CN109402211A - A kind of method of monomer ganoderic acid content in raising glossy ganoderma cell - Google Patents
A kind of method of monomer ganoderic acid content in raising glossy ganoderma cell Download PDFInfo
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- CN109402211A CN109402211A CN201811201185.7A CN201811201185A CN109402211A CN 109402211 A CN109402211 A CN 109402211A CN 201811201185 A CN201811201185 A CN 201811201185A CN 109402211 A CN109402211 A CN 109402211A
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- glossy ganoderma
- ganoderic acid
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- 229930182735 Ganoderic acid Natural products 0.000 title claims abstract description 28
- RDMQPKIDHAFXKA-JNORPAGFSA-N Ganoderic Acid Am1 Chemical compound C([C@@]12C)C[C@H](O)C(C)(C)[C@@H]1CC(=O)C1=C2C(=O)C[C@]2(C)[C@@H]([C@@H](CC(=O)CC(C)C(O)=O)C)CC(=O)[C@]21C RDMQPKIDHAFXKA-JNORPAGFSA-N 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 24
- 241000222336 Ganoderma Species 0.000 title claims abstract description 23
- 239000000178 monomer Substances 0.000 title claims abstract description 22
- 238000011218 seed culture Methods 0.000 claims abstract description 25
- 239000002609 medium Substances 0.000 claims abstract description 22
- 238000000855 fermentation Methods 0.000 claims abstract description 21
- 230000004151 fermentation Effects 0.000 claims abstract description 21
- 239000001963 growth medium Substances 0.000 claims abstract description 19
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 18
- 239000011324 bead Substances 0.000 claims abstract description 15
- HCWCAKKEBCNQJP-UHFFFAOYSA-N magnesium orthosilicate Chemical compound [Mg+2].[Mg+2].[O-][Si]([O-])([O-])[O-] HCWCAKKEBCNQJP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000000391 magnesium silicate Substances 0.000 claims abstract description 14
- 229910052919 magnesium silicate Inorganic materials 0.000 claims abstract description 14
- 235000019792 magnesium silicate Nutrition 0.000 claims abstract description 14
- 239000002245 particle Substances 0.000 claims abstract description 11
- 230000003213 activating effect Effects 0.000 claims abstract description 3
- 239000008187 granular material Substances 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 25
- 244000061456 Solanum tuberosum Species 0.000 description 12
- 235000002595 Solanum tuberosum Nutrition 0.000 description 12
- 240000008397 Ganoderma lucidum Species 0.000 description 10
- 235000001637 Ganoderma lucidum Nutrition 0.000 description 10
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 9
- 239000011691 vitamin B1 Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 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 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000001888 Peptone Substances 0.000 description 6
- 108010080698 Peptones Proteins 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 description 6
- 235000019319 peptone Nutrition 0.000 description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000011859 microparticle Substances 0.000 description 5
- 238000012807 shake-flask culturing Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
- RERVSJVGWKIGTJ-RQLZKMEDSA-N Ganoderic acid C2 Chemical compound C([C@@]12C)C[C@H](O)C(C)(C)[C@@H]1C[C@H](O)C1=C2C(=O)C[C@]2(C)[C@@H]([C@@H](CC(=O)C[C@@H](C)C(O)=O)C)C[C@H](O)[C@]21C RERVSJVGWKIGTJ-RQLZKMEDSA-N 0.000 description 3
- 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 description 3
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 230000000144 pharmacologic effect Effects 0.000 description 3
- 239000011574 phosphorus Substances 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 239000011591 potassium Substances 0.000 description 3
- 229910052700 potassium Inorganic materials 0.000 description 3
- 235000012015 potatoes Nutrition 0.000 description 3
- OTUZGGSAOMCYNC-AEGHVTTBSA-N Ganoderic acid Me Chemical compound CC1(C)[C@H](OC(C)=O)CC[C@]2(C)C3=CC[C@]4(C)[C@@H]([C@@H](CC\C=C(/C)C(O)=O)C)C[C@H](OC(C)=O)[C@@]4(C)C3=CC[C@H]21 OTUZGGSAOMCYNC-AEGHVTTBSA-N 0.000 description 2
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 2
- -1 alkaloid amino acid Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
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- 239000004615 ingredient Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 206010002660 Anoxia Diseases 0.000 description 1
- 241000976983 Anoxia Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- OCLVBEOPEKEKNM-CAXIOAJTSA-N Ganoderic Acid T Chemical compound CC1(C)[C@H](OC(C)=O)CC[C@]2(C)C3=CC[C@]4(C)[C@@H]([C@@H]([C@H](C\C=C(/C)C(O)=O)OC(C)=O)C)C[C@H](OC(C)=O)[C@@]4(C)C3=CC[C@H]21 OCLVBEOPEKEKNM-CAXIOAJTSA-N 0.000 description 1
- BCZACVDBVIYNMZ-DQPNMFNUSA-N Ganoderic acid Mk Natural products O([C@H]([C@@H](C)[C@H]1[C@@]2(C)[C@](C)([C@@H](O)C1)C=1C([C@@]3(C)[C@@H](C(C)(C)[C@H](OC(=O)C)CC3)CC=1)=CC2)C/C=C(\C(=O)O)/C)C(=O)C BCZACVDBVIYNMZ-DQPNMFNUSA-N 0.000 description 1
- BCZACVDBVIYNMZ-HRHDIOLXSA-N Ganoderic acid Mk Chemical compound CC(C(C\C=C(/C)C(O)=O)OC(C)=O)C1C[C@H](O)C2(C)C3=CCC4C(C)(C)[C@@H](CCC4(C)C3=CCC12C)OC(C)=O BCZACVDBVIYNMZ-HRHDIOLXSA-N 0.000 description 1
- FRRMMEJOPQSLSE-UHFFFAOYSA-N Ganoderic acid S Natural products CC1(C)C(O)CCC2(C)C3=CCC4(C)C(C(C(CC=C(C)C(O)=O)OC(C)=O)C)CCC4(C)C3=CCC21 FRRMMEJOPQSLSE-UHFFFAOYSA-N 0.000 description 1
- AQUHIKXTCOSRFY-GOEVOFJGSA-N Ganoderic acid S Chemical compound CC1(C)C(=O)CC[C@]2(C)C3=CC[C@]4(C)[C@@H]([C@@H](CC\C=C(\C)C(O)=O)C)CC[C@@]4(C)C3=CC[C@H]21 AQUHIKXTCOSRFY-GOEVOFJGSA-N 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- BYBLEWFAAKGYCD-UHFFFAOYSA-N Miconazole Chemical compound ClC1=CC(Cl)=CC=C1COC(C=1C(=CC(Cl)=CC=1)Cl)CN1C=NC=C1 BYBLEWFAAKGYCD-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010039897 Sedation Diseases 0.000 description 1
- OTUZGGSAOMCYNC-UHFFFAOYSA-N UNPD47180 Natural products CC1(C)C(OC(C)=O)CCC2(C)C3=CCC4(C)C(C(CCC=C(C)C(O)=O)C)CC(OC(C)=O)C4(C)C3=CCC21 OTUZGGSAOMCYNC-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 230000007953 anoxia Effects 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- OCLVBEOPEKEKNM-UHFFFAOYSA-N ganoderic acid T Natural products CC1(C)C(OC(C)=O)CCC2(C)C3=CCC4(C)C(C(C(CC=C(C)C(O)=O)OC(C)=O)C)CC(OC(C)=O)C4(C)C3=CCC21 OCLVBEOPEKEKNM-UHFFFAOYSA-N 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000002443 hepatoprotective effect Effects 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000013332 literature search Methods 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 229960002509 miconazole Drugs 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- DDBREPKUVSBGFI-UHFFFAOYSA-N phenobarbital Chemical compound C=1C=CC=CC=1C1(CC)C(=O)NC(=O)NC1=O DDBREPKUVSBGFI-UHFFFAOYSA-N 0.000 description 1
- 229960002695 phenobarbital Drugs 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000036280 sedation Effects 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 150000003648 triterpenes Chemical class 0.000 description 1
- AQUHIKXTCOSRFY-UHFFFAOYSA-N tyromycic acid Natural products CC1(C)C(=O)CCC2(C)C3=CCC4(C)C(C(CCC=C(C)C(O)=O)C)CCC4(C)C3=CCC21 AQUHIKXTCOSRFY-UHFFFAOYSA-N 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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Abstract
The invention discloses a kind of methods of monomer ganoderic acid content in raising glossy ganoderma cell; it is to be inoculated into the liquid seed culture medium containing bead to cultivate after activating ganoderma strain; obtain seed liquor; seed liquor is inoculated into the fermentation medium for being added to magnesium silicate granule or alumina particle; the constant-temperature shaking culture at 24-30 DEG C realizes the purpose for improving monomer ganoderic acid content in glossy ganoderma cell；Method of the invention can make GA-Mk in glossy ganoderma cell, GA-T, GA-S, and the content of GA-Me improves, and method is simple, easy to operate, be suitable for industrialized production and marketing application.
Description
Technical field
It is specifically a kind of to be improved in shaken cultivation glossy ganoderma cell by addition microparticle the invention belongs to field of biotechnology
The method of monomer ganoderic acid content.
Background technique
Ganoderma lucidum (Ganoderma lucidum) it is a kind of higher fungus, have thousands of years as a kind of traditional Chinese medicine
Medicinal history.Modern pharmacology research shows that ganoderma lucidum has an extensive pharmacological action, such as anti HIV-1 virus, anti anoxia and anti-
Aging effect, adjusting blood lipid, hypoglycemic, sedation, hepatoprotective effect etc..Ganoderma lucidum contains various active substance: polysaccharide, triterpene
Substance, alkaloid amino acid polypeptide etc.；Ganoderic acid has anti HIV-1 virus, anti-rotation as a kind of important triterpene compound
The effect of shifting, antitumor etc.；Different monomer ganoderic acids also shows different pharmacological activity, for example ganoderic acid T has induction
Increase Apoptosis of Lung Cancer Cells activity (Tang W, et al.Life Sciences, 2006；80,205-211), and ganoderic acid Me have suppression
Tumour processed diffusion (Chen NH, et al. Journal of Pharmacological Sciences, 2008；108,212-
216) and enhance IL-2 and IFN-γ expression and increase NK cell activity (Wang G, et al.International
Immunopharmacology, 2007；7,864-870).Ganoderic acid S is inhibited to the proliferation of cancer cell and invasion
(Liu RM, et al. Phytomedicine, 2011；18,349-355；Tang W, et al. Life Sciences,
2006；80,205-211) ganoderic acid Mk can be inhibited the proliferation of HeLa cell by mitochondria pathway and induce cell apoptosis (Liu
R M, et al. Latin American Journal of Pharmacy, 2012；31,43-50).Ganoderic acid is in cell
Low content and its difficulty isolated and purified seriously limit it extensive use and its mechanism of action further research.
Through the literature search of existing technologies, public in Chinese invention patent application prospectus CN103392510A
Second stage addition acetic acid during two stages liquid shake-flask fermentation has been opened the method that improves ganoderic acid content, compared to pair
According to improving 27.6%-156.2%.A kind of liquid is provided in Chinese invention patent application prospectus CN101692772A to shake
The method that bottle culture adds phenobarbital or Miconazole to improve ganoderic acid content with Liquid static culture and in stationary culture,
The method makes the content of monomer ganoderic acid improve 15%-60%；But monomer ganoderic acid is improved by addition micron order microparticle
Method there is not been reported.
Summary of the invention
In order to overcome the deficiencies of the prior art, the present invention provides one kind improves monomer in glossy ganoderma cell by addition microparticle
The method of ganoderic acid content；The method of the present invention is that the liquid seed culture medium containing bead is inoculated into after activating ganoderma strain
In cultivated, obtain seed liquor, seed liquor is inoculated into the fermentation medium for being added to magnesium silicate granule or alumina particle
In, the constant-temperature shaking culture at 24-30 DEG C realizes the purpose for improving ganoderic acid content in glossy ganoderma cell；By the method for the invention
So that the content of GA-Mk, GA-T, GA-S, GA-Me are up to 3.3,11.6,9.37,14.8 times compareed in glossy ganoderma cell.
The final concentration n of the magnesium silicate in the fermentation medium is 0 < n <, 20 g/L, and aluminium oxide is in the fermentation medium
Final concentration m be 0 < m <, 20 g/L.
The partial size of the magnesium silicate granule or alumina particle is 5-50 μm.
The partial size of the bead is 5mm.
The additive amount of the seed liquor is the 6% of fermentation medium volume.
The method of the present invention the following steps are included:
(1) ganoderma lucidum mycelium is cultivated in potato medium slant
The formula of potato culture medium: every liter of juice (addition 200g peeled potatoes boil 30 min acquisition), glucose 10g, anhydrous
Magnesium sulfate 1.5g, potassium dihydrogen phosphate 3g, vitamin B10.05g, agar 20g, pH 5.5.
(2) liquid seed culture medium is prepared
The formula of liquid seed culture medium: 1L water, glucose 35g, peptone 5g, yeast powder 2.5g, anhydrous magnesium sulfate 0.5g, phosphorus
Acid dihydride potassium 1g, vitamin B10.05g, pH 5.5；
(3) mycelium being grown in potato medium slant is accessed into the liquid seed culture medium containing 5 mm bead of diameter
In, stationary culture obtains first order seed culture solution；
(4) first order seed culture solution is taken, is accessed in the liquid seed culture medium containing 5 mm bead of diameter, stationary culture obtains
Obtain secondary seed culture solution；
(5) secondary seed culture solution is inoculated into the fermentation medium for being added to magnesium silicate granule and alumina particle, in 24-
Constant-temperature shaking culture at 30 DEG C realizes the purpose for improving ganoderic acid content in glossy ganoderma cell；
The formula of fermentation medium: 1L water, lactose 35g, peptone 5g, yeast powder 5g, anhydrous magnesium sulfate 0.5g, potassium dihydrogen phosphate
1g, vitamin B10.05g, pH 5.5.
The culture medium and cultivation temperature that the activation of lucidum strain used in the method for the present invention and culture use are conventional make
Condition of culture.
The present invention extracts the survey collected ganoderic acid and carry out monomer ganoderic acid content using conventional method from tunning
Fixed (Xu JW, et al.Applied Microbiology and Biotechnology, 2010；85,941-949；Xu JW,
Et al.Applied and Environmental Microbiology, 2012；78,7968-7976).
Ganoderma lucidum involved in the present invention (Ganoderma lucidum) CGMCC 5.616, it can be protected from Chinese microorganism strain
Hiding committee common micro-organisms center (CGMCC) buys, centre address: Xiaoyun Road, Chaoyang District, Beijing City 32.
Compared with prior art, the present invention has the advantage that
The present invention adds micron-sized magnesium silicate granule or alumina particle in ganoderma strain fermentation process, and ganoderma lucidum can be made thin
Monomer Lanost-8-en-26-oic acid,3,7,12-trihydroxy-11,15,23-trioxo-,(3BETA,7BETA,12BETA)- A-Mk, GA-T, GA-S, GA-Me content significantly improves in born of the same parents；When magnesium silicate adds final concentration of 5 g/L,
In every 100 mg dry cell weight GA-Mk, GA-T, GA-S, GA-Me content can be up to 34.46 ± 3.45,110.48 ± 10.50,
114.19 ± 11.90,76.54 ± 9.02 μ g are 3.3,11.6,9.37,14.8 times of control respectively；When aluminium oxide addition is dense eventually
When degree is 10 g/L, in every 100 mg dry cell weight GA-Mk, GA-T, GA-S, GA-Me content can be up to 35.05 ± 3.34,
67.63 ± 7.45,85.16 ± 9.97,50.25 ± 6.54 μ g are 3.5,6.5,7.3,8.1 times of control respectively, and of the invention
Method is simple, it is easy to operate, it is suitable for industrialized production and marketing application.
Specific embodiment
Below by embodiment, invention is further described in detail, but the contents of the present invention are not limited thereto, this
Method operating according to a conventional method unless otherwise specified in embodiment, agents useful for same unless otherwise specified use conventional reagent
Or the reagent configured according to a conventional method.
Embodiment 1: the method for ganoderic acid content in this raising glossy ganoderma cell, steps are as follows:
(1) cultivate ganoderma lucidum mycelium in potato medium slant, 30 DEG C constant incubator stationary culture 8 days；
The formula of potato culture medium: every liter of juice (addition 200g peeled potatoes boil 30 min acquisition), glucose 10g, anhydrous
Magnesium sulfate 1.5g, potassium dihydrogen phosphate 3g, vitamin B10.05g, agar 20g, pH 5.5.
(2) liquid seed culture medium is prepared
The formula of liquid seed culture medium: 1L water, glucose 35g, peptone 5g, yeast powder 2.5g, anhydrous magnesium sulfate 0.5g, phosphorus
Acid dihydride potassium 1g, vitamin B10.05g, pH 5.5；
(3) in 250 mL shaking flasks, it is packed into diameter 5mm bead, 50 mL liquid seed culture mediums are poured into, by potato culture medium
On inclined-plane mycelium access shaking flask in, 30 DEG C constant incubator stationary culture 5 days, daily jog bead obtains level-one kind
Sub- culture solution；
(4) in 250mL shaking flask, it is packed into diameter 5mm bead, 50mL first order seed culture solution is accessed into liquid seed culture medium
In, 30 DEG C constant incubator stationary culture 3 days, daily jog bead obtains secondary seed culture solution；
(5) fermentation medium for containing various concentration magnesium silicate granule (10 μm), the ingredient of fermentation medium, fermented and cultured are prepared
The formula of base: 1L water, lactose 35g, peptone 5g, yeast powder 5g, anhydrous magnesium sulfate 0.5g, potassium dihydrogen phosphate 1g, vitamin B1
0.05g, pH 5.5；Magnesium silicate granule final concentration of 0,5,10,15,20 g/L in the fermentation medium；
(6) in 250 mL shaking flasks, 3mL secondary seed culture solution is accessed in 47mL fermentation medium, shake flask culture, 30
DEG C, 150 rpm, cultivate 12 days, obtain fermented cells, 3 Duplicate Samples be set.
The analysis of monomer ganoderic acid:
The cell of 0 g/L: weighing the cell that 100 mg are ground, and 2 mL, 70% ethyl alcohol soaked overnight, 4 DEG C of ultrasounds 2.5 of low temperature are added
H, 12000 rpm are centrifuged 5 min, draw supernatant, and 40 DEG C of vacuum drying add 200 μ l methanol, are vortexed, with 0.22 μm of membrane filtration,
The content of four kinds of monomer ganoderic acids of efficient liquid phase chromatographic analysis.
The cell of 5-20 g/L: centrifugation obtains the fermented cells containing whole microparticles, weighs whole dry weights after drying,
Whole dry weights subtract the weight i.e. net (dry) weight of cell of addition microparticle；The ratio of 2 mL70 % ethyl alcohol is added in 100mg cell
Ethyl alcohol and soaked overnight are added, low temperature 4 DEG C of ultrasounds 2.5 h, 12000 rpm are centrifuged 5 min, draw 2 mL supernatants, 40 DEG C of vacuum
It is dry, add 200 μ l methanol, is vortexed, with 0.22 μm of membrane filtration, the content of four kinds of monomer ganoderic acids of efficient liquid phase chromatographic analysis.
It the results are shown in Table 1, shown in table, compared with the control, add magnesium silicate granule (10 μm) in shake flask culture, hair
Monomer Lanost-8-en-26-oic acid,3,7,12-trihydroxy-11,15,23-trioxo-,(3BETA,7BETA,12BETA)- A-Mk, GA-T, GA-S, GA-Me content is significantly increased in glossy ganoderma cell after ferment, and is in addition concentration
When 5g/L, GA-Mk, GA-T, GA-S, GA-Me content reaches 34.46 ± 3.45,110.48 ± 10.50,114.19 ± 11.90,
76.54 ± 9.02 μ g/100 mg DW(dry cell weights), it is 3.3,11.6,9.37,14.8 times of control respectively.
Table 1
。
Embodiment 2: the method for ganoderic acid content in this raising glossy ganoderma cell, steps are as follows:
(1) cultivate ganoderma lucidum mycelium in potato medium slant, 30 DEG C constant incubator stationary culture 8 days；
The formula of potato culture medium: every liter of juice (addition 200g peeled potatoes boil 30 min acquisition), glucose 10g, anhydrous
Magnesium sulfate 1.5g, potassium dihydrogen phosphate 3g, vitamin B10.05g, agar 20g, pH 5.5.
(2) liquid seed culture medium is prepared
The formula of liquid seed culture medium: 1L water, glucose 35g, peptone 5g, yeast powder 2.5g, anhydrous magnesium sulfate 0.5g, phosphorus
Acid dihydride potassium 1g, vitamin B10.05g, pH 5.5；
(3) in 250 mL shaking flasks, it is packed into diameter 5mm bead, 50 mL liquid seed culture mediums are poured into, by potato culture medium
On inclined-plane mycelium access shaking flask in, 30 DEG C constant incubator stationary culture 5 days, daily jog bead obtains level-one kind
Sub- culture solution；
(4) in 250mL shaking flask, it is packed into diameter 5mm bead, 50mL first order seed culture solution is accessed into liquid seed culture medium
In, 30 DEG C constant incubator stationary culture 3 days, daily jog bead obtains secondary seed culture solution；
(5) fermentation medium for containing various concentration alumina particle (10 μm), the ingredient of fermentation medium, fermented and cultured are prepared
The formula of base: 1L water, lactose 35g, peptone 5g, yeast powder 5g, anhydrous magnesium sulfate 0.5g, potassium dihydrogen phosphate 1g, vitamin B1
0.05g, pH 5.5；Magnesium silicate granule final concentration of 0,5,10,15,20 g/L in the fermentation medium；
(6) in 250mL shaking flask, 3mL secondary seed culture solution is accessed in 47mL fermentation medium, shake flask culture, 30
DEG C, 150 rpm, cultivate 12 days, obtain fermented cells, 3 Duplicate Samples be set.
The analysis method of monomer ganoderic acid is with embodiment 1, and the results are shown in Table 2, and oxidation is added in shake flask culture
Alumina particles (10 μm), monomer Lanost-8-en-26-oic acid,3,7,12-trihydroxy-11,15,23-trioxo-,(3BETA,7BETA,12BETA)- A-Mk, GA-T, GA-S, GA-Me content is significantly increased in glossy ganoderma cell after fermentation, and
And when adding concentration is 10 g/L, GA-Mk, GA-T, GA-S, GA-Me content reaches 35.05 ± 3.34,67.63 ± 7.45,
85.16 ± 9.97,50.25 ± 6.54 μ g/100 mg DW are 3.5,6.5,7.3,8.1 times of control respectively；
Table 2
。
Claims (4)
1. a kind of method for improving monomer ganoderic acid content in glossy ganoderma cell, it is characterised in that: be inoculated with after activating ganoderma strain
It is cultivated into the liquid seed culture medium containing bead, obtains seed liquor, seed liquor is inoculated into and is added to magnesium silicate
In the fermentation medium of particle or alumina particle, the constant-temperature shaking culture at 24-30 DEG C is realized and improves monomer in glossy ganoderma cell
The purpose of ganoderic acid content.
2. the method according to claim 1 for improving monomer ganoderic acid content in glossy ganoderma cell, it is characterised in that: magnesium silicate
Final concentration n in the fermentation medium is 0 < n <, 20 g/L, and the final concentration m of aluminium oxide in the fermentation medium is 0 < m < 20
g/L。
3. the method according to claim 1 for improving monomer ganoderic acid content in glossy ganoderma cell, it is characterised in that: magnesium silicate
The partial size of particle or alumina particle is 5-50 μm.
4. the method according to claim 1 for improving monomer ganoderic acid content in glossy ganoderma cell, it is characterised in that: bead
Partial size be 5mm.
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| Title |
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| TING LEI TAO等: "Improved mycelia and polysaccharide production of Grifola frondosa by controlling morphology with microparticle Talc", 《MICROBIAL CELL FACTORIES》 * |
| 徐军伟: "灵芝细胞在液体静置培养和振荡培养中单体灵芝酸的生产及差异表达基因的鉴定", 《中国博士学位论文全文数据库 农业科技辑》 * |
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| CN111304091A (en) * | 2019-11-28 | 2020-06-19 | 昆明理工大学 | Method for improving ganoderic acid content in ganoderma lucidum cell culture |
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