CN103988712A - High-yield polysaccharide cordyceps militaris cultivation method - Google Patents
High-yield polysaccharide cordyceps militaris cultivation method Download PDFInfo
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- CN103988712A CN103988712A CN201410234859.9A CN201410234859A CN103988712A CN 103988712 A CN103988712 A CN 103988712A CN 201410234859 A CN201410234859 A CN 201410234859A CN 103988712 A CN103988712 A CN 103988712A
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- 241001264174 Cordyceps militaris Species 0.000 title claims abstract description 154
- 150000004676 glycans Chemical class 0.000 title claims abstract description 43
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 43
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 43
- 238000012364 cultivation method Methods 0.000 title abstract 4
- 239000001963 growth medium Substances 0.000 claims abstract description 51
- 238000000034 method Methods 0.000 claims abstract description 28
- 239000000725 suspension Substances 0.000 claims abstract description 21
- 230000004913 activation Effects 0.000 claims abstract description 9
- 230000001954 sterilising effect Effects 0.000 claims description 33
- 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
- 235000013312 flour Nutrition 0.000 claims description 26
- 238000005286 illumination Methods 0.000 claims description 24
- 238000011218 seed culture Methods 0.000 claims description 24
- 235000013399 edible fruits Nutrition 0.000 claims description 20
- 239000000843 powder Substances 0.000 claims description 19
- 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 17
- 239000008103 glucose Substances 0.000 claims description 17
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- 239000000203 mixture Substances 0.000 claims description 16
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 16
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 16
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 16
- 238000004659 sterilization and disinfection Methods 0.000 claims description 16
- 239000001888 Peptone Substances 0.000 claims description 14
- 108010080698 Peptones Proteins 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 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
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- 241001052560 Thallis Species 0.000 description 3
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- 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
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- 229920001817 Agar Polymers 0.000 description 2
- 240000001307 Myosotis scorpioides Species 0.000 description 2
- 241001248610 Ophiocordyceps sinensis Species 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
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- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
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- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- KQLDDLUWUFBQHP-UHFFFAOYSA-N Cordycepin Natural products C1=NC=2C(N)=NC=NC=2N1C1OCC(CO)C1O KQLDDLUWUFBQHP-UHFFFAOYSA-N 0.000 description 1
- 241000190633 Cordyceps Species 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 235000002722 Dioscorea batatas Nutrition 0.000 description 1
- 235000006536 Dioscorea esculenta Nutrition 0.000 description 1
- 240000001811 Dioscorea oppositifolia Species 0.000 description 1
- 235000003416 Dioscorea oppositifolia Nutrition 0.000 description 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
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- 102000004895 Lipoproteins Human genes 0.000 description 1
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- 235000000640 Rosa roxburghii Nutrition 0.000 description 1
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- 230000001919 adrenal effect Effects 0.000 description 1
- 229960002478 aldosterone Drugs 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- SRBFZHDQGSBBOR-LECHCGJUSA-N alpha-D-xylose Chemical compound O[C@@H]1CO[C@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-LECHCGJUSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- OFEZSBMBBKLLBJ-BAJZRUMYSA-N cordycepin Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)C[C@H]1O OFEZSBMBBKLLBJ-BAJZRUMYSA-N 0.000 description 1
- OFEZSBMBBKLLBJ-UHFFFAOYSA-N cordycepine Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)CC1O OFEZSBMBBKLLBJ-UHFFFAOYSA-N 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
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- 230000007812 deficiency Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000019713 millet Nutrition 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Pest Control & Pesticides (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Mycology (AREA)
- Environmental Sciences (AREA)
- Mushroom Cultivation (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of edible fungi cultivation, in particular a high-yield polysaccharide cordyceps militaris cultivation method. The method includes the following steps that cordyceps militaris strains are subjected to activation cultivation, and spore suspension can be obtained; the spore suspension is inoculated to a cordyceps militaris seed medium with the volume percentage close to 3 percent to 8 percent for cultivation, and a seed solution is obtained; the seed solution is inoculated to a cordyceps militaris growth medium to be sequentially subjected to dark cultivation, hyphae color turning cultivation and fruiting body cultivation, and cordyceps militaris fruiting bodies are obtained. According to the high-yield cordyceps militaris cultivation method, the spore suspension is inoculated to the cordyceps militaris seed medium provided in the method for cultivation, and the seed solution is obtained; the seed solution is inoculated to the cordyceps militaris growth medium to be sequentially subjected to dark cultivation, hyphae color turning cultivation and fruiting body cultivation, the cultivation method is simple, short in cultivation period, the obtained cordyceps militaris fruiting bodies are high in cordyceps militaris polysaccharide content, and cordyceps militaris polysaccharide extracted from the cordyceps militaris fruiting bodies is high in purity.
Description
Technical field
The present invention relates to field of edible fungus culture, in particular to the cultural method of a kind of high polysaccharide Cordyceps militaris.
Background technology
Cordyceps militaris claims again northern Chinese caterpillar fungus, northern Chinese caterpillar Fungus, is the entomogenous fungi complex that fungi autoeciousness forms on the polypide of the insects such as Lepidoptera, is a kind of important and nutriment with tonic effect.The multiple efficacy of a drug effects such as that Cordyceps militaris has is antitumor, anti-inflammatory, it has extensively been used as invigorant and medicinal fungus in East Asia Region, and China Ministry of Public Health is formally classified as new resource food on March 16th, 2009.
The polysaccharide in Cordyceps militaris wherein, the polysaccharide mainly being formed by mannose, cordycepin, adenosine, galactose, arabinose, wood sugar essence, glucose, fucose.Experimental results show that, Cordyceps sinensis polysaccharide can improve immune function of human body, increasing leukocyte, improves respiratory system, can make suprarenal gland weight, blood plasma cortisol, aldosterone and suprarenal gland inner cholesterol content increase, there is the adrenal effect of promotion, suppress tumor growth, and there is antitumor, radioresistance, hypoglycemic and lipoprotein, cough-relieving, reduce phlegm, moistening lung and the pharmacological action such as delay senility, the clinical malignant tumour that has been used for the treatment of, therefore, Cordyceps sinensis polysaccharide has important value.
At present, Polysaccharides in Cultured Cordyceps militaris content mainly concentrates on by liquid deep layer fermenting and improves, and still, what submerged fermentation obtained is cordyceps mycelium, and it comprises outside Cordyceps militaris self also having some medium residues, affects the purity of Polysaccharides in Cultured Cordyceps militaris.
Summary of the invention
The object of the present invention is to provide the cultural method of Cordyceps militaris seed culture medium, Cordyceps militaris growth medium and a kind of high polysaccharide Cordyceps militaris, to solve the above problems.
Cordyceps militaris 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, dusty yeast 4-6 part, potassium dihydrogen phosphate 0.02-0.06 part, magnesium sulfate 0.02-0.06 part, sodium chloride 4-6 part, vitaminB10 .05-0.15 part, yam flour 8-12 part, water 800-1200 part.
Cordyceps militaris growth medium is also provided in an embodiment of the present invention, by weight, comprise following composition: corn flour 12-18 part, wheat powder 5-10 part, yam flour 3-8 part, dry Ribes burejense powder 2-5 part, peptone 0.02-0.05 part, potassium dihydrogen phosphate 0.02-0.04 part, magnesium sulfate 0.02-0.03 part, glucose 0.02-0.08 part, vitaminB10 .0005-0.001 part, water 30-40 part.
A cultural method for high polysaccharide Cordyceps militaris, comprises the following steps:
(a), by Cordyceps militaris spawn activation culture, making concentration is 1-3 * 10
7the spore suspension of CFU/ml;
(b) described spore suspension be take to ratio that percentage by volume is 3-8% and be inoculated in Cordyceps militaris seed culture medium claimed in claim 1 and cultivate, obtain seed liquor;
(c) described seed liquor is inoculated in Cordyceps militaris growth medium claimed in claim 2 secretly cultivate successively, mycelia annesl is cultivated and fruit body is cultivated, and obtains fruiting bodies of cordyceps militaris.
Preferably, in described step (b), condition of culture is: 25-30 ℃, 240-260rpm/min shaken cultivation.
Preferably, in described step (b), cultivate 3-5d, obtain described seed liquor.
Preferably, by described seed liquor dilution 20-30 doubly after, the percentage by volume of take is inoculated as 5-8%.
Preferably, in described step (c), described dark cultivation is cultivated between the cultivation of having sterilized, and condition of culture is: temperature 20-25 ℃, humidity 60-70%, cultivates 6-8d.
Preferably, in described step (c), the condition of culture that described mycelia annesl is cultivated is: temperature 15-20 ℃, and humidity 60-70%, the 1.5-2.5h that ventilates every day, illumination 8-12h, intensity of illumination is 250-300Lux, cultivates 5-8d.
Preferably, in described step (c), the condition of culture that described fruit body is cultivated is: ventilative cultivation, temperature is 20 ℃ ± 2 ℃, and humidity is 80-90%, and 1.5-2.5h ventilates every day, illumination 6-8h, intensity of illumination is 250-300Lux, and being cultured to fruit body length is 6-10cm, gathers and obtains described fruiting bodies of cordyceps militaris.
Preferably, described sterilization is: the water-bath that the container that peracetic acid soln is housed is placed in to 75-85 ℃ 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 ℃, humidity is 60%-80%.
The cultural method of the high polysaccharide Cordyceps militaris that the embodiment of the present invention provides, spore suspension is provided in the Cordyceps militaris seed culture medium providing in the present invention and cultivates, and obtains seed liquor; Seed liquor is inoculated in Cordyceps militaris growth medium provided by the invention secretly cultivate successively, mycelia annesl is cultivated and fruit body is cultivated, cultural method is simple, cultivation cycle is short, the fruiting bodies of cordyceps militaris Polysaccharides in Cultured Cordyceps militaris content obtaining is high, and the purity of the Polysaccharides in Cultured Cordyceps militaris extracting with this fruiting bodies of cordyceps militaris is high.
Embodiment
Below by specific embodiment, the present invention is described in further detail.
Cordyceps militaris 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, dusty yeast 4-6 part, potassium dihydrogen phosphate 0.02-0.06 part, magnesium sulfate 0.02-0.06 part, sodium chloride 4-6 part, vitaminB10 .05-0.15 part, yam flour 8-12 part, water 800-1200 part.
Further, by weight, comprise following composition: 1000 parts of glucose 9-10 parts, peptone 3-4 part, dusty yeast 5-6 part, potassium dihydrogen phosphate 0.03-0.05 part, magnesium sulfate 0.03-0.05 part, sodium chloride 4-5 part, vitaminB10 .05-0.15 part, yam flour 9-10 part, water.
The Cordyceps militaris seed culture medium comprehensive nutrition obtaining, medium component concentration and proportioning are suitable; The spore suspension of Cordyceps militaris 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.
Cordyceps militaris growth medium is also provided in an embodiment of the present invention, by weight, comprise following composition: corn flour 12-18 part, wheat powder 5-10 part, yam flour 3-8 part, dry Ribes burejense powder 2-5 part, peptone 0.02-0.05 part, potassium dihydrogen phosphate 0.02-0.04 part, magnesium sulfate 0.02-0.03 part, glucose 0.02-0.08 part, vitaminB10 .0005-0.001 part, water 30-40 part.
Further, by weight, comprise following composition: corn flour 14-16 part, wheat powder 6-8 part, yam flour 4-6 part, dry Ribes burejense powder 3-4 part, peptone 0.03-0.04 part, potassium dihydrogen phosphate 0.02-0.04 part, magnesium sulfate 0.02-0.03 part, glucose 0.03-0.06 part, vitaminB10 .0005-0.001 part, water 30-40 part.
The Cordyceps militaris growth medium comprehensive nutrition obtaining, medium component concentration and proportioning are suitable; The seed liquor of Cordyceps militaris is seeded to basal culture medium, is beneficial to spore and is grown to mycelium, obtain more mycelium, be beneficial to and obtain more fruiting bodies of cordyceps militaris, and the fruiting bodies of cordyceps militaris Polysaccharides in Cultured Cordyceps militaris content obtaining is high.
Wherein, the corn flour relating in Cordyceps militaris seed culture medium and Cordyceps militaris growth medium and the granularity of wheat powder are 1-3mm, yam flour and dry Ribes burejense powder are prepared in the following ways: fresh Chinese yam is dried, then pulverize, obtain the yam flour that granularity is 0.8-1.5mm; Fresh Rosa roxburghii is dried, go to pulverize after seed, obtaining granularity is the dry Ribes burejense powder of 0.8-1.5mm.The corn flour, wheat powder, yam flour and the dry Ribes burejense powder that use this granularity, it can be evenly distributed in sterilization process, the component distributing homogeneous in the medium obtaining after sterilizing.
The composition respectively Cordyceps militaris seed culture medium and Cordyceps militaris growth medium being contained separately takes rear mixing, all adopts autoclave sterilization, is specially 121 ℃, and sterilizing 20-30min, is cooled to room temperature after sterilizing, obtains being directly used in the medium of cultivating bacterial strain.
The cultural method of high polysaccharide Cordyceps militaris, comprises the following steps:
(a), by Cordyceps militaris spawn activation culture, making concentration is 1-3 * 10
7the spore suspension of CFU/ml;
(b) described spore suspension be take to ratio that percentage by volume is 3-8% and be inoculated in Cordyceps militaris seed culture medium claimed in claim 1 and cultivate, obtain seed liquor;
(c) described seed liquor is inoculated in Cordyceps militaris growth medium claimed in claim 2 secretly cultivate successively, mycelia annesl is cultivated and fruit body is cultivated, and obtains fruiting bodies of cordyceps militaris.
Adopt Cordyceps militaris seed culture medium provided by the invention and Cordyceps militaris growth medium to carry out the cultivation of Cordyceps militaris, cultural method is simple, and cultivation cycle is short, and cost is low, and the fruiting bodies of cordyceps militaris Polysaccharides in Cultured Cordyceps militaris content obtaining is high, in addition, available technology adopting liquid fermentation and culture Cordyceps militaris, the major part of Cordyceps militaris is in zymotic fluid, the materials such as secretion that self produce are mingled in zymotic fluid, thereby the Cordyceps militaris the obtaining composition that contains zymotic fluid, and Cordyceps militaris growth medium provided by the invention is solid culture medium, adopt solid culture Cordyceps militaris, the fruiting bodies of cordyceps militaris that harvesting obtains, impurity while having avoided adopting liquid fermentation and culture in medium is sneaked into the defect in fruiting bodies of cordyceps militaris, the Cordyceps militaris impurity obtaining still less, therefore, the purity of the polysaccharide that follow-up Cordyceps militaris is extracted is higher.
Cordyceps militaris spawn activation culture, Cordyceps militaris spawn 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 ℃ ± 2 ℃, obtain the spore of Cordyceps militaris, 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 dihydrogen phosphate 1g, agar 16g, water 1000ml; Compound method is: get the potato 100g having removed the peel, be cut into small pieces, add water 1000m1, boil 20min, by 4-6 layer filtered through gauze, then supply dehydration to 1000m1, add agar 16g to dissolve, and then add glucose 10g, potassium dihydrogen phosphate 1g, packing test tube, 121 ℃ 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 the sterile water of the Tween 80 of employing 0.05% is made concentration by spore
7the spore suspension of CFU/ml, the spore suspension spore making is evenly distributed; Then the ratio that the percentage by volume of take is 3-8% is seeded to Cordyceps militaris seed culture medium, and inoculum concentration is moderate, is beneficial at Cordyceps militaris seed culture medium Fast Growth.
Preferably, in described step (b), condition of culture is: 25-30 ℃, 240-260rpm/min shaken cultivation.Empirical tests, cultivation temperature is 25-30 ℃, Cordyceps militaris grows fast in Cordyceps militaris seed culture medium; Rotating speed 240-260rpm/min, in the situation that not damaging Cordyceps militaris spore, increase dissolved oxygen amount, be beneficial to spore and mycelial growth, Cordyceps militaris seed culture medium can be mixed, to prevent local Cordyceps militaris seed culture medium nutrient component deficiency, affect spore and mycelial growth simultaneously, therefore, the spore fast growth of Cordyceps militaris under this condition of culture, 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 described seed liquor dilution 20-30 doubly after, the percentage by volume of take is inoculated as 5-8%.In Cordyceps militaris growth medium, inoculate appropriate seed liquor, be beneficial to and obtain fruiting bodies of cordyceps militaris; In order to prevent the rear distributing inhomogeneity of inoculum concentration and inoculation, first seed liquor is diluted, then take percentage by volume as 5-8% inoculates, can obtain the Cordyceps militaris growth medium that seed liquor is evenly distributed, be beneficial to the growth of thalline.
Preferably, in described step (c), described dark cultivation is cultivated between the cultivation of having sterilized, and condition of culture is: temperature 20-25 ℃, humidity 60-70%, cultivates 6-8d.Under this condition of culture, thalli growth is quick, covers with mycelia in incubator, and cultivation cycle is short.
Preferably, in described step (c), the condition of culture that described mycelia annesl is cultivated is: temperature 15-20 ℃, and humidity 60-70%, the 1.5-2.5h that ventilates every day, illumination 8-12h, intensity of illumination is 250-300Lux, cultivates 5-8d.Under this condition of culture, thalli growth is quick, and mycelia starts to form the former base of millet shape in media surface, and mycelium annesl is effective, and cultivation cycle is short.
Preferably, in described step (c), the condition of culture that described fruit body is cultivated is: ventilative cultivation, temperature is 20 ℃ ± 2 ℃, and humidity is 80-90%, and 1.5-2.5h ventilates every day, illumination 6-8h, intensity of illumination is 250-300Lux, and being cultured to fruit body length is 6-10cm, gathers and obtains described fruiting bodies of cordyceps militaris.Ventilative cultivation is: on the film of culture vessel mouth, prick several holes, be beneficial to circulation of air, be beneficial to culture growth.And under this condition of culture, thalli growth is quick, and cultivation cycle is short, the fruiting bodies of cordyceps militaris Polysaccharides in Cultured Cordyceps militaris content obtaining is high.
Preferably, described sterilization is: the water-bath that the container that peracetic acid soln is housed is placed in to 75-85 ℃ 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 ℃, humidity is 60%-80%.Space to Cordyceps militaris growth carries out disinfection, and to prevent that it is subject to disease, and prevents the pollution of other microorganisms; Adopt Peracetic acid to sterilize between cultivating to stifling, sterilization is thorough, and to the injury of Cordyceps militaris culture nothing itself, in the training period, Cordyceps militaris culture growth conditions is good, and the fruiting bodies of cordyceps militaris fruiting bodies of cordyceps militaris Polysaccharides in Cultured Cordyceps militaris content pollution-free and that obtain obtaining is high.
The cultural method of the high polysaccharide Cordyceps militaris that the embodiment of the present invention provides, spore suspension is provided in the Cordyceps militaris seed culture medium providing in the present invention and cultivates, and obtains seed liquor; Seed liquor is inoculated in Cordyceps militaris growth medium provided by the invention secretly cultivate successively, mycelia annesl is cultivated and fruit body is cultivated, cultural method is simple, cultivation cycle is short, the fruiting bodies of cordyceps militaris Polysaccharides in Cultured Cordyceps militaris content obtaining is high, and the purity of the Polysaccharides in Cultured Cordyceps militaris extracting with this fruiting bodies of cordyceps militaris is high.
Embodiment 1
By weight, take following composition: 8 parts of glucose, 2 parts of peptones, 4 parts of dusty yeasts, 0.02 part of potassium dihydrogen phosphate, 0.02 part, magnesium sulfate, 4 parts, sodium chloride, vitaminB10 .05 part, 8 parts of yam flours, 800 parts, water, then 121 ℃, sterilizing 25min, after sterilizing, be cooled to room temperature, obtain Cordyceps militaris seed culture medium;
By weight, take following composition: 30 parts of 12 parts of corn flour, 5 parts, wheat powder, 3 parts of yam flours, 2 parts of dry Ribes burejense powders, 0.02 part of peptone, 0.02 part of potassium dihydrogen phosphate, 0.02 part, magnesium sulfate, 0.02 part of glucose, vitaminB10 .0005 part, water, then 121 ℃, sterilizing 25min, after sterilizing, be cooled to room temperature, obtain Cordyceps militaris growth medium;
The cultural method of high polysaccharide Cordyceps militaris, 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 be take to ratio that percentage by volume is 8% and be inoculated in Cordyceps militaris seed culture medium and cultivate, condition of culture is: 25 ℃, 240rpm/min shaken cultivation, cultivates 3d, obtains seed liquor;
(c) by after 20 times of seed liquor dilutions, take percentage by volume as 5% seed liquor is inoculated in Cordyceps militaris growth medium secretly cultivate successively, mycelia annesl is cultivated and fruit body is cultivated;
Dark cultivation cultivated between the cultivation of having sterilized, and condition of culture is: 20 ℃ of temperature, and humidity 60%, cultivates 6d;
The condition of culture that mycelia annesl is cultivated is: 15 ℃ of temperature, and humidity 60%, the 1.5h that ventilates every day, illumination 8h, intensity of illumination is 250Lux, cultivates 5d;
After mycelia annesl has been cultivated, culture is carried out to fruit body cultivation, condition of culture is: ventilative cultivation, and temperature is 20 ℃ ± 2 ℃, humidity is 80%, the 1.5h that ventilates every day, illumination 6h, intensity of illumination is 250Lux, and being cultured to fruit body length is 6-10cm, gathers and obtains fruiting bodies of cordyceps militaris;
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 ℃ is fumigated sterilizing between described cultivation, and fumigation time is 100min, and every cubic metre of space consumption of Peracetic acid is 3-3.2g; During sterilization, cultivate room temperature and be not less than 20 ℃, humidity is 60%-80%.
It is golden yellow that the fruiting bodies of cordyceps militaris obtaining is, anthrone colorimetric method for determining fruiting bodies of cordyceps militaris total sugar content, and the Polysaccharides in Cultured Cordyceps militaris content obtaining is 18%.
Embodiment 2
By weight, take following composition: 10 parts of glucose, 3 parts of peptones, 5 parts of dusty yeasts, 0.04 part of potassium dihydrogen phosphate, 0.04 part, magnesium sulfate, 5 parts, sodium chloride, vitaminB10 .10 part, 10 parts of yam flours, 1000 parts, water, then 121 ℃, sterilizing 30min, after sterilizing, be cooled to room temperature, obtain Cordyceps militaris seed culture medium;
By weight, take following composition: 35 parts of 15 parts of corn flour, 8 parts, wheat powder, 5 parts of yam flours, 4 parts of dry Ribes burejense powders, 0.04 part of peptone, 0.03 part of potassium dihydrogen phosphate, 0.02 part, magnesium sulfate, 0.05 part of glucose, vitaminB10 .0008 part, water, then 121 ℃, sterilizing 30min, after sterilizing, be cooled to room temperature, obtain Cordyceps militaris growth medium;
The cultural method of high polysaccharide Cordyceps militaris, 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 be take to ratio that percentage by volume is 5% and be inoculated in Cordyceps militaris seed culture medium and cultivate, condition of culture is: 27 ℃, 250rpm/min shaken cultivation, cultivates 4d, obtains seed liquor;
(c) by after 25 times of seed liquor dilutions, take percentage by volume as 6% seed liquor is inoculated in Cordyceps militaris growth medium secretly cultivate successively, mycelia annesl is cultivated and fruit body is cultivated;
Dark cultivation cultivated between the cultivation of having sterilized, and condition of culture is: 22 ℃ of temperature, and humidity 65%, cultivates 7d;
The condition of culture that mycelia annesl is cultivated is: 18 ℃ of temperature, and humidity 65%, the 2.0h that ventilates every day, illumination 10h, intensity of illumination is 280Lux, cultivates 7d;
After mycelia annesl has been cultivated, culture is carried out to fruit body cultivation, condition of culture is: ventilative cultivation, and temperature is 20 ℃ ± 2 ℃, humidity is 85%, the 2.0h that ventilates every day, illumination 7h, intensity of illumination is 280Lux, and being cultured to fruit body length is 6-10cm, gathers and obtains fruiting bodies of cordyceps militaris;
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 ℃ 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; During sterilization, cultivate room temperature and be not less than 20 ℃, humidity is 60%-80%.
It is golden yellow that the fruiting bodies of cordyceps militaris obtaining is, anthrone colorimetric method for determining fruiting bodies of cordyceps militaris total sugar content, and the Polysaccharides in Cultured Cordyceps militaris content obtaining is 20%.
Embodiment 3
By weight, take following composition: 12 parts of glucose, 4 parts of peptones, 6 parts of dusty yeasts, 0.06 part of potassium dihydrogen phosphate, 0.06 part, magnesium sulfate, 6 parts, sodium chloride, vitaminB10 .15 part, 12 parts of yam flours, 1200 parts, water, then 121 ℃, sterilizing 35min, after sterilizing, be cooled to room temperature, obtain Cordyceps militaris seed culture medium;
By weight, take following composition: 40 parts of 18 parts of corn flour, 10 parts, wheat powder, 8 parts of yam flours, 5 parts of dry Ribes burejense powders, 0.05 part of peptone, 0.04 part of potassium dihydrogen phosphate, 0.03 part, magnesium sulfate, 0.08 part of glucose, vitaminB10 .001 part, water, then 121 ℃, sterilizing 35min, after sterilizing, be cooled to room temperature, obtain Cordyceps militaris growth medium;
The cultural method of high polysaccharide Cordyceps militaris, 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 be take to ratio that percentage by volume is 3% and be inoculated in Cordyceps militaris seed culture medium and cultivate, condition of culture is: 30 ℃, 260rpm/min shaken cultivation, cultivates 5d, obtains seed liquor;
(c) by after 30 times of seed liquor dilutions, take percentage by volume as 8% seed liquor is inoculated in Cordyceps militaris growth medium secretly cultivate successively, mycelia annesl is cultivated and fruit body is cultivated;
Dark cultivation cultivated between the cultivation of having sterilized, and condition of culture is: 25 ℃ of temperature, and humidity 70%, cultivates 8d;
The condition of culture that mycelia annesl is cultivated is: 20 ℃ of temperature, and humidity 70%, the 2.5h that ventilates every day, illumination 12h, intensity of illumination is 300Lux, cultivates 8d;
After mycelia annesl has been cultivated, culture is carried out to fruit body cultivation, condition of culture is: ventilative cultivation, and temperature is 20 ℃ ± 2 ℃, humidity is 90%, the 2.5h that ventilates every day, illumination 8h, intensity of illumination is 300Lux, and being cultured to fruit body length is 6-10cm, gathers and obtains fruiting bodies of cordyceps militaris;
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 ℃ is fumigated sterilizing between described cultivation, and fumigation time is 90 min, 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 ℃, humidity is 60%-80%.
It is golden yellow that the fruiting bodies of cordyceps militaris obtaining is, anthrone colorimetric method for determining fruiting bodies of cordyceps militaris total sugar content, and the Polysaccharides in Cultured Cordyceps militaris content obtaining is 25%.
The cultural method of high polysaccharide Cordyceps militaris provided by the invention, cultural method is simple, at different cultivation stages, adopts different medium to cultivate, to guarantee the vigor of bacterial classification and to adapt to the demand of cultivating target; Condition of culture, the environmental condition of each cultivation stage are strictly controlled, and are beneficial to standardization and standardization that Cordyceps militaris is cultivated and manages; The fruiting bodies of cordyceps militaris Polysaccharides in Cultured Cordyceps militaris content obtaining is high, is 18-25%, and the purity of the Polysaccharides in Cultured Cordyceps militaris extracting with this fruiting bodies of cordyceps militaris 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 modification 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 seed culture medium, it is characterized in that, by weight, comprise following composition: glucose 8-12 part, peptone 2-4 part, dusty yeast 4-6 part, potassium dihydrogen phosphate 0.02-0.06 part, magnesium sulfate 0.02-0.06 part, sodium chloride 4-6 part, vitaminB10 .05-0.15 part, yam flour 8-12 part, water 800-1200 part.
2. Cordyceps militaris growth medium, it is characterized in that, by weight, comprise following composition: corn flour 12-18 part, wheat powder 5-10 part, yam flour 3-8 part, dry Ribes burejense powder 2-5 part, peptone 0.02-0.05 part, potassium dihydrogen phosphate 0.02-0.04 part, magnesium sulfate 0.02-0.03 part, glucose 0.02-0.08 part, vitaminB10 .0005-0.001 part, water 30-40 part.
3. a cultural method for high polysaccharide Cordyceps militaris, 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) described spore suspension be take to ratio that percentage by volume is 3-8% and be inoculated in Cordyceps militaris seed culture medium claimed in claim 1 and cultivate, obtain seed liquor;
(c) described seed liquor is inoculated in Cordyceps militaris growth medium claimed in claim 2 secretly cultivate successively, mycelia annesl is cultivated and fruit body is cultivated, and obtains fruiting bodies of cordyceps militaris.
4. the cultural method of high polysaccharide Cordyceps militaris according to claim 3, is characterized in that, in described step (b), condition of culture is: 25-30 ℃, 240-260rpm/min shaken cultivation.
5. the cultural method of high polysaccharide Cordyceps militaris according to claim 4, is characterized in that, in described step (b), cultivates 3-5d, obtains described seed liquor.
6. the cultural method of high polysaccharide Cordyceps militaris according to claim 5, is characterized in that, in described step (c), by described seed liquor dilution 20-30 doubly after, the percentage by volume of take is inoculated as 5-8%.
7. the cultural method of high polysaccharide Cordyceps militaris according to claim 6, is characterized in that, in described step (c), described dark cultivation is cultivated between the cultivation of having sterilized, and condition of culture is: temperature 20-25 ℃, humidity 60-70%, cultivates 6-8d.
8. the cultural method of high polysaccharide Cordyceps militaris according to claim 7, it is characterized in that, in described step (c), the condition of culture that described mycelia annesl is cultivated is: temperature 15-20 ℃, humidity 60-70%, the 1.5-2.5h that ventilates every day, illumination 8-12h, intensity of illumination is 250-300Lux, cultivates 5-8d.
9. the cultural method of high polysaccharide Cordyceps militaris according to claim 8, it is characterized in that, in described step (c), the condition of culture that described fruit body is cultivated is: ventilative cultivation, and temperature is 20 ℃ ± 2 ℃, humidity is 80-90%, the 1.5-2.5h that ventilates every day, illumination 6-8h, intensity of illumination is 250-300Lux, being cultured to fruit body length is 6-10cm, gathers and obtains described fruiting bodies of cordyceps militaris.
10. the cultural method of high polysaccharide Cordyceps militaris 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 ℃ 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 ℃, humidity is 60%-80%.
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| 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 |
| CN108633616A (en) * | 2018-05-11 | 2018-10-12 | 江苏圣福来生态农业有限公司 | A kind of culture implantation methods improving activity substance content in Cordyceps militaris |
| WO2020215609A1 (en) * | 2019-04-22 | 2020-10-29 | 江苏农林职业技术学院 | Liquid fermentation culture medium of high-yield antioxidant cordyceps cicadae mycelium, and method for producing antioxidant cordyceps cicadae mycelium granule formulation thereof |
| CN110122193A (en) * | 2019-06-17 | 2019-08-16 | 山西万海澳生物科技有限责任公司 | A kind of cordyceps militaris plantation method of stable high-content polysaccharide |
| CN110122193B (en) * | 2019-06-17 | 2021-07-09 | 山西万海澳生物科技有限责任公司 | Cordyceps militaris cultivation method capable of stabilizing high-content polysaccharide |
| CN112314325A (en) * | 2020-09-14 | 2021-02-05 | 上海国宝企业发展中心 | Method for culturing artificial cordyceps militaris sporocarp |
| CN112352945A (en) * | 2020-09-14 | 2021-02-12 | 上海国宝企业发展中心 | Artificial cordyceps militaris food with effects of resisting fatigue and improving immunity |
| CN112273142A (en) * | 2020-11-12 | 2021-01-29 | 江苏康能生物工程股份有限公司 | Culture method for increasing content of crude polysaccharide in cordyceps militaris sporocarp |
| CN112273142B (en) * | 2020-11-12 | 2022-04-29 | 江苏康能生物工程股份有限公司 | Culture method for increasing content of crude polysaccharide in cordyceps militaris sporocarp |
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| Publication number | Publication date |
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| WO2015180519A1 (en) | 2015-12-03 |
| CN103988712B (en) | 2016-05-11 |
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