CN108715870B - 一种提高桦褐孔菌发酵生产三萜产量的方法 - Google Patents
一种提高桦褐孔菌发酵生产三萜产量的方法 Download PDFInfo
- Publication number
- CN108715870B CN108715870B CN201810353156.6A CN201810353156A CN108715870B CN 108715870 B CN108715870 B CN 108715870B CN 201810353156 A CN201810353156 A CN 201810353156A CN 108715870 B CN108715870 B CN 108715870B
- Authority
- CN
- China
- Prior art keywords
- fermentation
- inonotus obliquus
- triterpene
- yield
- eucommia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000414067 Inonotus obliquus Species 0.000 title claims abstract description 62
- 150000003648 triterpenes Chemical class 0.000 title claims abstract description 57
- 238000000855 fermentation Methods 0.000 title claims abstract description 55
- 230000004151 fermentation Effects 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 29
- 239000002609 medium Substances 0.000 claims abstract description 7
- 239000007640 basal medium Substances 0.000 claims abstract description 5
- 239000000843 powder Substances 0.000 claims description 29
- 241000208688 Eucommia Species 0.000 claims description 19
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 5
- 239000007836 KH2PO4 Substances 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- 240000008042 Zea mays Species 0.000 claims description 5
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 5
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 5
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims description 5
- 235000005822 corn Nutrition 0.000 claims description 5
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 5
- 235000013312 flour Nutrition 0.000 claims description 5
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 5
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 5
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 5
- 239000011686 zinc sulphate Substances 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 2
- 230000008901 benefit Effects 0.000 abstract description 5
- 241000208689 Eucommia ulmoides Species 0.000 description 41
- 210000004027 cell Anatomy 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 10
- 241000758797 Pterocarya Species 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 238000011160 research Methods 0.000 description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 8
- 230000008859 change Effects 0.000 description 8
- 239000003814 drug Substances 0.000 description 8
- 239000002028 Biomass Substances 0.000 description 7
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 6
- 206010033128 Ovarian cancer Diseases 0.000 description 6
- 206010061535 Ovarian neoplasm Diseases 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 201000005202 lung cancer Diseases 0.000 description 6
- 208000020816 lung neoplasm Diseases 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000002994 raw material Substances 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- XBZYWSMVVKYHQN-MYPRUECHSA-N (4as,6as,6br,8ar,9r,10s,12ar,12br,14bs)-10-hydroxy-2,2,6a,6b,9,12a-hexamethyl-9-[(sulfooxy)methyl]-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylic acid Chemical compound C1C[C@H](O)[C@@](C)(COS(O)(=O)=O)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C XBZYWSMVVKYHQN-MYPRUECHSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 230000001093 anti-cancer Effects 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- -1 fuscoporianol Chemical compound 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 201000004624 Dermatitis Diseases 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000010903 husk Substances 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- TYFQFVWCELRYAO-UHFFFAOYSA-N suberic acid Chemical compound OC(=O)CCCCCCC(O)=O TYFQFVWCELRYAO-UHFFFAOYSA-N 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- BQPPJGMMIYJVBR-UHFFFAOYSA-N (10S)-3c-Acetoxy-4.4.10r.13c.14t-pentamethyl-17c-((R)-1.5-dimethyl-hexen-(4)-yl)-(5tH)-Delta8-tetradecahydro-1H-cyclopenta[a]phenanthren Natural products CC12CCC(OC(C)=O)C(C)(C)C1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21C BQPPJGMMIYJVBR-UHFFFAOYSA-N 0.000 description 1
- BDXXTCGLJBYHHM-ILLHTMCHSA-N (2r)-2-[(3s,5r,10s,13r,14r,17r)-3-hydroxy-4,4,10,13,14-pentamethyl-2,3,5,6,7,11,12,15,16,17-decahydro-1h-cyclopenta[a]phenanthren-17-yl]-6-methylhept-5-enal Chemical compound C([C@@]12C)C[C@H](O)C(C)(C)[C@@H]1CCC1=C2CC[C@]2(C)[C@@H]([C@H](C=O)CCC=C(C)C)CC[C@]21C BDXXTCGLJBYHHM-ILLHTMCHSA-N 0.000 description 1
- CHGIKSSZNBCNDW-UHFFFAOYSA-N (3beta,5alpha)-4,4-Dimethylcholesta-8,24-dien-3-ol Natural products CC12CCC(O)C(C)(C)C1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21 CHGIKSSZNBCNDW-UHFFFAOYSA-N 0.000 description 1
- XYTLYKGXLMKYMV-UHFFFAOYSA-N 14alpha-methylzymosterol Natural products CC12CCC(O)CC1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21C XYTLYKGXLMKYMV-UHFFFAOYSA-N 0.000 description 1
- BDXXTCGLJBYHHM-UHFFFAOYSA-N 3-Hydroxylanosta-8,24-dien-21-al Natural products CC12CCC(O)C(C)(C)C1CCC1=C2CCC2(C)C(C(C=O)CCC=C(C)C)CCC21C BDXXTCGLJBYHHM-UHFFFAOYSA-N 0.000 description 1
- FPTJELQXIUUCEY-UHFFFAOYSA-N 3beta-Hydroxy-lanostan Natural products C1CC2C(C)(C)C(O)CCC2(C)C2C1C1(C)CCC(C(C)CCCC(C)C)C1(C)CC2 FPTJELQXIUUCEY-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 206010000087 Abdominal pain upper Diseases 0.000 description 1
- 235000001553 Betula platyphylla Nutrition 0.000 description 1
- 241001313086 Betula platyphylla Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000747105 Fuscoporia Species 0.000 description 1
- BKLIAINBCQPSOV-UHFFFAOYSA-N Gluanol Natural products CC(C)CC=CC(C)C1CCC2(C)C3=C(CCC12C)C4(C)CCC(O)C(C)(C)C4CC3 BKLIAINBCQPSOV-UHFFFAOYSA-N 0.000 description 1
- 244000043261 Hevea brasiliensis Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- LOPKHWOTGJIQLC-UHFFFAOYSA-N Lanosterol Natural products CC(CCC=C(C)C)C1CCC2(C)C3=C(CCC12C)C4(C)CCC(C)(O)C(C)(C)C4CC3 LOPKHWOTGJIQLC-UHFFFAOYSA-N 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- CAHGCLMLTWQZNJ-UHFFFAOYSA-N Nerifoliol Natural products CC12CCC(O)C(C)(C)C1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21C CAHGCLMLTWQZNJ-UHFFFAOYSA-N 0.000 description 1
- JYDNKGUBLIKNAM-UHFFFAOYSA-N Oxyallobutulin Natural products C1CC(=O)C(C)(C)C2CCC3(C)C4(C)CCC5(CO)CCC(C(=C)C)C5C4CCC3C21C JYDNKGUBLIKNAM-UHFFFAOYSA-N 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- 241001558929 Sclerotium <basidiomycota> Species 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000000798 anti-retroviral effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- FVWJYYTZTCVBKE-ROUWMTJPSA-N betulin Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(CO)CC[C@@H](C(=C)C)[C@@H]5[C@H]4CC[C@@H]3[C@]21C FVWJYYTZTCVBKE-ROUWMTJPSA-N 0.000 description 1
- MVIRREHRVZLANQ-UHFFFAOYSA-N betulin Natural products CC(=O)OC1CCC2(C)C(CCC3(C)C2CC=C4C5C(CCC5(CO)CCC34C)C(=C)C)C1(C)C MVIRREHRVZLANQ-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 230000009702 cancer cell proliferation Effects 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- QBSJHOGDIUQWTH-UHFFFAOYSA-N dihydrolanosterol Natural products CC(C)CCCC(C)C1CCC2(C)C3=C(CCC12C)C4(C)CCC(C)(O)C(C)(C)C4CC3 QBSJHOGDIUQWTH-UHFFFAOYSA-N 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- CAHGCLMLTWQZNJ-RGEKOYMOSA-N lanosterol Chemical compound C([C@]12C)C[C@@H](O)C(C)(C)[C@H]1CCC1=C2CC[C@]2(C)[C@H]([C@H](CCC=C(C)C)C)CC[C@@]21C CAHGCLMLTWQZNJ-RGEKOYMOSA-N 0.000 description 1
- 229940058690 lanosterol Drugs 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 239000012907 medicinal substance Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920003052 natural elastomer Polymers 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229920001194 natural rubber Polymers 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 201000001245 periodontitis Diseases 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Images
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
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Epidemiology (AREA)
- Alternative & Traditional Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Medicines Containing Plant Substances (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了一种提高桦褐孔菌发酵生产三萜产量的方法，以桦褐孔菌为发酵菌，通过在液体发酵基础培养基中添加三萜产量促进物形成发酵培养基，三萜产量促进物的添加量为10‑60g/L，经液体深层发酵，从而提高三萜产量。本发明设备投资少，后处理简单，基本无污染，成本低，资源利用率高，能够明显提高桦褐孔菌液体深层发酵三萜的产量。
Description
技术领域
本发明涉及发酵工程技术领域，特别涉及一种提高桦褐孔菌发酵生产三萜产量的方法。
背景技术
桦褐孔菌,别名白桦茸,是一种生长于白桦树上的药用真菌,具有多种药效物质成分、较强的生物活性和广泛的药理作用。莫斯科医科院曾将桦褐孔菌认定为抗癌物质；美国将其归属为“特殊的天然物质”；日本将其作为一种治疗剂治疗艾滋病、肝癌和0-157大肠杆菌中毒。日本北方的阿伊努人饮用桦褐孔菌菌核的热水提取物来治疗胃痛和一些炎症；在俄罗斯北部和中部,桦褐孔菌提取物常用来降低病人的动脉和静脉血压,用来治疗牙周炎、湿疹、牛皮藓和皮炎,桦褐孔菌粉末用来治疗化脓性伤口且在治疗期间未发现毒性和药物使用的禁忌。此外，国外研究学者发现,桦褐孔菌在用于研制抗逆转录病毒药物中具有独特的功效且在临床测试中表明桦褐孔菌是一种安全、有效的疾病预防药物。因此，以其为原料开发新药将显示出巨大的发展潜力及经济效益！
随着中国经济的迅速发展，人类生活水平的不断提高，健康问题也越来越受到关注。据报道,截至2030年,全球将有2100万人患有癌症,其死亡人数达到1300万。近几年有研究表明，桦褐孔菌可以预防肿瘤细胞转移和复发,其代谢产物三萜类化合物能在体外抑制癌细胞的生长并诱导癌细胞的凋亡。目前桦褐孔菌三萜的抗癌研究正逐日升温,有关桦褐孔菌三萜抗卵巢癌、肺癌和间质瘤的研究报道尚不多见。研究开发新的抗癌药物已成为当前中医药界研究的热点,因此进一步研究开发桦褐孔菌三萜类化合物的抗肿瘤活性具有重大意义。
由于桦褐孔菌三萜类化合物提取效率低、产量较低和纯度不高等因素影响,导致桦褐孔菌三萜的药用价值开发利用严重不足。虽然通过液体发酵生产三萜类化合物可以大大缩短其生产周期,但其产量低是目前桦褐孔菌三萜研究与实际应用的限制性因素。有研究报道显示,通过改变培养基中碳源、氮源、无机盐、pH和发酵温度以及添加诱导剂对桦褐孔菌次级代谢产物的产量有较大影响。如果能够有效地提高三萜在液体深层发酵中的产量，那么，桦褐孔菌作为一种提取获得三萜的原料，就可以为人类造福，同时也降低了三萜类药物的获取成本，具有重要的战略意义。关于利用杜仲提高桦褐孔菌三萜类化合物产量的研究尚未见报道,众所周知,杜仲是我国特有的药用植物,资源丰富,杜仲皮是药用价值极高的中药，但杜仲翅果壳的药用价值不高，杜仲翅果壳提取天然橡胶的技术尚不成熟，导致杜仲翅果壳的利用率极低,从而造成了资源浪费的现象。因此本发明在基础培养基中添加杜仲翅果壳或杜仲皮，探索其对液体发酵桦褐孔菌三萜类化合物产生的促进作用。因而,本发明以杜仲翅果壳或杜仲皮粉末为发酵底物,不仅能提高桦褐孔菌三萜类化合物的产量，同时达到杜仲资源更好利用的目的。
发明内容
本发明的目的在于提供一种提高桦褐孔菌发酵生产三萜产量的方法，设备投资少，后处理简单，基本无污染，成本低，资源利用率高，能够明显提高桦褐孔菌液体深层发酵三萜的产量。
本发明解决其技术问题所采用的技术方案是：
一种提高桦褐孔菌发酵生产三萜产量的方法，以桦褐孔菌为发酵菌，通过在液体发酵基础培养基中添加三萜产量促进物形成发酵培养基，三萜产量促进物的添加量为10-60g/L，经液体深层发酵，从而提高三萜产量。
本发明在基础培养基中添加杜仲材料(杜仲翅果壳粉、杜仲皮粉)，探索其对液体发酵桦褐孔菌三萜类化合物产生的促进作用。经过研究发现，添加杜仲材料后能显著提高桦褐孔菌液体深层发酵三萜的产量。本发明以杜仲翅果壳或杜仲皮粉末为发酵底物,不仅能提高桦褐孔菌三萜类化合物的产量，同时达到杜仲资源更好利用的目的。
本发明主要可以获得白桦脂醇、木栓酸、桦褐孔菌醇、3β-hydroxy-lanosta-8,24-dien-21-al和羊毛甾醇等三萜类化合物。
作为优选，所述液体发酵基础培养基的组成为：玉米粉53g/L,蛋白胨3g/L,CoCl20.02g/L,KH2PO4 1g/L,MgSO4 0.2g/L,ZnSO4 0.01g/L,K2HPO40.5g/L,FeSO4 0.05g/L,CuSO40.02g/L,MnSO4 0.09g/L,pH自然。
作为优选，所述三萜产量促进物为杜仲翅果壳粉、杜仲皮粉或两者的混合物。杜仲翅果壳粉为杜仲翅果的壳的粉碎物，杜仲皮粉为杜仲皮的粉碎物。
作为优选，液体深层发酵的发酵条件为：发酵温度27-28℃,搅拌转速100-150转/分钟,起始pH和发酵过程pH均为自然,发酵周期为2-12天。
采用本发明提高桦褐孔菌发酵生产三萜产量的方法生产获得的三萜作为抗卵巢癌、肺癌和间质瘤药物原料的用途。本发明生产的三萜类化合物对肺癌细胞(A549和PC-9)和卵巢癌细胞(ES-2和SKOV-3)表现出强烈的抑制作用,且能有效抑制间质瘤细胞(MS924)的增殖。
本发明的有益效果是：
1、液体深层发酵具有设备投资少，后处理简单，发酵效率高，基本无污染，成本低等优点。
2、通过液体发酵技术生产桦褐孔菌三萜具有生产周期短,三萜含量相对稳定的特点。
3、发酵原料杜仲翅果壳、杜仲皮为天然产物,来源丰富,价格廉价,故而本发明所生产制备出来的三萜类化合物成本低。
4、杜仲翅果壳利用率低,在工业上的应用价值不高,造成了资源浪费现象,因此,本发明以此为发酵原料,有利于拓展杜仲翅果壳的加工利用途径并提高了其附加价值。
附图说明
图1杜仲翅果壳对液体深层发酵桦褐孔菌生物量动态变化的影响。
图2杜仲翅果壳对液体深层发酵桦褐孔菌总三萜产量动态变化的影响。
图3杜仲皮对液体深层发酵桦褐孔菌生物量动态变化的影响。
图4杜仲皮对液体深层发酵桦褐孔菌总三萜产量动态变化的影响。
图5桦褐孔菌三萜类化合物对肺癌细胞(A549和PC-9)增殖的抑制作用。
图6桦褐孔菌三萜类化合物对卵巢癌细胞(SKOV-3和ES-2)增殖的抑制作用。
图7桦褐孔菌三萜类化合物对间质瘤细胞(MS924)增殖的抑制作用。
具体实施方式
下面通过具体实施例，对本发明的技术方案作进一步的具体说明。
本发明中，若非特指，所采用的原料和设备等均可从市场购得或是本领域常用的。下述实施例中的方法，如无特别说明，均为本领域的常规方法。
由于桦褐孔菌三萜类化合物提取效率低、产量较低和纯度不高等因素影响,导致桦褐孔菌三萜的药用价值开发利用严重不足。利用杜仲降解转化对提高桦褐孔菌三萜类化合物产量的研究尚未见报道,本发明在液体发酵桦褐孔菌基础培养基中添加杜仲翅果壳粉末或杜仲皮粉末,探索其对三萜类化合物产生的促进作用。
实施例1：
液体发酵培养基：玉米粉53g/L,蛋白胨3g/L,CoCl2 0.02g/L,KH2PO4 1g/L,MgSO40.2g/L,ZnSO4 0.01g/L,K2HPO4 0.5g/L,FeSO4 0.05g/L,CuSO40.02g/L,MnSO4 0.09g/L,杜仲皮粉40g/L，pH自然。
桦褐孔菌10％的接种量，于28℃、136转/分钟恒温摇床培养。
杜仲皮对桦褐孔菌菌丝体生物量的动态变化：每隔24h取样测定桦褐孔菌菌丝体总量。添加杜仲皮培养基中，桦褐孔菌菌丝体生物量在第八天达到最大值为7.1g/L，对最大生物量的影响不显著(图1)。
杜仲皮对桦褐孔菌三萜类化合物产生的动态变化：每隔24h取样测定桦褐孔菌菌丝体总三萜含量。添加杜仲皮培养基中，桦褐孔菌总三萜产量在在第四天达到最大值为0.96g/L，比对照组(液体发酵基础培养基)提高了80.2％。(图2)。
三萜的提取分离：发酵结束后，将干燥得到的菌丝体用粉碎机磨成粉末状，并过60目筛子。称量1g粉末，向其加入20mL异丙醇，置于超声破碎仪中破壁4min(间歇时间为3s)，向其加入60mL异丙醇，置于50℃水中提取1天。取出、离心，收集上清液，所有提取物4000r/min离心5min后，浓缩冷冻干燥保存。
实施例2：
液体发酵培养基：玉米粉53g/L,蛋白胨3g/L,CoCl2 0.02g/L,KH2PO4 1g/L,MgSO40.2g/L,ZnSO4 0.01g/L,K2HPO4 0.5g/L,FeSO4 0.05g/L,CuSO40.02g/L,MnSO4 0.09g/L,杜仲翅果壳粉40g/L，pH自然。
桦褐孔菌10％的接种量，于28℃、136转/分钟恒温摇床培养。
杜仲翅果壳对桦褐孔菌菌丝体生物量的动态变化：每隔24h取样测定桦褐孔菌菌丝体总量。添加杜仲翅果壳培养基中，桦褐孔菌菌丝体生物量在第四天达到最大值为14.5g/L，比对照组(液体发酵基础培养基)提高了101.4％(图3)。
杜仲翅果壳对桦褐孔菌三萜类化合物产生的动态变化：每隔24h取样测定桦褐孔菌菌丝体总三萜含量。添加杜仲翅果壳培养基中，桦褐孔菌总三萜产量在第七天达到最大值约为1g/L，比对照组(液体发酵基础培养基)提高了87.6％(图4)。
三萜的提取分离：发酵结束后，将干燥得到的菌丝体用粉碎机磨成粉末状，并过60目筛子。称量1g粉末，向其加入20mL异丙醇，置于超声破碎仪中破壁4min(间歇时间为3s)，向其加入60mL异丙醇，置于50℃水中提取1天。取出、离心，收集上清液，所有提取物4000r/min离心5min后，浓缩冷冻干燥保存。
此外，液体发酵培养基：玉米粉53g/L,蛋白胨3g/L,CoCl2 0.02g/L,KH2PO4 1g/L,MgSO4 0.2g/L,ZnSO4 0.01g/L,K2HPO4 0.5g/L,FeSO4 0.05g/L,CuSO4 0.02g/L,MnSO40.09g/L,杜仲皮粉或杜仲翅果壳粉或杜仲皮粉与杜仲翅果壳粉的混合物10-60g/L，pH自然。发酵温度27-28℃,搅拌转速100-150转/分钟,发酵周期为2-12天。以上范围均是可以实施的。
本发明生产的三萜的抗癌活性：以每孔5000个的密度将处于对数生长期的癌细胞铺于96孔板中培养过夜。液体发酵桦褐孔菌三萜提取物(2、4、8、16、32、64μg/mL)分别处理肺癌细胞(A549,PC-9)、卵巢癌细胞(ES2，SKOV-3)、间质瘤细胞(MS924)至3天,达预设时相后，加入用PBS稀释的终浓度为0.5mg/mL的MTT工作液孵育4h，每孔加入150μL二甲基亚砜(DMSO),置摇床上低速振荡10min，在酶标仪490nm波长处读取吸光度OD(490)值,计算其IC50。桦褐孔菌三萜类化合物对肺癌细胞(A549和PC-9)(图5)和卵巢癌细胞(SKOV-3和ES-2)(图6)均表现出强烈的抑制活性，对间质瘤细胞(MS924)产生较好的抗癌效果(图7)。
以上所述的实施例只是本发明的一种较佳的方案，并非对本发明作任何形式上的限制，在不超出权利要求所记载的技术方案的前提下还有其它的变体及改型。
Claims (3)
1.一种提高桦褐孔菌发酵生产三萜产量的方法，其特征在于，以桦褐孔菌为发酵菌，通过在液体发酵基础培养基中添加三萜产量促进物形成发酵培养基，三萜产量促进物的添加量为10-60g/L，经液体深层发酵，从而提高三萜产量；
所述三萜产量促进物为杜仲翅果壳粉、杜仲皮粉或两者的混合物。
2.根据权利要求1所述的方法，其特征在于，所述液体发酵基础培养基的组成为：玉米粉 53g/L, 蛋白胨 3 g/L, CoCl2 0.02 g/L, KH2PO4 1 g/L, MgSO4 0.2 g/L, ZnSO4 0.01g/L, K2HPO4 0.5 g/L, FeSO4 0.05 g/L, CuSO4 0.02 g/L, MnSO4 0.09 g/L, pH自然。
3.根据权利要求1所述的方法，其特征在于，液体深层发酵的发酵条件为：发酵温度27-28℃, 搅拌转速100-150转/分钟, 起始pH和发酵过程pH均为自然, 发酵周期为2-12天。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810353156.6A CN108715870B (zh) | 2018-04-19 | 2018-04-19 | 一种提高桦褐孔菌发酵生产三萜产量的方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810353156.6A CN108715870B (zh) | 2018-04-19 | 2018-04-19 | 一种提高桦褐孔菌发酵生产三萜产量的方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108715870A CN108715870A (zh) | 2018-10-30 |
| CN108715870B true CN108715870B (zh) | 2021-05-14 |
Family
ID=63899281
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810353156.6A Active CN108715870B (zh) | 2018-04-19 | 2018-04-19 | 一种提高桦褐孔菌发酵生产三萜产量的方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108715870B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112972520B (zh) * | 2021-03-19 | 2022-12-09 | 浙江理工大学 | 一种利用桦褐孔菌液体深层发酵杜仲叶提高活性成分产量的方法 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106978465A (zh) * | 2017-04-06 | 2017-07-25 | 杭州娃哈哈科技有限公司 | 一种提高桦褐孔菌总三萜发酵产量的发酵方法 |
| CN107841523A (zh) * | 2017-10-19 | 2018-03-27 | 浙江大学 | 利用群体感应分子诱导从桦褐孔菌中提取三萜类物质方法 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9427008B2 (en) * | 2012-09-06 | 2016-08-30 | Mycotechnology, Inc. | Method of myceliation of agricultural substates for producing functional foods and nutraceuticals |
-
2018
- 2018-04-19 CN CN201810353156.6A patent/CN108715870B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106978465A (zh) * | 2017-04-06 | 2017-07-25 | 杭州娃哈哈科技有限公司 | 一种提高桦褐孔菌总三萜发酵产量的发酵方法 |
| CN107841523A (zh) * | 2017-10-19 | 2018-03-27 | 浙江大学 | 利用群体感应分子诱导从桦褐孔菌中提取三萜类物质方法 |
Non-Patent Citations (3)
| Title |
|---|
| Progress on Understanding the Anticancer Mechanisms of Medicinal Mushroom: Inonotus Obliquus;Fu-Qiang Song etal.;《ASIAN PACIFIC JOURNAL OF CANCER PREVENTION》;20131231;第14卷(第3期);第1571-1578页 * |
| 杜仲的化学成分及其药理作用研究进展;马博等;《Western Journal of Traditional Chinese Medicine》;20131231;第26卷(第12期);第153-159页 * |
| 液体深层发酵桦褐孔菌三萜类化合物的鉴定和生物合成的促进;张昕;《中国优秀硕士学位论文全文数据库》;20160715;正文第2-4页第1.1.2节、1.1.2.4节；正文第58页第4.1-4.2.1节、第60页第4.2.5-4.3.1节、第61-64页第4.4.1-4.4.2节、表4.3 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108715870A (zh) | 2018-10-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2020177390A1 (zh) | 一种猴头菌发酵制备含麦角硫因的化妆品原液的方法 | |
| Pokhrel et al. | Submerged culture conditions for mycelial yield and polysaccharides production by Lyophyllum decastes | |
| CN111317694B (zh) | 一种杜仲发酵提取液及其制备方法和其在化妆品中的应用 | |
| CN104856015A (zh) | 一种虫草酵素及其制备方法 | |
| CN105820956B (zh) | 一株牛樟芝菌株及牛樟芝液态发酵方法 | |
| Zhu et al. | Multi-fed batch culture integrated with three-stage light irradiation and multiple additions of copper ions for the hyperproduction of ganoderic acid and Ganoderma polysaccharides by the medicinal mushroom Ganoderma lucidum | |
| CN103598006B (zh) | 一种提高蛹虫草子实体中虫草菌素含量的方法 | |
| CN113150179A (zh) | 一种金耳多糖提取制备工艺 | |
| CN101445815A (zh) | 微生物合成γ-亚麻酸油脂的方法 | |
| CN108715870B (zh) | 一种提高桦褐孔菌发酵生产三萜产量的方法 | |
| CN117442510A (zh) | 一种基于Sirt信号通道的抗皱纹组合物及其制备方法和应用 | |
| CN112790310A (zh) | 一种雪莲多肽复合粉及其制备方法 | |
| CN103880826A (zh) | 一种异苯并呋喃酮化合物及其制备方法和应用 | |
| KR100844980B1 (ko) | 버섯 균사체 배양에 의한 기능성 뽕잎 건강식품의제조방법 | |
| TWM590969U (zh) | 具有仿生物間質系統的芸香科植物發酵液微粒結構 | |
| CN104894184B (zh) | 一种添加山药皮的姬松茸液体发酵培养基、发酵工艺及发酵制品 | |
| Wei et al. | Extraction and Antioxidant Activities of Polysaccharides Produced by Submerged Mycelia Culture of Oudemansiella raphanipes. | |
| CN113875989A (zh) | 一种功能化水溶性纳米硒、制备方法及应用 | |
| CN111789252A (zh) | 一种酶解发酵法提高玛卡补肾壮阳功效的方法 | |
| CN102188485A (zh) | 一种提高北五味子有效成分的微生物发酵方法 | |
| CN112251469A (zh) | 一种酶解发酵法提高玛卡抗氧化能力的方法 | |
| CN112079938A (zh) | 一种青稞多糖的提取方法、青稞多糖提取物及其应用 | |
| AU2020102037A4 (en) | A method of efficiently increasing the alpha-glucosidase inhibitor content in fresh mulberry leaves by the solid-state fermentation | |
| KR100320743B1 (ko) | 항산화, 미백, 보습, 면역증강 및 항여드름 작용을 나타내는 목질진흙버섯 자실체 및 배양균사체 추출물, 그 제조방법 및 용도 | |
| CN112553088B (zh) | 内生真菌a871提取物及其制备方法和在制备治疗痛风药物中的应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |