CN118006488A - Application of ant symbiotic actinomycetes and fermentation products thereof in inhibiting pathogenic fungi - Google Patents
Application of ant symbiotic actinomycetes and fermentation products thereof in inhibiting pathogenic fungi Download PDFInfo
- Publication number
- CN118006488A CN118006488A CN202410113821.XA CN202410113821A CN118006488A CN 118006488 A CN118006488 A CN 118006488A CN 202410113821 A CN202410113821 A CN 202410113821A CN 118006488 A CN118006488 A CN 118006488A
- Authority
- CN
- China
- Prior art keywords
- somamycin
- compound
- ant
- symbiotic
- went
- 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.)
- Pending
Links
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 21
- 244000053095 fungal pathogen Species 0.000 title claims abstract description 20
- 238000000855 fermentation Methods 0.000 title claims abstract description 9
- 230000004151 fermentation Effects 0.000 title claims abstract description 9
- 241000186361 Actinobacteria <class> Species 0.000 title abstract description 18
- 150000001875 compounds Chemical class 0.000 claims abstract description 36
- 241001225321 Aspergillus fumigatus Species 0.000 claims abstract description 27
- 229940091771 aspergillus fumigatus Drugs 0.000 claims abstract description 25
- 201000007336 Cryptococcosis Diseases 0.000 claims abstract description 16
- 241000221204 Cryptococcus neoformans Species 0.000 claims abstract description 16
- 241000222122 Candida albicans Species 0.000 claims abstract description 15
- 229940095731 candida albicans Drugs 0.000 claims abstract description 15
- 241001446247 uncultured actinomycete Species 0.000 claims abstract description 14
- 241001465754 Metazoa Species 0.000 claims abstract description 9
- 241000233866 Fungi Species 0.000 claims abstract description 7
- 239000001963 growth medium Substances 0.000 claims description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 21
- 239000004480 active ingredient Substances 0.000 claims description 13
- 239000002207 metabolite Substances 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- 241000187180 Streptomyces sp. Species 0.000 claims description 9
- 239000000287 crude extract Substances 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 8
- 229920001817 Agar Polymers 0.000 claims description 7
- 239000008272 agar Substances 0.000 claims description 7
- 238000004440 column chromatography Methods 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 7
- 239000000499 gel Substances 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 238000001228 spectrum Methods 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 6
- 235000013619 trace mineral Nutrition 0.000 claims description 6
- 239000011573 trace mineral Substances 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 229910052603 melanterite Inorganic materials 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 238000004809 thin layer chromatography Methods 0.000 claims description 5
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000012498 ultrapure water Substances 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 4
- 238000001704 evaporation Methods 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 4
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 4
- 244000005700 microbiome Species 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- 229910003208 (NH4)6Mo7O24·4H2O Inorganic materials 0.000 claims description 3
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 3
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 3
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 3
- 239000011565 manganese chloride Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 3
- 239000011686 zinc sulphate Substances 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 2
- 230000004913 activation Effects 0.000 claims description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 claims description 2
- 229910052564 epsomite Inorganic materials 0.000 claims description 2
- 238000002386 leaching Methods 0.000 claims description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Inorganic materials [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 2
- 238000001953 recrystallisation Methods 0.000 claims description 2
- 230000011218 segmentation Effects 0.000 claims description 2
- 238000010898 silica gel chromatography Methods 0.000 claims description 2
- 239000008107 starch Substances 0.000 claims description 2
- 235000019698 starch Nutrition 0.000 claims description 2
- 230000000843 anti-fungal effect Effects 0.000 abstract description 17
- 229940121375 antifungal agent Drugs 0.000 abstract description 14
- 241000244203 Caenorhabditis elegans Species 0.000 abstract description 10
- 238000011161 development Methods 0.000 abstract description 8
- 230000001580 bacterial effect Effects 0.000 abstract description 6
- 230000002147 killing effect Effects 0.000 abstract description 3
- 231100000331 toxic Toxicity 0.000 abstract description 3
- 230000002588 toxic effect Effects 0.000 abstract description 3
- 231100000419 toxicity Toxicity 0.000 abstract 1
- 230000001988 toxicity Effects 0.000 abstract 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 42
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 14
- 239000007788 liquid Substances 0.000 description 14
- 230000000694 effects Effects 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- 241000257303 Hymenoptera Species 0.000 description 12
- 241000244206 Nematoda Species 0.000 description 8
- 230000018109 developmental process Effects 0.000 description 7
- 229930014626 natural product Natural products 0.000 description 7
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 6
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000005481 NMR spectroscopy Methods 0.000 description 5
- 241000187747 Streptomyces Species 0.000 description 5
- 239000003085 diluting agent Substances 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 239000000872 buffer Substances 0.000 description 4
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical compound ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 230000035784 germination Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 208000031888 Mycoses Diseases 0.000 description 3
- 230000003042 antagnostic effect Effects 0.000 description 3
- 235000012000 cholesterol Nutrition 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 231100000517 death Toxicity 0.000 description 3
- 238000003113 dilution method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 241000993291 Armillariella Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 206010017533 Fungal infection Diseases 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 230000008485 antagonism Effects 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000002024 ethyl acetate extract Substances 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 229940050176 methyl chloride Drugs 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 108010023063 Bacto-peptone Proteins 0.000 description 1
- 241001132503 Camponotus pseudoirritans Species 0.000 description 1
- 240000008444 Celtis occidentalis Species 0.000 description 1
- 235000018962 Celtis occidentalis Nutrition 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108010049047 Echinocandins Proteins 0.000 description 1
- 241000223221 Fusarium oxysporum Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241001480007 Phomopsis Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241001655322 Streptomycetales Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- ITSGAQYTKYPNTH-SPNOXEBBSA-N [6-[6-[8-[(12E,18E)-3,5,7,11,21,23,27,31,33,35-decahydroxy-14,20,30-trimethyl-17,32-dioxo-16,37-dioxabicyclo[31.3.1]heptatriaconta-12,18-dien-15-yl]-5,7-dihydroxy-4,6-dimethylnonan-3-yl]oxy-4-hydroxy-2,4-dimethyloxan-3-yl]oxy-2-methyloxan-3-yl] 3-hydroxy-2-methyl-3-(6-methyl-5,8-dioxonaphthalen-2-yl)propanoate Chemical compound CCC(OC1CC(C)(O)C(OC2CCC(OC(=O)C(C)C(O)C3=CC=C4C(=O)C(C)=CC(=O)C4=C3)C(C)O2)C(C)O1)C(C)C(O)C(C)C(O)C(C)C1OC(=O)\C=C\C(C)C(O)CC(O)CCCC(O)CCC(C)C(O)C(=O)C2(O)CC(O)CC(CC(O)CC(O)CC(O)CCCC(O)\C=C\C1C)O2 ITSGAQYTKYPNTH-SPNOXEBBSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 150000003851 azoles Chemical class 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- ZYWFEOZQIUMEGL-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol;phenol Chemical compound ClC(Cl)Cl.CC(C)CCO.OC1=CC=CC=C1 ZYWFEOZQIUMEGL-UHFFFAOYSA-N 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 125000004218 chloromethyl group Chemical group [H]C([H])(Cl)* 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000005337 ground glass Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 230000036244 malformation Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 150000004291 polyenes Chemical class 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 230000004763 spore germination Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 108010082737 zymolyase Proteins 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/08—Bridged systems
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses an ant symbiotic actinomycete and application of a fermentation product thereof in inhibiting pathogenic fungi. The ant symbiotic actinomycetes are bacterial strains went and went, the fermentation products are compounds somamycin D, the compound has an excellent inhibiting effect on animal pathogenic fungi cryptococcus neoformans, candida albicans and aspergillus fumigatus, the Minimum Inhibiting Concentration (MIC) is 2-4 mug/mL, the compound toxicity is tested by taking caenorhabditis elegans as model organism, and the results show that somamycin D has no toxic and killing effect on caenorhabditis elegans at a concentration remarkably higher than the MIC of fungi, so that the active component somamycin D generated by the ant symbiotic actinomycetes went is safe and is an antifungal compound with development value.
Description
Technical Field
The invention relates to the technical field of microbiology, in particular to an application of ant symbiotic actinomycetes and a fermentation product thereof in inhibiting pathogenic fungi.
Background
Aspergillus fumigatus, candida albicans and Cryptococcus neoformans (Cryptococcus neoformans) are the most serious pathogenic fungi causing fungal infection diseases in humans. There are few classes and numbers of antifungal drugs currently in clinical use, and there are four major categories: azoles, echinocandins, polyenes and fluorocytones. This results in the development of resistance to pathogenic bacteria that is highly susceptible to the treatment of disease. In addition, the three fungi are quite common in the environment, and the types of agricultural antibiotics are the same as those of clinical medicines, so that drug-resistant strains are easier to generate, and the prevention and control of fungal infection and fungal diseases are difficult. The discovery of new antifungal compounds is therefore critical to the development of new drugs today.
Ants are social insects, and have complex life style including stuffy, soil or no fixed nest, which also complicates their feeding habits, even if some ants plant fungi themselves as food. In the face of complex natural environments, ants must mutilate with microorganisms to combat the threat of pathogenic fungi from the outside. Actinomycetes of the genus Streptomyces (Streptomyces) are the most common species of actinomycetes in soil, and the probability of contact with ants is very high, which makes the Streptomyces and ants easily form a mutually symbiotic relationship. Some actinomycetes symbiotic with ants have been found to produce high-activity antifungal natural products with low toxic and side effects, such as cyphomycin, which is a novel antifungal natural product found from streptomycete symbiotic with ants, and the in vitro activity of the actinomycetes symbiotic with ants is equivalent to that of the existing antifungal drugs, and after animal experiments, no toxic and side effects are found, so that the actinomycetes symbiotic with ants are a compound with development prospect. This also illustrates that actinomycetes symbiotic with ants are a good pool of antifungal natural products.
Most of the Streptomyces species producing antifungal compounds are now isolated from soil and are then found as endophytes in plants, while relatively few studies and reports of antifungal activity have been found in insects. Based on the existing literature reports, after comprehensive consideration, ants with different habitats and species are selected, and finally the actinomycetes are separated and screened from the avicula-like hackberry ants, so that the actinomycetes have great difference in strain sources from most strains, the antifungal active compounds generated by the actinomycetes are novel, and only a few documents have simple reports on the active ingredients (somamycinD). After sequencing and alignment, we initially identified the isolated strain as Streptomyces sp. After activity measurement and preliminary research, the compound is found to have better inhibitory activity on various animal pathogenic fungi. Therefore, somamycinD produced by this strain has potential to be developed as a novel antifungal compound.
Disclosure of Invention
The invention aims to provide actinomycetes went (Streptomyces sp.) capable of inhibiting various animal pathogenic fungi and separation and preparation of an active ingredient somamycin D thereof, wherein the compound somamycin D has good antifungal activity on one hand and good biological safety on the other hand.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
ant symbiotic actinomycete strain went (Streptomyces sp.) having deposit number GDMCC No.64177, date of deposit: 2023, 12 and 19, the deposit address is: building 59, accession number 100, guangzhou City martyr, accession number: the collection of microorganism strains from Guangdong province (GDMCC).
Further, the strain was used in the primary screening of activity using medium No. one of Gao's.
The invention also provides a metabolite of the ant symbiotic actinomycete strain went.
The invention also provides a compound with pathogenic fungi inhibiting effect, which is separated from the fermentation product of the ant symbiotic actinomycete strain went, wherein the preservation number of the ant symbiotic actinomycete strain went is GDMCC No.64177.
Further illustratively, the compound is somamycin D; the structural formula of the compound somamycin D is as follows:
Further, the pathogenic fungus is aspergillus fumigatus and/or candida albicans and/or cryptococcus neoformans.
The invention also provides a medicament against pathogenic fungi, which comprises the compound somamycin D as an active ingredient.
Further illustratively, the compound somamycin D has an antifungal concentration of 2-4 μg/mL.
The present invention also provides a process for preparing the above-mentioned compounds having pathogenic fungi inhibiting activity, which comprises the steps of:
1) After carrying out activation culture on the strain went in a YAG culture medium, transferring to a Gao's first solid culture medium, culturing for 14 days at 28 ℃, and cutting the thallus culture medium into small pieces by a knife;
2) Leaching the cut small pieces with mixed organic solvent (ethyl acetate: methanol: acetic acid=80:15:5, v: v), concentrating under reduced pressure, and evaporating to obtain crude extract;
3) Subjecting the obtained extract to silica gel column chromatography segmentation, and gradient eluting with dichloromethane-methanol system to obtain 5 eluting components Fr.1-Fr.5;
4) The components Fr.1-Fr.5 are separated and purified by comprehensively utilizing separation technologies such as recrystallization, silica gel/gel column chromatography, TLC (thin layer chromatography) preparation, rapid column chromatography, HPLC (high performance liquid chromatography) preparation and the like, and the compounds somamycin D are respectively obtained by utilizing modern spectrum technical means.
Further illustratively, the YAG medium has a formulation of: 2% glucose, 0.5% yeast extract, 0.1% 1000X TRACE ELEMENT and 1.6% agar ;1000×trace element：ZnSO4·7H2O 2.2g,H3BO31.1 g,MnCl2·4H2O 0.5g,FeSO4·7H2O 0.5g,CoCl2·5H2O 0.16g,CuSO40.16 g,(NH4)6Mo7O24·4H2O 0.11g,EDTA 5.0g, ultrapure water 100mL; the formula of the Gaoshi first culture medium is as follows: soluble starch 20g、KNO31 g、K2HPO40.5 g、MgSO4·7H2O 0.5g、NaCl 0.5g、FeSO4·7H2O 0.01g, agar 15g, distilled water 1L
The invention has the following beneficial effects:
The invention adopts a dilution plate method to separate 48 strains of symbiotic actinomycetes from a phomopsis hack ant (Camponotuspseudoirritans) collected in a Nanning region (North 22 DEG 51 '7', east 108 DEG 14 '10'), screens the obtained strains through a plate counter method, finally screens to obtain a strain went with excellent antagonism to aspergillus fumigatus, and performs DNA extraction, 16sRNA amplification and sequencing, and after the sequencing result is compared and analyzed in NCBI, the strain is initially identified as Streptomyces sp.
The invention tests the metabolite somamycin D of the screened bacterial strain went, finds out the MIC of the metabolite somamycin D to animal pathogenic fungi aspergillus fumigatus, candida albicans and cryptococcus neoformans, and finds out that the metabolite somamycin D has excellent inhibitory activity to all 3 animal pathogenic fungi, and has MIC value of 2-4 mug/mL, thus being an antifungal compound with development value.
In order to ensure the biological safety of the compound, after confirming the antifungal activity of the compound, the invention takes caenorhabditis elegans as model organism, tests the killing effect of the compound on caenorhabditis elegans, and the result shows that the compound does not have killing effect on caenorhabditis elegans at the selected treatment concentration (0-16 mug/mL), thus indicating that the compound has the biological safety guarantee.
Drawings
FIG. 1 is a colony morphology of strain went of the present invention. And (3) injection: left panel is went colony morphology; the right panel is a comparison of the results of the experiment of strain went against the other two strains, and the red frame of the right panel shows antagonism of strain went against A.fumigatus.
FIG. 2 is a graph showing the results of BLAST analysis of 16s RNA of strain went of the present invention in NCBI.
FIG. 3 is a graph showing the comparison of the inhibitory effect of the crude ethyl acetate extract of strain went of the present invention on the germination of Aspergillus fumigatus spores. Wherein, A is a control group and B is 100 mug/mL ethyl acetate crude extract treatment.
FIG. 4 is a mass spectrum of metabolite somamycin D of strain went of the present invention.
FIG. 5 is a graph of the hydrogen spectrum (1 H NMR) of metabolite somamycin D of strain went of the invention.
FIG. 6 is a carbon spectrum (13 C NMR) of metabolite somamycin D of strain went of the invention.
FIG. 7 shows the structure and name of the metabolite somamycin D of strain went of the present invention.
FIG. 8 is a graph showing the MIC effect of the metabolite somamycin D of strain went of the present invention on germination of Aspergillus fumigatus spores.
FIG. 9 is a graph comparing MIC effects of the metabolite somamycin D of strain went of the present invention on Candida albicans inhibition.
FIG. 10 is a graph comparing MIC effects of the metabolite somamycin D of strain went of the present invention on the inhibition of Cryptococcus neoformans.
Detailed Description
All of the features disclosed in this specification, or all of the steps in a method or process disclosed, may be combined in any combination, except for mutually exclusive features and/or steps.
Any feature disclosed in this specification (including any accompanying claims, abstract) may be provided with respect to each feature disclosed herein, unless otherwise indicated, as an example of a generic series of equivalent or similar features.
Example 1: isolation, screening and identification of Strain went9
Isolation of the Strain (one)
After the collected Armillariella pseudolaris (30) are treated with 75% alcohol for 30s, the Armillariella pseudolaris is washed with sterile water for 3 times, the surface excess water is absorbed, the solution is transferred into a sterile mortar, 10mL of sterile water is added, the solution is ground into a uniform slurry, the slurry is transferred into a 15mL sterile centrifuge tube, and the solution is subjected to gradient dilution to obtain the diluent with the concentration of 10 -1-10-5. Finally, 10 -1-10-3 -concentration diluent is smeared on a HV culture medium plate added with 2% potassium dichromate, 100 mu L of diluent is added into each plate, smeared uniformly by a sterile smearing rod, and the plates are placed in a 28 ℃ incubator for culturing for 1 week. And (3) picking the single colony of the grown actinomycetes into a fresh culture medium of Gao's first order for streak culture to obtain the ant symbiotic actinomycetes.
Screening of strains (II)
(1) Preparing aspergillus fumigatus spore liquid: 10mL of sterilized 0.2% Tween-20 solution is added into a plate for culturing aspergillus fumigatus, the bacterial colony surface is gently scraped by a sterile coating rod, spore liquid is sucked into a sterilized 2mL centrifuge tube by a pipette, then the spore liquid is subjected to gradient dilution for 4 times, fusarium oxysporum spores are counted by a blood cell counting plate, and finally the concentration of the aspergillus fumigatus spore liquid is prepared into 1X 10 6 spores/mL.
(2) Screening strains by a counter culture method: mu.L of the prepared spore liquid was pipetted into the center of a 90mm X90 mm YAG medium (2% glucose, 0.5% yeast extract, 0.1% 1000X TRACE ELEMENT and 1.6% agar ;1000×trace element：ZnSO4·7H2O 2.2g,H3BO31.1 g,MnCl2·4H2O 0.5g,FeSO4·7H2O 0.5g,CoCl2·5H2O 0.16g,CuSO40.16 g,(NH4)6Mo7O24·4H2O 0.11g,E DTA5.0 g, ultra pure water 100 mL) plate by a pipette, after the liquid droplets were dried, a single colony of the purified actinomycete strain was picked up and inoculated into the plate, and actinomycete was placed about 2cm from the edge of the plate. After inoculation, the strain is placed in a 28 ℃ incubator for 7 days to observe whether antagonistic bands appear or not and measure the widths of the antagonistic bands, and the strain with the widest antagonistic band is taken for identification and subsequent experiments (figure 1).
(III) extraction of Strain DNA
Single colonies of the strain to be identified were inoculated into 15ml of YAG medium for cultivation for 48 hours, centrifuged at 3000g for 10 minutes, the supernatant was discarded, the pellet was washed in sterile water for resuspension, and centrifuged at 3000g for 10 minutes, and the supernatant was discarded. The pellet was resuspended in 2ml buffer (20 mM Tris-HCl, pH 7.5,1.2M sorbitol, 10mM EDTA) and added to a 100. Mu. g zymolyase, incubated at 30℃for 30 minutes. 200. Mu.L of 10% SDS and 2mL of phenol-chloroform isoamyl alcohol were then added in sequence, and after thorough mixing, 6000g was centrifuged for 15 minutes to separate the layers. The clear aqueous phase in the supernatant was transferred to a new tube, 10. Mu.L of ribonuclease was added, and the temperature was kept constant at 37℃for 2 hours. 1mL of phenol-chloroform was added, and after thoroughly mixing, the mixture was centrifuged at 6000g for 15 minutes. The clear aqueous phase in the supernatant was transferred to a clean tube and one tenth of the volume of 3M sodium acetate and 2 volumes of absolute ethanol were added. After settling for 2 hours at-20 ℃, centrifugation was performed at 6000g for 15 minutes, and the supernatant was discarded. After the precipitate was washed with 70% ethanol 2 times, the DNA was air-dried at room temperature.
Molecular characterization of Strain (IV)
The extracted DNA was measured in concentration using nanadrop and diluted to an appropriate concentration for Polymerase Chain Reaction (PCR). The 16sRNA region universal primers 27F (AGAGTTTGATCCTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT) were amplified. Primers were synthesized by Jin Wei Intelligence. The PCR reaction system is described in Takara PRIMESTAR HS (Premix) polymerase, the reaction procedure is: pre-denaturation at 98 ℃ for 3min, then cycle, denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 10s, extension at 72 ℃ for 1min, 30 cycles total, and extension at 72 ℃ for 10min. The amplified product is separated by 1% agarose gel electrophoresis, the target fragment is recovered and purified by a DNA gel recovery kit and then sent to be sequenced by Jin Weizhi company, and the sequencing primer is 27F/1492R. Sequencing results were aligned on-line in the NCBI nucleic acid database using the BLAST analysis program, and BLAST results showed that most of the strains with high similarity to this strain were not specified to a specific species (fig. 2), and thus the strain was initially identified as Streptomyces sp.
Streptomyces sp. has now been designated as Ant symbiotic actinomycete strain went, and Ant symbiotic actinomycete strain went is deposited with the Guangdong province microorganism collection (GDMCC) under accession number GDMCC No.64177, date of deposition: 2023, 12 and 19, the deposit address is: building 59 of Guangzhou City martyr, road 100.
Example 2 preparation and identification of Strain went active ingredient
Large-scale culture of strains (one)
(1) Seed culture preparation: adding 5mL of sterilized water into a plate cultured with the strain went in an ultra-clean workbench, gently shaking the plate, gently scraping spores on the surface of a bacterial colony by using a sterile coating rod, sucking 200 mu L of spore liquid by using a pipette, adding into 100mL of sterilized YAG liquid culture medium, uniformly mixing, and placing into a shaking table at 28 ℃ for culturing for 48 hours to obtain the seed culture of the strain went 9.
(2) Mass culture of strain went: preparing 6L of solid culture medium of the first high-grade, pouring the solid culture medium into plates after sterilization, pouring 25mL of culture medium into each plate, sucking 100 mu L of cultured seed culture into each plate by a pipette after the culture medium is cooled and solidified, uniformly smearing bacterial liquid on the surfaces of the plates by a sterile coating rod, and finally placing the smeared plates into an incubator at 28 ℃ for culturing for 14 days.
(II) extraction of active ingredient
After culturing strain went at 28deg.C for 14 days, cutting the culture containing strain went 9 into small pieces with the size of about 1.5cm×1.5cm with a sterilized knife, filling each of the small pieces into a clean large-ground glass bottle with the volume of 5L, filling about two thirds of the total volume of the culture, adding a mixed organic solvent (ethyl acetate: methanol: acetic acid=80:15:5, V: V) into the glass bottle for soaking and extracting, stirring with a glass rod to discharge air in the gap of the culture, and finally adding the mixed organic solvent to exceed the culture by about 3-4 cm. After soaking for 3 days, filtering out an organic solvent, adding a new solvent into a glass bottle for secondary extraction according to a primary extraction mode, repeatedly extracting for 2 times, evaporating the organic solvent obtained by 3 times of filtration at 48 ℃ by using a rotary evaporator under reduced pressure to obtain a crude extract, adding 300mL of ultrapure water into the crude extract for dissolution, adding an equal volume of ethyl acetate for extracting a sample for 5 times, concentrating the obtained ethyl acetate extracted sample under reduced pressure, evaporating to dryness, and obtaining 2.472g of the ethyl acetate crude extract.
The activity detection method comprises the following steps: spore germination. (1) Firstly, preparing 2 XRPMI 1640 culture medium (RPMI 1640 powder 5.2g, MPOS17.27g, glucose 9g, adding ultrapure water to 200mL, after complete dissolution, adjusting pH to 7.0, fixing volume to 250mL, filtering with 0.22 μm filter membrane under aseptic condition) for later use; (2) Weighing a certain weight of crude extract to be detected, and dissolving the crude extract into a certain concentration by using DMSO (dimethyl sulfoxide) for later use; (3) Then preparing aspergillus fumigatus spore liquid with the concentration of 2 multiplied by 10 5/mL; (4) Taking 4 mu L of crude extract sample by a pipette, uniformly mixing with 96 mu L of 2 XRPMI 1640 medium and 100 mu L of aspergillus fumigatus spore liquid, and adding the mixture into a 96-well plate to prepare a blank control by adding pure DMSO (DMSO) with the same volume as the sample; (5) After the 96-well plate was placed in a 37℃incubator for 24 hours, the germination of Aspergillus fumigatus spores was observed under an inverted microscope (FIG. 3).
(III) separation and preparation of active ingredients
The crude ethyl acetate extract (2.472 g) was first dissolved in a methyl chloride solution (dichloromethane: methanol=1:1, V/V) and then the sample was purified by gel column chromatography with a gel column length of 100cm and a diameter of 2cm, the gel column packing being Sephadex LH-20, the mobile phase being methyl chloride solution (dichloromethane: methanol=1:1, V/V) and a flow rate of 1mL/min. After gel column chromatography, the samples were split into 5 sub-components (Fr.1-Fr.5) according to their color development on thin layer chromatography plates, where Fr.3 was the active component and 278.1mg total. Samples of this fraction were further purified by thin layer chromatography and applied to silica gel plates, which were then developed with a chloromethyl solution (dichloromethane: methanol=20:1, 1 drop of formic acid) system, after which the active ingredient-containing fraction of the silica gel on the plates was scraped off and eluted with dichloromethane methanol solution (dichloromethane: methanol=1:1, V/V). The samples were then purified by column chromatography on silica gel under reduced pressure, the mobile phase being gradient eluted with methylene chloride methanol (methylene chloride: methanol=10:1, 9:1,8:2 and 7:3), after purification they were divided into 5 sub-components (fr.3.1-fr.3.5) according to the color development of the samples on the thin layer chromatography plate, and after activity detection the active component was determined to be fr.3.3, total 28mg.
(IV) structural identification of active ingredient
10Mg of the pure active ingredient was weighed, completely dissolved in 0.6mL of deuterated DMSO, transferred into a nuclear magnetic resonance tube, and the hydrogen spectrum (1 H NMR) and the carbon spectrum (13 C NMR) of the active ingredient were measured by a 600 meganuclear magnetic resonance apparatus. About 1mg of the pure active ingredient is then weighed and the Mass Spectrum (MS) of the active ingredient is determined by means of a mass spectrometer. The data from the 3 spectra (FIGS. 4-6) were collated and analyzed and compared with the data from the micro-spectral database to finally identify the active substance produced by Streptomyces sp as somamycin D (FIG. 7).
Somamycin D: white amorphous powder, easy to dissolve in dimethyl sulfoxide ;ESI-MS m/z 513.4[M+H]+,C29H40N2O6;1H NMR(600MHz,DMSO-d6):δH 5.72(1H,d,J＝10.32Hz,H-2),5.87(1H,t,J＝10.26Hz,H-3),3.40(1H,m,H-4a),1.88(1H,m,H-4b),1.16(1H,m,H-5),1.62(1H,m,H-6),1.99(1H,m,H-7a),0.86(1H,m,H-7b),2.35(1H,m,H-8),2.00(1H,m,H-9a),0.78(1H,m,H-9b),1.31(1H,m,H-10),1.26(1H,m,H-11),1.75(1H,m,H-12),1.08(1H,m,H-13),3.22(1H,m,H-14),1.74(1H,m,H-15a),1.16(1H,m,H-15b),1.95(1H,m,H-16),6.21(1H,dd,J＝14.64,10.38Hz,H-17),7.28(1H,d,J＝15Hz,H-18),7.60(-NH,s,H-22),3.51(1H,m,H-23),3.77(1H,m,H-25),1.46(1H,m,H-26a),1.09(1H,m,H-26b),3.27(1H,m,H-27a),2.54(1H,m,H-27b),7.93(-NH,s,H-28),1.53(1H,m,H-29a),1.01(1H,m,H-29b),0.83(3H,t,J＝7.02Hz,H-30),1.03(3H,d,J＝5.82Hz,H-31);13C NMR(150MHz,DMSO-d6):δC 165.8(C-1),124.8(C-2),138.9(C-3),28.4(C-4),44.2(C-5),47.8(C-6),37.8(C-7),41.9(C-8),40.5(C-9),53.9(C-10),46.9(C-11),58.4(C-12),59.5(C-13),73.3(C-14),42.5(C-15,14-Me),44.6(C-16),143.9(C-17),129.4(C-18),181.5(C-19),101.6(C-20),178.3(C-21),67.4(C-23),193.2(C-24),70.9(C-25),31.5(C-26),36.4(C-27),26.3(C-29),13.1(C-30),18.8(C-31).
Example 3: determination of Minimum Inhibitory Concentration (MIC) of Compounds somamycin D isolated from fermentation extract of Strain went on Aspergillus fumigatus, candida albicans and Cryptococcus neoformans
(One) somamycin D Minimum Inhibitory Concentration (MIC) determination on aspergillus fumigatus:
(1) Firstly, preparing a 2 XRPMI 1640 culture medium required by activity measurement for later use; (2) Then weighing somamycin D with a certain weight, dissolving with DMSO to prepare mother solution with the concentration of 6.4 mu g/mu L, and then obtaining diluent with the concentration of 3.2 mu g/mu L, 1.6 mu g/mu L, 0.8 mu g/mu L, 0.4 mu g/mu L, 0.3 mu g/mu L, 0.25 mu g/mu L, 0.2 mu g/mu L, 0.15 mu g/mu L, 0.1 mu g/mu L and 0.05 mu g/mu L in sequence by a gradient dilution method for standby; (3) Then preparing aspergillus fumigatus spore liquid with the concentration of 2 multiplied by 10 5/mL; (4) Taking 4 mu L of each concentration sample diluent by a pipette, uniformly mixing with 96 mu L of 2 XRPMI 1640 medium and 100 mu L of aspergillus fumigatus spore liquid, and adding the mixture into a 96-well plate to prepare a blank control by adding pure DMSO (DMSO) with the same volume as the sample; (5) After the 96-well plate is placed in a 37 ℃ incubator for culturing for 24 hours, the germination condition of the aspergillus fumigatus spores is observed under an inverted microscope, and the concentration of the aspergillus fumigatus spores when the aspergillus fumigatus spores germinate into multiple branches is an MIC value. (6) The results showed that at somamycin D concentration of 3 μg/mL, aspergillus fumigatus spores exhibited a multi-branched malformation growth state, and at somamycin D concentration of 4 μg/mL, aspergillus fumigatus spores were unable to germinate, forming hyphae (FIG. 8). From this result, it was revealed that somamycin D had a MIC value of 4. Mu.g/mL for Aspergillus fumigatus. somamycin D is a natural compound having an excellent inhibitory effect on aspergillus fumigatus.
(II) somamycin D determination of Minimum Inhibitory Concentration (MIC) of Candida albicans, cryptococcus neoformans:
(1) Activating candida albicans and cryptococcus neoformans on YPD (glucose 20g, peptone 20g, yeast extract 10g, water 1000mL, agar 15 g) plates for 2-3d, and then transferring candida albicans and cryptococcus neoformans into YPD liquid culture medium for culturing overnight; (2) Preparing 2 XRPMI 1640 culture medium required by activity measurement for later use; (3) Transferring the bacterial liquid cultured with candida albicans or cryptococcus neoformans into a centrifuge tube, centrifuging (6000 rpm, 10 min) in the centrifuge tube, removing supernatant, adding 3-5ml of 2 xRPMI 1640 culture medium into thalli at the bottom of the centrifuge tube, uniformly shaking, performing gradient dilution and counting, and preparing 2 x10 5 candida albicans or cryptococcus neoformans per ml; (4) Then weighing somamycin D with a certain weight, dissolving with DMSO to prepare mother solution with the concentration of 6.4 mug/mu L, and then obtaining diluted solutions of 3.2 mug/mu L, 1.6 mug/mu L, 0.8 mug/mu L, 0.4 mug/mu L, 0.2 mug/mu L, 0.1 mug/mu L and 0.05 mug/mu L in sequence through a gradient dilution method for standby; (5) After 4. Mu.L of each concentration of sample dilutions were mixed with 96. Mu.L of 2 XPMI 1640 medium and 100. Mu.L of Aspergillus fumigatus spore liquid using a pipette, the mixture was added to a 96-well plate, and a sterile treatment group to which only the sample and the medium were added was set as a control (a blank group was required for MIC calculation). Adding pure DMSO which is equal to the sample in volume as a control group; (6) After all treatment groups were added to 96-well plates, they were incubated in an incubator at 37℃for 48h and 72h, the OD values of the respective groups were measured, and then the relative growth amounts were calculated according to the formula (OD ( Sample of +DMSO+ Bacteria (fungus) + Culture medium )-OD( Sample of +DMSO+ Culture medium )/OD(DMSO+ Bacteria (fungus) + Culture medium )-OD(DMSO+ Culture medium )) (FIGS. 9 to 10). (7) As can be seen from the relative growth amounts calculated by the formula, the growth of Candida albicans was completely inhibited at somamycin D concentration of 4. Mu.g/mL (FIG. 9), and the growth of Cryptococcus neoformans was completely inhibited at somamycin D concentration of 2. Mu.g/mL (FIG. 10), whereby the result showed that the MIC value of somamycin D for Candida albicans was 4. Mu.g/mL and the MIC value for Cryptococcus neoformans was 2. Mu.g/mL. somamycin D is a natural compound having excellent inhibitory effects on Candida albicans and Cryptococcus neoformans.
Therefore, somamycin D is an antifungal natural product with good development prospect.
Example 4: safety test of compound somamycin D separated from fermentation extract of strain went on animals, biological assay of poisoning effect of somamycin D by using caenorhabditis elegans as mode
(1) Weighing somamycin D with a certain weight, dissolving with DMSO to prepare a solution with a concentration of 1.6 mug/mu L, and sequentially obtaining diluted solutions of 0.8 mug/mu L, 0.4 mug/mu L, 0.2 mug/mu L, 0.1 mug/mu L and 0.05 mug/mu L by a gradient dilution method for later use; (2) Then culturing healthy and fresh caenorhabditis elegans of proper age on a nematode culture medium NGM flat plate (NGM culture medium: 3gNaCl,2.5g Bacto Peptone (BD 211677), 17g Gacto-agar,975mL ddH 2 O, autoclaving at 121 ℃ for 20min; after sterilization, 1mL5mg/mL ethanol cholesterol, 1mL 1M CaCl 2,1mL 1M MgSO4, 25mL 1M potassium phosphate buffer (1L): 45.6g K 2HPO4·3H2O,108.3g KH2PO4, pH 6.0, constant volume to 1L with distilled water, autoclaving at 121 ℃ for 20min.5mg/mL ethanol cholesterol solution: 0.25g cholesterol powder is dissolved in 50mL ethanol without sterilization); (3) Then, an appropriate amount of M9 buffer (12.12 g Na 2HPO4·12H2O,3.0g KH2PO4, 5.0g NaCl, volume-fixed to 1L with distilled water, sterilized at 121℃for 20min, and shaking well with 1mL 1M MgSO 4) was added to the NGM plate, the nematodes were suspended in the buffer, and the buffer with the nematodes was transferred to an 11 μm filter membrane by a pipette for filtration, and placed in a sterile 15mL centrifuge tube to give a large number of nematodes in L1 phase, counted, and finally the number of nematodes was counted to about 50 nematodes/10. Mu.L. (4) Then, 89 mu LM9 buffer solution is sucked into a 96-well plate by a liquid-transferring gun, 1 mu L of the prepared somamycin D solutions with different concentrations are added into the 96-well plate, the blank control group is added with the DMSO with the same volume, and then 10 mu L of nematode suspension is added into the 96-well plate and uniformly mixed. Finally, the 96-well plates were placed in a 20℃incubator for cultivation, and the number of nematode deaths was counted after 2 hours (Table 1) and 24 hours (Table 2). From the results in tables 1 and 2, it can be seen that somamycin D appears to be dead after 2h and 24h after treatment of caenorhabditis elegans at a concentration much higher than that of the fungus, indicating that somamycin D is substantially non-toxic to animals and is a safe antifungal natural product with the basis and potential to be used as a follow-up drug development.
Table 1 shows the mortality (death/total number) of the treatment groups after somamycin D treatment with caenorhabditis elegans for 2h
Table 2 shows the mortality (death/total number) of the various treatment groups 24h after somamycin D treatment of caenorhabditis elegans
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. Ant symbiotic actinomycete strain went (Streptomyces sp.) having deposit number GDMCC No.64177, date of deposit: 2023, 12 and 19, the deposit address is: building 59, accession number 100, guangzhou City martyr, accession number: the collection of microorganism strains from Guangdong province (GDMCC).
2. The ant symbiotic actinomycete strain went (Streptomyces sp.) of claim 1 that uses medium of No. one gao when active as a primary screen.
3. A metabolite of the ant symbiotic actinomycete strain went (Streptomyces sp.) of claim 1.
4. A compound having a pathogenic fungus inhibiting effect, wherein said compound is isolated from the fermentation product of an ant symbiotic actinomycete strain went, said ant symbiotic actinomycete strain went9 having a deposit number GDMCC No.64177.
5. A compound for inhibiting a plurality of animal pathogenic fungi according to claim 4, wherein the compound is somamycin D; the structural formula of the compound somamycin D is as follows:
6. A compound for inhibiting a pathogenic fungus according to claim 4 wherein the pathogenic fungus is aspergillus fumigatus and/or candida albicans and/or cryptococcus neoformans.
7. A medicament against pathogenic fungi, comprising the compound somamycin D according to claim 4 as an active ingredient.
8. The medicament of claim 7, wherein the concentration of compound somamycin D to inhibit fungi is 2-4 μg/mL.
9. A method for preparing a compound having a pathogenic fungus inhibiting effect according to claim 4, comprising the steps of:
1) After carrying out activation culture on the strain went in a YAG culture medium, transferring to a Gao's first solid culture medium, culturing for 14 days at 28 ℃, and cutting the thallus culture medium into small pieces by a knife;
2) Leaching the cut small pieces with mixed organic solvent (ethyl acetate: methanol: acetic acid=80:15:5, v: v), concentrating under reduced pressure, and evaporating to obtain crude extract;
3) Subjecting the obtained extract to silica gel column chromatography segmentation, and gradient eluting with dichloromethane-methanol system to obtain 5 eluting components Fr.1-Fr.5;
4) The components Fr.1-Fr.5 are separated and purified by comprehensively utilizing separation technologies such as recrystallization, silica gel/gel column chromatography, TLC (thin layer chromatography) preparation, rapid column chromatography, HPLC (high performance liquid chromatography) preparation and the like, and the compounds somamycin D are respectively obtained by utilizing modern spectrum technical means.
10. The method of claim 9, wherein the YAG medium is formulated as follows: 2% glucose, 0.5% yeast extract, 0.1% 1000X TRACE ELEMENT and 1.6% agar ;1000×trace element：ZnSO4·7H2O2.2g,H3BO31.1 g,MnCl2·4H2O 0.5g,FeSO4·7H2O 0.5g,CoCl2·5H2O 0.16g,CuSO40.16 g,(NH4)6Mo7O24·4H2O 0.11g,EDTA 5.0g, ultrapure water 100mL; the formula of the Gaoshi first culture medium is as follows: soluble starch 20g、KNO31 g、K2HPO40.5 g、MgSO4·7H2O 0.5g、NaCl 0.5g、FeSO4·7H2O 0.01g, agar 15g, distilled water 1L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410113821.XA CN118006488A (en) | 2024-01-26 | 2024-01-26 | Application of ant symbiotic actinomycetes and fermentation products thereof in inhibiting pathogenic fungi |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410113821.XA CN118006488A (en) | 2024-01-26 | 2024-01-26 | Application of ant symbiotic actinomycetes and fermentation products thereof in inhibiting pathogenic fungi |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118006488A true CN118006488A (en) | 2024-05-10 |
Family
ID=90951407
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410113821.XA Pending CN118006488A (en) | 2024-01-26 | 2024-01-26 | Application of ant symbiotic actinomycetes and fermentation products thereof in inhibiting pathogenic fungi |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118006488A (en) |
-
2024
- 2024-01-26 CN CN202410113821.XA patent/CN118006488A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| FI57781C (en) | FREQUENCY REARING FOR THE C-076-FOERING SYSTEM WITH ANTI-HELMINTISK NETWORK | |
| Ruanpanun et al. | Nematicidal activity of fervenulin isolated from a nematicidal actinomycete, Streptomyces sp. CMU-MH021, on Meloidogyne incognita | |
| KR100333017B1 (en) | Polycyclic anthelmintic agent, its preparation, its preparation, and compositions containing it | |
| Sun et al. | Antifungal and cytotoxic activities of the secondary metabolites from endophytic fungus Massrison sp. | |
| CN113142244B (en) | Synergistic prevention and treatment of common thrips by Beauveria bassiana SB063 and spinetoram | |
| CN107937453B (en) | Preparation method and antibacterial activity application of dichloro-substituted II-type halogenated polyketone compound | |
| CN115044505B (en) | Antibacterial lipopeptid produced by bacillus bailii and application of antibacterial lipopeptid in cosmetics and foods | |
| CN110684064B (en) | Compound with antibacterial activity and preparation method and application thereof | |
| CN108353906B (en) | Application of indole-3-formaldehyde and derivatives thereof in preventing and treating plant diseases caused by plant pathogenic fungi | |
| CN114317317B (en) | Bacillus belicus strain capable of resisting salt and producing lipopeptid at high yield and application thereof | |
| CN113444064B (en) | Xanthone compound, bacterial strain, preparation method and application | |
| CN118006488A (en) | Application of ant symbiotic actinomycetes and fermentation products thereof in inhibiting pathogenic fungi | |
| CN108102933B (en) | Streptomyces alboflavus strain and application thereof | |
| CN107488594B (en) | New penicillium and metabolite antacid A thereof | |
| CN116179436A (en) | Lipopeptide mixture with broad-spectrum antibacterial activity and application thereof | |
| US9624266B2 (en) | Brachiatin D and process for production thereof | |
| CN114982763A (en) | New use of geldanamycin and its analogue | |
| CN114292254A (en) | Tetrahydrotoxaanthone dimer compound and preparation method and application thereof | |
| CN108486011B (en) | Terphenyl compound, preparation method and application thereof | |
| CN107722032B (en) | Anhydride compound with prodenia litura poisoning activity and preparation method and application thereof | |
| CN109971655B (en) | Astragalus membranaceus endophytic Chaetomium sp HQ-1 and application thereof | |
| CN113402471B (en) | Siderophore compounds derived from plant endophytic fungi as well as preparation method and application of siderophore compounds | |
| US20100279861A1 (en) | Antifungal Agents | |
| CN115044488B (en) | Bacterial strain capable of degrading diuron and imidacloprid and application thereof | |
| CN110881466B (en) | Application of ixomycin compound in resisting tobacco brown spot and extraction method |
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 |