CN114426996B - Method for producing adenosine by cordyceps fermentation - Google Patents
Method for producing adenosine by cordyceps fermentation Download PDFInfo
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- CN114426996B CN114426996B CN202111388414.2A CN202111388414A CN114426996B CN 114426996 B CN114426996 B CN 114426996B CN 202111388414 A CN202111388414 A CN 202111388414A CN 114426996 B CN114426996 B CN 114426996B
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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 94
- 238000000855 fermentation Methods 0.000 title claims abstract description 86
- 230000004151 fermentation Effects 0.000 title claims abstract description 86
- 239000002126 C01EB10 - Adenosine Substances 0.000 title claims abstract description 47
- 229960005305 adenosine Drugs 0.000 title claims abstract description 47
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 22
- 241000190633 Cordyceps Species 0.000 title claims description 18
- 241001248610 Ophiocordyceps sinensis Species 0.000 claims abstract description 51
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 35
- 230000004913 activation Effects 0.000 claims abstract description 32
- 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 27
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000001963 growth medium Substances 0.000 claims description 52
- 238000011218 seed culture Methods 0.000 claims description 43
- 239000002609 medium Substances 0.000 claims description 39
- 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
- 108010080698 Peptones Proteins 0.000 claims description 30
- 239000008103 glucose Substances 0.000 claims description 30
- 239000002245 particle Substances 0.000 claims description 30
- 235000019319 peptone Nutrition 0.000 claims description 30
- 239000007788 liquid Substances 0.000 claims description 29
- 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
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-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 9
- 239000004111 Potassium silicate Substances 0.000 claims description 7
- 239000004115 Sodium Silicate Substances 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
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 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
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims 1
- 235000019797 dipotassium phosphate Nutrition 0.000 claims 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims 1
- 239000003630 growth substance Substances 0.000 abstract description 34
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000001994 activation Methods 0.000 description 30
- 238000012258 culturing Methods 0.000 description 21
- 239000000243 solution Substances 0.000 description 21
- 239000002054 inoculum Substances 0.000 description 14
- 239000000454 talc Substances 0.000 description 8
- 235000012222 talc Nutrition 0.000 description 8
- 229910052623 talc Inorganic materials 0.000 description 8
- 241001019659 Acremonium <Plectosphaerellaceae> Species 0.000 description 6
- 241000722941 Achillea Species 0.000 description 5
- 230000003213 activating effect Effects 0.000 description 5
- 239000003814 drug Substances 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 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
- HCWCAKKEBCNQJP-UHFFFAOYSA-N magnesium orthosilicate Chemical compound [Mg+2].[Mg+2].[O-][Si]([O-])([O-])[O-] HCWCAKKEBCNQJP-UHFFFAOYSA-N 0.000 description 3
- 239000000391 magnesium silicate Substances 0.000 description 3
- 229910052919 magnesium silicate Inorganic materials 0.000 description 3
- 235000019792 magnesium silicate Nutrition 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 208000003734 Supraventricular Tachycardia Diseases 0.000 description 2
- 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 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000012982 microporous membrane Substances 0.000 description 2
- 235000019794 sodium silicate Nutrition 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
- 240000000073 Achillea millefolium Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 206010028980 Neoplasm Diseases 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
- 239000004480 active ingredient Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000003056 antler Anatomy 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
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000012041 food component Nutrition 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
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 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
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 230000001954 sterilising effect Effects 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 a cordyceps sinensis artificial fermentation culture technology, and discloses a method for producing adenosine by cordyceps sinensis fermentation. The method for producing adenosine by cordyceps sinensis fermentation provided by the invention 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 sinensis, effectively promote the growth of the cordyceps sinensis, improve the yield of the cordyceps sinensis, save the production cost and is suitable for popularization and application.
Description
Technical Field
The invention relates to a cordyceps sinensis artificial fermentation culture technology, in particular to a method for producing adenosine by cordyceps sinensis fermentation.
Background
The natural Cordyceps is a complex of larva corpse and fungus sub-seat formed by infecting host insect larva with ascospores, conidium or hyphae of asexual strain. Modern medicine proves that Cordyceps sinensis has wide pharmacological actions of immunoregulation, oxidation resistance, aging resistance, fatigue resistance, bacteria resistance, tumor resistance, liver and kidney protection, and has good research value and research heat in the world when being combined with ginseng and pilose antler.
Adenosine, one of the main active ingredients of cordyceps, has been widely used in the pharmaceutical industry as a drug, has great commercial value, has become a first-line drug approved by the U.S. Food and Drug Administration (FDA) for treating paroxysmal supraventricular tachycardia (PSVT), and has subsequently been approved as a therapeutic drug in various other countries in the united kingdom, italy, and the like.
There are many methods for producing adenosine at present, but the methods can be mainly divided into two types, namely biosynthesis and chemical synthesis. The chemical synthesis method has the defects of complex process, high cost, low yield, large 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 cordyceps fermentation to produce adenosine, has the advantages of low cost, easiness in fermentation, no environmental pollution and the like, and can realize large-scale industrial production. However, the process of producing adenosine by fermentation method has to be improved in terms of adenosine production speed, adenosine yield and adenosine quality, etc., so as to lay a foundation for mass production by biological fermentation method of adenosine.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a method for producing adenosine by cordyceps sinensis fermentation, which can obviously improve the content of adenosine in cordyceps sinensis, effectively promote the growth of cordyceps sinensis and save the cost.
In order to achieve the above object, the present invention provides a method for producing adenosine by fermentation of Cordyceps sinensis, comprising 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 separately during the activation culture and the fermentation culture.
Preferably, the growth regulator is added in an amount of 15-25g/L during the activation culture.
Preferably, the growth regulator during the activation culture is talc.
Preferably, the addition amount of the growth regulator in the fermentation culture process is 1.5-30g/L.
Preferably, the growth regulator in the fermentation culture process is silicate and cellulose powder.
Preferably, the mass ratio of silicate to cellulose powder is 0.05-20:1.
preferably, 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 mesh.
Preferably, the process of activation culture comprises:
(1) Inoculating the Cordyceps sinensis into a first seed culture medium after slant activation to perform first seed culture to obtain a first seed liquid;
(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 medium and/or the secondary seed medium, more preferably the growth regulator is added to the primary seed medium.
Preferably, the primary seed 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 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 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.
Preferably, the conditions of the primary seed medium include: the temperature is 18-25 ℃, the rotating speed is 100-150rpm, and the time is 15-20 days.
Preferably, the conditions of the secondary seed 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% by volume.
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 100-150rpm, and the time is 15-20 days.
Through the technical scheme, the invention has the beneficial effects that:
by adopting the method for producing the adenosine by the cordyceps sinensis fermentation, the adenosine is effectively promoted to be formed in the growth metabolism process of the cordyceps sinensis by adding the growth regulator, the adenosine content in the cordyceps sinensis is obviously improved, the growth of the cordyceps sinensis is effectively promoted, the yield of the cordyceps sinensis is improved, the production cost is saved, and the method is suitable for popularization and application, thereby laying a foundation for large-scale production by the biological fermentation method of the adenosine.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to 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 research process of the cordyceps, the inventor of the invention surprisingly finds that the addition of the growth regulator in the growth metabolism process of the cordyceps can effectively promote the synthesis of adenosine in the cordyceps and improve the adenosine content in the cordyceps, and can promote the growth of the cordyceps, improve the yield of the cordyceps and save the production cost, and the finding lays a certain foundation for the mass production of the adenosine by the biological fermentation method.
According to the invention, the method for producing adenosine by cordyceps sinensis fermentation further comprises the step of extracting adenosine from the fermented cordyceps sinensis mycelia. Illustratively, the extraction process may specifically be: carrying out ultrasonic crushing on cordyceps mycelia: 200W,10min (start 1s pause 3 s), then carry out ultrasonic extraction: 100W,60 ℃,40min, and centrifuging for 10min at 11000rpm, and taking clear liquid; repeatedly extracting the precipitate for 2 times, mixing the clear solutions, fixing the volume with ultrapure water, and filtering with 0.22 μm microporous membrane to obtain an extract containing adenosine.
The adenosine content of the extracting solution containing adenosine is analyzed by high performance liquid chromatography, and the analysis process can be specifically as follows:
chromatographic column: reversed phase 18 high performance liquid chromatography column (Unitary, 250 mm. Times.4.6 mm,5 μm);
mobile phase: a is 20mmol/L NaH 2 PO 4 、Na 2 HPO 4 pH6.89; b is chromatographic purity-methanol; a90%, B10%;
sample injection amount: 10. Mu.L; column temperature: 30 ℃; detection wavelength: 260nm;
preparing an adenosine label: preparing adenosine targets with concentrations of 100, 75, 50, 25, 10 and 5mg/mL respectively, and performing high performance liquid chromatography under the same conditions;
and finally, quantifying the adenosine in the extracting solution containing the adenosine according to the peak area, and further qualitatively quantifying the adenosine in the mycelium.
In the invention, the cordyceps sinensis can be natural cordyceps sinensis or cordyceps sinensis obtained through manual screening or induction, and is preferably asexual type phyllosphaera virens of Xinjiang fine 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 method in the art.
According to the present invention, the growth regulator may be added during the activation culture, the growth regulator may be added during the fermentation culture, or the growth regulator may be added during both the activation culture and the fermentation culture. Preferably, the growth regulator is added separately during the activation culture and the fermentation culture. The inventors have found that in this preferred embodiment, the mycelium production during fermentation of Cordyceps can be further increased, increasing the yield of adenosine.
In the invention, the growth regulator can be added into the corresponding culture medium of the activation culture or the fermentation culture, or can be added at any time point in the activation culture stage or the fermentation culture stage of the cordyceps sinensis at one time or added in batches. Preferably, the growth regulator is added to the corresponding medium of the activation culture or the fermentation culture.
According to the invention, the addition amount of the growth regulator in the activation culture process is not particularly limited, and the purpose of promoting the generation of adenosine by cordyceps sinensis can be achieved. 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 between the two values.
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 and cellulose powder, talcum powder, cellulose powder and talcum powder, silicate and cellulose powder. Preferably, the growth regulator during the activation culture is talc. The inventor finds that under the preferred embodiment, more mycelia and spore amount are formed in the growth process of the cordyceps sinensis, so that the content of adenosine in the cordyceps sinensis can be improved, and the yield of the cordyceps sinensis can be improved.
According to the invention, the addition amount of the growth regulator in the fermentation culture process is not particularly limited, and the purpose of promoting the generation of adenosine by cordyceps sinensis can be achieved. 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, or can be a combination of talcum powder, silicate and cellulose powder, talcum powder, cellulose powder and talcum powder, silicate and cellulose powder. Preferably, the growth regulator in the fermentation culture process is silicate and cellulose powder. The inventors have found that in this preferred embodiment, it is advantageous to further increase the yield of adenosine in Cordyceps sinensis.
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 0.5-20g/L, more preferably 5-15g/L; the cellulose powder is added in an amount of 1 to 10g/L, more preferably 5 to 10g/L. The inventors have found that in this preferred embodiment, it is advantageous to further increase the yield of adenosine in Cordyceps sinensis.
According to the invention, the talc is a talc of the family of mineral talc of the magnesium silicate type, which is commercially available. Preferably, the average particle size of the talcum powder is 800-5000 meshes, talcum powder with corresponding particle size can be purchased directly, and the purchased large-particle talcum powder can be crushed to obtain the talcum powder. The inventor finds that under the preferred embodiment, the regulation effect of talcum powder on the cordyceps sinensis activation process can be improved, and the yield of mycelium and spores is further improved.
According to the present invention, the kind of the silicate is not particularly limited as long as the corresponding adjusting action can be formed. 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, all of which are commercially available.
According to the present invention, the kind of the cellulose powder is not particularly limited as long as the corresponding conditioning effect can be formed. Preferably, the molecular weight of the cellulose powder is 10000-30000, the average particle size is 800-5000 meshes, the cellulose powder with corresponding particle size can be directly purchased, and the purchased large-particle cellulose can be crushed to obtain the cellulose powder.
According to the present invention, preferably, the process of the activation culture includes:
(1) Inoculating the Cordyceps sinensis into a first seed culture medium after slant activation to perform first seed culture to obtain a first seed liquid;
(2) And inoculating the primary seed liquid to a secondary seed culture medium for secondary seed culture to obtain a secondary seed liquid.
In the invention, the inclined surface activation of the cordyceps sinensis can be performed by adopting a conventional inclined surface culture medium, such as PDA culture medium, and sterilizing at the high temperature of 121 ℃ for 20min. Placing the Cordyceps thallus in slant culture medium, culturing at 20-25deg.C for 15-30 days, and performing slant activation.
According to the present invention, when the growth regulator is added during the activation culture, the growth regulator may be added to the primary seed medium and/or the secondary seed medium. Preferably, the growth regulator is added to the primary seed medium. The inventor finds that under the preferred embodiment, the growth regulator is beneficial to promoting the condition action of the growth regulator on the cordyceps sinensis, improving the growth activity of the cordyceps sinensis in the secondary seed culture stage and obtaining more mycelium and spore quantity.
In the invention, the primary seed culture medium and the secondary seed culture medium are not particularly limited, and can provide nutritional components required in the growth and activation process of the cordyceps sinensis.
According to the present invention, preferably, the primary seed medium contains 15 to 30g/L of a carbon source I, 8 to 15g/L of a nitrogen source I, 0.5 to 2g/L of a magnesium salt I, and 2 to 4g/L of a potassium salt I.
According to the invention, the secondary seed 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 commercially available.
The primary seed medium illustratively contains 15-30g/L glucose, 8-15g/L, mgSO peptone 4 ·7H 2 O0.5-2 g/L and KH 2 PO 4 2-4g/L; the secondary seed culture medium contains glucose 15-30g/L and peptone 8-15g/L, mgSO 4 ·7H 2 O 0.5-2g/L、KH 2 PO 4 2-4g/L and yeast extract 4-8g/L.
According to the present invention, preferably, the conditions of the primary seed medium include: the temperature is 18-25deg.C, specifically 18 deg.C, 20 deg.C, 22 deg.C, 24 deg.C, 25 deg.C or any value between the two values; the rotation speed is 100-150rpm, and can be specifically 100rpm, 110rpm, 120rpm, 130rpm, 140rpm, 150rpm or any value between the two values; the time is 15-20 days, and can be 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or any value between the two values.
According to the present invention, preferably, the conditions of the secondary seed medium include: the temperature is 18-25deg.C, specifically 18 deg.C, 20 deg.C, 22 deg.C, 24 deg.C, 25 deg.C or any value between the two values; the rotation speed is 100-150rpm, and can be specifically 100rpm, 110rpm, 120rpm, 130rpm, 140rpm, 150rpm or any value between the two values; the time is 7-12 days, and can be 7 days, 8 days, 9 days, 10 days, 11 days, 12 days or any value between the two values.
According to the present invention, the inoculum size of the primary seed culture stage, the secondary seed culture stage and the fermentation culture stage is not particularly limited, as long as it enables the normal production of Cordyceps sinensis 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% by volume of the secondary seed culture medium; the inoculation amount of the secondary seed liquid in the culture medium of the fermentation culture is 5-10% by volume, namely the inoculation amount of the secondary seed liquid is 5-10% by volume of the culture medium of the fermentation culture.
According to the present invention, the culture medium for the fermentation culture is not particularly limited, and may provide nutrient components required in the growth and activation process of 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 commercially available.
The culture medium for fermentation culture contains glucose 15-30g/L and peptone 8-15g/L, mgSO 4 ·7H 2 O 0.5-2g/L、KH 2 PO 4 2-4g/L and yeast extract 4-8g/L.
According to the present invention, preferably, the conditions of the fermentation culture include: the temperature is 18-25deg.C, specifically 18 deg.C, 20 deg.C, 22 deg.C, 24 deg.C, 25 deg.C or any value between the two values; the rotation speed is 100-150rpm, and can be specifically 100rpm, 110rpm, 120rpm, 130rpm, 140rpm, 150rpm or any value between the two values; the time is 15-20 days, and can be 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or any value between the two values.
According to a particularly preferred embodiment of the present invention, the method for producing adenosine by fermentation of Cordyceps sinensis comprises the steps of:
(1) Inoculating the Cordyceps into a primary seed culture medium after slant activation, and culturing the primary seeds at 18-25deg.C and rotation speed of 100-150rpm for 15-20 days to obtain primary seed solution, wherein the primary seed culture medium contains glucose 15-30g/L and peptone 8-15g/L, mgSO 4 ·7H 2 O0.5-2g/L、KH 2 PO 4 2-4g/L and 15-25g/L talcum powder, wherein the average particle size of the talcum powder is 800-5000 meshes;
(2) Inoculating the primary seed solution into a secondary seed culture medium with an inoculum size of 5-10 vol%, and culturing the secondary seed at 18-25deg.C and a rotation speed of 100-150rpm for 7-12 days to obtain secondary seed solution, wherein the secondary seed culture medium contains glucose 15-30g/L and peptone 8-15g/L, mgSO 4 ·7H 2 O 0.5-2g/L、KH 2 PO 4 2-4g/L and yeast extract 4-8g/L;
(3) Inoculating the secondary seed liquid into a fermentation culture medium in an inoculum size of 5-10 vol%, and performing fermentation culture for 15-20 days at a temperature of 18-25 ℃ and a rotating speed of 100-150rpm to obtain fermentation mycelium, and extracting adenosine from the fermentation mycelium; wherein the fermentation medium containsHas glucose 15-30g/L and peptone 8-15g/L, mgSO 4 ·7H 2 O 0.5-2g/L、KH 2 PO 4 2-4g/L, 4-8g/L yeast extract, 0.5-20g/L silicate and 1-10g/L 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 by examples.
In the following examples, asexual type of Cordyceps sinensis, achillea californica, was offered by the university of Nanjing, food and pharmaceutical engineering college; talcum powder is purchased from Nanjing JiaKai technology Co., ltd, and has an average particle size of 800-5000 mesh; cellulose (molecular weight 10000-30000) is purchased from Nanj JiaKai technology Co., ltd, and has an average particle size of 800-5000 mesh; cellulose (molecular weight 40000-50000) is purchased from Nanjing JiaKai technology Co., ltd, and has an average particle size of 800-5000 mesh; other raw materials and reagents are all conventional commercial products.
In the following examples, PDA medium was used for the bevel activation.
Example 1
(1) Inoculating Achillea-containing strain on PDA slant culture medium, activating at 20deg.C for 30 days to obtain activated mycelium, inoculating the activated mycelium to primary seed culture medium (containing glucose 25g/L and peptone 12g/L, mgSO) 4 ·7H 2 O 1g/L、KH 2 PO 4 3g/L and 20g/L of talcum powder with the average particle size of 800-5000 meshes), and culturing the primary seeds for 18 days under the conditions of 20 ℃ and 120rpm of rotating speed to obtain primary seed liquid;
(2) The primary seed solution was inoculated to a secondary seed medium (the secondary seed medium contained 25g/L glucose, 12g/L, mgSO peptone) at an inoculum size of 10 vol% 4 ·7H 2 O 1g/L、KH 2 PO 4 3g/L and 6g/L of yeast extract), and culturing the secondary seeds for 10 days at the temperature of 20 ℃ and the rotating speed of 120rpm to obtain secondary seed liquid;
(3) The secondary seed solution was inoculated to a fermentation medium (fermentation medium containing 20g/L glucose, 12g/L, mgSO peptone) at an inoculum size of 10% by volume 4 ·7H 2 O 1g/L，KH 2 PO 4 3g/L, 6g/L of yeast extract, 10g/L of sodium silicate, 5g/L of cellulose powder with molecular weight of 10000-30000 and average particle size of 800-5000 meshes) and fermenting and culturing for 20 days at 20 ℃ and rotating speed of 120rpm to obtain fermentation mycelium.
Example 2
(1) Inoculating Achillea-containing strain on PDA slant culture medium, activating at 25deg.C for 20 days to obtain activated mycelium, inoculating the activated mycelium to primary seed culture medium (containing 15g/L glucose and 8g/L, mgSO peptone) 4 ·7H 2 O 0.5g/L、KH 2 PO 4 2g/L and 15g/L of talcum powder with the average particle size of 800-5000 meshes), and culturing the primary seeds for 15 days at the temperature of 18 ℃ and the rotating speed of 150rpm to obtain primary seed liquid;
(2) Inoculating the primary seed solution to a secondary seed culture medium (the secondary seed culture medium contains 15g/L glucose and 8g/L, mgSO peptone) at an inoculation amount of 8 vol% 4 ·7H 2 O 0.5g/L、KH 2 PO 4 2g/L and 4g/L of yeast extract), and culturing the secondary seeds for 7 days at the temperature of 18 ℃ and the rotating speed of 150rpm to obtain secondary seed liquid;
(3) The secondary seed solution was inoculated to a fermentation medium (fermentation medium containing 15g/L glucose, 8g/L, mgSO peptone) at an inoculum size of 8% by volume 4 ·7H 2 O 0.5g/L，KH 2 PO 4 2g/L, 4g/L of yeast extract, 5g/L of potassium silicate, 10g/L of cellulose powder with molecular weight of 10000-30000 and average particle size of 800-5000 meshes) and fermenting and culturing for 20 days at 18 ℃ and rotating speed of 150rpm to obtain fermentation mycelium.
Example 3
(1) Inoculating Achillea-containing strain on PDA slant culture medium, activating at 22deg.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 ·7H 2 O 2g/L、KH 2 PO 4 4g/L and 25g/L of talcum powder with average particle size of 800-5000 meshes) at 25 ℃ at a rotating temperatureCulturing the first seed for 20 days at the speed of 100rpm to obtain a first seed solution;
(2) Inoculating the primary seed solution to a secondary seed culture medium (the secondary seed culture medium contains 30g/L glucose, 15g/L, mgSO peptone) at an inoculum size of 5 vol% 4 ·7H 2 O 2g/L、KH 2 PO 4 4g/L and 8g/L of yeast extract), and culturing the secondary seeds at 25 ℃ and a rotating speed of 100rpm for 12 days to obtain secondary seed liquid;
(3) The secondary seed solution was inoculated to a fermentation medium (fermentation medium containing 30g/L glucose, 15g/L peptone, mgSO) at an inoculum size of 5% by volume 4 ·7H 2 O 2g/L，KH 2 PO 4 4g/L, 8g/L of yeast extract, 15g/L of sodium silicate, 3g/L of cellulose powder with molecular weight of 10000-30000 and average particle size of 800-5000 meshes) and fermenting and culturing for 20 days at 20 ℃ and rotating speed of 120rpm to obtain fermentation mycelium.
Example 4
The fermentation of Acremonium pinnatifida was carried out as in example 2 to obtain fermented mycelium, except that in step (3), potassium silicate was added in an amount of 0.5g/L, and cellulose powder having a molecular weight of 10000-30000 and an average particle diameter of 800-5000 mesh was 1g/L.
Example 5
The fermentation of Acremonium pinnatifida was performed as in example 2 to obtain fermented mycelium, except that in step (1), talc having an average particle size of 800 to 5000 mesh was replaced with talc having an average particle size of 100 to 200 mesh.
Example 6
The fermentation of Acremonium pinnatifida was carried out in the same manner as in example 2 to obtain fermented mycelium, except that in step (3), 5g/L of cellulose powder having a molecular weight of 10000-30000 and an average particle size of 800-5000 mesh was replaced with 5g/L of cellulose powder having a molecular weight of 30000-40000 and an average particle size of 100-200 mesh.
Example 7
The fermentation of Acremonium pinnata was performed as in example 2 to obtain fermented mycelium, except that step (2) was replaced with:
(2) The first stage is carried outThe seed solution was inoculated to a secondary seed medium (the secondary seed medium contained 15g/L glucose and 8g/L, mgSO peptone) at an inoculum size of 8 vol% 4 ·7H 2 O 0.5g/L、KH 2 PO 4 2g/L, 4g/L yeast extract, 10g/L talcum powder with the average particle size of 800-5000 meshes and 10g/L sodium silicate, and culturing the secondary seeds for 7 days at the temperature of 18 ℃ and the rotating speed of 150rpm to obtain secondary seed liquid.
Example 8
The fermentation of Acremonium pinnata was performed as in example 2 to obtain fermented mycelium, except that step (3) was replaced with:
(3) The secondary seed solution was inoculated to a fermentation medium (fermentation medium containing 15g/L glucose, 8g/L, mgSO peptone) at an inoculum size of 8% by volume 4 ·7H 2 O 0.5g/L，KH 2 PO 4 2g/L, 4g/L of yeast extract and 5g/L of potassium silicate) and fermenting and culturing for 20 days at the temperature of 18 ℃ and the rotating speed of 150rpm to obtain fermentation mycelium.
Example 9
The fermentation of Acremonium pinnata was performed as in example 2 to obtain fermented mycelium, except that step (3) was replaced with:
(3) The secondary seed solution was inoculated to a fermentation medium (fermentation medium containing 15g/L glucose, 8g/L, mgSO peptone) at an inoculum size of 8% by volume 4 ·7H 2 O 0.5g/L，KH 2 PO 4 3g/L and 4 g/L) of yeast extract, and fermenting and culturing at 20deg.C and rotational speed of 120rpm for 20 days to obtain fermented mycelium.
Example 10
(1) Inoculating Achillea-containing strain on PDA slant culture medium, activating at 25deg.C for 20 days to obtain activated mycelium, inoculating the activated mycelium to primary seed culture medium (containing 15g/L glucose and 8g/L, mgSO peptone) 4 ·7H 2 O0.5 g/L and KH 2 PO 4 2 g/L), culturing the first seed at 18 deg.C and 150rpm, adding 15g/L of talcum powder with average particle size of 800-5000 meshes after culturing for 5 days, and continuously culturing for 1Obtaining first-level seed liquid after 0 days;
(2) Inoculating the primary seed solution to a secondary seed culture medium (the secondary seed culture medium contains 15g/L glucose and 8g/L, mgSO peptone) at an inoculation amount of 8 vol% 4 ·7H 2 O 0.5g/L、KH 2 PO 4 2g/L and 4g/L of yeast extract), and culturing the secondary seeds for 7 days at the temperature of 18 ℃ and the rotating speed of 150rpm to obtain secondary seed liquid;
(3) The secondary seed solution was inoculated to a fermentation medium (fermentation medium containing 15g/L glucose, 8g/L, mgSO peptone) at an inoculum size of 8% by volume 4 ·7H 2 O 0.5g/L，KH 2 PO 4 2g/L, 4g/L of yeast extract, 5g/L of potassium silicate and 1g/L of cellulose powder with molecular weight of 10000-30000 and average particle size of 800-5000 meshes) are fermented and cultured at the temperature of 18 ℃ and the rotating speed of 150rpm, 5 days later, 5g/L of sodium silicate and 1g/L of cellulose powder with molecular weight of 10000-30000 and average particle size of 800-5000 meshes are added, and the culture is continued for 20 days to obtain fermented mycelium.
Comparative example 1
(1) Inoculating Achillea-containing strain on PDA slant culture medium, activating at 22deg.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 ·7H 2 O2 g/L and KH 2 PO 4 4 g/L), and culturing the first seed for 20 days at the temperature of 25 ℃ and the rotating speed of 100rpm to obtain first seed liquid;
(2) Inoculating the primary seed solution to a secondary seed culture medium (the secondary seed culture medium contains 30g/L glucose, 15g/L, mgSO peptone) at an inoculum size of 5 vol% 4 ·7H 2 O 2g/L、KH 2 PO 4 4g/L and 8g/L of yeast extract), and culturing the secondary seeds at 25 ℃ and a rotating speed of 100rpm for 12 days to obtain secondary seed liquid;
(3) The secondary seed solution was inoculated to a fermentation medium (fermentation medium containing 30g/L glucose, 15g/L peptone, mgSO) at an inoculum size of 5% by volume 4 ·7H 2 O 2g/L，KH 2 PO 4 4g/L and 8g/L of yeast extract) and fermenting and culturing at 20 ℃ and a rotating speed of 120rpm for 15 days to obtain fermentation mycelium.
Test case
The yield (dry weight) of the fermented mycelia obtained in example 1-example 10 and comparative example 1 was measured, and a part of mycelia was washed 3 times with ultrapure water, and after leaching treatment and filtration with a microporous membrane, the determination of the adenosine content was performed by high performance liquid chromatography, and the results are shown in Table 1.
TABLE 1
As can be seen from the results in Table 1, the method for producing adenosine by fermenting Cordyceps sinensis provided in examples 1-10 can significantly improve the content of adenosine in Cordyceps sinensis, can improve the yield of Cordyceps sinensis, saves the production cost, and is suitable for popularization and application.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (5)
1. A method for producing adenosine by cordyceps fermentation, which is characterized by comprising the following steps: performing activation culture on the cordyceps sinensis, and then performing fermentation culture;
the process of the activation culture comprises the following steps:
(1) Inoculating the Cordyceps sinensis into a first seed culture medium after slant activation to perform first seed culture to obtain a first seed liquid;
(2) Inoculating the primary seed liquid to a secondary seed culture medium for secondary seed culture to obtain a secondary seed liquid;
talcum powder is added into the primary seed culture medium, the adding amount of talcum powder is 15-25g/L, silicate and cellulose powder are added into the fermentation culture medium, the adding amount of silicate and cellulose powder is 1.5-30g/L, and the mass ratio of silicate to cellulose powder is 0.05-20:1, a step of; the average particle size of the talcum powder is 800-5000 meshes, the silicate is at least one selected from sodium silicate and potassium silicate, the molecular weight of the cellulose powder is 10000-30000, and the average particle size is 800-5000 meshes;
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; 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; the culture medium for 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.
2. The method according to claim 1, wherein 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 monopotassium phosphate, dipotassium phosphate, potassium chloride and potassium sulfate.
3. The method of claim 1, wherein the primary seed culture conditions comprise: the temperature is 18-25 ℃, the rotating speed is 100-150rpm, and the time is 15-20 days;
the conditions for the secondary seed culture include: the temperature is 18-25 ℃, the rotating speed is 100-150rpm, and the time is 7-12 days.
4. The method of claim 1, wherein the amount of seed liquid inoculated in 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% by volume.
5. The method according to claim 1, wherein the carbon source III is at least one selected from the group consisting of glucose, fructose, sucrose, lactose and maltose,
the nitrogen source III is at least one selected from peptone, yeast extract, corn steep liquor and soybean meal,
the magnesium salt III is at least one of 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;
the conditions of the fermentation culture include: the temperature is 18-25 ℃, the rotating speed is 100-150rpm, and the time is 15-20 days.
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