CN103804054B - Phellinus culture medium is optimized and short segment wood cultivated method - Google Patents
Phellinus culture medium is optimized and short segment wood cultivated method Download PDFInfo
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Abstract
The present invention relates to the cultivation of a kind of fungi plant, namely Phellinus culture medium is optimized and short segment wood cultivated method.Phellinus mother culture media: glucose 20g, soy peptone 2g, agar 18g, potassium primary phosphate 0.46g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, water 1000ml.The short segment wood cultivated method of Phellinus, step is as follows: (1) adopts fruit body tissue separation method to be separated Phellinus starter kind.(2) Phellinus Primary spawn.(3) Phellinus cultivar is cultivated.(4) preparation of short segment wood cultivated material.Select oak segment to make bacterium rod, the oak segment artificial culture Phellinus production means with low cost, output is high, and quality is good, and active constituent content is high, can obtain abundant economy return.
Description
Technical field
The present invention relates to the cultivation of a kind of fungi plant, namely Phellinus culture medium is optimized and short segment wood cultivated method.
Background technology
In the prior art, Phellinus is a kind of rare large-scale medicinal fungi, and its pharmaceutical use is just on the books as far back as " property of medicine opinion " of the Tang Dynasty.Compendium of Material Medica record, Phellinus can control addiction drink gather, metal-inflicted wound of suffering from abdominal pain; " Chinese medicine voluminous dictionary " describes it can treat internal medicine various diseases.Its sporophore is used as medicine, mildly bitter flavor, the sharp the five internal organs of energy, a surname's intestines gas, hemostasis, toxin expelling and stomach antidiarrheal.Modern medicine study shows; Phellinus except there is the pharmacological action in Traditional Chinese medical theory, also have anticancer, antitumor, anti-ageing, antifatigue, anti-oxidant, anti-fibrosis, hemopoietic function of bone marrow damage protection, immunomodulatory, protect the liver and the effect such as anti-liver cirrhosis.Phellinus is perennial wood-decay fungi; owing to being subject to the singularity of physiological ecological and the restriction of complicacy and external environment condition; the sporophore formed at occurring in nature is rare; add in recent years; the consumption of Phellinus strengthens day by day; particularly external purchase of China's wild resource being robbed to formula, wild resource is fewer and feweri.Therefore need to obtain its active substance by number of ways, as carried out artificial juggle cultivation, liquid and solid fermentation etc.Artificial juggle cultivation is at the early-stage, not yet has the concrete report about artificial juggle cultivation Phellinus, the domestic precedent of also not gathering of tame finished product Phellinus sporophore.
Summary of the invention
The object of the invention is for above-mentioned deficiency and provide one to improve substratum, realizing the feasible Phellinus culture medium of artificial culture Phellinus and optimize and short segment wood cultivated method.
Technical solution of the present invention is: a kind of Phellinus mother culture media, is made up: glucose 20g, soy peptone 2g, agar 18g, potassium primary phosphate 0.46g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, water 1000ml of the raw material of following weight.
A kind of Phellinus pedigree seed culture medium, is made up of the raw material of following weight: toothed oak wood chip 77%, wheat bran 10%, Semen Maydis powder 10%, glucose 2%, gypsum 1%.
A kind of Phellinus Cultivar culture medium, is made up of the raw material of following weight: toothed oak wood chip 50%, cotton seed hulls 39%, wheat bran 10%, gypsum 1%.
The short segment wood cultivated method of a kind of Phellinus, is characterized in that step is as follows:
(1) fruit body tissue separation method is adopted to be separated Phellinus starter kind: to carry out surface sterilization process to picking up from the wild Phellinus sporophore in Changbaishan area, hooking up a fritter Phellinus sporophore and being put on inclined-plane mother culture media; Be placed in 27 DEG C of-30 DEG C of thermostat containers and cultivate, treat to grow milk yellow mycelia around sporophore tissue block, being transplanted to new test tube at colony edge picking tip mycelium transplants on mother culture media inclined-plane, 27 DEG C of-30 DEG C of constant temperature culture, 10d mycelia can cover with test tube, so repeatedly transplant 2-3 time, obtained strains is as mother's kind; Described mother culture media is: glucose 20g, soy peptone 2g, agar 18g, potassium primary phosphate 0.46g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, water 1000ml.
(2) Phellinus Primary spawn: pedigree seed culture medium is toothed oak wood chip 77%, wheat bran 10%, Semen Maydis powder 10%, glucose 2%, gypsum 1%; In 28 DEG C of thermostatic chambers, darkroom is cultivated.
(3) Phellinus cultivar is cultivated: Cultivar culture medium is toothed oak wood chip 50%, cotton seed hulls 39%, wheat bran 10%, gypsum 1%; In 28 DEG C of thermostatic chambers, darkroom is cultivated.
(4) preparation of short segment wood cultivated material:
Toothed oak wood is selected to make bacterium rod, for subsequent use after autoclaving.Described bacterium rod selects toothed oak wood species as segment wood cultivated material, small-end diameter 4-8cm, is cut into the billet that 18-20cm is long, cleave in two, be combined into the Duan Mu Bales of about 15cm, load in low pressure polyethylene bacterium bag, one deck wood chip wheat bran skin nutrilite is filled at juggle two, wood chip, wheat bran ratio are 3:1, poach, ties sack, section wood bag is pricked twice simultaneously, what Duan Muyu plastics bag was fitted is comparatively tight, for subsequent use after autoclaving.
(5) inoculation of bacterium rod is cultivated with bacteria developing period:
By aseptic technique, the Phellinus cultivation strain selecting mycelial growth vigorous, adopts two inoculation method, inoculum size 20%, with bacterial classification capping charge level for degree.
Sending out culture temperature between bacterium incubation period is 28 DEG C, and shading is cultivated, and ventilates every day 1 time, incubation time 35-40d.
(6) the sporophore growth growth period cultivates:
When mycelia color in bacterium rod have pale yellow become deep yellow time, at bacterium rod both sides lateral incision mouth, opening proterties crescent, long 2cm, crescent width is less than 1cm, and each bacterium bag can open two mouths respectively in bacterium bag both sides.
Bacterium wood buries soil cultivation: first make cultivation bed on booth ground, covered with plastic film on canopy, sunshade net or straw screen or mat shade, designated port standing tree buries soil cultivation, first by dark 6cm, seeding row spacing 10cm × 10cm digs pit, then by 6cm place ring cutting at the bottom of the distance bag of bacterium bag one end, at the bottom of taking off bag, standing tree is put in hole, sandy soil in training, and then with blade, bacterium bag ring is drawn two cuttves, cut plastic film for degree, only mark twice fruiting mouth or seam.Cultivation bed can make three bacterium beds by the booth that 6m is wide, middle bacterium bedside 2m, and each 1.2m of both sides bacterium bed, stay 60cm effect road between bed, 20cm waterways is respectively stayed on canopy both sides.
Fruit body development period management: when mycelia color from pale yellow look enters the former base differentiation phase in bacterium rod, now adopt the method for day and night temperature, namely daytime culture temperature 28 DEG C, nocturnal temperature reduces to 20 DEG C, processes 3d continuously; Relative air humidity controls between 85%-95%; Give scattered light to irradiate.
The fruit body development stage, growth temperature 25 DEG C, relative air humidity remains between 85%-95%, ventilates every day 2 times, each 30min; Scattered light irradiates.
Sporophore is gathered: Phellinus sporophore answers 2-3 to gather once, adopt stay greatly little.
Advantage of the present invention is: 1, the present invention selects from Phellinus mother culture media, pedigree seed culture medium is selected, selection three aspect of Cultivar culture medium is set about; determine Phellinus substratum Reasonable Parameters; to obtaining excellent Phellinus bacterial classification, provide prerequisite guarantee for successfully cultivating Phellinus mushroom entity.2, select oak segment to make bacterium rod, adopt and bury soil cultivation, determine reasonable cultivation condition, be 28 DEG C as sent out culture temperature between bacterium incubation period, shading is cultivated, and ventilates every day 1 time, incubation time 35-40d; When entering the former base differentiation phase, now adopt the method for day and night temperature, namely daytime culture temperature 28 DEG C, nocturnal temperature reduces to 20 DEG C, processes 3d continuously; Relative air humidity controls between 85%-95%; Give scattered light to irradiate; The fruit body development stage, growth temperature 25 DEG C, relative air humidity remains between 85%-95%, ventilates every day 2 times, each 30min; Scattered light irradiates, and ensure that Phellinus artificial cultivation method is feasible, and suitability for industrialized production.3, the oak segment artificial culture Phellinus production means is with low cost, and output is high, and quality is good, and active constituent content is high; Abundant economy return can be obtained.4, this research is to save and this precious Resources of Medicinal Fungi of exploitation Phellinus bacterium; make up wild Phellinus bacterium because of natural resources finite sum excessively pluck caused species resource be on the verge of exhaustion, scientific experiment and research be difficult to exploitation and the market development utilize limited practical problems, there are great economic benefit, social benefit and ecological benefits.
Below in conjunction with embodiment, embodiments of the present invention are described in further detail.
Embodiment
Experimental example 1
The research of phellinus liteus culture condition
1 materials and methods
1.1 Phellinus bacterial strains (phelliuns igniarius Phellinusigniarius), pick up from the toothed oak wood (Mongolian oak) in the aged mountain range forest of Changbai Mountain system.
1.2 for examination substratum
Basic medium: glucose 20g, soy peptone 2g, agar 18g, potassium primary phosphate 0.46g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, water 1000ml.
1.3 experimental technique
1.3.1 strain separating and purifying are cultivated
Fruit body tissue separation method is adopted to be separated Phellinus starter kind.Under strict aseptic technique condition, carrying out surface sterilization process to picking up from the wild Phellinus sporophore in Changbaishan area, repeatedly embrocate sporophore surface with 75% alcohol and be placed in aseptic slat chain conveyor ware, then it is for subsequent use that culture dish is put into Bechtop.Longitudinally cut from Phellinus sporophore base portion with the scalper of flame calcination sterilizing; and then delineate several ' well ' word with scalper from section center at wish separated part, hook up a fritter Phellinus sporophore (mung bean size) with the inoculation hook of flame calcination sterilizing and be put on inclined-plane basic medium.Be placed in 28 DEG C of thermostat containers and cultivate.After 2 days, mycelia starts to sprout, treat to grow milk yellow mycelia around sporophore tissue block, be transplanted on new test tube basic medium inclined-plane at colony edge picking tip mycelium, 28 DEG C of constant temperature culture, 10d mycelia can cover with test tube, so repeatedly transplant 2-3 time, obtained strains is as mother's kind, and it is for subsequent use that mycelia covers with preservation in the rearmounted 4 DEG C of refrigerators of test tube.
1.3.2 inoculation culture and observation procedure
Poured in the aseptic slat chain conveyor ware of diameter 9cm by the test tube basic medium of sterilizing, each culture dish adds basic medium 15ml; After culture medium solidifying, in culture dish, connect the bacterium block of a diameter 5mm, each process inoculation 3 culture dish (namely repeating for three times).Measure a colony diameter every 3 days, calculate mycelial growth rate, and observe bacterium colony change and growing way.
1.3.3 data statistical approach
The process of raw data, correlation analysis and chart, use Excel software processes, variance analysis SPSS12.0 software processes, the significance of difference (p < 0.05) between application Duncan one-way analysis of variance respectively processes.
1.3.4 different carbon source impact that phellinus liteus is grown
Respectively with the glucose in maltose, sucrose, lactose, N.F,USP MANNITOL replacement basic medium, 28 DEG C, cultivate under dark condition, the impact that observation different carbon source grows phellinus liteus.
1.3.5 different nitrogen sources impact that phellinus liteus is grown
Respectively with the soy peptone in extractum carnis, yeast extract paste, ammonium sulfate, ammonium chloride replacement basic medium, 28 DEG C, cultivate under dark condition, the impact that observation different nitrogen sources grows phellinus liteus.
1.3.6 differing temps impact that phellinus liteus is grown
Use basic medium, respectively at 18 DEG C, 21 DEG C, 24 DEG C, 27 DEG C, 30 DEG C, 33 DEG C, cultivate under dark condition, the impact that observation differing temps grows phellinus liteus.
1.3.7 different pH impact that phellinus liteus is grown
Use basic medium, adjust pH at 5.0,5.5,6.0,6.5,7.0,7.5,28 DEG C, is cultivated under dark condition respectively, observes the impact that different pH value grows phellinus liteus.
2 results and analysis
The impact (the results are shown in Table 1-1) that 2.1 different carbon source grow phellinus liteus
Impact that table 1-1 different carbon source grows phellinus liteus (
± S.Dn ﹦ 3)
As can be seen from table 1-1: Phellinus all can grow in 5 kinds of carbon sources for examination, but different carbon source affects significant difference (p < 0.05) to mycelial growth.When taking glucose as carbon source, mycelial growth rate is the fastest, 4.2mm/d; Thereafter be N.F,USP MANNITOL, sucrose, maltose, lactose successively; From growth growing way, when glucose is carbon source, bacterium colony mycelia is dense, growth potential is vigorous, during with N.F,USP MANNITOL and sucrose for carbon source, bacterium colony mycelia is elongated, denser, growth potential is more vigorous, and during with lactose and maltose for carbon source, bacterium colony mycelia is sparse, growth potential is more weak (see table 1-1).As can be seen here, the optimum carbon source of this bacterium mycelial growth is glucose.
The impact (the results are shown in Table 1-2) that 2.2 different nitrogen sources grow phellinus liteus
Impact that table 1-2 different nitrogen sources grows phellinus liteus (
± S.Dn ﹦ 3)
As can be seen from table 1-2: Phellinus all can grow in 5 kinds of nitrogenous sources of trial-product, but different nitrogen sources affects significant difference (p < 0.05) to mycelial growth.When taking peptone as nitrogenous source, mycelial growth rate is the fastest; Thereafter be yeast extract paste, ammonium chloride, saltpetre, extractum carnis successively; From growth potential, when peptone is nitrogenous source, bacterium colony mycelia is dense, growth potential is vigorous, during with yeast extract paste and extractum carnis for nitrogenous source, bacterium colony mycelia is denser, growth potential is more vigorous, but, when taking extractum carnis as nitrogenous source, mycelial growth rate is slow, with ammonium chloride and saltpetre for bacterium colony mycelia during nitrogenous source is sparse, and growth potential more weak (see table 1-2).As can be seen here, the utilization of the nitric nitrogen (saltpetre, ammonium chloride) during this bacterium is inorganic nitrogen-sourced to two kinds is without marked difference, and more inorganic nitrogen-sourced, this bacterium can utilize organonitrogen better, and the suitableeest nitrogenous source of mycelial growth is soy peptone.
The impact (the results are shown in Table 1-3) that 2.3 differing tempss grow phellinus liteus
The impact (colony diameter mm) that table 1-3 differing temps grows phellinus liteus
As can be seen from table 1-3: what differing temps grew phellinus liteus affects significant difference (p < 0.05).When culture temperature is 18 DEG C, mycelial growth rate is slow, is only 1.2mm/d; Along with the rising of temperature, mycelial growth rate is progressively accelerated; When temperature is 27 DEG C, the speed of growth of mycelia is the fastest, is 3.8mm/d; When temperature rises to 33 DEG C, although mycelia can grow, the speed of growth obviously slows down.Significance of difference test result shows, it is remarkable that temperature 27 DEG C and 30 DEG C affect difference to mycelial growth rate, but have significant difference between both with other Temperature Treatment.Therefore, the growth temperature that mycelia is suitable is 27 DEG C-30 DEG C.
The impact (the results are shown in Table 1-4) that 2.4 different pH grow phellinus liteus
The impact that the different pH of table 1-4 grows phellinus liteus
As can be seen from table 1-4 in: the basic medium of phellinus liteus within the scope of pH5.5-7.5 all can grow, but different pH phellinus liteus is grown affect significant difference (p < 0.05).During basic medium pH5.0, mycelial growth rate is 3.2mm/d; During basic medium pH5.5, mycelial growth rate is very fast, and be 3.6mm/d, bacterium colony mycelia is denser, growth potential is more vigorous; During basic medium pH6.0, mycelial growth rate is the fastest, and be 3.8mm/d, bacterium colony mycelial growth is dense, and growth potential is vigorous; When basic medium pH is greater than 6.0, mycelial growth rate increases with pH and slows down, and growth potential is also more weak; During basic medium pH7.5, mycelial growth rate is down to 2.6mm/d.Phellinus is suitable for growing in slant acidity environment as can be seen here.Significance of difference test result shows simultaneously, and pH6.0 has significant difference between the impact of the phellinus liteus speed of growth and process.Therefore, in this experiment, pH6.0 is the optimum pH of phellinus liteus growth.
Experimental example 2
Phellinus original seed, Cultivar culture medium Quality Research
The production of phellinus igniarius strain is a very important ring in whole Phellinus cultivation.Only have excellent bacterial classification, then be equipped with good cultivation condition, just may successfully obtain Phellinus sporophore.The key that excellent species obtains except species, most critical be exactly the selection of substratum or culture medium, only have suitable culture medium could obtain stalwartness, great-hearted excellent species.This experiment is selected from mother culture media, pedigree seed culture medium is selected, selection three aspect of cultivar culture material is set about, and to obtaining excellent Phellinus bacterial classification, provides prerequisite guarantee for successfully cultivating Phellinus mushroom entity.
The screening of 1 Phellinus bacterium pedigree seed culture medium
1.1 materials and methods
1.1.1 strains tested
Phellinus bacterial strain (phelliuns igniarius phelliusingiarius), picks up from the toothed oak wood (Mongolian oak) in the forest of mountain range, Xi Lao ridge, Changbai Mountain for 2004.
1.1.2 experimental technique
Select five kinds of different pedigree seed culture mediums:
1) corn culture medium: by dry corn bubble after 4 hours, puts into after boiling water is boiled and pulls out, and control water, adds 1% gypsum, stirring and evenly mixing.
2) mulberry wood chips substratum: mulberry wood chips 77%, wheat bran 10%, Semen Maydis powder 10%, glucose 2%, gypsum 1%, stirring and evenly mixing.
3) toothed oak sawdust medium: toothed oak wood chip 77%, wheat bran 10%, Semen Maydis powder 10%, glucose 2%, gypsum 1%, stirring and evenly mixing.
4) poplar sawdust medium: poplar bits 77%, wheat bran 10%, Semen Maydis powder 10%, glucose 2%, gypsum 1%, stirring and evenly mixing.
5) birch sawdust medium: birch bits 77%, wheat bran 10%, Semen Maydis powder 10%, glucose 2%, gypsum 1%, stirring and evenly mixing.
Prepared respectively by substratum, fill high 10cm, diameter 8cm Cans cultivate Phellinus bacterium, each formula (process) fills 30 bottles, and every 10 bottles is 1 group, and repeat for 3 times, adopt high pressure steam sterilization, pressure 0.11 MPa (temperature 121 DEG C) continues 2.5h.Inoculate under aseptic condition, after inoculation, under 25 DEG C of-28 DEG C of conditions, airtight shading is cultivated, and phellinus liteus sprouting state, mycelial growth rate on each substratum of observed and recorded, filter out the suitableeest pedigree seed culture medium of Phellinus bacterial classification.
1.1.3 data statistical approach
The process of raw data, correlation analysis and chart, use Excel software processes, variance analysis SPSS12.0 software processes, the significance of difference (p < 0.05) between application Duncan one-way analysis of variance respectively processes.
1.2 results and analysis
The growing state of table 2-1 Phellinus in 5 kinds of Primary spawn matrix
As can be seen from table 2-1 in: phellinus liteus all can grow in 5 kinds of Primary spawn matrix, but different culture media confrontation phellinus liteus growth affect significant difference (p < 0.05).In corn culture medium, Phellinus bacterium growing way is best, fast growth, and 30d just can cover with seed bottle, and mycelia color is brown color, and surperficial mycelia kink, has very thick lawn; Toothed oak sawdust medium growing way is fine, and mycelia all has unanimously, and 37d covers with seed bottle substantially, and color is pale brown; Poplar bits and birch sawdust medium phellinus liteus grow intensive, and growing way is better, newborn mycelium color cadmium yellow, and mycelial growth rate is relatively slow; Mulberry wood chips substratum mycelium growth vigor is good, and surperficial mycelia is intensive, and newborn mycelia color is light yellow, but mycelial growth rate is slow, and 50d can cover with seed bottle.In sum: compare phellinus liteus growth growing way and speed on these 5 kinds of pedigree seed culture mediums, corn culture medium is optimum, but cost is the highest, in four kinds of sawdust mediums, the research of toothed oak wood chip is more suitable for making Phellinus bacterium original seed.
The screening of 2 Phellinus bacteria cultivation kind matrix
2.1 materials and methods
2.1.1 strains tested
Phellinus bacterial strain ((phelliuns igniarius phelliusingiarius)) picks up from the toothed oak wood (Mongolian oak) in the forest of mountain range, Xi Lao ridge, Changbai Mountain for 2004.
2.1.2 experimental technique
The selection of Phellinus artificial culture matrix, according to pedigree seed culture medium the selection result, reduces further and expands numerous cost, according to the principle of gathering materials on the spot, and the following 5 kinds of Cultivar culture medium of design:
1) wood chip wheat, bran substratum: toothed oak wood chip 80%, wheat bran 18%, sucrose 1%, gypsum 1%;
2) wood chip rice chaff substratum: toothed oak wood chip 77%, thin rice chaff 22%, gypsum 1%;
3) cotton seed hulls sawdust medium: toothed oak wood chip 50%, cotton seed hulls 39%, wheat bran 10%, gypsum 1%;
4) wood chip corn cob substratum: toothed oak wood chip 65%, corn cob 34%, gypsum 1%;
5) corn cob wheat bran medium: corn cob 80%, wheat bran 19%, gypsum 1%.
Prepare planting material matrix respectively, the culture material prepared is loaded high 10cm, diameter 8cm Cans, seal bottleneck with polypropylene, adopt high pressure steam sterilization, pressure 0.11 MPa (temperature 121 DEG C) continues 2.5h.Inoculate under aseptic condition, after inoculation, under 25 DEG C of-28 DEG C of conditions, airtight shading is cultivated, and observation phellinus liteus sends out bacterium situation.By investigating bacterium situation of sending out, mycelia feature, the full bottle number of days of record mycelia, filter out the cultivar formula that Phellinus bacterium is suitable.
2.2 results and analysis
The growing state of table 2-2 Phellinus on 5 kinds of culture mediumes
The impact of different culture media formula on cultivar mycelial growth, from the display of table 2-2 result: on 5 kinds of cultivation base, mycelial growth rate exists significant difference, phellinus liteus speed of growth in cotton seed hulls wood chip wheat bran medium is the fastest, it covers with bag time is 36d, and growth potential is strong, mycelia is closeer, faint yellow; In wood chip wheat bran medium, the speed of growth is taken second place, and it covers with bag time is 39d, and in corn cob wheat bran medium, the speed of growth is the slowest, and 51d covers with bacterium bag, and growing way is weak, and mycelia is more sparse.Therefore, according to overall target, cotton seed hulls wood chip wheat bran medium is cultivar optimal medium.
The research of three short segment wood cultivated technology
1 materials and methods
1.1 test materials
1.1.1 strains tested and source thereof
Phellinus bacterial strain ((phelliuns igniarius phelliusingiarius)) picks up from the toothed oak wood (Mongolian oak) in the forest of mountain range, Xi Lao ridge, Changbai Mountain for 2004.
1.1.2 for the preparation of examination original seed (secondary kind) substratum
Test proves that original seed selects toothed oak wood chip to do substratum as well, adds 1% terra alba mixing during preparation in grain, for subsequent use after bottling autoclaving.
1.1.3 for the preparation of examination cultivar (three grades of kinds) substratum
For subsequent use selected the culture medium prescription bottling autoclaving of cultivar by testing sieve after.
1.1.4 the screening of section wood species is cultivated
1.1.4.1 for preparation and the preparation of the segment wood cultivated material of examination
According to the seeds that wild Phellinus grows under field conditions (factors), the mulberry tree selecting Changbaishan area common, toothed oak tree (Mongolian oak), willow (mountain white poplar) and birch (white birch) are planting material.The age of tree is at 6-10.
1.1.4.2 the preparation of segment wood cultivated material (i.e. bacterium rod)
Select above 4 kinds of different seeds as segment wood cultivated material, contrast it to phellinus liteus and the growth of sporophore thereof and the impact of output.Slightly head diameter 4-8cm, be cut into the billet that 18-20cm is long, cleave in two (thin does not split), is combined into the Duan Mu Bales of about 15cm, loads in low pressure polyethylene bacterium bag, each seeds 50 bags.One deck wood chip wheat bran skin nutrilite (wood chip, wheat bran ratio are 3:1, add suitable quantity of water and mix thoroughly) is filled at juggle two, ties sack, section wood bag is pricked twice simultaneously, and what Duan Muyu plastics bag was fitted is comparatively tight, for subsequent use after autoclaving.
1.2 test method
1.2.1 original seed (secondary kind) is cultivated
Strictly observe aseptic technique requirement by Phellinus slant strains access pedigree seed culture medium (a slant tube mother plants and connects 500g Cans 6 bottles), in 28 DEG C of thermostatic chambers, darkroom is cultivated, and 30d mycelia can cover with seed bottle.
1.2.2 the cultivation of cultivar (three grades of kinds)
Aseptically access Phellinus grain second class inoculum, 1 bottle of second class inoculum can connect 750ml Cans 20 bottles, and in 28 DEG C of thermostatic chambers, darkroom is cultivated, and 40d bacterial classification can cover with bottle.
1.2.3 the inoculation of bacterium rod is cultivated with bacteria developing period
Strictly observe aseptic technique, the Phellinus cultivation strain selecting mycelial growth vigorous, adopt two inoculation method, inoculum size 20%, with bacterial classification capping charge level for degree.
Sending out culture temperature between bacterium incubation period is 28 DEG C, and shading is cultivated, and treats that mycelia is covered with bacterium rod and changes yellow gradually into and can enter physiological differentiation stage.
1.2.4 the sporophore growth growth period cultivates
When mycelia color in bacterium rod have pale yellow become deep yellow time, at bacterium rod both sides lateral incision mouth, opening proterties crescent, long 2cm, crescent width is less than 1cm, and each bacterium bag can open two mouths respectively in bacterium bag both sides.
2 results and analysis
2.1 bacteria developing period management
Send out bacterium stage culture temperature 28 DEG C, do not need illumination, darkroom is cultivated, and ventilates every day 1 time, incubation time 35-40d.
2.2 bacterium wood bury soil cultivation
First make cultivation bed on ground, the wide booth of 6m can make three bacterium beds, middle bacterium bedside 2m, each 1.2m of both sides bacterium bed, and stay 60cm effect road between bed, 20cm waterways is respectively stayed on canopy both sides, and on canopy, covered with plastic film (12) and two-layer sunshade net (or straw screen or mat) shade.Bed surface whole good after, adopt de-bag, designated port standing tree buries soil cultivation, first by dark 6cm, seeding row spacing 10cm × 10cm digs pit, then by bacterium bag one end apart from 6cm place ring cutting at the bottom of bag, at the bottom of taking off bag, standing tree is put in hole, sandy soil in training, and then with blade, bacterium bag ring is drawn two cuttves (cutting plastic film for degree), only mark twice fruiting mouth (seam), not de-bag, is beneficial to moisturizing.
2.3 fruit body development period managements
When mycelia color from pale yellow look enters the former base differentiation phase in bacterium rod, now adopt the method for day and night temperature, namely daytime culture temperature 28 DEG C, nocturnal temperature reduces to 20 DEG C, processes 3d continuously, can accelerate Phellinus sporophore and occur.
Found by test, relative air humidity is comparatively large to Phellinus sporophore growth speed and quality influence thereof, for this reason, has investigated the impact of different gradient relative air humidity on Phellinus sporophore growth speed and quality.Result shows, is controlled by relative air humidity between 85%-95%, be conducive to Phellinus sporophore growth, and the Phellinus quality obtained also reaches best.Temperature is another the important environmental factor affecting Phellinus sporophore growth velometer quality.Temperature is too high, although Phellinus sporophore looks fast, and sporophore lighter in weight, and temperature is too high adds high humidity, is easy to bacteria infection, thus reduce output.In view of this, we have also investigated the impact of differing temps on Phellinus sporophore growth and quality.Considering between temperature and relative air humidity under interactional prerequisite, finally finding, temperature being controlled take into account Phellinus sporophore growth speed and quality at 25 DEG C.Reach between 85%-95% at relative air humidity, Phellinus sporophore bacteria infection phenomenon is less.
In addition, found by test: when there being sufficient oxygen, be conducive to Phellinus sporophore growth; For this reason, culturing room early, once, each 30min, can accelerate Phellinus sporophore growth like this in late respectively ventilation, can also ensure Phellinus quality simultaneously.
2.4 sporophores are gathered
Phellinus is perennial medicinal fungi, and sporophore growth is comparatively slow, answers 2-3 to gather once, adopt stay greatly little.
The Phellinus sporophore shape of a hoof of artificial wood section cultivation or kidney shape, wooden hard, monolithic or two, three stacked lifes, base portion is thicker, and edge is thin blunt circle gradually, and cap is deep yellow to light coffee color.No matter segment wood cultivated Phellinus is in sporophore shape feature, or Phellinus seed output and quality is all better than the Phellinus of plastics cultivation, and segment wood cultivated Phellinus sporophore is similar to wild Phellinus.
When the not regrowth of Phellinus cap, and see when having a small amount of spore to distribute, just enter the sporophore ripening stage.Gather and to cut off from Phellinus sporophore base portion with cutter, principle adopts to stay greatly little, and limit is ripe, gather in limit.
2.5 different sections of wood species cultivation Phellinus Yield compari@
The growing state of table 3-1 Phellinus on 5 kinds of different tree species
| Seeds | Connect bacterium quantity (bag) | Cover with bacterium rod time (d) | Growth potential | Survive quantity (bag) | Fruit-body formation time (d) | The excellent output (g) of average list | Ultimate production (Second Year receipts) |
| Mulberry | 50 | 60 | Weak | 23 | 45 | 58 | 1334 |
| Toothed oak | 50 | 48 | By force | 46 | 25 | 110 | 5060 |
| Poplar | 50 | 45 | By force | 48 | 30 | 80 | 3840 |
| Birch | 50 | 52 | More weak | 45 | 32 | 78 | 3510 |
The linden of different tree species is on the impact of phellinus liteus and sporophore growth and output; result as can be seen from table 3-1: mycelia, sporophore growth speed and output thereof exist obvious difference on the juggle of 4 different tree species; Phellinus grows comparatively fast on toothed oak wood; 48d covers with bacterium wood substantially; after burying soil cultivation, 35d just produces sporophore at scarfing place or sack successively; within 2 years, to gather the first damp sporophore after autumn; adopt stay greatly little; juggle average per unit area yield Phellinus sporophore (dry product) 110g; output; sporophore quality is hard, and form is close with wild person.Although mycelial growth rate fast (45d covers with bacterium wood) in poplar section; fruit-body formation is (after burying soil cultivation, 30d produces successively) comparatively early; but its output is not as good as oak segment (mean yield is 80g/ section), and Phellinus sporophore quality is more loose, and quality product is taken second place.Mulberry cultivation due to bark thinner, mycelium growth vigor is weak, and long speed is slow, generally needs 60d to cover with bacterium wood, and has part juggle not produce Phellinus sporophore after cultivation, surviving rate less than 50%, average per unit area yield 58g/ section; However, the Phellinus product that mulberry grows is called superfine product, deeply by consumers.Therefore, we still continue to inquire into and further investigation employing mulberry section cultivation Phellinus, wish the output that can improve mulberry section cultivation Phellinus as early as possible.
3 discuss
Found by experiment in cultivation: Phellinus bacterium is the higher type fungi of middle temperature, fruiting body differentiation temperature, between 25 DEG C-35 DEG C, continues more than 35 DEG C and less than 18 DEG C and all can not break up.Meanwhile, Phellinus bacterium also belongs to alternating temperature firm type fungi, and the row that thermal stimulation is conducive to Phellinus sporophore becomes.
Opening shape and size affect shape and the formation of Phellinus mushroom entity.Find through test, adopt ring section wood designated port (only the not de-bag of standardized bar seam, can ventilate, not dehydration), favourable fruiting is best Ways of fruiting.Square aperture or circular open easily make Phellinus ear dentification time lag, and its possible cause is large owing to exposing aerial mycelia area, competitive strong between mycelia, and are unfavorable for the formation of Phellinus sporophore, and the Phellinus sporophore profile now formed is bad.
Relative air humidity is also one of important factor affecting Phellinus bacterium fruit-body formation, and relative air humidity is too low can accelerate the evaporation of Phellinus mushroom solid object surface moisture, and Medium Culture moisture is run off in a large number, even makes Phellinus fruit body primordium withered and dead.Relative air humidity is excessive, easily makes again respiration be obstructed, and suppresses Phellinus sporophore growth to be grown.Because hot and humid culture condition causes Phellinus bacterium wood to suffer infecting of Neurospora, so will note observing at any time mycelia colour-change in bacterium bag, find that living contaminants should be isolated, immediately in case infect other bacterium bag.Phellinus should control between 85%-95% in fruiting stage air relative humidity, and temperature controls at 25 DEG C, and this conditions favouring Phellinus growth, fruiting is good.
Ventilation also can not be ignored the formation of Phellinus sporophore, and Medium Culture anoxic can suppress the respiration of phellinus liteus, and make mycelial growth slow, mycelia is weak, and time serious, phellinus liteus stops growing, even dead.Phellinus fruiting at least ventilates 2 times every day in stage, each 30min.If Phellinus sporophore stage oxygen is not enough, easily causes Phellinus sporophore that a large amount of deformity occurs, affect Phellinus grade.
Phellinus does not need light at vegetative stage, darkroom constant temperature culture.After the former base of Phellinus is formed, should be stimulated with suitable scattered light, the differentiation of Phellinus fruit body primordium can be accelerated.Phellinus liteus also can form fruit body primordium in dark surrounds, but Phellinus fruit-body color is more shallow, for faint yellow; Giving the Phellinus sporophore quality stimulated with scattered light better, is deep yellow.
Describe above, just the specific embodiment of the present invention, various illustrating is not construed as limiting flesh and blood of the present invention.
Claims (4)
1. a Phellinus mother culture media, is characterized in that being made up of the raw material of following weight: glucose 20g, soy peptone 2g, agar 18g, potassium primary phosphate 0.46g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, water 1000mL.
2. a Phellinus pedigree seed culture medium, is characterized in that being made up of the raw material of following weight: toothed oak wood chip 77%, wheat bran 10%, Semen Maydis powder 10%, glucose 2%, gypsum 1%.
3. a Phellinus Cultivar culture medium, is characterized in that being made up of the raw material of following weight: toothed oak wood chip 50%, cotton seed hulls 39%, wheat bran 10%, gypsum 1%.
4. the short segment wood cultivated method of Phellinus, is characterized in that step is as follows:
(1) fruit body tissue separation method is adopted to be separated Phellinus starter kind: to carry out surface sterilization process to picking up from the wild Phellinus sporophore in Changbaishan area, hooking up a fritter Phellinus sporophore and being put on inclined-plane mother culture media; Be placed in 27 DEG C of-30 DEG C of thermostat containers and cultivate, treat to grow milk yellow mycelia around sporophore tissue block, being transplanted to new test tube at colony edge picking tip mycelium transplants on mother culture media inclined-plane, 27 DEG C of-30 DEG C of constant temperature culture, 10d mycelia can cover with test tube, so repeatedly transplant 2-3 time, obtained strains is as mother's kind; Described mother culture media is: glucose 20g, soy peptone 2g, agar 18g, potassium primary phosphate 0.46g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, water 1000mL;
(2) Phellinus Primary spawn: pedigree seed culture medium is toothed oak wood chip 77%, wheat bran 10%, Semen Maydis powder 10%, glucose 2%, gypsum 1%; In 28 DEG C of thermostatic chambers, darkroom is cultivated;
(3) Phellinus cultivar is cultivated: Cultivar culture medium is toothed oak wood chip 50%, cotton seed hulls 39%, wheat bran 10%, gypsum 1%; In 28 DEG C of thermostatic chambers, darkroom is cultivated;
(4) preparation of short segment wood cultivated material:
Toothed oak wood is selected to make bacterium rod, for subsequent use after autoclaving; Described bacterium rod selects toothed oak wood species as segment wood cultivated material, small-end diameter 4-8cm, is cut into the billet that 18-20cm is long, cleave in two, be combined into the Duan Mu Bales of about 15cm, load in low pressure polyethylene bacterium bag, one deck wood chip wheat bran skin nutrilite is filled at juggle two, wood chip, wheat bran ratio are 3:1, poach, ties sack, section wood bag is pricked twice simultaneously, what Duan Muyu plastics bag was fitted is comparatively tight, for subsequent use after autoclaving;
(5) inoculation of bacterium rod is cultivated with bacteria developing period:
By aseptic technique, the Phellinus cultivation strain selecting mycelial growth vigorous, adopts two inoculation method, inoculum size 20%, with bacterial classification capping charge level for degree;
Sending out culture temperature between bacterium incubation period is 28 DEG C, and shading is cultivated, and ventilates every day 1 time, incubation time 35-40d;
(6) the sporophore growth growth period cultivates:
When mycelia color in bacterium rod have pale yellow become deep yellow time, at bacterium rod both sides lateral incision mouth, each bacterium bag can open two mouths respectively in bacterium bag both sides;
Bacterium wood buries soil cultivation: first make cultivation bed on booth ground, covered with plastic film on canopy, sunshade net or straw screen or mat shade, and designated port standing tree buries soil cultivation; Described cultivation bed can make three bacterium beds by the booth that 6m is wide, middle bacterium bedside 2m, each 1.2m of both sides bacterium bed, and stay 60cm effect road between bed, 20cm waterways is respectively stayed on canopy both sides; Described designated port standing tree buries soil cultivation and refers to first by dark 6cm, seeding row spacing 10cm × 10cm digs pit, again by 6cm place ring cutting at the bottom of the distance bag of bacterium bag one end, at the bottom of taking off bag, standing tree is put in hole, sandy soil in training, and then with blade, bacterium bag ring is drawn two cuttves, cut plastic film for degree, only mark twice fruiting mouth or seam;
Fruit body development period management: when mycelia color from pale yellow look enters the former base differentiation phase in bacterium rod, now adopt the method for day and night temperature, namely daytime culture temperature 28 DEG C, nocturnal temperature reduces to 20 DEG C, processes 3d continuously; Relative air humidity controls between 85%-95%; Give scattered light to irradiate;
The fruit body development stage, growth temperature 25 DEG C, relative air humidity remains between 85%-95%, ventilates every day 2 times, each 30min; Scattered light irradiates;
Sporophore is gathered: Phellinus sporophore answers 2-3 to gather once, adopt stay greatly little.
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