95%, glucose 3.49%, yeast extract paste 1.2%, potassium primary phosphate 0.1%, sal epsom 0.2%, vitaminize B in addition
10.01%;
4. sorghum rice 45%, Semen Maydis grit 40%, millet 10%, sucrose 2%, peptone 2%, yeast powder 0.8%, potassium primary phosphate 0.1%, sal epsom 0.1%;
5. rice 50%, wheat bran 25%, Semen Maydis powder 9.99%, glucose (or sucrose) 2%, wood chip 10%, dried silkworm chrysalis meal 2%, urea 0.1%, sal epsom 0.9%, vitaminize B in addition
10.01%;
(2) spice bottling; (3) sterilization; (4) inoculation; (5) culturing room cultivates; (6) final-period management; (7) gather.
7, Coragyceps militaris bacterium culture method according to claim 6 is characterized in that described female the kind prepares, and makes culture medium prescription and be one of two kinds:
(1) glucose 20 grams, agar 20 grams, peeled potatoes 200 gram (liquor), 1000 milligrams in water (PDA substratum); (2) rolled oats 30 gram, agar 20 grams, water are 1000 milliliters;
Test tube is female, and to plant the enlarged culturing based formulas be one of following three kinds:
(1) peptone 10 grams, glucose 10 grams contain fat silkworm chrysalis 10 grams, whole milk powder 10 grams, potassium primary phosphate 15 grams, agar 20 grams, 1000 milliliters in water;
(2) glucose 10 grams, peptone 10 grams, yeast extract paste 1 gram, potassium primary phosphate 1 gram, sal epsom 0.5 gram, agar 20 grams, 5 milliliters in growth hormone, 1000 milliliters in water;
(3) peeled potatoes 200 grams (liquor), glucose 20 grams (or sucrose 40 grams), potassium primary phosphate 1.5 grams, agar 20 grams, vitamins B
110 grams increase spirit 0.5 gram, 1000 milliliters in water.
8, Coragyceps militaris bacterium culture method according to claim 7 is characterized in that female substratum that changes original seed and cultivar of planting is one of following:
(1) sucrose 10 grams, peptone 10 grams, agar 15 grams, sal epsom 0.5 gram, vitamins B
15 1000 milliliters;
(2) glucose 20 grams, agar 20 grams, potato 200 grams (liquor), vitamins B
13, peptone 5 grams, potassium primary phosphate (reagent) 0.5 gram.
9, Coragyceps militaris bacterium culture method according to claim 8 is characterized in that suspension culture of strains base is one of following:
(1) potassium primary phosphate 3 grams, salt 3 grams, vitamins B
15, glucose sugar 15 grams, sal epsom 0.5 gram, monosodium glutamate 0.2 gram, analysis for soybean powder 15 grams, 1000 milliliters in water;
(2) Semen Maydis powder 12 grams, yeast powder 3 grams, potassium primary phosphate 1 gram, glucose 22 grams, analysis for soybean powder 10 grams, vitamins B
1The 180-200 milli, sal epsom 0.5 gram, 1000 milliliters in water;
(3) glucose sugar 35 grams, peptone 25 grams, potassium primary phosphate 1 gram, salt 2.5 grams, vitamins B
13.
10, Coragyceps militaris bacterium culture method according to claim 9, the substratum that it is characterized in that liquid spawn preparation are one of following:
(1) peptone 12 grams, yeast powder 25 grams, Semen Maydis powder 10 grams, analysis for soybean powder 10 grams, potassium primary phosphate 0.5 gram, sal epsom 0.3 gram, monosodium glutamate 0.1 gram, glucose 20 grams, salt 0.3 gram, vitamins B
11000 milliliters of 100 milligrams, water;
(2) glucose 10 gram, potassium primary phosphate 0.5 gram, sal epsom 1 gram, calcium chloride 1 gram, amino acid composite powder 5 grams, peeled potatoes 250 grams (liquor), cicada pupa 20 grams, water are 1000 milliliters;
(3) potassium primary phosphate 0.05 gram, vitamins B
11000 milliliters of 1150 milligrams, peptone 15 grams, glucose 25 grams, yeast powder 10 grams, sal epsom 0.05 gram, salt 8 grams, water.
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