CN103981105A - Method for cultivating cordyceps militaris with high yield of cordycepic acid - Google Patents
Method for cultivating cordyceps militaris with high yield of cordycepic acid Download PDFInfo
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- 241001264174 Cordyceps militaris Species 0.000 title claims abstract description 96
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 title claims abstract description 30
- 239000001963 growth medium Substances 0.000 claims abstract description 51
- 239000000725 suspension Substances 0.000 claims abstract description 21
- 238000005286 illumination Methods 0.000 claims description 36
- 230000001954 sterilising effect Effects 0.000 claims description 30
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 28
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 claims description 26
- 238000011218 seed culture Methods 0.000 claims description 24
- 238000012258 culturing Methods 0.000 claims description 20
- 241000190633 Cordyceps Species 0.000 claims description 19
- 230000003203 everyday effect Effects 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 17
- 239000000843 powder Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 16
- 229910019142 PO4 Inorganic materials 0.000 claims description 16
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 16
- 240000006394 Sorghum bicolor Species 0.000 claims description 16
- 235000011684 Sorghum saccharatum Nutrition 0.000 claims description 16
- 235000013312 flour Nutrition 0.000 claims description 16
- 239000008103 glucose Substances 0.000 claims description 16
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 16
- 239000010452 phosphate Substances 0.000 claims description 16
- 239000011591 potassium Substances 0.000 claims description 16
- 229910052700 potassium Inorganic materials 0.000 claims description 16
- 239000001888 Peptone Substances 0.000 claims description 14
- 108010080698 Peptones Proteins 0.000 claims description 14
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 14
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 14
- 235000019319 peptone Nutrition 0.000 claims description 14
- 238000004659 sterilization and disinfection Methods 0.000 claims description 13
- 235000011201 Ginkgo Nutrition 0.000 claims description 10
- 235000008100 Ginkgo biloba Nutrition 0.000 claims description 10
- 230000004913 activation Effects 0.000 claims description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 7
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 claims description 7
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 238000003958 fumigation Methods 0.000 claims description 6
- 244000194101 Ginkgo biloba Species 0.000 claims 1
- 241000233866 Fungi Species 0.000 abstract description 8
- 239000002609 medium Substances 0.000 abstract description 6
- 238000004040 coloring Methods 0.000 abstract 2
- 230000003213 activating effect Effects 0.000 abstract 1
- 238000012364 cultivation method Methods 0.000 abstract 1
- -1 0.02 part Chemical compound 0.000 description 11
- 230000009286 beneficial effect Effects 0.000 description 10
- 241000218628 Ginkgo Species 0.000 description 9
- 244000061456 Solanum tuberosum Species 0.000 description 6
- 235000002595 Solanum tuberosum Nutrition 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 239000012535 impurity Substances 0.000 description 4
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical group C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 3
- 240000001307 Myosotis scorpioides Species 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 238000004737 colorimetric analysis Methods 0.000 description 3
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical class CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 3
- 229940066779 peptones Drugs 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 239000012533 medium component Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 206010008132 Cerebral thrombosis Diseases 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 201000001429 Intracranial Thrombosis Diseases 0.000 description 1
- 244000062793 Sorghum vulgare Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
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- 230000003035 anti-peroxidant effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
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- 235000013305 food Nutrition 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
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- 208000014674 injury Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
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- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
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- 230000001256 tonic effect Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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Abstract
The invention relates to the field of edible fungi cultivation, and particularly relates to a method for cultivating cordyceps militaris with high yield of cordycepic acid. The method comprises the following steps: activating and cultivating the culture of the cordyceps militaris to prepare a spore suspension; inoculating the spore suspension into a cordyceps militaris seed medium for cultivation according to a volume percent of 3-8% so as to obtain a seed solution; inoculating the seed solution into a cordyceps militaris growth medium for sequentially carrying out hypha growth cultivation, hypha coloring cultivation and sporocarp cultivation so as to obtain cordyceps militaris sporocarp. According to the method, the spore suspension is inoculated into the cordyceps militaris seed medium for cultivation so as to obtain the seed solution, the seed solution is inoculated into the cordyceps militaris growth medium for sequentially carrying out hypha growth cultivation, hypha coloring cultivation and sporocarp cultivation; the cultivation method is simple and has a short cultivation cycle; the obtained cordyceps militaris sporocarp has high content of cordycepic acid, and the cordycepic acid extracted from the cordyceps militaris sporocarp has high purity.
Description
Technical field
The present invention relates to field of edible fungus culture, in particular to a kind of high yield cordycepic acid cordyceps culturing method.
Background technology
Cordyceps militaris (L.) Link. claims again Cordyceps militaris, Cordyceps militaris (L.) Link., is the entomogenous fungi complex body that fungi autoeciousness above forms in the polypide of the insects such as lepidopteran, is a kind of important and nutritious prod with tonic effect.The multiple efficacy of a drug effects such as that Cordyceps militaris (L.) Link. has is antitumor, anti-inflammatory, it has extensively been used as invigorant and medicinal fungus in East Asia Region, and the Ministry of Health of China is formally classified as new resource food on March 16th, 2009.
Cordycepic acid is one of main active ingredient of Cordyceps militaris (L.) Link., and the height of cordycepic acid content is one of main standard of weighing Chinese caterpillar fungus quality, it is generally acknowledged that the pharmaceutical use of the Chinese caterpillar fungus that cordycepic acid content is high is high.Cordycepic acid can prevent and treat cerebral thrombosis, hematencephalon, myocardial infarction, long-term exhaustion; Anti-liver tissue fibrosis, anti peroxidation of lipid, strengthens because the enhancing immunologic function of Chinese caterpillar fungus makes the detoxification of liver simultaneously, thereby can effectively protect liver cell.
At present, Cordyceps militaris (L.) Link. cordycepic acid content mainly concentrates on by liquid submerged fermentation and improves, but, the mycelium for Cordyceps militaris (L.) Link. that submerged fermentation obtains, the content of the cordycepic acid of its generation is generally in 3-4.5% left and right, and the culture that fermentation obtains not only comprises Cordyceps militaris (L.) Link. self, also contains some substratum residues, affects the purity of cordycepic acid.
Summary of the invention
The object of the present invention is to provide Cordyceps militaris (L.) Link. seed culture medium, Cordyceps militaris (L.) Link. growth medium and a kind of high yield cordycepic acid cordyceps culturing method, to solve the above problems.
A kind of Cordyceps militaris (L.) Link. seed culture medium is provided in an embodiment of the present invention, by weight, comprise following composition: glucose 8-12 part, peptone 2-4 part, yeast powder 4-6 part, potassium primary phosphate 0.02-0.06 part, magnesium sulfate 0.02-0.06 part, sodium-chlor 4-6 part, vitaminB10 .05-0.15 part, sorghum flour 8-12 part, water 800-1200 part.
A kind of Cordyceps militaris (L.) Link. growth medium is also provided in an embodiment of the present invention, by weight, comprise following composition: sorghum flour 24-26 part, ginkgo leaf powder 4-6 part, peptone 0.04-0.06, seminose 0.04-0.08 part, potassium primary phosphate 0.02-0.04 part, magnesium sulfate 0.02-0.03 part, glucose 0.04-0.06 part, vitaminB10 .0005-0.001 part, water 30-40 part.
A kind of high yield cordycepic acid cordyceps culturing method, comprises the following steps:
(a), by Cordyceps militaris spawn activation culture, making concentration is 1-3 × 10
7the spore suspension of CFU/ml;
(b) by described spore suspension, the ratio taking percent by volume as 3-8% is inoculated in Cordyceps militaris (L.) Link. seed culture medium claimed in claim 1 and cultivates, and obtains seed liquor;
(c) described seed liquor is inoculated in Cordyceps militaris (L.) Link. growth medium claimed in claim 2 and carries out successively mycelial growth cultivation, the cultivation of mycelia annesl and sporophore cultivation, obtain Cordyccps-militaris-(L.)-link. Sporophore.
Preferably, in described step (b), culture condition is: 25-30 DEG C, 240-260rpm/min shaking culture.
Preferably, in described step (b), cultivate 3-5d, obtain described seed liquor.
Preferably, by after 20-30 times of described seed liquor dilution, inoculate taking percent by volume as 5-8%.
Preferably, in described step (c), described mycelial growth is cultivated and is cultivated between the cultivation of having sterilized, and culture condition is: temperature 20-25 DEG C, and illumination every day 2-4h, intensity of illumination is 250-300Lux, humidity 60-70% cultivates 5-7d.
Preferably, in described step (c), the culture condition that described mycelia annesl is cultivated is: temperature 14-18 DEG C, and humidity 70-80%, the 1.5-2.5h that ventilates every day, illumination 8-12h, intensity of illumination is 250-300Lux, cultivates 3-5d.
Preferably, in described step (c), the culture condition that described sporophore is cultivated is: ventilative cultivation, temperature is 20 DEG C ± 2 DEG C, and humidity is 80-90%, and 1.5-2.5h ventilates every day, blue light illumination 12-14h, intensity of illumination is 250-300Lux, and being cultured to sporophore length is 7-8cm, gathers and obtains described Cordyccps-militaris-(L.)-link. Sporophore.
Preferably, described sterilization is: the water-bath that the container that peracetic acid soln is housed is placed in to 75-85 DEG C is fumigated sterilizing between described cultivation, and fumigation time is 90-100min, and every cubic metre of space consumption of described Peracetic Acid is 3-3.2g; During sterilization, cultivate room temperature and be not less than 20 DEG C, humidity is 60%-80%.
The high yield cordycepic acid cordyceps culturing method that the embodiment of the present invention provides, spore suspension is provided in the Cordyceps militaris (L.) Link. seed culture medium providing in the present invention and cultivates, and obtains seed liquor; Seed liquor is inoculated in Cordyceps militaris (L.) Link. growth medium provided by the invention, carry out successively mycelial growth cultivation, mycelia annesl cultivates and sporophore is cultivated, cultural method is simple, culture cycle is short, the Cordyccps-militaris-(L.)-link. Sporophore cordycepic acid content obtaining is high, and the purity of the cordycepic acid extracting with this Cordyccps-militaris-(L.)-link. Sporophore is high.
Embodiment
Below by specific embodiment, the present invention is described in further detail.
A kind of Cordyceps militaris (L.) Link. seed culture medium is provided in an embodiment of the present invention, by weight, comprise following composition: glucose 8-12 part, peptone 2-4 part, yeast powder 4-6 part, potassium primary phosphate 0.02-0.06 part, magnesium sulfate 0.02-0.06 part, sodium-chlor 4-6 part, vitaminB10 .05-0.15 part, sorghum flour 8-12 part, water 800-1200 part.
Further, by weight, comprise following composition: 1000 parts of glucose 9-11 parts, peptone 3-4 part, yeast powder 4-5 part, potassium primary phosphate 0.04-0.05 part, magnesium sulfate 0.04-0.05 part, sodium-chlor 4-5 part, vitaminB10 .10-0.15 part, sorghum flour 9-10 part, water.
The Cordyceps militaris (L.) Link. seed culture medium comprehensive nutrition obtaining, medium component concentration and proportioning are suitable; The spore suspension of Cordyceps militaris (L.) Link. is seeded to basal culture medium, and part spore grows into mycelium, and spore vitality is vigorous, and spore count amplification is fast, and obtains part mycelium, and the seed liquor obtaining contains spore and mycelium, is beneficial to subsequent growth.
A kind of Cordyceps militaris (L.) Link. growth medium is also provided in an embodiment of the present invention, by weight, comprise following composition: sorghum flour 24-26 part, ginkgo leaf powder 4-6 part, peptone 0.04-0.06, seminose 0.04-0.08 part, potassium primary phosphate 0.02-0.04 part, magnesium sulfate 0.02-0.03 part, glucose 0.04-0.06 part, vitaminB10 .0005-0.001 part, water 30-40 part.
Further, by weight, comprise following composition: sorghum flour 25-26 part, ginkgo leaf powder 4-5 part, peptone 0.04-0.05, seminose 0.06-0.07 part, potassium primary phosphate 0.03-0.04 part, magnesium sulfate 0.02-0.03 part, glucose 0.04-0.05 part, vitaminB10 .0005-0.001 part, water 30-40 part.
The Cordyceps militaris (L.) Link. growth medium comprehensive nutrition obtaining, medium component concentration and proportioning are suitable; The seed liquor of Cordyceps militaris (L.) Link. is seeded to basal culture medium, is beneficial to spore and is grown to mycelium, obtain more mycelium, be beneficial to and obtain more Cordyccps-militaris-(L.)-link. Sporophore, and the Cordyccps-militaris-(L.)-link. Sporophore cordycepic acid content obtaining is high.
Wherein, the sorghum flour relating in Cordyceps militaris (L.) Link. seed culture medium and Cordyceps militaris (L.) Link. growth medium was that 20-30 mesh sieve obtains, and ginkgo leaf powder is that dry Folium Ginkgo powder is pulverized as the big or small 0.5-1.5mm × 0.5-1.5mm of being of sheet.The sorghum flour and the ginkgo leaf powder that use this granularity, it can be evenly distributed in sterilization process, the composition profiles homogeneous in the substratum obtaining after sterilizing.
The composition respectively Cordyceps militaris (L.) Link. seed culture medium and Cordyceps militaris (L.) Link. growth medium being contained separately takes rear mixing, all adopts autoclave sterilization, is specially 121 DEG C, and sterilizing 20-30min, is cooled to room temperature after sterilizing, obtains being directly used in the substratum of cultivating bacterial strain.
A kind of high yield cordycepic acid cordyceps culturing method, comprises the following steps:
(a), by Cordyceps militaris spawn activation culture, making concentration is 1-3 × 10
7the spore suspension of CFU/ml;
(b) by described spore suspension, the ratio taking percent by volume as 3-8% is inoculated in Cordyceps militaris (L.) Link. seed culture medium claimed in claim 1 and cultivates, and obtains seed liquor;
(c) described seed liquor is inoculated in Cordyceps militaris (L.) Link. growth medium claimed in claim 2 and carries out successively mycelial growth cultivation, the cultivation of mycelia annesl and sporophore cultivation, obtain Cordyccps-militaris-(L.)-link. Sporophore.
Adopt Cordyceps militaris (L.) Link. seed culture medium provided by the invention and Cordyceps militaris (L.) Link. growth medium to carry out the cultivation of Cordyceps militaris (L.) Link., cultural method is simple, and culture cycle is short, and cost is low, and the Cordyccps-militaris-(L.)-link. Sporophore cordycepic acid content obtaining is high, in addition, available technology adopting liquid fermentation and culture Cordyceps militaris (L.) Link., the major part of Cordyceps militaris (L.) Link. is in fermented liquid, the materials such as the secretory product that self produces are mingled in fermented liquid, thereby the Cordyceps militaris (L.) Link. the obtaining composition that contains fermented liquid, and Cordyceps militaris (L.) Link. growth medium provided by the invention is solid medium, adopt solid culture Cordyceps militaris (L.) Link., the Cordyccps-militaris-(L.)-link. Sporophore that harvesting obtains, impurity while having avoided adopting liquid fermentation and culture in substratum is sneaked into the defect in Cordyccps-militaris-(L.)-link. Sporophore, the Cordyceps militaris (L.) Link. impurity obtaining still less, therefore, the purity of the cordycepic acid that follow-up Cordyceps militaris (L.) Link. is extracted is higher.
Cordyceps militaris spawn activation culture, the Cordyceps militaris spawn using is purchased from Chinese Typical Representative culture collection center, preserving number is CCTCC M2013056, Cordyceps militaris spawn is inoculated in to potato slant medium, cultivate 6-8d for 27 DEG C ± 2 DEG C, obtain the spore of Cordyceps militaris (L.) Link., with spore under the aseptic washing that contains 0.05% tween 80, making concentration is 1-3 × 10
7the spore suspension of CFU/ml.Wherein, potato slant medium composition comprises potato 100g, glucose 10g, potassium primary phosphate 1g, agar 16g, water 1000ml; Compound method is: get the potato 100g having removed the peel, be cut into small pieces, the 1000m1 that adds water, boils 20min, by 4-6 layer filtered through gauze, then supply dehydration to 1000m1, add agar 16g to dissolve, and then add glucose 10g, potassium primary phosphate 1g, packing test tube, 121 DEG C of autoclaving 25-30min, prepare potato slant medium.Adopt potato slant medium activation Cordyceps militaris spawn, method is simple, and the spore obtaining is many.It is 1-3 × 10 that spore is made concentration by the sterilized water of the tween 80 of employing 0.05%
7the spore suspension of CFU/ml, the spore suspension spore making is evenly distributed; Then the ratio taking percent by volume as 3-8% is seeded to Cordyceps militaris (L.) Link. seed culture medium, and inoculum size is moderate, is beneficial at Cordyceps militaris (L.) Link. seed culture medium Fast Growth.
Preferably, in described step (b), culture condition is: 25-30 DEG C, 240-260rpm/min shaking culture.Empirical tests, culture temperature is 25-30 DEG C, Cordyceps militaris (L.) Link. grows fast in Cordyceps militaris (L.) Link. seed culture medium; Rotating speed 240-260rpm/min, in the situation that not damaging Cordyceps militaris (L.) Link. spore, increase dissolved oxygen amount, be beneficial to spore and mycelial growth, Cordyceps militaris (L.) Link. seed culture medium can be mixed, affect spore and mycelial growth to prevent local Cordyceps militaris (L.) Link. seed culture medium nutritive ingredient deficiency simultaneously, therefore, the spore fast growth of Cordyceps militaris (L.) Link. under this culture condition, the mycelium of formation is more, saves incubation time.
Preferably, in described step (b), cultivate 3-5d, obtain described seed liquor.Cultivate 3-5d, the seed liquor thalline content obtaining is high, and it is vigorous to grow.
Preferably, by after 20-30 times of described seed liquor dilution, inoculate taking percent by volume as 5-8%.In Cordyceps militaris (L.) Link. growth medium, inoculate appropriate seed liquor, be beneficial to and obtain Cordyccps-militaris-(L.)-link. Sporophore; In order to prevent that inoculum size difference is large and to inoculate rear distributing inhomogeneity, first seed liquor is diluted, then inoculate taking percent by volume as 5-8%, can obtain the Cordyceps militaris (L.) Link. growth medium that seed liquor is evenly distributed, be beneficial to the growth of thalline.
Preferably, in described step (c), described mycelial growth is cultivated and is cultivated between the cultivation of having sterilized, and culture condition is: temperature 20-25 DEG C, and illumination every day 2-4h, intensity of illumination is 250-300Lux, humidity 60-70% cultivates 5-7d.Under this culture condition, thalli growth is quick, and culture vessel covers with mycelia, and culture cycle is short.
Preferably, in described step (c), the culture condition that described mycelia annesl is cultivated is: temperature 14-18 DEG C, and humidity 70-80%, the 1.5-2.5h that ventilates every day, illumination 8-12h, intensity of illumination is 250-300Lux, cultivates 3-5d.Under this culture condition, thalli growth is quick, starts to form the former base of millet shape in media surface, and mycelium annesl is effective, and culture cycle is short.
Preferably, in described step (c), the culture condition that described sporophore is cultivated is: ventilative cultivation, temperature is 20 DEG C ± 2 DEG C, and humidity is 80-90%, and 1.5-2.5h ventilates every day, blue light illumination 12-14h, intensity of illumination is 250-300Lux, and being cultured to sporophore length is 7-8cm, gathers and obtains described Cordyccps-militaris-(L.)-link. Sporophore.
Ventilative cultivation is: Cordyceps militaris (L.) Link. growth medium 35-45ml puts into triangle culturing bottle (200-250ml), and its mouthful seals with sealing film, then cultivates, by prick 3-5 hole on sealing film, the cultivation of breathing freely, is beneficial to circulation of air, is beneficial to culture growth.And under this culture condition, thalli growth is quick, and culture cycle is short, the Cordyccps-militaris-(L.)-link. Sporophore cordycepic acid content obtaining is high.
In addition, the Cordyceps militaris (L.) Link. that solid culture obtains, directly cutting can obtain, and has prevented that the impurity in substratum from sneaking in the Cordyccps-militaris-(L.)-link. Sporophore obtaining, and the Cordyceps militaris (L.) Link. impurity obtaining is still less.The purity of the cordycepic acid that therefore, follow-up Cordyceps militaris (L.) Link. is extracted is higher.
Preferably, described sterilization is: the water-bath that the container that peracetic acid soln is housed is placed in to 75-85 DEG C is fumigated sterilizing between described cultivation, and fumigation time is 90-100min, and every cubic metre of space consumption of described Peracetic Acid is 3-3.2g.Space to Cordyceps militaris (L.) Link. growth carries out disinfection, and to prevent that it is subject to disease, and prevents other microbiological contamination; Adopt Peracetic Acid to sterilize between cultivating to stifling, sterilization is thorough, and to the injury of Cordyceps militaris (L.) Link. culture nothing itself, in the training period, Cordyceps militaris (L.) Link. culture growth conditions is good, and the Cordyccps-militaris-(L.)-link. Sporophore Cordyccps-militaris-(L.)-link. Sporophore cordycepic acid content pollution-free and that obtain obtaining is high.
The high yield cordycepic acid cordyceps culturing method that the embodiment of the present invention provides, spore suspension is provided in the Cordyceps militaris (L.) Link. seed culture medium providing in the present invention and cultivates, and obtains seed liquor; Seed liquor is inoculated in Cordyceps militaris (L.) Link. growth medium provided by the invention, carry out successively mycelial growth cultivation, mycelia annesl cultivates and sporophore is cultivated, cultural method is simple, culture cycle is short, the Cordyccps-militaris-(L.)-link. Sporophore cordycepic acid content obtaining is high, and the purity of the cordycepic acid extracting with this Cordyccps-militaris-(L.)-link. Sporophore is high.
Embodiment 1
By weight, take following composition: 8 parts of glucose, 2 parts of peptones, 4 parts of yeast powders, 0.02 part of potassium primary phosphate, 0.02 part, magnesium sulfate, 4 parts, sodium-chlor, vitaminB10 .05 part, 8 parts of sorghum flours, 800 parts, water, then 121 DEG C, sterilizing 25min, after sterilizing, be cooled to room temperature, obtain Cordyceps militaris (L.) Link. seed culture medium;
By weight, take following composition: 30 parts of 24 parts of sorghum flours, 4 parts of ginkgo leaf powders, peptone 0.04,0.04 part of seminose, 0.02 part of potassium primary phosphate, 0.02 part, magnesium sulfate, 0.04 part of glucose, vitaminB10 .0005 part, water, then 121 DEG C, sterilizing 25min, after sterilizing, be cooled to room temperature, obtain Cordyceps militaris (L.) Link. growth medium;
High yield cordycepic acid cordyceps culturing method, comprises the following steps:
(a), by Cordyceps militaris spawn activation culture, making concentration is 1 × 10
7the spore suspension of CFU/ml;
(b) spore suspension is cultivated as 8% ratio is inoculated in Cordyceps militaris (L.) Link. seed culture medium taking percent by volume, culture condition is: 25 DEG C, 240rpm/min shaking culture, cultivates 3d, obtains seed liquor;
(c) by after 20 times of seed liquor dilutions, taking percent by volume as 5% seed liquor is inoculated in Cordyceps militaris (L.) Link. growth medium, carry out successively mycelial growth cultivation, mycelia annesl cultivates and sporophore is cultivated;
Mycelial growth is cultivated and is cultivated between the cultivation of having sterilized, and culture condition is: 20 DEG C of temperature, and illumination every day 4h, intensity of illumination is 250Lux, humidity 60% is cultivated 7d;
The culture condition that mycelia annesl is cultivated is: 14 DEG C of temperature, and humidity 70%, the 1.5h that ventilates every day, illumination 12h, intensity of illumination is 250Lux, cultivates 3d;
After mycelia annesl has been cultivated, culture is carried out to sporophore cultivation, culture condition is: ventilative cultivation, temperature is 20 DEG C ± 2 DEG C, and humidity is 80%, and 1.5h ventilates every day, blue light illumination 14h, intensity of illumination is 250Lux, and being cultured to sporophore length is 7-8cm, gathers and obtains described Cordyccps-militaris-(L.)-link. Sporophore;
Wherein, between cultivating, carry out disinfection in the following ways: the water-bath that the container that peracetic acid soln is housed is placed in to 75 DEG C is fumigated sterilizing between described cultivation, and fumigation time is 100min, and every cubic metre of space consumption of described Peracetic Acid is 3-3.2g; During sterilization, cultivate room temperature and be not less than 20 DEG C, humidity is 60%-80%.
It is golden yellow that the Cordyccps-militaris-(L.)-link. Sporophore obtaining is, the content of colorimetric method for determining cordycepic acid, and obtaining cordycepic acid content is 18%.
Embodiment 2
By weight, take following composition: 10 parts of glucose, 3 parts of peptones, 5 parts of yeast powders, 0.04 part of potassium primary phosphate, 0.04 part, magnesium sulfate, 5 parts, sodium-chlor, vitaminB10 .10 part, 10 parts of sorghum flours, 1000 parts, water, then 121 DEG C, sterilizing 30min, after sterilizing, be cooled to room temperature, obtain Cordyceps militaris (L.) Link. seed culture medium;
By weight, take following composition: 35 parts of 25 parts of sorghum flours, 5 parts of ginkgo leaf powders, peptone 0.05,0.06 part of seminose, 0.03 part of potassium primary phosphate, 0.03 part, magnesium sulfate, 0.05 part of glucose, vitaminB10 .001 part, water, then 121 DEG C, sterilizing 30min, after sterilizing, be cooled to room temperature, obtain Cordyceps militaris (L.) Link. growth medium;
High yield cordycepic acid cordyceps culturing method, comprises the following steps:
(a), by Cordyceps militaris spawn activation culture, making concentration is 2 × 10
7the spore suspension of CFU/ml;
(b) spore suspension is cultivated as 5% ratio is inoculated in Cordyceps militaris (L.) Link. seed culture medium taking percent by volume, culture condition is: 27 DEG C, 250rpm/min shaking culture, cultivates 4d, obtains seed liquor;
(c) by after 25 times of seed liquor dilutions, taking percent by volume as 6% seed liquor is inoculated in Cordyceps militaris (L.) Link. growth medium, carry out successively mycelial growth cultivation, mycelia annesl cultivates and sporophore is cultivated;
Mycelial growth is cultivated and is cultivated between the cultivation of having sterilized, and culture condition is: 22 DEG C of temperature, and illumination every day 3h, intensity of illumination is 280Lux, humidity 65% is cultivated 6d;
The culture condition that mycelia annesl is cultivated is: 16 DEG C of temperature, and humidity 75%, the 2.0h that ventilates every day, illumination 10h, intensity of illumination is 280Lux, cultivates 4d;
After mycelia annesl has been cultivated, culture is carried out to sporophore cultivation, culture condition is: ventilative cultivation, temperature is 20 DEG C ± 2 DEG C, and humidity is 85%, and 2.0h ventilates every day, blue light illumination 13h, intensity of illumination is 280Lux, and being cultured to sporophore length is 7-8cm, gathers and obtains described Cordyccps-militaris-(L.)-link. Sporophore;
Wherein, between cultivating, carry out disinfection in the following ways: the water-bath that the container that peracetic acid soln is housed is placed in to 80 DEG C is fumigated sterilizing between described cultivation, and fumigation time is 95min, and every cubic metre of space consumption of described Peracetic Acid is 3-3.2g.
It is golden yellow that the Cordyccps-militaris-(L.)-link. Sporophore obtaining is, the content of colorimetric method for determining cordycepic acid, and obtaining cordycepic acid content is 20%.
Embodiment 3
By weight, take following composition: 12 parts of glucose, 4 parts of peptones, 6 parts of yeast powders, 0.06 part of potassium primary phosphate, 0.06 part, magnesium sulfate, 6 parts, sodium-chlor, vitaminB10 .15 part, 12 parts of sorghum flours, 1200 parts, water, then 121 DEG C, sterilizing 35min, after sterilizing, be cooled to room temperature, obtain Cordyceps militaris (L.) Link. seed culture medium;
By weight, take following composition: 40 parts of 26 parts of sorghum flours, 6 parts of ginkgo leaf powders, peptone 0.06,0.08 part of seminose, 0.04 part of potassium primary phosphate, 0.03 part, magnesium sulfate, 0.06 part of glucose, vitaminB10 .001 part, water, then 121 DEG C, sterilizing 35min, after sterilizing, be cooled to room temperature, obtain Cordyceps militaris (L.) Link. growth medium;
High yield cordycepic acid cordyceps culturing method, comprises the following steps:
(a), by Cordyceps militaris spawn activation culture, making concentration is 3 × 10
7the spore suspension of CFU/ml;
(b) spore suspension is cultivated as 3% ratio is inoculated in Cordyceps militaris (L.) Link. seed culture medium taking percent by volume, culture condition is: 30 DEG C, 260rpm/min shaking culture, cultivates 5d, obtains seed liquor;
(c) by after 30 times of seed liquor dilutions, taking percent by volume as 8% seed liquor is inoculated in Cordyceps militaris (L.) Link. growth medium, carry out successively mycelial growth cultivation, mycelia annesl cultivates and sporophore is cultivated;
Mycelial growth is cultivated and is cultivated between the cultivation of having sterilized, and culture condition is: 25 DEG C of temperature, and illumination every day 2h, intensity of illumination is 300Lux, humidity 70% is cultivated 5d;
The culture condition that mycelia annesl is cultivated is: temperature 14-18 DEG C, and humidity 70-80%, the 2.5h that ventilates every day, illumination 8h, intensity of illumination is 300Lux, cultivates 5d;
After mycelia annesl has been cultivated, culture is carried out to sporophore cultivation, culture condition is: ventilative cultivation, temperature is 20 DEG C ± 2 DEG C, and humidity is 90%, and 2.5h ventilates every day, blue light illumination 12h, intensity of illumination is 300Lux, and being cultured to sporophore length is 7-8cm, gathers and obtains described Cordyccps-militaris-(L.)-link. Sporophore;
Wherein, between cultivating, carry out disinfection in the following ways: the water-bath that the container that peracetic acid soln is housed is placed in to 85 DEG C is fumigated sterilizing between described cultivation, and fumigation time is 90min, and every cubic metre of space consumption of described Peracetic Acid is 3-3.2g.
It is golden yellow that the Cordyccps-militaris-(L.)-link. Sporophore obtaining is, the content of colorimetric method for determining cordycepic acid, and obtaining cordycepic acid content is 25%.
High yield cordycepic acid cordyceps culturing method provided by the invention, cultural method is simple, adopts different substratum to cultivate at different cultivation stages, to guarantee the vigor of bacterial classification and the demand of adaptation cultivation target; Culture condition, the envrionment conditions of each cultivation stage are strictly controlled, and are beneficial to standardization and stdn that Cordyceps militaris (L.) Link. is cultivated and manages; The Cordyccps-militaris-(L.)-link. Sporophore Cordyceps militaris (L.) Link. cordycepic acid content obtaining is high, is 18-25%, and the purity of the cordycepic acid extracting with this Cordyccps-militaris-(L.)-link. Sporophore is high.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. Cordyceps militaris (L.) Link. seed culture medium, it is characterized in that, by weight, comprise following composition: glucose 8-12 part, peptone 2-4 part, yeast powder 4-6 part, potassium primary phosphate 0.02-0.06 part, magnesium sulfate 0.02-0.06 part, sodium-chlor 4-6 part, vitaminB10 .05-0.15 part, sorghum flour 8-12 part, water 800-1200 part.
2. Cordyceps militaris (L.) Link. growth medium, it is characterized in that, by weight, comprise following composition: sorghum flour 24-26 part, ginkgo leaf powder 4-6 part, peptone 0.04-0.06, seminose 0.04-0.08 part, potassium primary phosphate 0.02-0.04 part, magnesium sulfate 0.02-0.03 part, glucose 0.04-0.06 part, vitaminB10 .0005-0.001 part, water 30-40 part.
3. a high yield cordycepic acid cordyceps culturing method, is characterized in that, comprises the following steps:
(a), by Cordyceps militaris spawn activation culture, making concentration is 1-3 × 10
7the spore suspension of CFU/ml;
(b) by described spore suspension, the ratio taking percent by volume as 3-8% is inoculated in Cordyceps militaris (L.) Link. seed culture medium claimed in claim 1 and cultivates, and obtains seed liquor;
(c) described seed liquor is inoculated in Cordyceps militaris (L.) Link. growth medium claimed in claim 2 and carries out successively mycelial growth cultivation, the cultivation of mycelia annesl and sporophore cultivation, obtain Cordyccps-militaris-(L.)-link. Sporophore.
4. high yield cordycepic acid cordyceps culturing method according to claim 3, is characterized in that, in described step (b), culture condition is: 25-30 DEG C, 240-260rpm/min shaking culture.
5. high yield cordycepic acid cordyceps culturing method according to claim 4, is characterized in that, in described step (b), cultivates 3-5d, obtains described seed liquor.
6. high yield cordycepic acid cordyceps culturing method according to claim 5, is characterized in that, in described step (c), by after 20-30 times of described seed liquor dilution, inoculates taking percent by volume as 5-8%.
7. high yield cordycepic acid cordyceps culturing method according to claim 6, it is characterized in that, in described step (c), described mycelial growth is cultivated and is cultivated between the cultivation of having sterilized, culture condition is: temperature 20-25 DEG C, and illumination every day 2-4h, intensity of illumination is 250-300Lux, humidity 60-70%, cultivates 5-7d.
8. high yield cordycepic acid cordyceps culturing method according to claim 7, it is characterized in that, in described step (c), the culture condition that described mycelia annesl is cultivated is: temperature 14-18 DEG C, humidity 70-80%, the 1.5-2.5h that ventilates every day, illumination 8-12h, intensity of illumination is 250-300Lux, cultivates 3-5d.
9. high yield cordycepic acid cordyceps culturing method according to claim 8, it is characterized in that, in described step (c), the culture condition that described sporophore is cultivated is: ventilative cultivation, and temperature is 20 DEG C ± 2 DEG C, humidity is 80-90%, the 1.5-2.5h that ventilates every day, blue light illumination 12-14h, intensity of illumination is 250-300Lux, being cultured to sporophore length is 7-8cm, gathers and obtains described Cordyccps-militaris-(L.)-link. Sporophore.
10. high yield cordycepic acid cordyceps culturing method according to claim 7, it is characterized in that, described sterilization is: the water-bath that the container that peracetic acid soln is housed is placed in to 75-85 DEG C is stifling to sterilizing between described cultivation, fumigation time is 90-100min, and every cubic metre of space consumption of described Peracetic Acid is 3-3.2g; During sterilization, cultivate room temperature and be not less than 20 DEG C, humidity is 60%-80%.
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| CN104686208A (en) * | 2015-03-26 | 2015-06-10 | 北京美拉德中药饮片科技有限公司 | Usage and method for detection, processing and artificial cultivation of tussah cordyceps militaris taken as traditional Chinese medicinal materials |
| WO2015180520A1 (en) * | 2014-05-29 | 2015-12-03 | 熊艳 | Method for cultivating high-cordyceps-acid cordyceps militaris |
| CN105418192A (en) * | 2015-12-04 | 2016-03-23 | 正源堂（天津）生物科技有限公司 | Cordyceps militaris strain culture medium and preparation method thereof |
| CN105503375A (en) * | 2015-12-05 | 2016-04-20 | 正源堂（天津）生物科技有限公司 | Efficient cordyceps militaris strain medium |
| CN106867791A (en) * | 2017-04-13 | 2017-06-20 | 刘勇 | The preparation method of Cordceps militaris health liquor |
| CN107347446A (en) * | 2017-07-25 | 2017-11-17 | 南江宏信生物科技有限公司 | The cultural method of Cordceps militaris |
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| CN113348962A (en) * | 2021-06-03 | 2021-09-07 | 徐州工程学院 | Artificial cultivation method for improving cordycepic acid content in cordyceps sobolifera sporocarp |
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| CN103981105B (en) * | 2014-05-29 | 2016-02-17 | 熊艳 | The cultural method of a kind of high yield cordycepic acid Cordyceps militaris (L.) Link. |
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| US3892629A (en) * | 1972-02-10 | 1975-07-01 | Ici Ltd | Process for growing a fungus |
| CN1772876A (en) * | 2005-09-30 | 2006-05-17 | 袁有宝 | Formulation of aweto culture medium and oxygenating culture method of aweto |
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| WO2015180520A1 (en) * | 2014-05-29 | 2015-12-03 | 熊艳 | Method for cultivating high-cordyceps-acid cordyceps militaris |
| CN104686208A (en) * | 2015-03-26 | 2015-06-10 | 北京美拉德中药饮片科技有限公司 | Usage and method for detection, processing and artificial cultivation of tussah cordyceps militaris taken as traditional Chinese medicinal materials |
| CN105418192A (en) * | 2015-12-04 | 2016-03-23 | 正源堂（天津）生物科技有限公司 | Cordyceps militaris strain culture medium and preparation method thereof |
| CN105503375A (en) * | 2015-12-05 | 2016-04-20 | 正源堂（天津）生物科技有限公司 | Efficient cordyceps militaris strain medium |
| CN106867791A (en) * | 2017-04-13 | 2017-06-20 | 刘勇 | The preparation method of Cordceps militaris health liquor |
| CN107347446A (en) * | 2017-07-25 | 2017-11-17 | 南江宏信生物科技有限公司 | The cultural method of Cordceps militaris |
| CN110915542A (en) * | 2019-12-04 | 2020-03-27 | 山西万海澳生物科技有限责任公司 | Cordyceps militaris cultivation method with stable high-content cordycepic acid |
| CN112913572A (en) * | 2019-12-06 | 2021-06-08 | 宏润生物科技股份有限公司 | Method for culturing cordyceps militaris, culture product and pharmaceutical composition |
| CN112913572B (en) * | 2019-12-06 | 2022-08-02 | 陈衫润 | Method for culturing cordyceps militaris, culture product and pharmaceutical composition |
| JP2021132637A (en) * | 2020-02-24 | 2021-09-13 | 広東省科学院動物研究所 | Application of methylfarnesyl ester in promoting generation of germinated spores of cordy ceps sinensis sacc |
| JP7006988B2 (en) | 2020-02-24 | 2022-01-24 | 広東省科学院動物研究所 | Use of methyl farnesyl ester in promoting germination spore production of Cordyceps sinensis |
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