CN114426996A - Method for producing adenosine by fermenting cordyceps sinensis - Google Patents
Method for producing adenosine by fermenting cordyceps sinensis Download PDFInfo
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- CN114426996A CN114426996A CN202111388414.2A CN202111388414A CN114426996A CN 114426996 A CN114426996 A CN 114426996A CN 202111388414 A CN202111388414 A CN 202111388414A CN 114426996 A CN114426996 A CN 114426996A
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- culture
- silicate
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- potassium
- magnesium
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- 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 title claims abstract description 92
- 239000002126 C01EB10 - Adenosine Substances 0.000 title claims abstract description 46
- 229960005305 adenosine Drugs 0.000 title claims abstract description 46
- 241001248610 Ophiocordyceps sinensis Species 0.000 title claims abstract description 43
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 21
- 238000000855 fermentation Methods 0.000 claims abstract description 77
- 230000004151 fermentation Effects 0.000 claims abstract description 77
- 238000000034 method Methods 0.000 claims abstract description 52
- 239000003630 growth substance Substances 0.000 claims abstract description 40
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 32
- 230000004913 activation Effects 0.000 claims abstract description 32
- 241000190633 Cordyceps Species 0.000 claims abstract description 31
- 230000008569 process Effects 0.000 claims abstract description 29
- FPAFDBFIGPHWGO-UHFFFAOYSA-N dioxosilane;oxomagnesium;hydrate Chemical compound O.[Mg]=O.[Mg]=O.[Mg]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O FPAFDBFIGPHWGO-UHFFFAOYSA-N 0.000 claims abstract description 26
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 claims abstract description 23
- 238000011218 seed culture Methods 0.000 claims description 61
- 239000001963 growth medium Substances 0.000 claims description 58
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 36
- 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 30
- 239000001888 Peptone Substances 0.000 claims description 30
- 239000008103 glucose Substances 0.000 claims description 30
- 239000007788 liquid Substances 0.000 claims description 30
- 239000002609 medium Substances 0.000 claims description 30
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 30
- 108010080698 Peptones Proteins 0.000 claims description 29
- 235000019319 peptone Nutrition 0.000 claims description 29
- 239000002245 particle Substances 0.000 claims description 24
- 229940041514 candida albicans extract Drugs 0.000 claims description 23
- 239000012138 yeast extract Substances 0.000 claims description 23
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 18
- 229910052799 carbon Inorganic materials 0.000 claims description 18
- 159000000003 magnesium salts Chemical class 0.000 claims description 18
- 229910052757 nitrogen Inorganic materials 0.000 claims description 18
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 claims description 18
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 12
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 12
- YIXJRHPUWRPCBB-UHFFFAOYSA-N magnesium nitrate Chemical compound [Mg+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O YIXJRHPUWRPCBB-UHFFFAOYSA-N 0.000 claims description 12
- 238000011081 inoculation Methods 0.000 claims description 11
- 230000003213 activating effect Effects 0.000 claims description 10
- 239000004111 Potassium silicate Substances 0.000 claims description 7
- 239000004115 Sodium Silicate Substances 0.000 claims description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- 229910052913 potassium silicate Inorganic materials 0.000 claims description 7
- 235000019353 potassium silicate Nutrition 0.000 claims description 7
- NNHHDJVEYQHLHG-UHFFFAOYSA-N potassium silicate Chemical compound [K+].[K+].[O-][Si]([O-])=O NNHHDJVEYQHLHG-UHFFFAOYSA-N 0.000 claims description 7
- 229910052911 sodium silicate Inorganic materials 0.000 claims description 7
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 claims description 7
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 6
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 6
- 229930091371 Fructose Natural products 0.000 claims description 6
- 239000005715 Fructose Substances 0.000 claims description 6
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 6
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 6
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 6
- 235000019764 Soybean Meal Nutrition 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 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 claims description 6
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- 239000008101 lactose Substances 0.000 claims description 6
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
- 239000001103 potassium chloride Substances 0.000 claims description 6
- 235000011164 potassium chloride Nutrition 0.000 claims description 6
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 6
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 6
- 235000011151 potassium sulphates Nutrition 0.000 claims description 6
- 239000004455 soybean meal Substances 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- 239000000454 talc Substances 0.000 claims description 5
- 235000012222 talc Nutrition 0.000 claims description 5
- 229910052623 talc Inorganic materials 0.000 claims description 5
- HCWCAKKEBCNQJP-UHFFFAOYSA-N magnesium orthosilicate Chemical compound [Mg+2].[Mg+2].[O-][Si]([O-])([O-])[O-] HCWCAKKEBCNQJP-UHFFFAOYSA-N 0.000 claims description 4
- 239000000391 magnesium silicate Substances 0.000 claims description 4
- 229910052919 magnesium silicate Inorganic materials 0.000 claims description 4
- 235000019792 magnesium silicate Nutrition 0.000 claims description 4
- GNKTZDSRQHMHLZ-UHFFFAOYSA-N [Si].[Si].[Si].[Ti].[Ti].[Ti].[Ti].[Ti] Chemical compound [Si].[Si].[Si].[Ti].[Ti].[Ti].[Ti].[Ti] GNKTZDSRQHMHLZ-UHFFFAOYSA-N 0.000 claims description 3
- 235000019794 sodium silicate Nutrition 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 4
- 238000001994 activation Methods 0.000 description 28
- 239000007836 KH2PO4 Substances 0.000 description 24
- 239000000243 solution Substances 0.000 description 17
- 239000002054 inoculum Substances 0.000 description 10
- 239000003814 drug Substances 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 229910052564 epsomite Inorganic materials 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 241000223678 Aureobasidium pullulans Species 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 208000003734 Supraventricular Tachycardia Diseases 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 206010047302 ventricular tachycardia Diseases 0.000 description 2
- 241000223600 Alternaria Species 0.000 description 1
- 241000221377 Auricularia Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000208672 Lobelia Species 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000002929 anti-fatigue Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 210000003056 antler Anatomy 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 150000004760 silicates Chemical class 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
-
- C—CHEMISTRY; METALLURGY
- 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
Abstract
The invention relates to an artificial fermentation culture technology of cordyceps sinensis, and discloses a method for producing adenosine by fermenting cordyceps sinensis. The method for producing adenosine by fermenting cordyceps comprises the following steps: performing activation culture on the cordyceps sinensis, and then performing fermentation culture; wherein, a growth regulator is added in the process of the activation culture and/or the fermentation culture, and the growth regulator is at least one of talcum powder, silicate and cellulose powder. The method provided by the invention can obviously improve the adenosine content in the cordyceps, effectively promote the growth of the cordyceps, improve the yield of the cordyceps, save the production cost and is suitable for popularization and application.
Description
Technical Field
The invention relates to an artificial fermentation culture technology of cordyceps sinensis, in particular to a method for producing adenosine by fermenting cordyceps sinensis.
Background
Natural cordyceps is a larva corpse and fungal stroma complex formed by infecting host insect larvae with ascospores, conidia or hyphae of an anamorphic strain of the natural cordyceps. Modern medicine proves that the cordyceps sinensis has wide pharmacological actions of immunoregulation, antioxidation, anti-aging, anti-fatigue, antibiosis, anti-tumor, liver and kidney protection and the like, is called as 'three treasures of traditional Chinese medicine' together with ginseng and pilose antler, and has better research value and research heat in the world.
Adenosine, one of the major active ingredients of cordyceps, has been widely used in the pharmaceutical industry as a drug with great commercial value, has become a first-line drug approved by the Food and Drug Administration (FDA) for the treatment of paroxysmal supraventricular tachycardia (PSVT), and has subsequently been agreed as a therapeutic drug in various other countries such as the uk, italy, etc.
There are many methods for producing adenosine, but they can be mainly classified into two types, i.e., biosynthesis and chemical synthesis. The chemical synthesis method has the defects of complex process, high cost, low yield, great environmental pollution and the like, so that the chemical synthesis method is difficult to apply to large-scale industrial production; the biosynthesis method mainly utilizes the cordyceps fermentation to produce the adenosine, has the advantages of low cost, easy fermentation, no environmental pollution and the like, and can realize large-scale industrial production. However, the process of producing adenosine by fermentation still needs to be improved in aspects of adenosine production speed, adenosine yield, adenosine quality and the like, and a foundation is laid for large-scale production of adenosine by a biological fermentation method.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a method for producing adenosine by fermenting cordyceps, which can obviously improve the content of adenosine in the cordyceps, effectively promote the growth of the cordyceps and save the cost.
In order to realize the aim, the invention provides a method for producing adenosine by fermenting cordyceps, which comprises the following steps: performing activation culture on the cordyceps sinensis, and then performing fermentation culture; wherein, a growth regulator is added in the process of the activation culture and/or the fermentation culture, and the growth regulator is at least one of talcum powder, silicate and cellulose powder.
Preferably, the growth regulator is added during the activation culture and the fermentation culture separately.
Preferably, the addition amount of the growth regulator in the activation culture process is 15-25 g/L.
Preferably, the growth regulator in the activation culture process is talcum powder.
Preferably, the addition amount of the growth regulator in the fermentation culture process is 1.5-30 g/L.
Preferably, the growth regulators in the fermentation culture process are silicates and cellulose powder.
Preferably, the mass ratio of the silicate to the cellulose powder is 0.05 to 20: 1.
preferably, the average particle size of the talcum powder is 800-5000 meshes.
Preferably, the silicate is selected from at least one of titanium silicate, sodium silicate, potassium silicate and magnesium silicate.
Preferably, the molecular weight of the cellulose powder is 10000-30000, and the average particle size is 800-5000 meshes.
Preferably, the process of activation culture comprises:
(1) activating the cordyceps sinensis by a slant, and then inoculating the cordyceps sinensis to a first-stage seed culture medium to perform first-stage seed culture to obtain a first-stage seed solution;
(2) inoculating the primary seed liquid to a secondary seed culture medium for secondary seed culture to obtain a secondary seed liquid;
preferably, the growth regulator is added to the primary seed culture medium and/or the secondary seed culture medium, and more preferably, the growth regulator is added to the primary seed culture medium.
Preferably, the primary seed culture medium contains 15-30g/L of carbon source I, 8-15g/L of nitrogen source I, 0.5-2g/L of magnesium salt I and 2-4g/L of potassium salt I.
Preferably, the secondary seed culture medium contains 15-30g/L of carbon source II, 12-25g/L of nitrogen source II, 0.5-2g/L of magnesium salt II and 2-4g/L of potassium salt II.
Preferably, the carbon source I and the carbon source II are independently selected from at least one of glucose, fructose, sucrose, lactose and maltose, the nitrogen source I and the nitrogen source II are independently selected from at least one of peptone, yeast extract, corn steep liquor and soybean meal, the magnesium salt I and the magnesium salt II are independently selected from at least one of magnesium sulfate, magnesium chloride and magnesium nitrate, and the potassium salt I and the potassium salt II are independently selected from at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium chloride and potassium sulfate.
Preferably, the conditions of the primary seed culture medium include: the temperature is 18-25 ℃, the rotating speed is 100-.
Preferably, the conditions of the secondary seed culture medium include: the temperature is 18-25 ℃, the rotating speed is 100-150rpm, and the time is 7-12 days.
Preferably, the inoculation amount of the primary seed liquid in the secondary seed culture medium is 5-10% by volume; the inoculation amount of the secondary seed liquid in the culture medium of the fermentation culture is 5-10 vol%.
Preferably, the medium of the fermentation culture contains 15-30g/L of carbon source III, 12-25g/L of nitrogen source III, 0.5-2g/L of magnesium salt III and 2-4g/L of potassium salt III.
Preferably, the carbon source III is selected from at least one of glucose, fructose, sucrose, lactose and maltose, the nitrogen source III is selected from at least one of peptone, yeast extract, corn steep liquor and soybean meal, the magnesium salt III is selected from at least one of magnesium sulfate, magnesium chloride and magnesium nitrate, and the potassium salt III is selected from at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium chloride and potassium sulfate.
Preferably, the conditions of the fermentation culture include: the temperature is 18-25 ℃, the rotating speed is 100-.
Through the technical scheme, the invention has the beneficial effects that:
by adopting the method for producing adenosine by fermenting the cordyceps, the growth regulator is added, so that the adenosine formation of the cordyceps in the growth and metabolism process is effectively promoted, the content of the adenosine in the cordyceps is obviously improved, the growth of the cordyceps is effectively promoted, the yield of the cordyceps is improved, the production cost is saved, the method is suitable for popularization and application, and the foundation is laid for large-scale production of the adenosine biological fermentation method.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The invention provides a method for producing adenosine by cordyceps fermentation, which comprises the following steps: performing activation culture on the cordyceps sinensis, and then performing fermentation culture; wherein, a growth regulator is added in the process of the activation culture and/or the fermentation culture, and the growth regulator is at least one of talcum powder, silicate and cellulose powder.
In the process of researching the cordyceps sinensis, the inventor of the invention unexpectedly discovers that the growth regulator is added in the growth and metabolism process of the cordyceps sinensis and is regulated in the activation culture and/or fermentation culture stage, so that the synthesis of adenosine in the cordyceps sinensis can be effectively promoted, the content of adenosine in the cordyceps sinensis can be improved, the growth of the cordyceps sinensis can be promoted, the yield of the cordyceps sinensis can be improved, and the production cost can be saved.
According to the invention, the method for producing adenosine by cordyceps fermentation further comprises the step of extracting adenosine from the fermented cordyceps mycelia. Illustratively, the extraction process may specifically be: carrying out ultrasonic crushing on cordyceps sinensis mycelia: 200W, 10min (start 1s pause 3s), and then carrying out ultrasonic extraction: centrifuging at the rotation speed of 11000rpm for 10min at the temperature of 60 ℃ for 40min under the condition of 100W, and taking clear liquid; repeatedly extracting the precipitate for 2 times, mixing the clear liquids, diluting with ultrapure water to desired volume, and filtering with 0.22 μm microporous membrane to obtain extractive solution containing adenosine.
Analyzing the content of adenosine in the extracting solution containing adenosine by using high performance liquid chromatography, wherein the analyzing process can specifically comprise the following steps:
a chromatographic column: reverse phase 18 high performance liquid chromatography column (Unit, 250 mm. times.4.6 mm, 5 μm);
mobile phase: a is 20mmol/L NaH2PO4、Na2HPO4pH6.89; b is chromatographically pure-methanol; a90%, B10%;
sample injection amount: 10 mu L of the solution; column temperature: 30 ℃; detection wavelength: 260 nm;
preparing an adenosine standard product: preparing adenosine standard substance with concentration of 100, 75, 50, 25, 10, 5mg/mL respectively, and performing high performance liquid chromatography analysis under the same conditions;
finally, the adenosine in the extracting solution containing adenosine is quantified according to the peak area, and further the adenosine in mycelium is qualitatively and quantitatively determined.
In the invention, the cordyceps can be natural cordyceps, also can be cordyceps obtained by manual screening or induction, and preferably anamorphic lobelia of Xinjiang cordyceps sinensis. The culture medium and culture conditions used in the activation culture and fermentation culture of Cordyceps can be those used in the conventional culture methods in the art.
According to the invention, a growth regulator can be added in the process of the activation culture, or in the process of the fermentation culture, or in both the processes of the activation culture and the fermentation culture. Preferably, the growth regulator is added during the activation culture and the fermentation culture separately. The inventor finds that the production of mycelium in the cordyceps fermentation process can be further increased and the yield of adenosine can be improved under the preferred embodiment.
In the present invention, the growth regulator may be added to the corresponding culture medium for activation culture or fermentation culture, or may be added at once or in batches at any time point in the activation culture stage or fermentation culture stage of Cordyceps sinensis. Preferably, the growth regulator is added to the medium corresponding to the activation culture or fermentation culture.
According to the invention, the addition amount of the growth regulator in the activation culture process is not particularly limited, and the aim of promoting the cordyceps sinensis to generate adenosine can be fulfilled. Preferably, the addition amount of the growth regulator in the activation culture process is 15-25g/L, and specifically may be 15g/L, 17g/L, 19g/L, 21g/L, 23g/L, 25g/L or any value therebetween.
According to the invention, the growth regulator in the activation culture process can be a single component of talcum powder, silicate or cellulose powder, or can be a combination of talcum powder + silicate, silicate + cellulose powder, talcum powder + silicate + cellulose powder. Preferably, the growth regulator in the activation culture process is talcum powder. The inventor finds that under the preferred embodiment, the method is more suitable for forming more mycelium and spore amount in the growth process of the cordyceps sinensis, and is beneficial to improving the content of adenosine in the cordyceps sinensis and improving the yield of the cordyceps sinensis.
According to the invention, the addition amount of the growth regulator in the fermentation culture process is not particularly limited, and the aim of promoting the cordyceps sinensis to generate adenosine can be fulfilled. Preferably, the addition amount of the growth regulator in the fermentation culture process is 1.5-30g/L, and specifically may be 1.5g/L, 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L or any value between the two values.
According to the invention, the growth regulator in the fermentation culture process can be a single component of talcum powder, silicate or cellulose powder, and also can be a combination of talcum powder + silicate, silicate + cellulose powder, talcum powder + silicate + cellulose powder. Preferably, the growth regulators in the fermentation culture process are silicate and cellulose powder. The inventors have found that in this preferred embodiment, it is advantageous to further increase the production of adenosine in the cordyceps.
According to the present invention, preferably, the mass ratio of the silicate to the cellulose powder is 0.05 to 20: 1. illustratively, the silicate is added in an amount of 0.5 to 20g/L, more preferably 5 to 15 g/L; the addition amount of the cellulose powder is 1 to 10g/L, more preferably 5 to 10 g/L. The inventors have found that in this preferred embodiment, it is advantageous to further increase the production of adenosine in the cordyceps.
According to the invention, the talc is of the magnesium silicate mineral talc family, which is commercially available. Preferably, the average particle size of the talcum powder is 800-5000 meshes, and the talcum powder with the corresponding particle size can be directly purchased or obtained by crushing large-particle talcum powder purchased. The inventor finds that under the preferred embodiment, the regulation effect of the talcum powder on the activation process of the cordyceps can be improved, and the yield of mycelium and spores can be further improved.
According to the invention, the type of substance of the silicate is not particularly limited, as long as a corresponding conditioning effect can be achieved. For example, the silicate is selected from at least one of titanium silicate, sodium silicate, potassium silicate and magnesium silicate, which are conventional choices in the art and are commercially available.
According to the present invention, the kind of the cellulose powder is not particularly limited as long as a corresponding regulating action can be formed. Preferably, the cellulose powder has a molecular weight of 10000-30000 and an average particle size of 800-5000 meshes, and the cellulose powder with the corresponding particle size can be directly purchased or obtained by crushing large-particle cellulose.
According to the present invention, preferably, the process of activation culture comprises:
(1) activating the cordyceps sinensis by a slant, and then inoculating the cordyceps sinensis to a first-stage seed culture medium to perform first-stage seed culture to obtain a first-stage seed solution;
(2) and inoculating the primary seed solution to a secondary seed culture medium for secondary seed culture to obtain a secondary seed solution.
In the present invention, the slant culture medium for the activation of Cordyceps sinensis can be conventional slant culture medium, such as PDA culture medium, and sterilized at 121 deg.C for 20 min. Placing the thallus of the cordyceps sinensis in a slant culture medium, and culturing for 15-30 days at 20-25 ℃ to carry out slant activation.
According to the invention, when the growth regulator is added in the activation culture process, the growth regulator can be added in the primary seed culture medium and/or the secondary seed culture medium. Preferably, the growth regulator is added to the primary seed culture medium. The inventor finds that under the preferred embodiment, the condition effect of the growth regulator on the cordyceps sinensis is promoted, the growth activity of the cordyceps sinensis in the secondary seed culture stage is improved, and more mycelium and spore amount is obtained.
In the invention, the primary seed culture medium and the secondary seed culture medium are not particularly limited, and can provide nutrient components required in the growth and activation process of the cordyceps sinensis.
According to the invention, preferably, the primary seed culture medium contains 15-30g/L of carbon source I, 8-15g/L of nitrogen source I, 0.5-2g/L of magnesium salt I and 2-4g/L of potassium salt I.
According to the invention, the secondary seed culture medium preferably contains 15-30g/L of carbon source II, 12-25g/L of nitrogen source II, 0.5-2g/L of magnesium salt II and 2-4g/L of potassium salt II.
According to the present invention, preferably, the carbon source I and the carbon source II are each independently selected from at least one of glucose, fructose, sucrose, lactose and maltose, the nitrogen source I and the nitrogen source II are each independently selected from at least one of peptone, yeast extract, corn steep liquor and soybean meal, the magnesium salt I and the magnesium salt II are each independently selected from at least one of magnesium sulfate, magnesium chloride and magnesium nitrate, and the potassium salt I and the potassium salt II are each independently selected from at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium chloride and potassium sulfate.
The above materials are conventional choices in the art and are all commercially available.
Illustratively, the primary seed medium contains glucose 15-30g/L, peptone 8-15g/L, MgSO4·7H2O0.5-2 g/L and KH2PO42-4 g/L; the secondary seed culture medium contains glucose 15-30g/L and peptone 8-15g/L, MgSO4·7H2O 0.5-2g/L、KH2PO42-4g/L and 4-8g/L of yeast extract.
According to the present invention, preferably, the conditions of the primary seed culture medium include: the temperature is 18-25 deg.C, and can be 18 deg.C, 20 deg.C, 22 deg.C, 24 deg.C, 25 deg.C or any value between the above two values; the rotation speed is 100-150rpm, and specifically can be 100rpm, 110rpm, 120rpm, 130rpm, 140rpm, 150rpm or any value between the two values; the time period is 15-20 days, specifically 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or any value between the above two values.
According to the present invention, preferably, the conditions of the secondary seed culture medium include: the temperature is 18-25 deg.C, specifically 18 deg.C, 20 deg.C, 22 deg.C, 24 deg.C, 25 deg.C or any value between the above two values; the rotation speed is 100-150rpm, and specifically can be 100rpm, 110rpm, 120rpm, 130rpm, 140rpm, 150rpm or any value between the two values; the time period is 7-12 days, specifically, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days or any value between the above two values.
According to the invention, the inoculation amount of the first-stage seed culture stage, the second-stage seed culture stage and the fermentation culture stage is not particularly limited, and only the cordyceps can be normally produced in the corresponding culture medium. Preferably, the inoculation amount of the primary seed liquid in the secondary seed culture medium is 5-10% by volume, namely the inoculation amount of the primary seed liquid is 5-10% of the volume of the secondary seed culture medium; the inoculation amount of the secondary seed liquid in the culture medium for fermentation culture is 5-10% by volume, namely the inoculation amount of the secondary seed liquid is 5-10% of the culture medium for fermentation culture.
According to the invention, the culture medium for fermentation culture is not particularly limited, and can provide nutrient components required in the growth and activation process of the cordyceps sinensis. Preferably, the medium of the fermentation culture contains 15-30g/L of carbon source III, 12-25g/L of nitrogen source III, 0.5-2g/L of magnesium salt III and 2-4g/L of potassium salt III.
According to the present invention, preferably, the carbon source III is selected from at least one of glucose, fructose, sucrose, lactose and maltose, the nitrogen source III is selected from at least one of peptone, yeast extract, corn steep liquor and soybean meal, the magnesium salt III is selected from at least one of magnesium sulfate, magnesium chloride and magnesium nitrate, and the potassium salt III is selected from at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium chloride and potassium sulfate. The above materials are conventional choices in the art and are all commercially available.
Illustratively, the fermentation medium contains glucose 15-30g/L, peptone 8-15g/L, MgSO4·7H2O 0.5-2g/L、KH2PO42-4g/L and 4-8g/L of yeast extract.
According to the present invention, preferably, the conditions of the fermentation culture include: the temperature is 18-25 deg.C, specifically 18 deg.C, 20 deg.C, 22 deg.C, 24 deg.C, 25 deg.C or any value between the above two values; the rotation speed is 100-150rpm, and specifically can be 100rpm, 110rpm, 120rpm, 130rpm, 140rpm, 150rpm or any value between the two values; the time period is 15-20 days, specifically 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or any value between the above two.
According to a particularly preferred embodiment of the invention, the method for producing adenosine by fermenting cordyceps comprises the following steps:
(1) activating the cordyceps sinensis by a slant, inoculating the cordyceps sinensis to a first-level seed culture medium, and performing first-level seed culture for 15-20 days at the temperature of 18-25 ℃ and the rotating speed of 100-150rpm to obtain a first-level seed solution, wherein the first-level seed culture medium contains 15-30g/L of glucose and 8-15g/L, MgSO of peptone4·7H2O0.5-2g/L、KH2PO42-4g/L and 15-25g/L of talcum powder, wherein the average grain diameter of the talcum powder is 800-5000 meshes;
(2) inoculating the primary seed liquid to a secondary seed culture medium with an inoculation amount of 5-10 vol%, and performing secondary seed culture at 18-25 deg.C and rotation speed of 100-150rpm for 7-12 days to obtain a secondary seed liquid, wherein the secondary seed culture medium contains glucose 15-30g/L and peptone 8-15g/L, MgSO g/L4·7H2O 0.5-2g/L、KH2PO42-4g/L and 4-8g/L of yeast extract;
(3) inoculating the second-stage seed liquid into a fermentation culture medium at an inoculation amount of 5-10 vol%, performing fermentation culture at 18-25 deg.C and rotation speed of 100-150rpm for 15-20 days to obtain fermented mycelia, and extracting from the fermented myceliaObtaining adenosine; wherein the fermentation medium contains glucose 15-30g/L and peptone 8-15g/L, MgSO4·7H2O 0.5-2g/L、KH2PO42-4g/L, 4-8g/L of yeast extract, 0.5-20g/L of silicate and 1-10g/L of cellulose powder, wherein the molecular weight of the cellulose powder is 10000-30000, and the average particle size is 800-5000 meshes.
The present invention will be described in detail below by way of examples.
In the following examples, the anamorphic type of Alternaria lobioides of Sinkiang Cordyceps sinensis is provided by the institute of food and pharmaceutical engineering of Nanjing university; the talcum powder is purchased from Nanjing Jia Kai science and technology Limited, and the average grain diameter is 800-; cellulose (molecular weight 10000-; cellulose (molecular weight 40000-50000) is purchased from Nanjing Jia Kai technology ltd, and the average grain diameter is 800-5000 meshes; other raw materials and reagents are conventional commercial products.
In the following examples, PDA medium was used for slant activation.
Example 1
(1) Inoculating Leucoporia obliqua on PDA slant culture medium, activating at 20 deg.C for 30 days to obtain activated mycelium, inoculating the activated mycelium into primary seed culture medium (containing glucose 25g/L and peptone 12g/L, MgSO)4·7H2O 1g/L、KH2PO43g/L and 20g/L of talcum powder with the average grain diameter of 800-;
(2) the primary seed solution was inoculated into a secondary seed medium (secondary seed medium containing 25g/L glucose, 12g/L, MgSO peptone) at an inoculum size of 10 vol%4·7H2O 1g/L、KH2PO43g/L and 6g/L of yeast extract), and performing secondary seed culture for 10 days at the temperature of 20 ℃ and the rotation speed of 120rpm to obtain secondary seed liquid;
(3) the secondary seed liquid was inoculated into a fermentation medium containing 20g/L glucose and 12g/L, MgSO peptone at an inoculum size of 10 vol%4·7H2O 1g/L，KH2PO43g/L of yeast extract, 6g/L of sodium silicate, 10g/L of sodium silicate and 5g/L of cellulose powder with the molecular weight of 10000-30000 and the average particle size of 800-5000 meshes) under the conditions of the temperature of 20 ℃ and the rotating speed of 120rpm for 20 days to obtain the fermentation mycelium.
Example 2
(1) Inoculating Leucoporia obliqua on PDA slant culture medium, activating at 25 deg.C for 20 days to obtain activated mycelium, inoculating the activated mycelium to primary seed culture medium (containing glucose 15g/L and peptone 8g/L, MgSO)4·7H2O 0.5g/L、KH2PO42g/L and 15g/L of talcum powder with the average grain diameter of 800-;
(2) the primary seed solution was inoculated into a secondary seed medium (secondary seed medium containing 15g/L glucose, 8g/L, MgSO peptone) at an inoculum size of 8 vol%4·7H2O 0.5g/L、KH2PO42g/L and 4g/L of yeast extract), and performing secondary seed culture for 7 days at the temperature of 18 ℃ and the rotation speed of 150rpm to obtain secondary seed liquid;
(3) the secondary seed liquid was inoculated into a fermentation medium containing 15g/L glucose and 8g/L, MgSO peptone at an inoculum size of 8 vol%4·7H2O 0.5g/L，KH2PO42g/L, 4g/L of yeast extract, 5g/L of potassium silicate and 10g/L of cellulose powder with the molecular weight of 10000-30000 and the average particle size of 800-5000 meshes) under the conditions of the temperature of 18 ℃ and the rotating speed of 150rpm for 20 days to obtain the fermentation mycelium.
Example 3
(1) Inoculating Leucoporia obliqua on PDA slant culture medium, activating at 22 deg.C for 15 days to obtain activated mycelium, inoculating the activated mycelium to primary seed culture medium (containing glucose 30g/L and peptone 15g/L, MgSO)4·7H2O 2g/L、KH2PO44g/L and the average particle diameter of 800-25g/L of talcum powder), and performing primary seed culture for 20 days at the temperature of 25 ℃ and the rotating speed of 100rpm to obtain primary seed liquid;
(2) the primary seed solution was inoculated into a secondary seed medium (secondary seed medium containing 30g/L glucose, 15g/L, MgSO peptone) at an inoculum size of 5 vol%4·7H2O 2g/L、KH2PO44g/L and 8g/L of yeast extract), and performing secondary seed culture for 12 days at the temperature of 25 ℃ and the rotation speed of 100rpm to obtain secondary seed liquid;
(3) inoculating the secondary seed liquid into a fermentation medium (the fermentation medium contains 30g/L glucose, 15g/L peptone and MgSO)4·7H2O 2g/L，KH2PO44g/L, 8g/L of yeast extract, 15g/L of sodium silicate and 3g/L of cellulose powder with the molecular weight of 10000-30000 and the average particle size of 800-5000 meshes) under the conditions of the temperature of 20 ℃ and the rotating speed of 120rpm for 20 days to obtain fermentation mycelium.
Example 4
The fermentation of Aureobasidium pullulans was carried out in the same manner as in example 2 except that the amount of potassium silicate added in step (3) was 0.5g/L and 1g/L of cellulose powder having a molecular weight of 10000-30000 and an average particle size of 800-5000 mesh.
Example 5
The fermentation of the Aureobasidium pullulans was carried out in the same manner as in example 2 to obtain fermented mycelia, except that talc having an average particle size of 800-5000 mesh was replaced with talc having an average particle size of 100-200 mesh in step (1).
Example 6
The fermentation of the Aureobasidium pullulans was carried out according to the method of example 2 to obtain fermentation mycelia, except that in the step (3), 5g/L of the cellulose powder with the molecular weight of 10000-30000 and the average particle size of 800-5000 meshes was replaced with 5g/L of the cellulose powder with the molecular weight of 30000-40000 and the average particle size of 100-200 meshes.
Example 7
The fermentation of the stalk of the Lumbriae was carried out according to the method of example 2, except that the step (2) was replaced with:
(2) the primary seed solution was inoculated into a secondary seed medium (secondary seed medium containing 15g/L glucose, 8g/L, MgSO peptone) at an inoculum size of 8 vol%4·7H2O 0.5g/L、KH2PO42g/L, 4g/L of yeast extract, 10g/L of talcum powder with the average particle size of 800-.
Example 8
The fermentation of the stalk of the Lumbriae was carried out according to the method of example 2, except that the step (3) was replaced with:
(3) the secondary seed liquid was inoculated into a fermentation medium containing 15g/L glucose and 8g/L, MgSO peptone at an inoculum size of 8 vol%4·7H2O 0.5g/L，KH2PO42g/L, 4g/L yeast extract and 5g/L potassium silicate), and fermenting and culturing for 20 days at 18 ℃ and 150rpm to obtain the fermentation mycelium.
Example 9
The fermentation of the stalk of the Lumbriae was carried out according to the method of example 2, except that the step (3) was replaced with:
(3) the secondary seed liquid was inoculated into a fermentation medium containing 15g/L glucose and 8g/L, MgSO peptone at an inoculum size of 8 vol%4·7H2O 0.5g/L，KH2PO43g/L and 4g/L of yeast extract) under the conditions of 20 ℃ and 120rpm for 20 days to obtain fermentation mycelium.
Example 10
(1) Inoculating Leucoporia obliqua on PDA slant culture medium, activating at 25 deg.C for 20 days to obtain activated mycelium, inoculating the activated mycelium to primary seed culture medium (containing glucose 15g/L and peptone 8g/L, MgSO)4·7H2O0.5 g/L and KH2PO42g/L), performing primary seed culture at 18 deg.C and rotation speed of 150rpm, adding medium with average particle diameter of 80 after 5 days of culture15g/L of 0-5000 meshes of talcum powder, and continuously culturing for 10 days to obtain a first-class seed solution;
(2) the primary seed liquid was inoculated into a secondary seed medium (containing 15g/L glucose and 8g/L, MgSO peptone) at an inoculation amount of 8 vol%4·7H2O 0.5g/L、KH2PO42g/L and 4g/L of yeast extract), and performing secondary seed culture for 7 days at the temperature of 18 ℃ and the rotation speed of 150rpm to obtain secondary seed liquid;
(3) the secondary seed liquid was inoculated into a fermentation medium containing 15g/L glucose and 8g/L, MgSO peptone at an inoculum size of 8 vol%4·7H2O 0.5g/L，KH2PO42g/L, 4g/L of yeast extract, 5g/L of potassium silicate and 1g/L of cellulose powder with the molecular weight of 10000-30000 and the average particle size of 800-5000 meshes) at the temperature of 18 ℃ and the rotation speed of 150rpm, adding 5g/L of sodium silicate and 1g/L of cellulose powder with the molecular weight of 10000-30000 and the average particle size of 800-5000 meshes after 5 days of culture, and continuously culturing for 20 days to obtain the fermentation mycelium.
Comparative example 1
(1) Inoculating Auricularia into PDA slant culture medium, activating at 22 deg.C for 15 days to obtain activated mycelium, inoculating the activated mycelium into primary seed culture medium (containing glucose 30g/L and peptone 15g/L, MgSO)4·7H2O2 g/L and KH2PO44g/L), performing primary seed culture for 20 days at the temperature of 25 ℃ and the rotating speed of 100rpm to obtain primary seed liquid;
(2) the primary seed solution was inoculated into a secondary seed medium (secondary seed medium containing 30g/L glucose, 15g/L, MgSO peptone) at an inoculum size of 5 vol%4·7H2O 2g/L、KH2PO44g/L and 8g/L of yeast extract), and performing secondary seed culture for 12 days at the temperature of 25 ℃ and the rotation speed of 100rpm to obtain secondary seed liquid;
(3) the secondary seed solution was inoculated into a fermentation medium (fermentation medium containing 30g/L glucose, 15g/L peptone,MgSO4·7H2O 2g/L，KH2PO44g/L and 8g/L of yeast extract) under the conditions of 20 ℃ and 120rpm for 15 days to obtain fermentation mycelium.
Test example
The yields (dry weight) of the fermentation tubes obtained in examples 1 to 10 and comparative example 1 were measured, and a part of the fermentation tubes was washed with ultrapure water 3 times, subjected to leaching treatment and microfiltration membrane filtration, and then subjected to adenosine content measurement by high performance liquid chromatography, and the results are shown in Table 1.
TABLE 1
The results in table 1 show that, by adopting the method for producing adenosine by fermenting cordyceps sinensis provided by the invention in the embodiments 1 to 10, the content of adenosine in the cordyceps sinensis can be obviously increased, the yield of the cordyceps sinensis can be increased, the production cost can be saved, and the method is suitable for popularization and application.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. The method for producing adenosine by fermenting cordyceps is characterized by comprising the following steps of: performing activation culture on the cordyceps sinensis, and then performing fermentation culture; wherein, a growth regulator is added in the process of the activation culture and/or the fermentation culture, and the growth regulator is at least one of talcum powder, silicate and cellulose powder.
2. The method of claim 1, wherein the growth regulator is added during the activation culture and the fermentation culture separately.
3. The method according to claim 1, wherein the growth regulator is added in an amount of 15-25g/L during the activation culture;
preferably, the growth regulator in the activation culture process is talcum powder.
4. The method according to claim 1, wherein the growth regulator is added in an amount of 1.5-30g/L during the fermentation culture;
preferably, the growth regulators in the fermentation culture process are silicate and cellulose powder;
preferably, the mass ratio of the silicate to the cellulose powder is 0.05 to 20: 1.
5. the method as claimed in any one of claims 1 to 4, wherein the talc has an average particle size of 800-5000 mesh;
preferably, the silicate is selected from at least one of titanium silicate, sodium silicate, potassium silicate and magnesium silicate;
preferably, the molecular weight of the cellulose powder is 10000-30000, and the average particle size is 800-5000 meshes.
6. The method according to any one of claims 1 to 4, wherein the activating culture comprises:
(1) activating the cordyceps sinensis by a slant, inoculating the cordyceps sinensis to a first-stage seed culture medium, and performing first-stage seed culture to obtain a first-stage seed solution;
(2) inoculating the primary seed liquid to a secondary seed culture medium for secondary seed culture to obtain a secondary seed liquid;
preferably, the growth regulator is added to the primary seed culture medium and/or the secondary seed culture medium, more preferably, the growth regulator is added to the primary seed culture medium.
7. The method of claim 6, wherein the primary seed culture medium comprises 15-30g/L of carbon source I, 8-15g/L of nitrogen source I, 0.5-2g/L of magnesium salt I, and 2-4g/L of potassium salt I;
preferably, the secondary seed culture medium contains 15-30g/L of carbon source II, 12-25g/L of nitrogen source II, 0.5-2g/L of magnesium salt II and 2-4g/L of potassium salt II;
preferably, the carbon source I and the carbon source II are each independently selected from at least one of glucose, fructose, sucrose, lactose and maltose,
the nitrogen source I and the nitrogen source II are respectively and independently selected from at least one of peptone, yeast extract, corn steep liquor and soybean meal,
the magnesium salt I and the magnesium salt II are respectively and independently selected from at least one of magnesium sulfate, magnesium chloride and magnesium nitrate,
the potassium salt I and the potassium salt II are respectively and independently selected from at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium chloride and potassium sulfate.
8. The method of claim 6, wherein the conditions of the primary seed culture comprise: the temperature is 18-25 ℃, the rotating speed is 100-;
preferably, the conditions of the secondary seed culture include: the temperature is 18-25 ℃, the rotating speed is 100-150rpm, and the time is 7-12 days.
9. The method of claim 6, wherein the amount of primary seed fluid inoculated into the secondary seed medium is 5-10% by volume;
the inoculation amount of the secondary seed liquid in the culture medium of the fermentation culture is 5-10 vol%.
10. The method according to any one of claims 1 to 4, wherein the medium of the fermentation culture contains 15-30g/L of carbon source III, 12-25g/L of nitrogen source III, 0.5-2g/L of magnesium salt III and 2-4g/L of potassium salt III;
preferably, the carbon source III is selected from at least one of glucose, fructose, sucrose, lactose and maltose,
the nitrogen source III is at least one of peptone, yeast extract, corn steep liquor and soybean meal,
the magnesium salt III is at least one selected from magnesium sulfate, magnesium chloride and magnesium nitrate,
the potassium salt III is at least one selected from potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium chloride and potassium sulfate;
preferably, the conditions of the fermentation culture include: the temperature is 18-25 ℃, the rotating speed is 100-.
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