CN103493685A - Method for cultivating phellinus igniarius by using wild living tree stumps - Google Patents

Abstract

Disclosed is a method for cultivating phellinus igniarius by using wild living tree stumps. The method includes the steps that first, mother seeds, stock seeds and cultivation seeds are manufactured through a traditional method; second, mulberry seedlings are cultivated, 100-1000mu of mulberry trees are planted, and 1/5 of mulberry bark is cut away with a knife when the diameter of the mulberry trees reaches 10cm according to the standard of enabling the mulberry trees to grow as well as preventing the mulberry trees from dying; third, when surface drying occurs to the xylem of the positions where the bark is cut away, holes with the depth of 5.5-6cm are punched through a hollow punch or electric drill, the space between every two holes is made to be 16-18cm, and then the holes are inoculated with phellinus igniarius strains and sealed by yellow mud; phellinus igniarius fruit can be picked within as long as 10 years through one time of inoculation till the mulberry trees die of aging and rot. By means of the method for cultivating the phellinus igniarius by using the wild living tree stumps, forest land resources can be fully utilized for cultivating phellinus igniarius products, the method is simple and easy to realize, the produced phellinus igniarius products are large in size and can be artificially cultivated in quantity, and therefore market demands can be fully met.

Description

A kind of method of utilizing wild stub cultivation Phellinus alive

Technical field

The present invention relates to a kind of method of cultivating Phellinus, relate in particular to a kind of method of utilizing wild stub cultivation Phellinus alive, belong to Chinese medicine and cultivate technical field.

Background technology

Phellinus, the monkey eye, the mulberry ear, phellinus, the fruit body stockless, the flat hemispherical of cap or the shape of a hoof, 2-12*3-21 centimetre, thick 1.5-10 centimetre, wooden, shallow liver brown to lead or black, often be full of cracks always, without cot, there is trickle fine hair at initial stage, rear change is without hair, the concentric ring rib is arranged. edge is blunt, dark cinnamon is to light coffee color, downside is without sporiferous layer, the bacterial context dark brown, firmly, wooden. tube and bacterial context are closely homochromy, multilayer, but level is not obvious, old tube layer is full of white hypha. and mouth of pipe rust brown is to dark reddish brown, circular, every millimeter 4-5. spore is subsphaeroidal, smooth, colourless, the 5-6*3-4 micron. the bristle top is sharp-pointed, base portion expands, the 10-25*5-7 micron. mycelia is branch not, without tabula, diameter 3-5 micron.Be born on the trunks such as poplar, willow, birch, oak the ground such as distribution northeast, North China, northwest and Sichuan, Yunnan.

Modern study confirms that Phellinus can improve the immunity of human body, alleviates the side effect of anticancerogenics, so can be used to the radiation and chemotherapy of auxiliary tumour patient, in addition, Phellinus is also effective in cure to gynecological diseases such as female irregular menstruations.

Phellinus is the best a kind of mushroom of anticancer effect in known higher fungi, and its anticancer mechanism also is familiar with by people gradually, therefore its market prospects are very optimistic.Fruit body in Korea S's Phellinus is sold as rare medicinal herbs always, and its price is up to 2300 dollars/kilogram; Medicinal also beginning of Japanese Phellinus, come into the market.Although China's application Phellinus is cured the disease the history of more than 2000 year is arranged, it is slower in marketing development, may topmost reason be exactly inadequate to its pharmacological research degree of depth, and the cultivation method shortcoming.

Summary of the invention

The objective of the invention is for a kind of method of efficient, energy-saving and environmental protection, the ecological wild stub cultivation Phellinus alive of utilization is provided.

A kind of method of utilizing wild stub cultivation Phellinus alive, step is as follows:

The first step, make female kind, original seed, cultivated species by traditional method;

Second step, cultivate mulberry saplings, cultivates the mulberry tree woods of 100 mu-1000 mu, grows to diameter until mulberry tree and reach 10cm when thick, by the paper mulberry bark of hilt 1/5, prunes, and not only can grow but also be unlikely to dead tree;

The 3rd step, when the bark place xylem of pruning has dry tack free, dark with belt puncher or electric drill punching 5.5-6cm, interval 16-18cm between Kong Yukong, then, in Phellinus bacterial classification access hole, seal with yellow mud; Once inoculation, can pluck the Phellinus many decades, until old dead the rotting of mulberry tree.

Adopt the beneficial effect of technique scheme to be:

The present invention can take full advantage of forest land resource cultivation Phellinus product, and simple, the Phellinus of output is individual large, in a large number artificial cultivation, the fully demand of satisfying the market.

Embodiment

A kind of method of utilizing wild stub cultivation Phellinus alive, step is as follows:

The first step, make female kind, original seed, cultivated species by traditional method;

Second step, cultivate mulberry saplings, cultivates the mulberry tree woods of 100 mu-1000 mu, grows to diameter until mulberry tree and reach 10cm when thick, by the paper mulberry bark of hilt 1/5, prunes, and not only can grow but also be unlikely to dead tree;

The 3rd step, when the bark place xylem of pruning has dry tack free, dark with belt puncher or electric drill punching 5.5-6cm, interval 16-18cm between Kong Yukong, then, in Phellinus bacterial classification access hole, seal with yellow mud; Once inoculation, can pluck the Phellinus many decades, until old dead the rotting of mulberry tree.

The described first step is made female the kind by traditional method, and method is as follows:

1.1.1 Phellinus

The Phellinus fruit body is picked up from nature reserve, Ningshan County, Shaanxi Province height above sea level approximately on the birch withered tree of 1800 about m.Select not yet lignification, without the fruit body of any breakage, paper using is wrapped and is taken back laboratory.

1.1.2 isolation medium

Add rich PDA medium: potato 200g, glucose 20g, peptone 5g, KH2PO31 g, MgSO4 0.5g, VB11 0mg, agar 20g, distill 1000 mL, the pH nature.

1.2.3 carbon source is measured basal medium

Peptone 5g, KH2PO41g, MgSO40.5g, VB11 0mg, agar 20g, distillation 1000mL, pH nature.

1.2.4 nitrogenous source is measured basal medium

Glucose 20g, KH2PO41g, MgSO40.5g, VB11 0 mg, agar 20 g, distilled water 1000 mL, pH nature.

1.1.5 culture medium for cultivating

Mulberry tree wood sawdust medium: mulberry tree wood sawdust 100%, water content 60% ~ 62%.Fresh pure mulberry tree wood sawdust is added to water in proportion, mix between, webs agglomerating to gently pinching with have gentle hands, water is arranged and do not flow, touch loose, the 500mL wide-mouth bottle of then packing into, every bottled siccative 150g, bottleneck ties with the additional one deck plastic moistureproof of two-layer thin paper, 0.1 MPa sterilizing 2h.

Bright mulberry tree branch juggle medium: the fresh mulberry tree branch of cut-off footpath 3 ~ 5cm, be sawn into the long juggle of 4cm, split two for every, the wide-mouth bottle (bottleneck diameter 7.5 cm, high 12 cm) of packing into, every bottled 12, bottleneck ties with the additional one deck plastic moistureproof of two-layer thin paper equally, 0.1 MPa sterilizing 2h.

1.2 method

1.2.1 the Phellinus fruit body is processed

Get fresh Phellinus fruit body, brush away the impurity of mushroom surface with clean brush, the alcohol with 75% is cleaned the solid outer surface and is carried out disinfection for 5 times, then is placed in aseptic beaker standby.

1.2.2 Phellinus tissue isolation

The processed fruit body disinfected is torn on superclean bench; cut in bacterium sheet central authorities 1, the tissue that the grain of rice is large with sterilized scalpel; put into aseptic dull and stereotyped culture dish surface; be inverted in 25 ℃ of insulating boxs and cultivate; after growing mycelia; get the mycelia piece that growth is fast, mycelia is long; moving into another aseptic dull and stereotyped culture dish surface cultivates; after growing mycelia; get growth fast, without the mycelia piece of living contaminants; immigration slant medium surface, 25 ℃ of cultivation 12d, mycelia are covered with inclined-plane and, without miscellaneous bacteria, are Phellinus starter kind.

The described first step is made original seed by traditional method, and method is as follows: original seed is the bacterial classification of using in breeding and culturing, and original seed is planted and bred out by mother.The test tube obtained through fruit body tissue separation is female plants, and growth is good more also must just can be further used as producing again of expanding production kind through Primary spawn, observation, evaluation, determines Optimality and the stability of bacterial classification.One, introduce the different formulations of several pedigree seed culture mediums below pedigree seed culture medium.1. sawdust medium formula: wood sawdust 78%, gypsum 1%, wheat bran 20%, white sugar 1%.Better, wherein Yi Qing hilllock tree wood sawdust is best (pine, cypress, camphor tree and should not use containing the wood sawdust of aromatic oil) to the wood sawdust shoulder that wood sawdust generally adopts broad-leaved tree and so on, requires freshly, and nothing is gone mouldy.2. cotton seed hulls 44%, wood chip 44%, and wheat bran l 0%, land plaster 1%, urea 0.5%:, superphosphate 0.5%.3. cotton seed hulls 78%～79%, corn flour 20%, land plaster 1%～2%, water content 65%.4. wood chip 73%, wheat bran 25%, sugar 1%, land plaster 1%.Manufacturing process: the above-mentioned raw materials formula is mixed, after white sugar dissolves with clear water, add in people's composts or fertilisers of cultivating and fully mix thoroughly, adding again clear water, the composts or fertilisers of cultivating water content is reached between 55%～65%. water content is too high. and composts or fertilisers of cultivating is airtight, and mycelial growth is slowly or not long, water content is too low, composts or fertilisers of cultivating humidity is inadequate, and mycelia is difficult growth also, and the conversion of various nutrient components must have certain humidity just can carry out.In order to grasp water content accurately, best limit spice, the limit water spray, limit is measured, in case use dilutional hyponatremia.Method is very simple, with hand, pinches a composts or fertilisers of cultivating, and between the finger seam, water breakthrough does not drip be advisable (being that water content is 65%).Two, the sterilizing of sterilizing pedigree seed culture medium can adopt autoclaving or two kinds of methods of normal-pressure sterilization.General raw material preferably adopts autoclaving, and because its quantity is few, quality requirement is higher, and at first sterilizing must be thoroughly strict.Autoclaving is put into pressure cooker by raw material, tighten the pot cover screw, fill up water level, then fire and heat, when gauge hand rises to 0.5 pound (1 pound=0.453592 kilogram), start to open vent valve, get rid of the cold air in pot, close (the automatic exhausting cold air of meeting when general high voltage pot press pin rises to 0.5 pound while making gauge hand automatically return original position, automatically close), continue subsequently to heat, while making press pin rise to 1.5 pounds, keep being advisable in 1.5 hours, then allow its automatic cooling rear taking-up inoculate.Three, the inoculation of inoculation Phellinus can determine according to the original seed quantity, and quantity is many can adopt the transfer room inoculation, and quantity can adopt the inoculating hood inoculation less.The general quantity of original seed is all not many, and the most handy inoculating hood connects, although because the inoculating hood operating difficulties, inoculation speed is slow, is difficult for being infected by bacteria, and survival rate is high.Inoculating hood sterilization, clean up inoculating hood before inoculation in advance, and the alcohol with 75% or liquor potassic permanganate are cleaned one time, then seed bottle, reaches other apparatus and all clean one time and put people's inoculating hood.Sterilization method can adopt two kinds of methods to carry out simultaneously.Ultra violet lamp, potassium permanganate and formaldehyde are mixed into promoting the circulation of qi sterilization inoculation in 30 minutes.During inoculation, after both hands are first used 75% alcohol disinfecting, then stretch into inoculating hood, light alcolhol burner, transfer needle is placed on flame and burns the red cool rear use of drying in the air.Female tampon epidermis of planting test tube can burn on Alcohol Flame, pull out again tampon, with a piece large invisible spectro mother culture media picking broad bean of inoculation hook, be placed on the medium in original seeds bottle, long have that face medium of mycelia should be upward, be conducive to hyphal development contact sawdust medium, tampon beyond the Great Wall after inoculation, then carry out second bottle of operation.Every female kind of test tube can connect 5～8 bottles, original seed.4-24 tetra-, cultivate under the constant temperature that postvaccinal original seed is placed on 25 ℃ of-28 ℃ of left and right and cultivate 5-7 days after, just can see that white female the kind on piece in surface of medium grows white fine hair mycelia, to the medium deep or surrounding diffusion development.Whether now should check at any time bacteria infection, if occur, miscellaneous bacteria should eliminate in time, until mycelia is covered with media surface.After original seed inoculation, at normal temperature, at the bottom of generally can covering with about 25 days bottle, 7-1 8 day left and right mycelia of l grows to a half of medium, and surface there will be fruit body primordium.How bacterial classification as original seed is grown and all can not be pulled out bottle stopper regardless of fruit body, in order to avoid miscellaneous bacteria enters in bottleneck.Mycelia is in process of growth, and because respiration will be distributed certain heat, often bottle temperature is higher than 2 ℃～3 ℃ of room temperature or natural temperatures, and therefore, seed bottle is unsuitable stacking too high or excessively crowded when sending out bacterium, in order to avoid excess Temperature, and strain quality F is fallen.Five, after the preservation Phellinus original seed mycelia of original seed is covered with bottle, should use in time, should not deposit for a long time, can preserve at normal temperatures about one month, as long as fruit body does not stop growing.Do not have the dried-up expanding species of all can doing to use, cell age shortens to as far as possible in a word.The store method of original seed generally adopts under low temperature and keeps in Dark Place better, and temperature is controlled at 5 ℃～7 ℃ and saves as suitablely, and the available refrigerator in place with good conditionsi is preserved.The described first step of 4-25 is made cultivated species by traditional method, and method is as follows:

Cultivated species is to expand with original seed the bacterial classification that system is directly used in cultivation.Cultivated species has two kinds of preparation methods, and a kind of is the bacterial classification of wood sawdust and so on, and another kind is the wooden unit bacterial classification.The bacterial classification of wood sawdust is more suitable for bottle cultivation and plastic sack cultivation, and the wooden unit bacterial classification is for segment wood cultivated, and selecting materials of cultivated species made substantially similar with method of operating to original seed.One, wooden unit, corncob medium are segment wood cultivated because the cultivation of Phellinus adopts substantially, therefore, below introduce the composition of raw materials of wooden unit cultivated species.Wooden unit, corncob culture medium prescription: 1000 kilograms of wooden units, 2 kilograms of superphosphate, 18 kilograms of corncobs, pH6～6.5,10 kilograms, wheat bran, water is appropriate, 2 kilograms, gypsum.This culture medium prescription sugar content is higher, do not need separately to add white sugar, in case meta-acid,, the later stage fruit body development is bad.Concrete operations: after raw material is mixed by the formula rate of selecting, then add the white sugar dissolved with clear water to mix thoroughly, keeping the composts or fertilisers of cultivating water content is 65%.The assay method of water content is measured identical with original seed, and then is adjusted to needed pH scope with sodium hydroxide or watery hydrochloric acid.Two, bottling method is packed the material mixed in bottle, and the limit rim is shaken seed bottle up and down, makes progressively consolidation of composts or fertilisers of cultivating, but can not be too tight.Can not composts or fertilisers of cultivating be pushed in order to avoid cause degree of tightness irregular in bottle with other things, require the composts or fertilisers of cultivating degree of tightness in whole bottle to be applicable to, unanimous between the higher and lower levels.In general composts or fertilisers of cultivating fills too loosely, and mycelial growth is fast, but very rare, occur that fruit body is slow; The composts or fertilisers of cultivating tension, mycelial growth is slow, closely, occurs that fruit body primordium early.The irregular composts or fertilisers of cultivating of degree of tightness can aggravate the mycelia teratogenesis.After composts or fertilisers of cultivating is filled to bottleneck, then with spades, media surface is flattened, water washes away the medium sticked inside and outside bottle wall, and tampon, carry out high-temperature sterilization beyond the Great Wall.In general the bottle preferably filled the same day just carried out high-temperature sterilization a same day, because there are a lot of microorganisms constantly to carry out activity in composts or fertilisers of cultivating, carried out not in time high-temperature sterilization, and other miscellaneous bacterias will be bred, and affect the normal nutrient component of medium.Three, sterilizing methods production cultivated species is the same with the original seed sterilizing, but Common Cultivation kind quantity is larger, and the quantity of high-pressure sterilizing pot dress is few, preferably adopts native rice steamer sterilizing.How many large I of soil rice steamer decides according to output, generally adopts brick and cement to build up, and native rice steamer sterilizing requires sealing tighter.It is square better than circle that the soil rice steamer is made employing, and square native rice steamer is convenient to put bottle or sack, generally should be deeply unsuitable shallow.The space of one cube of density can fill 800 left and right of bottle of 750 grams, the about 250 bags of left and right of plastic sack, and the method for putting of bottle and plastic sack is all adopted angle and is kept flat, and places gap vertically too large.In a large rice steamer, do not put tens bottles and carry out high-temperature sterilization, quantity is very few, and the rice steamer internal pore is excessive, and adding native rice steamer does not have certain high-pressure installation, just can not reach the purpose of thorough sterilizing.During sterilizing, should keep enough firepower 4 ~ 6 hours from water boil, require space temperature in rice steamer to reach I00.More than C, notice that firepower size and time combination of long drives and drop shots arrange.No matter adopt autoclaving or local method sterilizing, sterilizing pressure can not too high or overlong time, hypertonia or the time hear and longly can destroy the nutrient in the nutrition base, acid-base value is descended, be unfavorable for the phellinus liteus growth.Sterilization time is too short or temperature is too low, and the miscellaneous bacteria in medium and other microorganisms do not kill, and will breed in the future, to production, brings heavy losses.Must grasp temperature, pressure and the time of sterilizing.After sterilizing, do not take out at once, be preferably in a vexed night in pot, second day takes out and treats that a bottle temperature drops to 30.After C, then inoculated.Four, inoculation production kind of inoculation quantity is large, adopts inoculating hood inoculation speed too slow, preferably adopts transfer room to inoculate, and at first transfer room requires the clean life of meeting, and air does not flow, and is conducive to like this thorough disinfection sterilizing.The transfer room sterilization: inoculating the previous day, in advance transfer room is cleaned up, then with running water, the surroundings wall of transfer room is rinsed well, indoor humidity is increased, air is inactive state.Mix sterilization with formaldehyde and potassium permanganate again, dosage is calculated by 10 milliliters, space requirement formaldehyde, potassium permanganate 5 grams of every cubic metre.During sterilization, should close the doors and windows vexed one day or a night, second day put people's sterilizing bottle, original seed, with its etc.Carry out drug disinfection once again after putting well, can adopt ultra violet lamp to inoculate after half an hour simultaneously.The inoculation Job readiness: the staff should finish changing in advance the inoculation clothing between the buffering of transfer room outside, puts on mouth mask, emgloves, cap etc., then enters the transfer room inoculation.Transfer room inoculation operation is more convenient than inoculating hood, speed.The transfer room air conditions is poor, oxygen is few, the inoculation working time is oversize, influential to human body respiration, therefore entering transfer room must make the best use of time to operate. inoculation operation: first light alcolhol burner, then the original seeds bottle tampon is pulled out, original seed surface one deck is approximately removed on the medium top layer of 1 cm thick, again with inoculation hook picking finger large l-2 be placed on rapidly and produce on kind of medium, tampon beyond the Great Wall.Every bottle of original seed approximately can connect 100 bottles of left and right of cultivated species.After having connect, open door and window, move postvaccinal bottle to culturing room and go to cultivate.Five, cultivate cultivated species in incubation, main work is that temperature treatment and miscellaneous bacteria are observed.If adopt the natural temperature in 5～September to cultivate, just do not need manually heat or lower the temperature, if produce April to Second Year September then, natural temperature is too low can not meet the needed temperature of Phellinus, just must manually heat.Although phellinus liteus all can be grown at 3 ℃～4 ℃, preferably temperature is controlled to 20 ℃～30 ℃ left and right, is conducive to like this normal growth of mycelia.Postvaccinal miscellaneous bacteria is observed, and when general phellinus liteus starts to sprout, miscellaneous bacteria also just starts growth.The former reason many factors of the growth of miscellaneous bacteria causes have the transfer room space disinfection not tight, and the miscellaneous bacteria that when operation bring into is arranged, and has high-temperature sterilization halfway former thereby cause miscellaneous bacteria to infect, and also has original seed whether miscellaneous bacteria infection etc. is arranged.These reasons can identify, if high-temperature sterilization is not thorough, the miscellaneous bacteria of whole bacterium culture medium is to occur from bottle middle part or bottom; If itself has miscellaneous bacteria original seed, after inoculation, the strain culture-material surface is miscellaneous bacteria entirely, but can miscellaneous bacteria not occur from bottle bottom or middle part; If the inoculation misoperation, just the part bottle surface is grown miscellaneous bacteria, and this situation is whole bacteria infections not.Also has another kind of situation, postvaccinal kind of piece neither grown miscellaneous bacteria, do not sprout again mycelia, this class situation will check from many aspects: whether bacterial classification is aging, and whether inoculation temperature is too high, and whether the medium acid-base value is suitable, whether cultivation temperature is normal, whether medium nutrient content meets the requirements, and whether the original seed of use is reliable etc., and these all can cause mycelia not sprout.The pure mycelial growth rate of Phellinus is neither fast nor slow, growth is evenly neat, pure white, and the mycelia stalwartness, general mycelia grows at 1/3 o'clock, and media surface just there will be fruit body primordium, the expansion of constantly growing of former base.In the time of at the bottom of mycelia grows to bottle, fruit body can grow to bottleneck tampon place, but can not allow it grow tampon, in order to avoid the nutrition in too much decomposition medium.Generally from being inoculated into mycelial growth, finish approximately to need about 25～30 days, after covering with, put at low temperatures and properly save backup.

Claims (1)

1. one kind is utilized wild stub alive to cultivate the method for Phellinus, and it is characterized in that: step is as follows:

The first step, make female kind, original seed, cultivated species by traditional method;

Second step, cultivate mulberry saplings, cultivates the mulberry tree woods of 100 mu-1000 mu, grows to diameter until mulberry tree and reach 10cm when thick, by the paper mulberry bark of hilt 1/5, prunes, and not only can grow but also be unlikely to dead tree;

The 3rd step, when the bark place xylem of pruning has dry tack free, dark with belt puncher or electric drill punching 5.5-6cm, interval 16-18cm between Kong Yukong, then, in Phellinus bacterial classification access hole, seal with yellow mud; Once inoculation, can pluck the Phellinus many decades, until old dead the rotting of mulberry tree.