CN117683645B - Ganoderma lucidum strain L4914 and cultivation method and application thereof - Google Patents
Ganoderma lucidum strain L4914 and cultivation method and application thereof Download PDFInfo
- Publication number
- CN117683645B CN117683645B CN202410156270.5A CN202410156270A CN117683645B CN 117683645 B CN117683645 B CN 117683645B CN 202410156270 A CN202410156270 A CN 202410156270A CN 117683645 B CN117683645 B CN 117683645B
- Authority
- CN
- China
- Prior art keywords
- ganoderma lucidum
- cultivation
- strain
- culture medium
- bag
- 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
- 240000008397 Ganoderma lucidum Species 0.000 title claims abstract description 141
- 235000001637 Ganoderma lucidum Nutrition 0.000 title claims abstract description 135
- 238000012364 cultivation method Methods 0.000 title claims abstract description 17
- 241000233866 Fungi Species 0.000 claims abstract description 42
- 244000005700 microbiome Species 0.000 claims abstract description 9
- 238000004321 preservation Methods 0.000 claims abstract description 4
- 239000001963 growth medium Substances 0.000 claims description 64
- 239000007788 liquid Substances 0.000 claims description 31
- 239000000843 powder Substances 0.000 claims description 28
- 241000222336 Ganoderma Species 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 238000012258 culturing Methods 0.000 claims description 22
- 238000000855 fermentation Methods 0.000 claims description 22
- 230000004151 fermentation Effects 0.000 claims description 22
- 239000002689 soil Substances 0.000 claims description 19
- 238000009631 Broth culture Methods 0.000 claims description 16
- 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 15
- 239000008103 glucose Substances 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 239000001888 Peptone Substances 0.000 claims description 14
- 108010080698 Peptones Proteins 0.000 claims description 14
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 14
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 14
- 235000019319 peptone Nutrition 0.000 claims description 14
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 13
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 13
- 238000011218 seed culture Methods 0.000 claims description 12
- 238000003306 harvesting Methods 0.000 claims description 11
- 238000011081 inoculation Methods 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 10
- 239000002023 wood Substances 0.000 claims description 10
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 7
- 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 7
- 239000005720 sucrose Substances 0.000 claims description 7
- 244000068988 Glycine max Species 0.000 claims description 6
- 235000010469 Glycine max Nutrition 0.000 claims description 6
- 239000000428 dust Substances 0.000 claims description 6
- 239000010440 gypsum Substances 0.000 claims description 6
- 229910052602 gypsum Inorganic materials 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 235000015099 wheat brans Nutrition 0.000 claims description 6
- 229920002261 Corn starch Polymers 0.000 claims description 5
- 239000008120 corn starch Substances 0.000 claims description 5
- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- 235000013312 flour Nutrition 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 13
- 150000004676 glycans Chemical class 0.000 abstract description 8
- 229920001282 polysaccharide Polymers 0.000 abstract description 8
- 239000005017 polysaccharide Substances 0.000 abstract description 8
- 150000003648 triterpenes Chemical class 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 3
- 235000019658 bitter taste Nutrition 0.000 abstract description 3
- 235000015097 nutrients Nutrition 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 125000003118 aryl group Chemical group 0.000 abstract description 2
- 239000000796 flavoring agent Substances 0.000 abstract description 2
- 235000019634 flavors Nutrition 0.000 abstract description 2
- 230000012010 growth Effects 0.000 description 20
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 18
- 238000012360 testing method Methods 0.000 description 12
- 235000013372 meat Nutrition 0.000 description 11
- 239000001965 potato dextrose agar Substances 0.000 description 11
- 238000003752 polymerase chain reaction Methods 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000001962 electrophoresis Methods 0.000 description 9
- 229910052757 nitrogen Inorganic materials 0.000 description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 8
- 229910052799 carbon Inorganic materials 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 7
- 230000003042 antagnostic effect Effects 0.000 description 7
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 7
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 7
- 238000007476 Maximum Likelihood Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 239000007640 basal medium Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 229910017053 inorganic salt Inorganic materials 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000000499 gel Substances 0.000 description 5
- 238000007423 screening assay Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 230000002936 tranquilizing effect Effects 0.000 description 5
- 241000221198 Basidiomycota Species 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 4
- 101000597041 Gallus gallus Transcriptional enhancer factor TEF-3 Proteins 0.000 description 4
- 101000755643 Homo sapiens RIMS-binding protein 2 Proteins 0.000 description 4
- 101000756365 Homo sapiens Retinol-binding protein 2 Proteins 0.000 description 4
- 101000653735 Homo sapiens Transcriptional enhancer factor TEF-1 Proteins 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 102100022371 RIMS-binding protein 2 Human genes 0.000 description 4
- 102100029898 Transcriptional enhancer factor TEF-1 Human genes 0.000 description 4
- 239000002246 antineoplastic agent Substances 0.000 description 4
- 229940041181 antineoplastic drug Drugs 0.000 description 4
- 239000002585 base Substances 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000007400 DNA extraction Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 241001489091 Ganoderma sinense Species 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000012268 genome sequencing Methods 0.000 description 3
- 239000002932 luster Substances 0.000 description 3
- 238000013081 phylogenetic analysis Methods 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 208000002109 Argyria Diseases 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 230000009418 agronomic effect Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000008485 antagonism Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 239000004922 lacquer Substances 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000003147 molecular marker Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- -1 triterpene compounds Chemical class 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- 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 description 1
- MIJYXULNPSFWEK-GTOFXWBISA-N 3beta-hydroxyolean-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[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 MIJYXULNPSFWEK-GTOFXWBISA-N 0.000 description 1
- 101150033839 4 gene Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 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 description 1
- 206010002660 Anoxia Diseases 0.000 description 1
- 241000976983 Anoxia Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical class [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102100039303 DNA-directed RNA polymerase II subunit RPB2 Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- JKLISIRFYWXLQG-UHFFFAOYSA-N Epioleonolsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4CCC3C21C JKLISIRFYWXLQG-UHFFFAOYSA-N 0.000 description 1
- 241000416536 Euproctis pseudoconspersa Species 0.000 description 1
- 241001475335 Ganoderma sp. Species 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000669831 Homo sapiens DNA-directed RNA polymerase II subunit RPB2 Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 240000007049 Juglans regia Species 0.000 description 1
- 235000009496 Juglans regia Nutrition 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 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 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 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 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- YBRJHZPWOMJYKQ-UHFFFAOYSA-N Oleanolic acid Natural products CC1(C)CC2C3=CCC4C5(C)CCC(O)C(C)(C)C5CCC4(C)C3(C)CCC2(C1)C(=O)O YBRJHZPWOMJYKQ-UHFFFAOYSA-N 0.000 description 1
- MIJYXULNPSFWEK-UHFFFAOYSA-N Oleanolinsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C MIJYXULNPSFWEK-UHFFFAOYSA-N 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 241001489088 Tomophagus colossus Species 0.000 description 1
- 241000223259 Trichoderma Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 230000007953 anoxia Effects 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 238000012365 batch cultivation Methods 0.000 description 1
- 210000003323 beak Anatomy 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- 230000002457 bidirectional effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000037237 body shape Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 235000013877 carbamide Nutrition 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000007799 cork Substances 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000008098 formaldehyde solution Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 230000028644 hyphal growth Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000003810 lymphokine-activated killer cell Anatomy 0.000 description 1
- 235000018977 lysine Nutrition 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 235000006109 methionine Nutrition 0.000 description 1
- 238000012543 microbiological analysis Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 229940100243 oleanolic acid Drugs 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000003234 polygenic effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- HZLWUYJLOIAQFC-UHFFFAOYSA-N prosapogenin PS-A Natural products C12CC(C)(C)CCC2(C(O)=O)CCC(C2(CCC3C4(C)C)C)(C)C1=CCC2C3(C)CCC4OC1OCC(O)C(O)C1O HZLWUYJLOIAQFC-UHFFFAOYSA-N 0.000 description 1
- 230000022558 protein metabolic process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 235000004400 serine Nutrition 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 235000020234 walnut Nutrition 0.000 description 1
- 238000004804 winding Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Mushroom Cultivation (AREA)
Abstract
The invention relates to a ganoderma lucidum strain L4914 and a cultivation method and application thereof, belonging to the technical field of microorganisms. The ganoderma lucidum strain L4914 is characterized by being preserved in the microorganism strain preservation center of Guangdong province in China at the year 10 and the month 31 of 2023, and the preservation number is GDMCC No:63947. the ganoderma lucidum strain L4914 has the characteristics of excellent fruiting body property, high yield, high cultivation stability, high fruiting rate, high cap lacquer-like glossiness, high conversion rate and the like; the ganoderma lucidum has the advantages of aromatic fungus flavor, no bitter taste, high triterpene and polysaccharide content, rich nutrient substances, higher yield than that of the ganoderma lucidum of a reference golden field, higher application value and better market prospect.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a ganoderma lucidum strain L4914 and a cultivation method and application thereof.
Background
Ganoderma Ganoderma lucidum is one of the important species of Ganoderma genus. The fungus cap has lacquer-like high gloss, white fungus flesh and high content of active ingredients such as polysaccharide, triterpene and the like, and is regarded as a high-quality ganoderma lucidum type, and has been recorded by a plurality of national formulary. At present, ganoderma lucidum is cultivated in batches in various places of the country, the main cultivated varieties are ganoderma lucidum G.lucidum 'lingzhi' and ganoderma sinense G.sinesis, the cultivation mode is 2 kinds of segmented wood cultivation and material substitution cultivation, the cultivation technology is mature day by day, the yield is increased year by year, and good economic and social benefits are obtained. Because the requirements for the preparation and culture of the ganoderma lucidum strains are high, in the actual production process, the phenomena that the strains are easy to pollute, the quality of the strains is uneven and the fruiting body quality and the yield are unstable frequently occur because the fruiting body cannot normally grow, the fruiting is deformed and the fruiting body is dead after cultivation due to irregular production of the strains in workshops. The key technical specifications of the cultivation variety such as the matched production, cultivation season, fruiting management requirement and the like are deficient, and finally the ganoderma yield and the bioconversion rate are seriously influenced, thus preventing the large-scale popularization of ganoderma cultivation industry.
The ganoderma lucidum strain is a novel strain collected and separated in Nanhua county of Chuxiong, yunnan province, and is not reported by research.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a ganoderma lucidum strain L4914, a cultivation method and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
ganoderma lucidum strain L4914 was deposited at the microorganism strain collection, cantonese, china, 10-31 th year, under accession number GDMCC No:63947.
The invention also provides a cultivation method of the ganoderma lucidum strain L4914, which comprises the following steps:
Taking glossy ganoderma strain L4914 tissue under the aseptic condition, inoculating the tissue to a PDA culture medium, sealing and placing the tissue in an incubator at 22-25 ℃ for constant temperature culture until bacterial colonies grow on a culture dish;
Step (2), inoculating the mycelium blocks cultured in the step (1) into a sterilized shake flask seed culture medium, and culturing for 5-8 days at 25 ℃ to obtain seed liquid;
wherein, shake flask seed culture medium is: corn flour or corn starch 1% -1.5%, glucose 1.9% -2.1%, peptone 0.2% -0.3%, yeast powder 0.3% -0.5%, potassium dihydrogen phosphate 0.09% -0.11%, ferric trichloride 0.058% -0.062%, the balance water, and the pH is natural;
step (3), inoculating the seed liquid obtained in the step (2) into a fermentation broth culture medium, and culturing for 7-10 days to obtain liquid strains;
Wherein, the fermentation broth culture medium is: 2.0 to 2.5 percent of soybean powder, 0.29 to 0.31 percent of yeast powder, 0.19 to 0.21 percent of peptone, 1.9 to 2.1 percent of glucose, 0.09 to 0.11 percent of ferric trichloride, 0.09 to 0.11 percent of potassium dihydrogen phosphate, and the balance of water, wherein the pH value is 6.2 to 7.0;
Inoculating the liquid strain cultured in the step (3) into a cultivation bag, and placing the cultivation bag in a 22-26 ℃ cultivation room for dark cultivation until hypha grows up to the cultivation bag, and keeping the air humidity at 60-65% during dark cultivation;
the culture medium in the culture bag comprises the following components in percentage by mass: 77-79 parts of miscellaneous wood dust, 19-21 parts of wheat bran, 0.9-1.1 parts of sucrose and 0.9-1.1 parts of gypsum powder; the water content is 55 to 60 percent
Step (5), conveying the cultivation bags cultivated in the step (4) to an economic forest for soil covering cultivation, and carrying out fruiting management after soil covering; harvesting when fruiting body grows to maturity.
Further, in the step (1), taking a tissue of a ganoderma lucidum strain L4914 with a cubic side length of 1cm-1.5cm, and inoculating the tissue into a PDA culture medium; culturing for 5d-8d until the colony grows to be full of the culture dish.
In the step (2), 4-6 mycelium blocks with sides of 1cm-1.5cm cubic are inoculated into 500-1000 mL of triangular flask culture medium.
Further, in the step (3), the seed solution is inoculated into the fermentation broth culture medium according to the volume ratio of 0.95% -1.05%.
Further, in the step (4), the dark culture is carried out for 25 to 30 days until hypha grows up to the cultivation bag; the mass ratio of the inoculation volume of the liquid strain to the dry material of each bag of culture medium is 10ml:550g.
Further, in the step (5), when the ganoderma lucidum is produced and managed, the temperature is controlled to be 18-28 ℃, and the relative humidity of air is 80% -90%.
Further, in the step (5), the specific method for soil-covered cultivation under the economic forests comprises the following steps: selecting a economic forest land with a moisture retention degree of Lin Muyu of 0.6-0.7, digging a groove with a depth of 30cm-32cm and a width of 95cm-105cm on the ground, digging a furrow with a depth of 30cm-32cm, a length of 39cm-41cm and a width of 15cm-18cm at a position where the forest is sparse and can not be ditched according to the forest, opening a fungus bag shoulder on the upper surface of the cultivating bag cultivated in the step (4) by 2cm-3cm and a small Kong Shuli, placing the furrow or furrow, and covering soil by 2cm-4cm for cultivation.
The invention also provides application of the mycelium, fermentation liquor or fruiting body of the ganoderma lucidum strain L4914 in preparing antitumor drugs or tranquillizing and sleep-promoting drugs.
In the invention, after the cultivation bag is subjected to after-ripening cultivation for 10-15 days after the cultivation is finished, cultivation is started to enter the fruiting stage for management, and the relative air humidity is increased to 80-90% at the moment, so that the formation of the glossy ganoderma buds is promoted. Keeping the bed moist after soil covering, controlling the temperature at 18-28 ℃, and covering soil for 10-15 days to obtain young ganoderma lucidum.
In the invention, the ganoderma lucidum maturation harvesting standard is: the ganoderma lucidum fungus cover stops growing, a circle of bright yellow or white growth circles at the edge of the fungus cover completely disappear, a monocotyledonous entity is changed from light reddish brown to dark reddish brown, the fungus cover starts to be innovated, the pore below the fungus cover starts to spray basidiospores outwards, and the basidiospores are timely collected when released and the color deepens. During harvesting, the stipe can be cut by a sharp blade, a basal part of about 0.5cm is left, the culture material and impurities at the root part are removed in time after harvesting, water spraying is maintained within 1d-2d after harvesting, moisture preservation and ventilation management is enhanced, hyphae are enabled to resume growing as soon as possible, bacterial bud formation is promoted, and total harvesting can be carried out for 2 batches to 3 batches.
The ganoderma lucidum strain L4914 contains high-content ganoderan and ganoderma lucidum triterpene compounds. The method for detecting the ganoderma lucidum by using a ganoderma lucidum polysaccharide assay and ganoderma lucidum triterpene assay (calculated by oleanolic acid) in the 2020 edition of Chinese pharmacopoeia is adopted for detection, the content of polysaccharide in a wild ganoderma lucidum strain L4914 is 1.07%, the content of triterpene is 2.37%, the content of polysaccharide and triterpene in cultivated ganoderma lucidum strain L4914 is obviously improved, the content of polysaccharide is 1.20%, the content of triterpene is 2.90%, and the ganoderma lucidum polysaccharide has wide pharmacological activity, and can reduce blood sugar, blood fat, resist thrombus, resist oxidation, remove free radicals, resist aging, resist radiation, resist tumor, promote blood circulation, regulate immunity, regulate nucleic acid and protein metabolism, promote DNA synthesis and promote human umbilical blood LAK cell proliferation. The Ganoderma triterpene compound has antiinflammatory, analgesic, tranquilizing, antiaging, tumor cell killing, and anoxia resisting effects.
The invention discloses a molecular marker of ganoderma lucidum strain L4914, which is characterized in that an SSR primer is developed based on a genome sequencing result, a PCR (polymerase chain reaction) amplification primer of the molecular marker is YLLZ SSR-02, and the primer sequence is as follows: 5'-caagtacgagacggcggtg-3',5'-tggttgcgcatgtactcgat-3'.
The ganoderma lucidum strain L4914 is obtained by separating wild ganoderma lucidum, and is characterized by excellent fruiting body character, high yield, high cultivation stability, high fruiting rate, high cap lacquer gloss, high conversion rate and the like. The basidiomycete has elliptic shape, light yellow brown color, double wall, smooth outer wall and rough punctiform grain on the inner wall, and the size is 10.0-13.5X17.0-9.0 μm. The production period is short, the growth period of the cultivation bag is 25-40 days, and the average fruiting period is 87 days. In 2021-2023, in the Panlong district of Kunming, wujicounty of Chuxiong, paoshan, the culture of 4 ten thousand bags of ganoderma lucidum strain L4914 is carried out, the average fresh weight of fruiting body is 120g, the average dry weight is 44.79g, the average cultivation period is 87 days, and the bioconversion rate is 18.09%. The average dry weight of the fruiting body of the ganoderma lucidum in the control is 29.68g, the cultivation period is 120 days, the bioconversion rate is 11.38%, and the yield is increased by 50.9% compared with the control. The average dry weight of the fruiting body of the control white ganoderma lucidum is 25.16g, the cultivation period is 98 days, the bioconversion rate is 11.07%, and the yield is increased by 56.2% compared with the control. Therefore, the ganoderma lucidum strain L4914 has high yield, popularization value and market potential.
Drawings
FIG. 1 is a morphological diagram of fruiting body of Ganoderma strain L4914; wherein a-c: a fruiting body; d: pore surfaces; e: cutting the fungus cover; f: cortical cells; g: a fungus meat skeleton hypha; h: bacterial tube reproduction hypha; i: a stipe cortex cell; j: the fungus tube is combined with hypha; k-n: basidiospore; scale bar: 20mm (f-g); 10 [ mu ] m (h-m);
FIG. 2 is a phylogenetic tree of Ganoderma lucidum strain L4914;
FIG. 3 is a gel diagram of SSR molecular markers;
FIG. 4 is an antagonistic test picture, the left antagonistic line is L4914 hypha, and the right antagonistic line is Ganoderma lucidum hypha;
FIG. 5 is a photograph of pure culture of mycelium of Ganoderma strain L4914;
FIG. 6 "fruiting body of Ganoderma lucidum strain L4914 during different growth periods;
FIG. 7 is a graph showing the effect of Ganoderma strain L4914 on greenhouse cultivation in the Panlong district of Kunming, demonstrated;
FIG. 8 is a graph showing the effect of ganoderma lucidum strain L4914 on cultivation under walnut forests in the exemplary Zhou Wu Ding county, chuxiong;
FIG. 9 is a graph showing the effect of ganoderma lucidum strain L4914 on the primary forest of the TongchongGaogong mountain.
The ganoderma lucidum strain L4914 is named in a classified way: ganoderma sp.31, 2023, was deposited with the China center for type culture Collection, accession number GDMCC No:63947. the preservation address is building 5 of Guangzhou City martyr, highway 100, university 59, guangdong national academy of sciences of microbiology.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The materials or equipment used are conventional products available from commercial sources, not identified to the manufacturer.
Example 1: ganoderma lucidum strain L4914 was deposited at the microorganism strain collection, cantonese, china, 10-31 th year, under accession number GDMCC No:63947.
The cultivation method of the ganoderma lucidum strain L4914 comprises the following steps:
Taking glossy ganoderma strain L4914 tissue under the aseptic condition, inoculating the tissue to a PDA culture medium, sealing and placing the tissue in an incubator at 24 ℃ for constant temperature culture until bacterial colonies grow on a culture dish;
Step (2), inoculating the mycelium blocks cultured in the step (1) into a sterilized shake flask seed culture medium, and culturing for 6 days at 25 ℃ to obtain seed liquid;
Wherein, shake flask seed culture medium is: corn flour 1.2%, glucose 2%, peptone 0.25%, yeast powder 0.4%, monopotassium phosphate 0.1%, ferric trichloride 0.06%, and water in balance, and the pH is natural;
Step (3), inoculating the seed liquid obtained in the step (2) into a fermentation broth culture medium, and culturing for 8 days to obtain liquid strains;
wherein, the fermentation broth culture medium is: 2.3% of soybean powder, 0.3% of yeast powder, 0.2% of peptone, 2% of glucose, 0.1% of ferric trichloride, 0.1% of monopotassium phosphate and the balance of water, wherein the pH value is 6.5;
Inoculating the liquid strain cultured in the step (3) into a cultivation bag, and placing the cultivation bag in a 24 ℃ cultivation room for dark cultivation until hypha grows up in the cultivation bag, and keeping the air humidity to be 62% -64% during dark cultivation;
The culture medium in the culture bag comprises the following components in percentage by mass: 75 parts of miscellaneous wood dust, 20 parts of wheat bran, 1 part of sucrose, 1 part of gypsum powder and 57-59% of water content;
And (5) transporting the cultivation bags cultivated in the step (4) to the under-economic-forest soil-covering cultivation, performing fruiting management after soil covering, and harvesting when fruiting bodies grow to maturity.
Example 2: ganoderma lucidum strain L4914 was deposited at the microorganism strain collection, cantonese, china, 10-31 th year, under accession number GDMCC No:63947.
The cultivation method of the ganoderma lucidum strain L4914 comprises the following steps:
step (1): under aseptic condition, taking the tissue of the ganoderma lucidum strain L4914, inoculating the tissue to a PDA culture medium, sealing and placing the tissue in an incubator at 22 ℃ for constant temperature culture until the colony grows on a culture dish;
Step (2): inoculating the mycelium blocks cultured in the step (1) into a sterilized shake flask seed culture medium, and culturing for 5 days at 25 ℃ to obtain seed liquid;
wherein, shake flask seed culture medium is: corn starch 1%, glucose 1.9%, peptone 0.2%, yeast powder 0.3%, potassium dihydrogen phosphate 0.09%, ferric trichloride 0.058%, and water in balance, and the pH is natural;
Step (3): inoculating the seed liquid obtained in the step (2) into a fermentation broth culture medium, and culturing for 7 days to obtain liquid strains;
Wherein, the fermentation broth culture medium is: 2.0% of soybean powder, 0.29% of yeast powder, 0.19% of peptone, 1.9% of glucose, 0.09% of ferric trichloride, 0.09% of monopotassium phosphate and the balance of water, wherein the pH value is 6.2-6.5;
Step (4): inoculating the liquid strain cultured in the step (3) into a cultivation bag, and placing the cultivation bag in a 22 ℃ cultivation room for dark cultivation until mycelia grow on the cultivation bag, and keeping the air humidity at 60% -62% during dark cultivation;
The culture medium in the culture bag comprises the following components in percentage by mass: 77 parts of miscellaneous wood dust, 19 parts of wheat bran, 0.9 part of sucrose, 0.9 part of gypsum powder and 55% -57% of water content;
step (5): and (3) conveying the cultivation bags cultivated in the step (4) to an economic forest for soil covering cultivation, performing fruiting management after soil covering, and harvesting when fruiting bodies grow to maturity.
In the step (1), taking a tissue of a ganoderma lucidum strain L4914 with a cubic side length of 1cm-1.2cm, and inoculating the tissue into a PDA culture medium; culturing for 5d until the colony grows to be full of the culture dish.
In the step (2), 4 pieces of mycelium blocks with the side length of 1cm-1.2cm and a cube shape are taken and inoculated into 500mL of triangular flask culture medium.
In the step (3), the seed solution is inoculated into the fermentation broth culture medium according to the volume ratio of 0.95 percent.
In the step (4), the dark culture is carried out for 25 days until hypha grows up to the culture bag; the mass ratio of the inoculation volume of the liquid strain to the dry material of each bag of culture medium is 10ml:550g.
In the step (5), when the ganoderma lucidum is produced and managed, the temperature is controlled to be 18-23 ℃ and the relative humidity of air is 80-85%.
In the step (5), the specific method for soil-covering cultivation under the economic forests comprises the following steps: selecting a economic forest land with a water retention capacity of Lin Muyu and a closure degree of 0.6-0.62, digging a groove with a depth of 30cm and a width of 95cm on the ground, digging a furrow with a depth of 30cm, a length of 39cm and a width of 15cm at a position where the forest is sparse and can not be ditched, putting the groove or furrow at a position where the fungus bag shoulder on the upper surface of the cultivating bag cultivated in the step (4) is opened by 2cm and a small Kong Shuli, and covering soil for 2cm cultivation.
The mycelium, fermentation liquor or fruiting body of the Ganoderma lucidum strain L4914 can be used for preparing antitumor drugs or tranquillizing and sleep promoting drugs.
Example 3: ganoderma lucidum strain L4914 was deposited at the microorganism strain collection, cantonese, china, 10-31 th year, under accession number GDMCC No:63947.
The cultivation method of the ganoderma lucidum strain L4914 comprises the following steps:
taking glossy ganoderma strain L4914 tissue under the aseptic condition, inoculating the tissue to a PDA culture medium, sealing and placing the tissue in an incubator at 25 ℃ for constant temperature culture until bacterial colonies grow on a culture dish;
Step (2), inoculating the mycelium blocks cultured in the step (1) into a sterilized shake flask seed culture medium, and culturing for 8 days at 25 ℃ to obtain seed liquid;
Wherein, shake flask seed culture medium is: corn starch 1.5%, glucose 2.1%, peptone 0.3%, yeast powder 0.5%, monopotassium phosphate 0.11%, ferric trichloride 0.062%, the balance water, and natural pH;
step (3), inoculating the seed liquid obtained in the step (2) into a fermentation broth culture medium, and culturing for 10 days to obtain liquid strains;
wherein, the fermentation broth culture medium is: 2.5% of soybean powder, 0.31% of yeast powder, 0.21% of peptone, 2.1% of glucose, 0.11% of ferric trichloride, 0.11% of monopotassium phosphate and the balance of water, wherein the pH value is 6.8-7.0;
Inoculating the liquid strain cultured in the step (3) into a cultivation bag, and placing the cultivation bag in a 26 ℃ cultivation room for dark cultivation until hypha grows up in the cultivation bag, and keeping the air humidity to be 63% -65% during dark cultivation;
The culture medium in the culture bag comprises the following components in percentage by mass: 79 parts of miscellaneous wood dust, 21 parts of wheat bran, 1.1 parts of sucrose and 1.1 parts of gypsum powder; the water content is 58 to 60 percent
Step (5), conveying the cultivation bags cultivated in the step (4) to an economic forest for soil covering cultivation, and carrying out fruiting management after soil covering; harvesting when fruiting body grows to maturity.
In the step (1), taking a tissue of a ganoderma lucidum strain L4914 with a cubic side length of 1.3cm-1.5cm, and inoculating the tissue into a PDA culture medium; culturing for 8d until the colony grows to be full of the culture dish.
In the step (2), 6 pieces of mycelia with a cubic side length of 1.3cm-1.5cm are inoculated into 1000mL of triangular flask culture medium.
In the step (3), the seed solution is inoculated into the fermentation broth culture medium according to the volume ratio of 1.05 percent.
In the step (4), the dark culture is carried out for 30 days until hypha grows up to the culture bag; the mass ratio of the inoculation volume of the liquid strain to the dry material of each bag of culture medium is 10ml:550g.
In the step (5), when the ganoderma lucidum is produced and managed, the temperature is controlled to be 25-28 ℃, and the relative humidity of air is 85% -90%.
In the step (5), the specific method for soil-covering cultivation under the economic forests comprises the following steps: selecting a economic forest land with a Lin Muyu closure degree of 0.68-0.7, digging a groove with a depth of 32cm and a width of 105cm on the ground, digging a furrow with a depth of 32cm, a length of 41cm and a width of 18cm at a position where the forest is sparse and can not be ditched according to the length of the forest, opening a 3cm small Kong Shuli at the shoulder of the fungus bag on the upper surface of the cultivation bag cultivated in the step (4), placing the groove or furrow, and covering soil for 4cm cultivation.
The mycelium, fermentation liquor or fruiting body of the Ganoderma lucidum strain L4914 can be used for preparing antitumor drugs or tranquillizing and sleep promoting drugs.
Example 4: ganoderma lucidum strain L4914 was deposited at the microorganism strain collection, cantonese, china, 10-31 th year, under accession number GDMCC No:63947.
The cultivation method of the ganoderma lucidum strain L4914 comprises the following steps:
Taking glossy ganoderma strain L4914 tissue under the aseptic condition, inoculating the tissue to a PDA culture medium, sealing and placing the tissue in an incubator at 23 ℃ for constant temperature culture until bacterial colonies grow on a culture dish;
Step (2), inoculating the mycelium blocks cultured in the step (1) into a sterilized shake flask seed culture medium, and culturing for 7 days at 25 ℃ to obtain seed liquid;
wherein, shake flask seed culture medium is: corn starch 1.2%, glucose 2%, peptone 0.25%, yeast powder 0.4%, monopotassium phosphate 0.1%, ferric trichloride 0.06%, and water in balance, and the pH is natural;
Step (3), inoculating the seed liquid obtained in the step (2) into a fermentation broth culture medium, and culturing for 8 days to obtain liquid strains;
Wherein, the fermentation broth culture medium is: 2.4% of soybean powder, 0.3% of yeast powder, 0.2% of peptone, 2% of glucose, 0.1% of ferric trichloride, 0.1% of monopotassium phosphate and the balance of water, wherein the pH value is 6.3-6.8;
Inoculating the liquid strain cultured in the step (3) into a cultivation bag, and placing the cultivation bag in a 23 ℃ cultivation room for dark cultivation until hypha grows up in the cultivation bag, and keeping the air humidity at 61% -64% during dark cultivation;
the culture medium in the culture bag comprises the following components in percentage by mass: 78 parts of miscellaneous wood dust, 20 parts of wheat bran, 1 part of sucrose and 1 part of gypsum powder; the water content is 56-59%
Step (5), conveying the cultivation bags cultivated in the step (4) to an economic forest for soil covering cultivation, and carrying out fruiting management after soil covering; harvesting when fruiting body grows to maturity.
In the step (1), taking a tissue of a ganoderma lucidum strain L4914 with a cubic side length of 1.2cm-1.4cm, and inoculating the tissue into a PDA culture medium; culturing for 7d until the colony grows to be full of the culture dish.
In the step (2), 5 pieces of mycelia with a cubic side length of 1.2cm-1.4cm are inoculated into 800mL of triangular flask culture medium.
In the step (3), the seed solution is inoculated into the fermentation broth culture medium according to the volume ratio of 1 percent.
In the step (4), the dark culture is carried out for 28 days until hypha grows up to the culture bag; the mass ratio of the inoculation volume of the liquid strain to the dry material of each bag of culture medium is 10ml:550g.
In the step (5), when the ganoderma lucidum is produced and managed, the temperature is controlled to be 20-25 ℃, and the relative humidity of air is 82-88%.
In the step (5), the specific method for soil-covering cultivation under the economic forests comprises the following steps: selecting a economic forest land with a water retention capacity of Lin Muyu and a closure degree of 0.62-0.68, digging a groove with a depth of 31cm and a width of 100cm on the ground, digging a furrow with a depth of 31cm, a length of 40cm and a width of 17cm at a position where the wood is sparse and can not be ditched, putting the groove or furrow at a position where the fungus bag shoulder on the upper surface of the cultivating bag cultivated in the step (4) is opened by 2.5cm and a width of Kong Shuli, and covering soil for 3cm cultivation.
The mycelium, fermentation liquor or fruiting body of the Ganoderma lucidum strain L4914 can be used for preparing antitumor drugs or tranquillizing and sleep promoting drugs.
Application example: 1. fruiting body shape characteristics of Ganoderma lucidum strain L4914: the basidiomycete fruit has one year old, has stalks, and is light and soft after being dried, and the cork is made into suppository without smell and with slightly bitter taste. The fungus cap shell is semicircular to circular, has a length of 6.6-15.5 cm and a width of 5.5-12.7 cm, and has a thickness of 1.6cm, and the fungus cap surface has a lacquer-like luster, and is yellow brown to red brown. The surface of the fungus cover is provided with concentric ring grooves which are shallow or deep, and the fungus cover is not cracked. The edges are obvious, sharp or blunt, and sometimes slightly curled, changeable in color, pale yellow when young, light brown to reddish brown. The fungus flesh can reach 0.5cm in thickness, the color is generally layered, the upper layer is milky white or light brown, the lower layer is light brown or brown, wood is in the shape of a bolt to spongy, occasionally has concentric rings, and no black shell line exists in the mature basidiomycete fruits. The length of the fungus tube can reach 0.8cm, the fungus tube is light brown or brown, and the wood is in a shape of a suppository without layering. 5-8 fungus holes per millimeter, and the fungus holes are angular, round or angular, the tube wall is slightly thick or thick, and most of fungus holes are all edges; the orifice surface is white, turns brown after being touched, and sometimes turns pale yellow. The stipe is 5-17 multiplied by 1.3-2.7 cm, is laterally or partially grown, is flat or nearly cylindrical, has smooth surface, has lacquer-like high gloss, and is reddish brown to dark reddish brown.
A mycelium three system: the diameter of the reproduction mycelium is 1-2.5 mu m, the reproduction mycelium is provided with lock-shaped combination, is thin-walled and colorless, and is unusual; the diameter of the skeleton hypha is 2-6 mu m, the skeleton hypha is thick to be sub-solid, the skeleton hypha is branched into more than two branches and is brown yellow, and the skeleton hypha accounts for most of the skeleton hypha; winding hypha with a diameter of 2-4 mu m, thick walls, often interweaving branches, light brown, thick walls and narrow inner cavities, and being less solid and rare; the 3 types of hyphae are IKI-, CB+; the tissue color darkened in the KOH reagent.
And (3) a capsid cell: the length and width are 30-40 x 7-11 um, the club shape is bright yellow to be sub-solid, and the shell cells in the mature basidiomycetes generally have strong starchy reaction.
Basidiospore: (60/3/2) (10.0) 11.0-12.5-13.5× (7.0) 7.5-8.2-9.0 μm, q= (1.36) 1.39-1.71 (1.74), qm = 1.530.11 (including umbilical protrusions); elliptic, the top beak drops off when ripe, takes the shape of a frustum, is light yellow brown, KI-, CB+ has starchy reaction, double-layer wall, smooth outer wall and rough punctiform texture on the surface of the inner wall. No basics or pseudobasics are seen.
The habitat: the wild fruiting body generally occurs in summer and autumn, the host plants and habitat are various, the wild fruiting body mainly grows on saplings, inversed woods or living tree bases of broad-leaved woods, and is distributed in various regions in Yunnan province, so that the wild fruiting body is more common.
The morphology is shown in figure 1.
2. Molecular identification: scraping proper amount of pure culture mycelium, grinding with liquid nitrogen (or grinding instrument), and extracting total DNA by modified CTAB method or plant genome DNA extraction kit (Shanghai Biotechnology Co., ltd.). The corresponding sequences were amplified using RNA Polymerase Chain Reaction (PCR) techniques. The PCR reaction system (25. Mu.l) included: 2.5 Mu.l of PCR reaction buffer, 2.5. Mu.l of 0.2% BSA, 2. Mu.l of dNTPs (2.5 mmol), 0.5. Mu.l of each of the upstream and downstream primers at a concentration of 100 pmol/. Mu.l, 1. Mu.l of DNA solution and 16. Mu.l of sterile ddH2O.
Four gene segments selected: ITS, nrLSU, TEF-1 alpha and RBP2 (the sequences are shown in SEQ ID NO. 9-SEQ ID NO. 12), and the specific primer comprises the following base compositions:
ITS primer pair:
ITS1-F：5'-cttggtcatttagaggaagtaa-3'；（SEQ ID NO.1）
ITS4：5'-tcctccgcttattgatatgc-3'；（SEQ ID NO.2）
nrLSU primer pair:
LR0R：5'-acccgctgaacttaagc-3'；（SEQ ID NO.3）
LR5：5'-tcctgagggaaacttcg-3'；（SEQ ID NO.4）
TEF-1. Alpha. Primer pair:
983F：5'-gcyccygghcaycgtgayttyat-3'；（SEQ ID NO.5）
1567R：5'-achgtrccrataccaccratctt-3'；（SEQ ID NO.6）
RBP2 primer pair:
fRrbp2-6F：5'-tggggyatggtntgyccygc-3'；（SEQ ID NO.7）
fRrbp2-7cR：5'-cccatrgcttgyttrcccat-3'。（SEQ ID NO.8）
note that: bases include a, t, c, g, and the presence of other letters in the primer represents a degenerate base.
The reaction procedure is 94 ℃ initial denaturation 5 min; repeating 35 cycles at 94 ℃ for 30 seconds, 53 ℃ for 30 seconds, 72 ℃ for 50 seconds; finally, the temperature was prolonged by 10 min at 72℃and the amplified product was stored at 4℃with agarose 1g/100mL of 0.5 XTBE (10 XTBE diluted with distilled water). 1g of agarose is precisely weighed by using an electronic balance and dissolved in 100mL of TBE, the TBE is put into a microwave oven, the TBE is heated by using middle fire to enable the TBE to reach a clear and transparent state, 1 mu L of nucleic acid dye is added when the temperature reaches 65 ℃, the TBE is fully and evenly shaken, the TBE is poured into a silica gel plate to be cooled for standby, the TBE is put into an electrophoresis tank, a pipette is used for sample application, the sample application amount is 5 mu L, and the voltage is 160V, the current is 90A and the time is 15min during electrophoresis. Electrophoresis in 1.0 XTBE buffer, observation, photographing and recording result in ultraviolet gel imager, detecting extracted DNA concentration, and sequencing positive product in Shanghai worker. The successful PCR products were sent to sequencing by bioengineering limited using forward primers. When the sequence has heterozygote INDEL or ambiguous sites, the sample is subjected to bidirectional sequencing, so that amplified regions are spliced or ambiguous sites are verified. The ITS fragments are aligned in NCBI database, and sequences with higher similarity are downloaded for alignment, and the ganoderma lucidum strain L4914 is determined to be ganoderma lucidum (Ganoderma lucidum).
ITS is :acctgcggaggatcattatcgagtcctgactgggttgtagctggccttccgaggcacgtgcacgccctgctcatccactctacacctgtgcacttactgtgggtttcagatctgtgaagcgtgccccttgcggggcttcgtgaagcgcgtctgtgcctgcgtttatcacaaactccataaagtattagaatgtgtattgcgatgtaacgcatctatatacaactttcagcaacggatctcttggctctcgcatcgatgaagaacgcagcgaaatgcgataagtaatgtgaattgcagaattcagtgaatcatcgaatctttgaacgcaccttgcgctccttggtattccgaggagcatgcctgtttgagtgtcatgaaatcttcaacctacaagcttttgcggtttgtaggcttggacttggaggcttgtcggccctttgtcggtcggctcctcttaaatgcattagcttgattccttgcggatcggctctcggtgtgataatgtctacgccgcgaccgtgaagcgtttggcgagcttctaaccgtcttcgcttgaagacagctttatgacctctgacctcaaatcaggtaggactacccgctgaacttaagcatatcaataagcccgaaagg（SEQ ID NO.9）
NrLSU is :ataagcatatcaataagcggaggaaaagaaactaacaaggattcccctagtaactgcgagtgaagcgggaaaagctcaaatttaaaatctggcggtcttcggccgtccgagttgtagtctggagaagtgctttccgcgctggaccgtgtataagtctcttggaacagagcgtcatagagggtgagaatcccgtctttgacacggactaccagtgctttgtgatgcgctctcaaagagtcgagttgtttgggaatgcagctcaaaatgggtggtgaattccatctaaagctaaatattggcgagagaccgatagcgaacaagtaccgtgagggaaagatgaaaagcactttggaaagagagttaaacagtacgtgaaattgctgaaagggaaacgcttgaagtcagtcgcgtcgtccggaactcagccttgcttycgcttggtgcactttccggatgacgggtcagcatcgattttgaccgtcggaaaagggctggagtaatgtggcacctccgggtgtgttatagactctagtcgcatacggcggttgggatcgaggaacgcagcgcgccgcaaggcaggggttcgcccactttcgcgcttaggatgctggcataatggctttaaacgacccgtcttgaaacacggaccaaggagtctaacatacctgcgagtgtttgggtggaaaacccgagcgcgtaatgaaagtgaaagttgagacctctgtcgtggagggcatcgacgcccggacctgacgttctctgaaggatccgcggtagagcatgtatgttgggacccgaaagatggtgaactatgcctgaatagggtgaagccagaggaaactctggtggaggctcgtagcgattctgacgtgcaaatcgatcgtcaaatttgggtataggggcgaaagactaatcgaaccatctagtagctggttcctgccga （SEQ ID NO.10）
TEF-1 alpha is ：atcaagaacatgatcactggtacctcgcaggctgactgtgctatcctcatcatcgccgctggtaccggtgagttcgaggctggtatctctaaagatggccagacccgcgagcacgcccttcttgccttcaccctcggtgtcaggcagctcatcgtcgccgtcaacaagatggacaccaccaaggtttgtcgtcaggtgcatcagcatacccgcctgctctgactcccgttcgcagtggtccgaggaccgtttcaacgaaatcatcaaggagacgtccaccttcatcaagaaggtcggttacaacccgaaagcggttgcgttcgttcccatctctggctggcacggcgacaacatgttggaggagtccagcaagtgagtgtatgcactatactgtctgatgactcgtcaccctgacccttatgctttagcatgacctggtacaagggttggacgaaggagaccaaggctggtgttgtcaaggggaagacccttttggacgctattgatgctattgagccccccgtccgtccctccgacaagcccctccgtctccctctccaggatgtctacaagat（SEQ ID NO.11）
RBP2 is ：gccgagacgccagaaggacaggcctgcggtctcgtcaagaacttgtcgcttatgtcttgcatatccgtcggcaccctctctgcacccgtcatcgaattcttggaggagtggggcctggagtctctggaggagaatgctcatgcctcaacgccttgcacgaaggtcttcgtgaatggcgtttggatgggcgtccatcgagatcctgtgaagctcgtcagcacgctcaggaagctccgtcgcaaagatgacatcaactgcgaggtatccgtcgtccgtgacattcgagaacgcgagctccgtctctacacggatgctggtcgcgtctgccgaccgctcttcatcgtcgagaaccagcagctccttatccagaagaaacatatcgagagcttggtccgtgccaaggaagacccgacgttgtcctacaactgggacagcctcctcaaggacggtgtcatcgagctgctagatgccgaggaagaggagacggttatgatatgcatgacaccggaggatttggagaattcgaggctccaggctgccggtatcgacccccatgcggacgaggagaacgacccctcagctcgattgaaggcgccgacctccgcgcatacgtggacgcactgcgagattcacccgagtatgatcttgggtgtctgtgccagtatcattccgttccccgatcacaatcaggtaagctcaggttaggaagtaaacttcagtcgaagtactaacgtgcgtctagtcgcctcgtaacacgtaccaatctgccat （SEQ ID NO.12）
The 4 polygene sequences of the mycelium of the ganoderma lucidum strain L4914 are spliced in forward and reverse directions and are compared in an NCBI database, and the result shows that the similarity of the ITS sequence of the ganoderma lucidum strain L4914 and the Ganoderma lucidum (HKAS 123773) sequence reaches 100 percent; the nrLSU sequence has 99.89 percent similarity with Ganoderma lucidum (LGAM 408,408) sequence. The similarity between the TEF-1 alpha sequence and Ganoderma lucidum (Dai 20017) sequence reaches 99.81 percent; the RBP2 sequence has 99.48 percent of similarity with Ganoderma lucidum (MUCL 35119) sequence. The sequence comparison result shows that the ganoderma lucidum strain L4914 and the ganoderma lucidum Ganoderma lucidum have higher similarity, and the ganoderma lucidum strain L4914 is initially determined to be ganoderma lucidum Ganoderma lucidum according to the fact that the similarity of the large fungus genes is more than 97 percent and is generally considered to be the same species.
3. Ganoderma lucidum strain L4914 phylogenetic tree: according to the comparison result of 4 gene sequences NCBI, downloading related reference sequences from GenBank, selecting the gene sequences of 26 species of ganoderma lucidum, taking Tomophagus colossus as an outer group, and carrying out polygenic phylogenetic analysis based on ITS+ nrLSU +TEF 1-alpha+RPB2 sequences. Maximum Likelihood (ML) phylogenetic analysis, the combined dataset analysis of RAxML generates an optimal scoring tree with a final Maximum Likelihood (ML) optimal likelihood value of-17967.975231. The aligned matrix has 1063 different alignment patterns, of which 38.54% of the characters or gaps are completely indeterminate. The fundamental frequency is ：A=0.221494,C=0.254336,G=0.269140,T=0.255030;rate AC=0.085035,AG=5.575773,AT=1.093575,CG=1.301105,CT=7.523430,GT=1.000000; gamma distribution model: α= 0.236017. Pilot values with a Maximum Likelihood (ML) of greater than 70% are displayed at the nodes (fig. 2).
Phylogenetic analysis shows that the ganoderma lucidum strain L4914 forms a single branch with the reported ganoderma lucidum G. The combined form determines that the ganoderma lucidum strain L4914 is ganoderma lucidum Ganoderma lucidum.
4. Biological characterization study: basic culture medium (200 g potato extract, glucose 20 g, peptone 2g, magnesium sulfate 0.2 g, potassium dihydrogen phosphate 0.2 g, agar powder 15 g, distilled water 1L) is used as strain activation culture medium, and after transfer, the colony grows on a culture dish (60 mm) for mycelium biological property research experiment. Ganoderma strain mycelium blocks are respectively inoculated in the center of each condition culture medium by adopting a puncher with the diameter of 0.7cm, and other single factor conditions except for a temperature gradient experiment are placed in a constant temperature incubator with the temperature of 22 ℃ for culture, and each treatment is repeated for 5 times. Measuring colony diameters at intervals of 24 hours by a cross scribing method; hypha growth rate (mm/d) = (hypha diameter-pellet diameter at inoculation)/day of calculation, observation and recording of hypha morphology and growth vigor, data results were analyzed statistically using Excel 2019 and SPSS 20.0.
Carbon source single factor screening assay: taking a basic culture medium as a control, respectively setting 5 carbon sources: glucose, sucrose, maltose, lactose, and soluble starch were all used as treatment groups at a concentration of 20g/L (i.e., the other components in the basal medium were unchanged, only the carbon source was changed).
Nitrogen source single factor screening assay: setting 5 nitrogen sources by taking a basal medium as a control: peptone, ammonium chloride, ammonium sulfate, urea, yeast powder were used as treatment groups at concentrations of 2g/L (i.e., other components in the basal medium were unchanged, only the nitrogen source was changed).
Inorganic salt single factor screening assay: setting 5 inorganic salts by taking a basal medium as a control: magnesium sulfate, ferric trichloride, calcium carbonate, ferrous sulfate and sodium chloride are all used as treatment groups, and the concentration is 0.5g/L (namely, other components in the basic culture medium are unchanged and only inorganic salts are changed).
Temperature single factor screening assay: after inoculation with basal medium, the dishes were placed separately: culturing at 20deg.C, 22deg.C, 24deg.C, 26deg.C, and 28deg.C in a constant temperature incubator.
PH single factor screening assay: the basic culture medium formula is prepared by adjusting acid with 1.0 mol/L HCl and alkali with 1.0 mol/L NaOH, and 5 initial pH gradients are respectively adjusted: 5.0, 5.5, 6.0, 6.5, 7.0 (i.e., the other components in the basal medium are unchanged, only the pH is changed).
Orthogonal experiment: 4 factors with great influence on the hypha growth speed are selected from a single factor experiment, and 3 levels with good effect are selected from each factor. The L9 (3 4) orthogonal table is adopted to carry out a 4-factor 3 horizontal orthogonal test, and a carbon source, a nitrogen source, inorganic salt and temperature which are obvious in hypha growth are directly factors and combined into a test culture medium.
As can be seen from the orthogonal visual analysis of the 4 factor 3 level, the L4914 strain inorganic salt is the greatest, r=1.26, followed by temperature, carbon source and nitrogen source. The maximum growth rate of T1 in the carbon source is 6.71mm/d, the maximum growth rate of T2 in the nitrogen source is 6.91mm/d, the maximum growth rate of T2 in the inorganic salt is 7.12mm/d, and the maximum growth rate of T3 in the temperature is 6.74mm/d. In combination with analysis of variance, the significant differences of 4 factors are, in order from large to small, inorganic salt > temperature > carbon source > nitrogen source. Analysis of comprehensive data results shows that the mycelium culture of the L4914 strain has optimal growth conditions: the carbon source is glucose, the nitrogen source is yeast powder, the inorganic salt is ferric chloride, and the temperature is 22 ℃ (shown in table 1). Through the pH test of ganoderma lucidum mycelia, the mycelia of the strain L4914 are mainly suitable for the growth under the meta-acid condition, and when the pH exceeds 7.0, the mycelia germinate but grow slowly. The most suitable pH for hypha growth is 6.0, and the hypha growth is rapid and dense.
TABLE 1 visual analysis of orthogonal experimental results for hyphal growth by L4914
Note that: the data are the average of 8 times; kn represents the average growth rate of hypha on the level of the ganoderma lucidum strain with n, and R is very bad; "+", "++", "+++". Indicating hyphae growth vigor is enhanced in turn
5. SSR molecular markers: (1) DNA extraction: transferring the strain to be tested onto potato dextrose agar solid culture medium, culturing at 25 ℃ for 15d, and collecting hypha; the genome DNA of hypha is extracted from the Jinsha organism DE711-50 kit, and the purity and concentration of the DNA are detected by agarose gel electrophoresis and a biological spectrophotometry.
(2) Genome sequencing: after DNA extraction, purification and library establishment, the sample is sequenced based on a sequencing platform by adopting a sequencing technology. The SSR locus was searched in the sequence of the genome of Ganoderma lucidum strain L4914.
(3) Developing SSR primers based on genome sequencing results: based on the genome data of ganoderma lucidum, 4 pairs of SSR primers YLLZ, YLLZ, YLLZ, YLLZ and YLLZ are used for developing and screening, wherein the ganoderma lucidum strain L4914 is distinguished from the ganoderma lucidum and ganoderma lucidum L4439 which are used for controlling, and the marking detailed information is shown in table 2.
TABLE 2 SSR labeled primer information
(4) And (3) PCR amplification: performing PCR (polymerase chain reaction) amplification of SSR markers on the extracted DNA by adopting SSR molecular markers, wherein a PCR amplification system is as follows: total volume 25ul, comprising: 1uL (10 uM) each of the forward primer and the reverse primer of the SSR marker, 2uL of the template DNA and 8.5 uL of sterile ultrapure water were prepared by 1.5 uL of Process 2X San Taq PCR Mix (with Bule Dye). PCR reaction conditions of 95 ℃ 2 min;95℃30 second,53℃30 second,72℃30 second,36 cycles; and at 72℃for 5min. To ensure accuracy of the identification, three replicates were performed. The above conditions were used with Ganoderma sinense, and repeated three times.
(5) Electrophoresis: electrophoresis was performed using polyacrylamide gel, and silver staining developed after electrophoresis, showing bands: 48mL of 1 XTBE solution; acr-Bis (19:1) 12mL; 1mL of 10% (W/V) APS solution; 10% TEMED 100 uL; pouring glue immediately after configuration, and discharging bubbles. After the gel is solidified, electrophoresis is carried out; the loading was 2 uL per well and the electrophoresis conditions were 270V constant pressure 1h. After electrophoresis is completed, washing the gel with distilled water for 2 times; 400ml of 10% (W/V) silver nitrate solution is added, and the shaking table oscillates 8 min; washing twice after silver staining is completed; sodium hydroxide and formaldehyde (NaOH 6g, distilled water 400mL, formaldehyde solution 2 mL) were added for color development.
TABLE 3 comparison of amplified fragment sizes of primers in "L4914" and control
As can be seen from the results of fig. 3 and table 3, using SSR primers YLLZ, YLLZ, YLLZ, YLLZ, wherein YLLZ, 3502, SSR-02 can distinguish ganoderma strain L4914 from control CK, the amplified specific fragment size of ganoderma strain L4914 is (105, 108) bp, and when the control CK fragment size is 108 bp, ganoderma strain L4914 is heterokaryon, and the control CK is homokaryon, indicating that both have genetic differences.
6. Antagonism test
The method is operated by referring to national industry standard (NY/T1845-2010) of distinguishing identification antagonistic reaction of edible fungus strains. Inoculating Ganoderma strain L4914 and control Ganoderma strain into PDA culture medium, activating, taking 7d bacterial blocks with diameter of 0.5cm with puncher, spacing two inoculating blocks at 30mm, respectively placing at 15mm from the center of plate, culturing at 25deg.C for 4d with mycelium upward, repeating for 3 times, and observing antagonism of mycelium at colony boundary between strains.
The antagonistic test of the isolated ganoderma lucidum strain L4914 and the control ganoderma lucidum shows that the pure ganoderma lucidum strain L4914 and the ganoderma lucidum have stronger antagonistic reaction and no affinity, and form a 'bump' -shaped antagonistic line (figure 4).
7. Low temperature and high temperature resistance test
As shown in Table 4, the mycelium growth rate of "Ganoderma strain L4914" was faster than that of the control Ganoderma lucidum at 5℃to 26℃under the conditions of 5℃10 ℃, 15 ℃, 20 ℃, 22 ℃, 24 ℃, 26 ℃, 28 ℃, 30 ℃, 32 ℃ and 35℃for 6 days, and the mycelium was dense as a whole; the mycelium of the ganoderma lucidum strain L4914 is obviously stronger in germination and growth than ganoderma lucidum with gold after being cultured for 6 days at the low temperature of 5 ℃ and 10 ℃.
TABLE 4 growth rates of "Ganoderma lucidum strain L4914", "Jindi Ganoderma lucidum" hyphae (unit: mm/d)
According to the combination test result, the optimal temperature for culturing the mycelium of the ganoderma lucidum strain L4914 is 24 ℃, the mycelium starts to degenerate and brown at the temperature exceeding 30 ℃, and the mycelium cannot grow at the temperature of 35 ℃; the optimal temperature for culturing mycelia of Ganoderma strain of control golden land is 28deg.C, and mycelia start to degenerate and grow slowly above 35deg.C. The ganoderma lucidum strain L4914 is a medium-low temperature fungus, and is suitable for popularization and planting in mountain areas of Yunnan plateau.
In the antibacterial test of ganoderma lucidum on penicillium and trichoderma, the antibacterial rate of the ganoderma lucidum strain L4914 is higher than that of the ganoderma lucidum strain of the control ganoderma lucidum, which shows that the impurity resistance of the ganoderma lucidum strain L4914 is stronger. The seed pollution rate is low in the seed production process, the feeding speed is high, the seeds are not easy to infect mixed bacteria, and the impurity resistance is high; and the occurrence of mixed bacterial infection and plant diseases and insect pests is less in the breeding multi-batch cultivation test. Therefore, the ganoderma lucidum strain L4914 has stronger antibacterial capability.
8. Quality analysis
And (3) carrying out statistical analysis on nutrient substances and functional components of the ganoderma lucidum strain L4914 and the control ganoderma lucidum.
TABLE 5 statistics of Ganoderma lucidum Strain L4914 and comparative "Jindi Ganoderma lucidum" composition
Note that: * Essential amino acids
The fruiting bodies of the ganoderma lucidum L4914 and the ganoderma lucidum in the mature period of 2023 cultivation are dried and then sent to a microbiological analysis and detection center for determination in Guangdong province. The content of aspartic acid, serine, glutamic acid, alanine, methionine, lysine, total 16 amino acids, ganoderan, etc. of Ganoderma strain L4914 is higher than that of Ganoderma sinense.
9. Specificity, consistency and stability
Specificity: the fruiting temperature of the ganoderma lucidum strain L4914 is 10-30 ℃, the temperature of the control ganoderma lucidum is 18-35 ℃, compared with the temperature of the control ganoderma lucidum, the uniformity of the ganoderma lucidum strain L4914 is high, the mushroom shape is large, the fruiting body is lacquer-like and high in gloss, the meat quality is thick, the average length of the fungus cover is 13.24cm, the width of the fungus cover is 10.37cm, the fungus meat is grey-white to grey-brown, the average thickness of the fungus meat is 1.67cm, no black shell line exists, the texture is softer, the average dry weight is 44.79g, and the biological conversion rate is 18.09%. The control golden-land ganoderma lucidum has smaller mushroom shape, more uneven branches and irregular fruiting, the average mushroom cap length is 11.07cm, and the mushroom cap width is 6.59cm; the fungus meat is reddish brown to dark reddish brown, the average thickness of the fungus meat is 1.11cm, the biological conversion rate is 11.38%, and the average dry weight of fruiting body single mushroom is 29.68g; the ganoderma lucidum strain L4914 has short production period, one-crop period of 87 days, and contrast of 120 days (after the collection of spore powder). The SSR primer YLLZ, the SSR primer YLLZ, the SSR primer 02, the SSR primer YLLZ, the SSR primer 03 and the SSR primer YLLZ, and the SSR primer YLLZ, the SSR primer YLLZ, the SSR primer YLLZ, the SSR primer YLLZ and the SSR primer 04 can be distinguished.
Consistency: through the observation of 3 years cultivation demonstration, the ganoderma lucidum strain L4914 has consistency.
Stability: "Ganoderma lucidum strain L4914" has consistency and in principle stability.
10. Cultivation method
(1) Demonstration test design
The ganoderma lucidum strain L4914 is cultivated in a bag with soil, and according to the requirements of GB/T21125-2007 edible fungus variety breeding technical specification, the number of cultivation bags in each group is 10000 bags (3 times of repetition at each point, and about 3600 bags of repeated cultivation) in an exemplary cultivation test design. 550g of each bag of dry material is inoculated by liquid strain, and the inoculation amount of each bag is 10ml, so that the inoculation amount of each group is 36.0L, and the inoculation amount of each group of bag cultivation dry material is 1980kg. The market separated strain 'golden ganoderma' is used as a control. Each point adopts the same batch of strains and cultivation bags, and ensures the same strain quantity and consistent demonstration cultivation management technology.
(2) Demonstration site selection
The Panlong district of Kunming, yunnan province, wu-Ji county of Chuxiong, baoshan, tengchong.
(3) Results and analysis
TABLE 6 demonstration Point yield and cultivation period of Ganoderma lucidum strain L4914
TABLE 7 ganoderma strain L4914 demonstrates major agronomic trait performance
Table 8 controls the expression of major agronomic traits of Ganoderma lucidum
Exemplary results for multiple points from 2021 to 2023 indicate: as shown in tables 6-8, compared with the control 'golden ganoderma', the bright ganoderma strain L4914 has the characteristics of good fruiting body property, high yield, high cultivation stability, high fruiting rate, high lacquer gloss of fungus caps, high conversion rate and the like. The average cultivation period was 87 days. The fruiting body is reddish brown, and the average length of the fungus cover is 13.24cm; the fungus meat is grey to brown, has an average thickness of 1.67cm, has black shell threads and has softer texture; the stipe is brown, the average length is 6.38cm, the color is darker than that of the pileus, the average diameter is 2.63cm, the average fresh weight of each bag entity is 120g, the average dry weight is 44.79g, and the biological conversion rate is 18.09%. Compared with the control "golden field ganoderma lucidum", "ganoderma lucidum strain L4914" in which the fruiting body is lacquer-like in luster, the fungus flesh is grey white to light brown, the fungus handle is dark in color, the whole fruiting effect is good, the period is short, and the mushroom is large and the meat is thick; the average length of the fungus caps of the fruiting bodies of the golden ganoderma lucidum bags is 11.07cm, the average thickness of the meat layer is 1.11cm, the fungus meat is reddish brown to dark brown, the average dry weight of the fruiting bodies is 29.68g, and the biological efficiency is 11.38%; therefore, the 'ganoderma lucidum strain L4914' can be used as a new ganoderma lucidum variety, not only can solve the problem of lack of good ganoderma lucidum varieties in Yunnan, but also can realize short-period high-yield high-quality under-forest and facility cultivation of ganoderma lucidum.
The ganoderma lucidum strain L4914 is used for preparing strains, so that the mycelia are thick and strong, the fermentation is fast, the pollution is less, and the impurity resistance is strong; the fruiting body is reddish brown, has high lacquer-like luster and stable property; the mushroom shape is larger, the fruit body has thick meat quality, and the grey white color is light brown; the fungus has aromatic flavor, no bitter taste, high triterpene and polysaccharide content, rich nutrient substances, higher yield and active ingredients than the contrast, higher application value and better market prospect.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. A cultivation method of a ganoderma lucidum strain L4914 is characterized in that the ganoderma lucidum strain (Ganoderma lucidum) L4914 is preserved in the microorganism strain preservation center of Guangdong province in China at the month of 10 and 31 of 2023, with the preservation number GDMCC No:63947;
The cultivation method of the ganoderma lucidum strain L4914 comprises the following steps:
Taking glossy ganoderma strain L4914 tissue under the aseptic condition, inoculating the tissue to a PDA culture medium, sealing and placing the tissue in an incubator at 22-25 ℃ for constant temperature culture until bacterial colonies grow on a culture dish;
Step (2), inoculating the mycelium blocks cultured in the step (1) into a sterilized shake flask seed culture medium, and culturing for 5-8 days at 25 ℃ to obtain seed liquid;
wherein, shake flask seed culture medium is: corn flour or corn starch 1% -1.5%, glucose 1.9% -2.1%, peptone 0.2% -0.3%, yeast powder 0.3% -0.5%, potassium dihydrogen phosphate 0.09% -0.11%, ferric trichloride 0.058% -0.062%, the balance water, and the pH is natural;
step (3), inoculating the seed liquid obtained in the step (2) into a fermentation broth culture medium, and culturing for 7-10 days to obtain liquid strains;
Wherein, the fermentation broth culture medium is: 2.0 to 2.5 percent of soybean powder, 0.29 to 0.31 percent of yeast powder, 0.19 to 0.21 percent of peptone, 1.9 to 2.1 percent of glucose, 0.09 to 0.11 percent of ferric trichloride, 0.09 to 0.11 percent of potassium dihydrogen phosphate, and the balance of water, wherein the pH value is 6.2 to 7.0;
Inoculating the liquid strain cultured in the step (3) into a cultivation bag, and placing the cultivation bag in a 22-26 ℃ cultivation room for dark cultivation until hypha grows up to the cultivation bag, and keeping the air humidity at 60-65% during dark cultivation;
The culture medium in the culture bag comprises the following components in percentage by mass: 77-79 parts of miscellaneous wood dust, 19-21 parts of wheat bran, 0.9-1.1 parts of sucrose and 0.9-1.1 parts of gypsum powder; the water content is 55 to 60 percent
Step (5), conveying the cultivation bags cultivated in the step (4) to an economic forest for soil covering cultivation, and carrying out fruiting management after soil covering; harvesting when fruiting bodies grow to maturity;
In the step (5), when the ganoderma lucidum is produced and managed, the temperature is controlled to be 18-28 ℃, and the relative humidity of air is 80% -90%.
2. The cultivation method of ganoderma lucidum strain L4914 according to claim 1, wherein in step (1), ganoderma lucidum strain L4914 tissue with a side length of 1cm-1.5cm is inoculated into PDA culture medium; culturing for 5d-8d until the colony grows to be full of the culture dish.
3. The method according to claim 1, wherein in the step (2), 4 to 6 pieces of mycelia having a cubic shape with a side length of 1cm to 1.5cm are inoculated into 500mL to 1000mL of a flask culture medium.
4. The cultivation method of ganoderma lucidum strain L4914 according to claim 1, wherein in step (3), the seed solution is inoculated into the fermentation broth medium at a volume ratio of 0.95% -1.05%.
5. The cultivation method of Ganoderma lucidum strain L4914 according to claim 1, wherein in step (4), the cultivation is performed in dark for 25-30 days until the mycelia grow over the cultivation bag; the mass ratio of the inoculation volume of the liquid strain to the dry material of each bag of culture medium is 10ml:550g.
6. The cultivation method of ganoderma lucidum strain L4914 according to claim 1, wherein in step (5), the specific method of cultivation under economic forests by earthing is: selecting a economic forest land with a shade water retention capacity of Lin Muyu of 0.6-0.7, digging a groove with a depth of 30-32cm and a width of 95-105cm on the ground, digging a furrow with a depth of 30-32cm, a length of 39-41cm and a width of 15-18cm at a position where the forest is sparse and can not be ditched according to the length of the forest, opening a small Kong Shuli of 2-3cm at the shoulder of the fungus bag on the upper surface of the cultivating bag cultivated in the step (4), placing the cultivating bag into the groove or the furrow, and covering soil for 2-4cm cultivation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410156270.5A CN117683645B (en) | 2024-02-04 | 2024-02-04 | Ganoderma lucidum strain L4914 and cultivation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410156270.5A CN117683645B (en) | 2024-02-04 | 2024-02-04 | Ganoderma lucidum strain L4914 and cultivation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117683645A CN117683645A (en) | 2024-03-12 |
| CN117683645B true CN117683645B (en) | 2024-05-24 |
Family
ID=90139493
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410156270.5A Active CN117683645B (en) | 2024-02-04 | 2024-02-04 | Ganoderma lucidum strain L4914 and cultivation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117683645B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992010562A1 (en) * | 1990-12-04 | 1992-06-25 | Il-Yang Pharmaceutical Co., Ltd. | Ganoderma lucidium iy009 which produces proteoglycan (g009) having effect of enhancing antitumor immunity |
| CN114107072A (en) * | 2021-12-21 | 2022-03-01 | 四川省中医药科学院 | Ganoderma lucidum strain and application thereof |
| WO2022127943A1 (en) * | 2020-12-15 | 2022-06-23 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Low-spore variety of ganoderma lucidum having high polysaccharide yield and artificial cultivation method therefor |
| CN115975817A (en) * | 2022-04-22 | 2023-04-18 | 云南省农业科学院生物技术与种质资源研究所 | Ganoderma lucidum strain L5478 and artificial cultivation method thereof |
| CN116948836A (en) * | 2023-06-05 | 2023-10-27 | 北京林业大学 | Ganoderma lucidum strain with high flavone yield, culture medium, culture method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7135183B1 (en) * | 2001-08-06 | 2006-11-14 | Academia Sinica | Immuno-modulating antitumor activities of Ganoderma lucidum (Reishi) polysaccharides |
-
2024
- 2024-02-04 CN CN202410156270.5A patent/CN117683645B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992010562A1 (en) * | 1990-12-04 | 1992-06-25 | Il-Yang Pharmaceutical Co., Ltd. | Ganoderma lucidium iy009 which produces proteoglycan (g009) having effect of enhancing antitumor immunity |
| WO2022127943A1 (en) * | 2020-12-15 | 2022-06-23 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Low-spore variety of ganoderma lucidum having high polysaccharide yield and artificial cultivation method therefor |
| CN114107072A (en) * | 2021-12-21 | 2022-03-01 | 四川省中医药科学院 | Ganoderma lucidum strain and application thereof |
| CN115975817A (en) * | 2022-04-22 | 2023-04-18 | 云南省农业科学院生物技术与种质资源研究所 | Ganoderma lucidum strain L5478 and artificial cultivation method thereof |
| CN116948836A (en) * | 2023-06-05 | 2023-10-27 | 北京林业大学 | Ganoderma lucidum strain with high flavone yield, culture medium, culture method and application thereof |
Non-Patent Citations (9)
| Title |
|---|
| 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence * |
| and large subunit ribosomal RNA gene, partial sequence.《Genbank Database》.Accession NO.ON994248.1. * |
| He,j..Ganoderma lucidum voucher HKAS 123773 internal transcribed spacer 1, partial sequence * |
| Jiahui Lu等.Molecular mechanisms of bioactive polysaccharides from Ganoderma lucidum (Lingzhi), a review.《International Journal of Biological Macromolecules》.第150卷第765-774页. * |
| 我国灵芝种质资源及生产技术研究进展;李钦艳等;《中国食用菌》;20160115;第35卷(第1期);第8-12页 * |
| 王守东等.《灵芝百问》.中国中医药出版社,2015,(第978-7-5132-2427-7版),第57页. * |
| 陈国良等.《食用菌治百病》.上海科学技术文献出版社,2014,(第978-7-5439-5910-1版),第39页. * |
| 陈惠等.《食用菌与健康》.上海科学普及出版社,2021,(第978-7-5427-8000-3版),第81页. * |
| 陈明岭等.《皮肤病常用中药药理及临床》.中国科学技术出版社,2017,(第978-7-5046-7609-2版),第459-460页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117683645A (en) | 2024-03-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112662566B (en) | Ganoderma lucidum spore-less variety with high yield of polysaccharide and artificial cultivation method thereof | |
| US20220369648A1 (en) | Endophytic falciphora oryzae fo-r20 and its application | |
| CN102037856B (en) | Simple cordyceps militaris strain rejuvenation method | |
| CN1704469A (en) | Method for producing Chinese aweto fungus | |
| CN106399132B (en) | One plant of Irpex lacteus and its application | |
| CN104789480A (en) | Aspergillus flavus strain and mixed flora not producing aflatoxin and application thereof | |
| CN108865895A (en) | Paecilomyces hepiali chen ZJB18001 and its application | |
| CN108293599A (en) | A kind of Lepista sordida and its separation expanding propagation method and soil covering culture method | |
| CN109964731B (en) | High-yield and high-efficiency culture method of Armillaria mellea | |
| CN110343621A (en) | A kind of Trichoderma asperellum strain and its application | |
| CN110447457B (en) | Haematococcus strain and artificial cultivation method and application thereof | |
| CN114766285B (en) | Ganoderma lucidum strain L4495 and cultivation method and application thereof | |
| CN117683645B (en) | Ganoderma lucidum strain L4914 and cultivation method and application thereof | |
| CN106635822B (en) | A kind of elm mushroom bacterial strain and its cross breeding method | |
| CN114085781B (en) | Ganoderma GZ and application thereof | |
| CN113136352B (en) | Biocontrol strain and application thereof in preventing and treating rice sheath blight and promoting rice growth | |
| CN108719336A (en) | Carry on a shoulder pole the method that Pseudomonas fungi promotes orchid growth in angle | |
| CN114292759A (en) | Fusarium oxysporum with effect of preventing and treating continuous cropping obstacle of tobacco | |
| CN110117548B (en) | New strain of phellinus linteus as well as artificial cultivation method and application thereof | |
| CN112673900A (en) | Amauroderma rude strain and cultivation, picking and preservation method thereof | |
| CN110172411A (en) | A kind of scab shape Xylaria strain ZJ1811 and its cultural method and application | |
| CN115039638B (en) | Resin ganoderma lucidum strain H63 and application thereof | |
| CN109566271A (en) | It is a kind of for cultivating the culture medium of wild crimping net gill fungus parent species | |
| CN115820425B (en) | DSE strain capable of improving liquorice growth and salt tolerance and application thereof | |
| CN115948253B (en) | Dictyophora rubrovalvata strain Qian PR12 and application thereof |
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 |