CN103891523B - A kind of Inonotus obliquus artificial cultivation method - Google Patents
A kind of Inonotus obliquus artificial cultivation method Download PDFInfo
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Abstract
The invention discloses a kind of Inonotus obliquus artificial cultivation method, 1) separation of Inonotus obliquus bacterial strain, purifying; 2) select carbon-free basal medium, without nitrogen basal medium, take maltose as the medium of carbon source, take sucrose as the medium of carbon source, analysis for soybean powder is nitrogenous source, and C/N ratio 20:1, pH are 7, and temperature is 30 DEG C; 3) Inonotus obliquus artificial domesticating cultivation.The present invention can form Inonotus obliquus sporophore.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of Inonotus obliquus artificial cultivation method.
Background technology
Inonotus obliquus has very high medical value.(1) antitumor, antitumaous effect: the extract of Inonotus obliquus all has result for the treatment of to liver cancer, lung cancer, cancer of the stomach, cervical carcinoma etc.; its polysaccharide and amino acid can play the effect protected with promoting to body immune system; improve the immunity of human body, maintain the vitality of human normal cell and metabolism.(2) prevent and treat diabetes: its polysaccharose product (beta glucan, heteroglycan and albumen composition) can strengthen the glycometabolism process in the function of islet cells, balanced human, thus reduce blood sugar, and 3 ~ 48 hours can be maintained.(3) acquired immune deficiency syndrome (AIDS), antivirus action is prevented and treated: β in extract components, 3-D-glucan has obvious inhibitory action to SARS class infective virus, and the lignin derivative of high molecular weight in water soluble ingredient can impedance HIV-I protease activity so that be used for the treatment of acquired immune deficiency syndrome (AIDS).(4) antioxidant and anti-aging: the catechol in Inonotus obliquus has strong antioxidant action, and has protective effect to gene.(5) other effects: the American-European countries such as Finland, Canada application Inonotus obliquus alcohol high standard extract possesses effect of enhancing human body immunity to treat influenza.Inonotus obliquus is the cleaning agent of blood, obvious therapeutic action is had to food poisoning, gastrointestinal dysfunction, hepatitis, ephritis, hypertension, appetite, alleviating pain can also be increased and improve allergic constitution, market there are the tea that Inonotus obliquus is special and medicinal wine always, also it are made aerosol and be used for the treatment of various disease.
Along with going deep into active substance research in large-scale integration of drinking and medicinal herbs fungi, the medical value of Inonotus obliquus, nutritive value and economic worth have caused the extensive concern of the people, become the popular development field of industry-by-industry.Multidisciplinary synthesis exploitation should be studied in different field such as basic theory, clinical practice, developments simultaneously.Fuscoporia obliqua polysaccharide, triterpene, Polyphenols natural anti-cancer drugs material can as the primary raw materials of health medicine, healthy food, health drink, microcapsules, its natural colouring matter can be applied in the deep processing of food additives, tealeaves, cosmetic industry, in addition, Inonotus obliquus also will have very wide application prospect in interdisciplinary researchs such as tobacco business, textile industry, feedstuff industry and product development.
Wild Inonotus obliquus price very expensive because of the ratio of this wood decay fungi in birch woods approximately only have ten thousand/, due to a large amount of collections, make wild Inonotus obliquus resource increasingly exhausted, therefore the culture technique of Inonotus obliquus becomes important field of research.
At present, because the grow mechanism understanding of people to the fruit body of Inonotus obliquus is not enough, the condition of artificial creation and the microorganism environment difference in the Nature is very large, therefore cause the certain difficulty of domestication and cultivation, the domestication about inonotus obliquus sclerotium has had certain achievement and scale.But up to now, there is no the precedent forming fruit body, also there is the difficult problem that some urgent needs will be captured in inonotus obliquus cultivation technology, prior art can only promote the generation of sclerotium, and does not form fruit body.
Summary of the invention
The object of the invention is to the defect overcoming the existence of above-mentioned technology, a kind of Inonotus obliquus artificial cultivation method is provided, optimize cultivation formula, according to the biological characteristic of wild Inonotus obliquus, utilize intermittent warming, successfully promote the formation of fruit body.Its concrete technical scheme is:
A kind of Inonotus obliquus artificial cultivation method, comprises the steps:
1) separation of Inonotus obliquus bacterial strain, purifying: first use the calcium hypochlorite supernatant of 1: 14 to carry out surface sterilization to fruit body, middle about 5mm blockage tissue is got again with the tweezers of high-temperature sterilization, moved the central authorities being connected to PDA culture medium flat plate, during cutting tissue, inoculation 1d is placed in 25 DEG C of constant temperature darkrooms and cultivates, check strain growth situation at any time, the contaminated flat board of timely eliminating, cover with after flat board until mycelia, proceed to and PDA slant tube medium is preserved purebred, the refrigerator being then placed in 4 DEG C after covering with saves backup;
2) medium based on carbon-free medium, nitrogen-free agar, the carbon source filtering out Inonotus obliquus mycelial growth is sucrose, and nitrogenous source is analysis for soybean powder, and C/N ratio 20: 1, pH is 7, and temperature is 30 DEG C;
3) Inonotus obliquus artificial domesticating cultivation:
On PDA medium, under 35 DEG C of conditions, cultivate 15d, cultivate 20d under placing it in room temperature condition, on medium, position to the right, center has grown Inonotus obliquus sporophore;
Culture medium for cultivating: birch wood chip 78%, analysis for soybean powder 20%, gypsum 1%, sucrose 1%, the moisture content of medium is 65%, cultivates 30 days under 35 DEG C of incubators, and under room temperature, opening does not place 20-50d, creates fruit body equally at cultivation sack and base portion.
Further preferably, the nucleotide sequence of described Inonotus obliquus bacterial strain is as shown in SEQIDNO.1.
Compared with prior art, beneficial effect of the present invention is: the present invention can form Inonotus obliquus sporophore, after large-scale production, will produce obvious economic benefit.
Accompanying drawing explanation
Fig. 1 be different nitrogen sources to Inonotus obliquus growing state, Fig. 1 a is analysis for soybean powder, Fig. 1 b is dusty yeast, Fig. 1 c is peptone, Fig. 1 d is glycine, Fig. 1 e is ammonium sulfate, Fig. 1 f is the impact grown Inonotus obliquus without nitrogen;
Fig. 2 be different C/N ratio to Inonotus obliquus growing state, Fig. 2 a is C/N ratio 5: 1, Fig. 2 b is 10: 1, Fig. 2 c is 20: 1, Fig. 2 d is 30: 1, Fig. 2 e is 40: 1, Fig. 2 f be 50: 1 on Inonotus obliquus growth impacts;
Fig. 3 is that different pH values is to Inonotus obliquus growing state; Fig. 3 a-Fig. 3 f is followed successively by the impact of pH3,8,5,6,7 and 4 on Inonotus obliquus growth;
Fig. 4 is the fruit body that synthetic medium produces.Fig. 4 a-Fig. 4 c is followed successively by fruit-body formation situation, fruit-body formation situation, mycelial growth situation.
Embodiment
Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is described in more detail.
Test material be the irregular warty of pitchy in 2011 with picking up from May, 2012 in the wild Inonotus obliquus sporophore in Daxing ' anling, heilongjiang Huzhong Nature Reserve, and surperficial hard, quality is firmly crisp, and fruit body outside is more dry, cut out tangent plane place feel moistening with axe.Bacterial context suberin, has ambiguous ring grain, khaki.The mycelia cultivated on the bacterium colony of 7 days in PDA solid plate is dense, neat, grows from center to surrounding; Bacterium colony center color is yellowish-brown, thin out gradually from the center to edge yellow, peripheral pure white; Dull and stereotyped back side bacterium colony has obvious light brown ring grain.Bacterial classification through separation and purification goes on PDA inclined-plane, cultivates 5 days full packages, bacterium colony initial stage pure white, after gradually become light yellow, turn yellow brown gradually.Bacterial classification receives PDA liquid nutrient medium, and at cabinet type constant-temperature table medium speed 120rpm, cultivate 7 days at 28 DEG C, bacterium ball edge is spininess; Mycelia band on shaking flask wall is thicker, and part is shaken to cause drop in liquid; In shaking flask, liquid color gradually becomes dark brown by yellowish, and the hyphal knot come off is combined into circular or avette mycelium pellet.Observe under an optical microscope after the vegetative mycelium compressing tablet of picking flat board, mycelia is half arc, transparent, thread.Nutrient hypha multi-branched, visible mycelia tabula, without clamp connection.The object fragment length that the ITS-rDNA of wild strain obtains through order-checking is 761bp, comprises 18SrDNA partial sequence, 306bpITS1 sequence, 160bp5.8SrDNA total length, 243bpITS2 total length, and part 28SrDNA sequence.Through BLAST comparison on GeneBank, have the similitude of 99% with the ITS-rDNA sequence of Inonotusobliquus, combining form is observed, and determines that adopted wild-type strain is Inonotus obliquus.Sequence has submitted the EST storehouse on NCBI to, and its number of logging on GenBank is: KC312697 (GI:461725870).
By the analysis of BLAST software, the ITS-rDNA sequence downloaded in the sequence record Inonotus obliquus bacterial strain and GenBank carries out cluster analysis, the bacterial classification (targetedsequence) that the present invention gathers is nearest with InonotusobliquusisolateFS656163 and InonotusobliquusstrainMDJCBS88 autoploidy, these two is Inonotus obliquus different strains, and with InonotuslinteusstrainPL08121 and InonotusrickiiisolatePF241 affinity farthest, all belong to fine pore fungi belong to.) Inonotus obliquus sampling position is in the natural one-tenth of white birch, overmature forest, Forestry University forest Pathology Lab is separated and obtains northeastward.Strain separating adopts conventional organization isolation technics, first uses the calcium hypochlorite supernatant of 1: 14 to carry out surface sterilization to fruit body.Get middle about 5mm blockage tissue with the tweezers of high-temperature sterilization again, moved the central authorities being connected to PDA culture medium flat plate.During cutting tissue, inoculation 1d is placed in 25 DEG C of constant temperature darkrooms and cultivates, and checks strain growth situation at any time, gets rid of contaminated flat board in time.Cover with after flat board until mycelia, then proceed to and PDA slant tube medium is preserved purebred, the refrigerator being then placed in 4 DEG C after covering with saves backup.
Carbon-free basal medium: glycine 1g, KH
2pO
40.5g, magnesium sulfate 0.25g, agar 10g, water 500ml.
Without nitrogen basal medium: glucose 10g, potassium dihydrogen phosphate 0.5g, magnesium sulfate 0.25g, agar 10g, water 500ml.
Carbon source: according to bought experimental drug specification being learnt glucose, maltose, fructose, sucrose, soluble starch phosphorus content are respectively 40%, 42.1%, 40%, 42.1%, 44.4%.With glucose, maltose, fructose, sucrose, soluble starch, as carbon source, distinguishes 10g, 9.5g, 10g, 9.5g, 9.01g adds in carbon-free basal medium, separately establishes a sugar-free to compare, and repeats 6 totally 36 process, at 25 DEG C, under dark condition, adopt plating method, quantitatively inoculate at culture dish central authorities 0.5mm card punch, be inverted in 25 DEG C of insulating boxs and cultivate.Latter 3rd day of inoculation, with every 24 the hour record colony growth diameters of right-angled intersection method, and note mycelia color, lawn thickness, colony edge feature, after this adopt Dunca inspection in Spssl1.5 software to carry out significance of difference analysis, in Table 1-2. to mycelial growth rate
Nitrogenous source: according to bought experimental drug specification being learnt the nitrogen content of glycine, yeast leaching powder, peptone, ammonium sulfate is respectively 18.65%, 8 ~ 9%, 14.5%, 21%.Analysis for soybean powder and corn gluten meal matter content are 32.7% and 8.7%.According to Kjeldahl's method, in any biological sample, every gram of nitrogen is equivalent to 6.25g protein, and the nitrogen content that can calculate analysis for soybean powder and corn flour is 5.232% and 1.392%.At 25 DEG C, under dark condition, take dusty yeast 1.10g respectively with analytical balance, analysis for soybean powder 1.7823g, peptone 0.6431g, glycine 0.5g, ammonium sulfate 0.444g, add without in nitrogen basal medium, separately establish one to compare without nitrogen.Inoculation is tested with carbon source process above with measuring method, and experimental result is in Table 1-3.
C/N ratio: 25 DEG C, under no light condition, the content of glucose in immobilizing foundation medium, the content of conversion analysis for soybean powder, is mixed with C/N ratio and is respectively 5: 1,10: 1,20: 1,30: Isosorbide-5-Nitrae 0: 1, the medium of 50: 1.Often process 0.5mm card punch is quantitatively connected in the medium of different C/N ratio respectively, repeats 6 totally 36 process, and inoculation is tested with carbon source process above with measuring method, and experimental result is in Table 1-4.
PH value: on medium PDA, regulate the pH value of medium to be respectively with sodium hydrogen phosphate-citrate buffer solution: 3,4,5,6,7,8, repeat 6 totally 36 process, inoculation is tested with carbon source process above with measuring method, and experimental result is in Table 1-5.
Temperature: on medium PDA, access Inonotus obliquus, establishes 5 DEG C respectively, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, repeats 7 totally 42 process, and inoculation is tested with carbon source process above with measuring method, and experimental result is in Table 1-6.
Screening is female plants: different acquisition place or the bacterial strain of different acquisition time carried out purifying, expand and cultivate with for subsequent use.PDA medium is inoculated, and method is with the experiment of carbon source process above, and outcome measurement is also as the experiment of carbon source process above, and experimental result is in Table 1-6.
Composts or fertilisers of cultivating screens: female kind, pedigree seed culture medium all adopt conventional PDA medium; Two bacterial strains that the activity that cultivated species obtains in using the female kind of screening to test is good, get its long PDA inclined-plane of 1-2cm as cultivated species, with silver birch wood chip for major ingredient, analysis for soybean powder, corn flour, white granulated sugar, gypsum, lime etc. become cultivated species composts or fertilisers of cultivating for auxiliary material by different ratio, carry out cultivation bag Screening of compost formula, each formula composition and numbering are in Table 1-1.By conventional to major ingredient and auxiliary material spice pack, select specification to be the polyethylene knuckle bag of 17 × 33cm, pack is placed on sterilizing 120min in high-pressure sterilizing pot (121 DEG C).Each formula 30 process of each bacterial strain, amounts to 180 bacterium bags.Inoculation is placed on light culture in 25 DEG C of constant incubators, and bacterium bag situation is observed in timing, and clean out contaminated bacterium bag at any time, observe mycelial growth situation after hyphal colonization, size, density, number appear in sclerotium.Best culture material formula is selected to carry out next step domestication research by data analysis and growing way situation.
Table 1 composts or fertilisers of cultivating component (siccative %)
Results and analysis
Different carbon source is on the impact of Inonotus obliquus mycelial growth
Carbon source is the most important nutrition of microorganism, and it is as the important component part of carbohydrate and protein, provides the energy and form the skeleton of cell for fungi.Inonotus obliquus belongs to organic nutrition type heterotroph on nutrient type, direct carbonic acid gas can not be utilized to carry out photosynthesis and maintain normal vital movement.Under natural growthing condition, the carbohydrate lignin of plant origin, starch and various carbohydrate are that Inonotus obliquus provides a large amount of abundant carbon sources.Nearly all fungi can absorb polysaccharide as starch, dextrin, cellulose, and monose is if glucose, fructose, galactose etc., disaccharides are as maltose etc.
Table 2 different carbon source is on the impact of Inonotus obliquus cultural hypha characteristic
Can observe out from experimental result: with maltose be carbon source medium Inonotus obliquus hyphal development the fastest, be secondly soluble starch and sucrose; But three's difference is not remarkable; Fructose is then beneficial to most the growth of Inonotus obliquus.Take sucrose as the medium of carbon source, dark in the middle of colony colour, both sides are shallow, and quality is thick.This and Jiang Yuji etc. (2004) think that ground rice is the conclusion difference to some extent of optimum carbon source, and the author thinks and causes the reason of this difference to be the difference due to bacterial strain and the difference with reference to sample.In Jiang Yuji literary composition, bacterial strain sample is from Korea S, and reference standard is dry mycelial weight; On the other hand, this experimental result should have been demonstrate,proved Chen Yanqiu (2005) and thought that maltose and glucose are the conclusion of optimum carbon source.Visible, different bacterial strains and different reference standards affect to some extent on experimental result.
Different nitrogen sources is on the impact of Inonotus obliquus mycelial growth
The effect of nitrogenous source is only second to nitrogenous source concerning growth of microorganism, and it is the essential element forming necessary protein, vitamin, amino acid and nucleic acid in organism.Nitrogen is divided into organic nitrogen and inorganic nitrogen.Inorganic nitrogen commonly uses nitrate and ammonium salt, and the availability of general fungi to ammonium salt is higher, has then showed adaptation to the utilization of nitrate, and some fungi can absorb nitrite, and some then cannot.Organic nitrogen commonly uses peptone, beef extract, yeast extract.Nitrogen content in each timber is little, in 0.03 ~ 0.1% scope, slightly dislikes not enough to the growth of mushroom.
Table 3 different nitrogen sources is on the impact of Inonotus obliquus cultural hypha characteristic
As can be seen from the table: the medium taking analysis for soybean powder as nitrogenous source, mycelial growth rate is the fastest, secondly be dusty yeast, both do not have difference on duna detection level, but take analysis for soybean powder as darker, the intensive stalwartness of mycelia color of nitrogenous source and lawn is thicker, experimentally result is learnt thus: analysis for soybean powder is optimum nitrogen source; Secondly be dusty yeast, peptone, glycine, ammonium sulfate.The mycelial growth of Inonotus obliquus must have nitrogenous source, grows hardly without Inonotus obliquus under the condition of nitrogen; Inonotus obliquus is greater than the utilizing status to inorganic nitrogen to the utilizing status of organic nitrogen in mycelium growth vigor and growth rate, the most difficultly utilizes ammonium sulfate.Chen Yanqiu etc. (2005) experiment think the nitrogenous source of different disposal on its mycelial growth to affect difference then not remarkable; Jiang Yuji etc. (2004) think that wheat skin is optimum nitrogen source; Difference the author of this experimental result thinks relevant with criterion with the difference of experimental strain.On the other hand, the optimum nitrogen source result of study of Zhang Liqiu (2006) is similarly analysis for soybean powder.This phenomenon again demonstrates different strains and has this viewpoint of different growth characteristics.
Different C/N ratio is on the impact of Inonotus obliquus mycelial growth
In timber, lignocellulose accounts for more than 90% of dry weight of wood, containing carbon rich, nitrogen pole plaque is weary, its C/N ratio can reach 350: 1 ~ 1250: 1, the C/N ratio of white birch is subject to the impact of the factors such as kind, geographical position, the age of tree, and the low nitrogen level that contains in white birch wood is the factor that restriction Inonotus obliquus is surely grown and grown.At present, shortage is compared in the research of researcher to Inonotus obliquus optimum carbon nitrogen ratio.
The different C/N ratio of table 4 is on the impact of Inonotus obliquus cultural hypha characteristic
Can obtain from experimental result: the colony colour of all 6 ratios does not have marked difference, be all yellowish-brown substantially.From mycelia day growth speed, time C/N ratio is 5: 1, Inonotus obliquus mycelial growth is the fastest, 10: 1,20: 1,30: 1,40: 1 and 50: 1 all do not have marked difference in 0.01 level, front four do not have marked difference in 0.05 level yet, and 40: 1 and 50: 1 do not have marked difference in 0.05 level.From lawn thickness and colony edge color, the lawn of 20: 1 is the thickest, and colony edge is the most neat, and mycelia is healthy and strong, and the suitableeest C/N ratio is 20: 1.The C/N ratio of more than 20: 1 is higher, and bacterium colony is more sparse, and colony edge is more irregular.Less than 20: 1 then C/N ratio is larger, and bacterium colony is more healthy and stronger.Obtain thus, C/N ratio is less, and the growth rate of mycelia is faster; But from lawn thickness and mycelia robustness: be optimum carbon nitrogen ratio at 20: 1.This studies from dry mycelial weight angle the optimum carbon nitrogen ratio obtained with Jiang Yuji (2005) is to be consistent at 20: 1.
Different pH values is on the impact of Inonotus obliquus mycelial growth
In fungus lives, adaptable soda acid scope is comparatively wide, generally can survive when pH value is 3 ~ 9.But for most bacterial classification, the optimum pH of their growths is generally slant acidity, is between 5.5 ~ 6.5.The existence of the fungi of most harm timber no matter in its spore development stage, or during mycelial growth, is suitable for living in (Lv Wenhua etc., 2002 in acid medium all significantly; Zhou Huiming, 1991).
Table 1-5 different pH values is on the impact of Inonotus obliquus cultural hypha characteristic
Under the initial pH of difference, the colony diameter of Inonotus obliquus and mycelium growth vigor present different upgrowth situations.Time pH is 7, mycelial growth rate is the fastest, and secondly pH is 8; PH be 6 with pH be 5 time there is no significant difference, time pH is 4 or 3, mycelial growth rate is the poorest; Bacterium colony quality aspect, pH is higher, and quality is thinner.Can draw from experimental result: Inonotus obliquus the most suitable growth acid-base value is 7, and acid condition is conducive to the formation of lawn, and neutrality contributes to the growth rate of mycelia.This result and Ji is grand, Chen Yanqiu, Zhang Liqiu, Jiang Yuji
[21-24]etc. coming to the same thing.
Different temperatures is on the impact of Inonotus obliquus mycelial growth
Inonotus obliquus cold resistance is comparatively strong, and can survive in the low temperature environment of-40 DEG C, the sprouting of basidiospore, the whole growth cycle of mycelia are all subject to the Limitation and Confinement of temperature.Under optimum temperature, its vital movement is also vigorous.And the vital movement under minimum or maximum temperature is the slowest.
Table 6 different temperatures is on the impact of Inonotus obliquus cultural hypha characteristic
Can obtain from experimental result: temperature mycelial growth rate in 30 DEG C is the fastest, and be secondly 25 DEG C and 20 DEG C, both do not have marked difference in 0.05 and 0.01 level, and secondly in 15 DEG C, 35 DEG C of growth rate slowly; Grow hardly at 10 DEG C; Can find out that Inonotus obliquus growth preference temperature is 30 DEG C, grow hardly at too high or too low temperature.In the formation of mycelia pigment, 30 DEG C then do not have marked difference with 25 DEG C, and temperature is more than 30 DEG C or all have influence on the formation of Inonotus obliquus pigment lower than 25 DEG C.The result of study that this conclusion and Korea S state-run farm institute open the profound chief of a tribe and Chen Yanqiu conforms to.
The generation of Inonotus obliquus sporophore on synthetic medium
Exploring different temperatures in the impact experiment of Inonotus obliquus, by on Inonotus obliquus hz3 bacterial strain access PDA medium, cultivate 15d under 35 DEG C of conditions after, cultivate 20d under placing it in room temperature condition, on medium, position to the right, center has grown Inonotus obliquus sporophore.This is that first time discovery produces the situation of fruit body under culture conditions in Inonotus obliquus correlative study.Inonotus obliquus sporophore under natural conditions, generally rarely found, completely calm under live standing tree bark, or on the sapwood of dead wood, be difficult to be separated from matrix.Situation about producing on synthetic medium about Inonotus obliquus sporophore is both at home and abroad reported without scholar.Inonotus obliquus sporophore is found first herein in synthetic medium matter, by measuring to obtain the thick about 5mm of fruit body, light brown; Tube, than short under natural conditions, only has 3 ~ 5mm, the front end of tube also in cracking state, large under bacterium boring ratio natural conditions, every millimeter reaches 1 ~ 3, is generally shallow white or bright yellow, rear dimmed brown; Bristle (see Fig. 4) can be observed under microscope.
Contrast with the fruit body on synthetic medium under table 7 natural conditions
Bacterial strain screening result
There is certain difference in the activity of separate sources ground, the bacterial strain of different acquisition time.Through field acquisition, indoor strain isolation cultivates and purifying agaric obtains 5 bacterial strains, and each bacterial strain experimental result is as follows: hz3 and th1 mycelial growth growing way is better, and next is followed successively by th2, hz1, hz2.Colony colour aspect does not then have significant difference.
Table 8 different strains is on the impact of Inonotus obliquus cultural hypha characteristic
Using the PDA test tube slant of the hz3 of preservation as cultivated species, be seeded in different cultivation culture material formula respectively, cultivate 30 days under 35 DEG C of incubators, 3 formulas cover with mycelia substantially, mycelial growth rate is large without significant difference, but formula 1 and 2 sclerotium output are less, and head is smaller, and sclerotium generation place mainly concentrates on above bacterium bag; And 3 bacterium bag late growing stage speed of filling a prescription are fast, product sclerotium number is many and maximum, wherein maximum sclerotium diameter 4.52cm.By above-mentioned bacterium bag at room temperature not opening place 20-50d, cultivation sack and base portion create fruit body equally.This is the report first planting material producing Inonotus obliquus sporophore.
The above; be only the present invention's preferably embodiment; protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the simple change of the technical scheme that can obtain apparently or equivalence are replaced and are all fallen within the scope of protection of the present invention.
Claims (2)
1. an Inonotus obliquus artificial cultivation method, is characterized in that, comprises the steps:
1) separation of Inonotus obliquus bacterial strain, purifying: first use the calcium hypochlorite supernatant of 1: 14 to carry out surface sterilization to fruit body, middle about 5mm blockage tissue is got again with the tweezers of high-temperature sterilization, moved the central authorities being connected to PDA culture medium flat plate, during cutting tissue, inoculation 1d is placed in 25 DEG C of constant temperature darkrooms and cultivates, check strain growth situation at any time, the contaminated flat board of timely eliminating, cover with after flat board until mycelia, proceed to and PDA slant tube medium is preserved purebred, the refrigerator being then placed in 4 DEG C after covering with saves backup;
2) medium based on carbon-free medium, nitrogen-free agar, the carbon source filtering out Inonotus obliquus mycelial growth is sucrose, and nitrogenous source is analysis for soybean powder, and C/N ratio 20: 1, pH is 7, and temperature is 30 DEG C;
3) Inonotus obliquus artificial domesticating cultivation:
On PDA medium, under 35 DEG C of conditions, cultivate 15d, cultivate 20d under placing it in room temperature condition, on medium, position to the right, center has grown Inonotus obliquus sporophore;
Culture medium for cultivating: birch wood chip 78%, analysis for soybean powder 20%, gypsum 1%, sucrose 1%, the moisture content of medium is 65%, cultivates 30 days under 35 DEG C of incubators, and under room temperature, opening does not place 20-50d, creates fruit body equally at cultivation sack and base portion.
2. Inonotus obliquus artificial cultivation method according to claim 1, is characterized in that, the nucleotide sequence of described Inonotus obliquus bacterial strain is as shown in SEQIDNO.1.
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| CN108293618B (en) * | 2016-08-18 | 2020-07-03 | 天明制药股份有限公司 | Inonotus obliquus mycelium culture flask device |
| CN106748043A (en) * | 2016-12-05 | 2017-05-31 | 江苏师范大学 | A kind of promotion Inonotus obliquus orient the culture medium prescription and method to form fructification |
| CN112106589A (en) * | 2020-07-09 | 2020-12-22 | 上海华泓生物科技有限公司 | Cultivation method for sclerotium of pseudoecological inonotus obliquus |
| CN114747423B (en) * | 2022-05-19 | 2023-05-30 | 山西运奕道生物科技有限公司 | Inonotus obliquus culture method and culture product thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| TWI631214B (en) * | 2017-04-27 | 2018-08-01 | 天明製藥股份有限公司 | Culture method for improving amount of triterpenoids in inonotus obliquus |
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