CN115029252B - Neurospora crassa WC2022 strain and application thereof - Google Patents
Neurospora crassa WC2022 strain and application thereof Download PDFInfo
- Publication number
- CN115029252B CN115029252B CN202210676359.5A CN202210676359A CN115029252B CN 115029252 B CN115029252 B CN 115029252B CN 202210676359 A CN202210676359 A CN 202210676359A CN 115029252 B CN115029252 B CN 115029252B
- Authority
- CN
- China
- Prior art keywords
- strain
- soybean isoflavone
- neurospora crassa
- aglycone
- glycoside
- 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
- 241000221961 Neurospora crassa Species 0.000 title claims abstract description 32
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 70
- 244000068988 Glycine max Species 0.000 claims abstract description 69
- GOMNOOKGLZYEJT-UHFFFAOYSA-N isoflavone Chemical compound C=1OC2=CC=CC=C2C(=O)C=1C1=CC=CC=C1 GOMNOOKGLZYEJT-UHFFFAOYSA-N 0.000 claims abstract description 47
- CJWQYWQDLBZGPD-UHFFFAOYSA-N isoflavone Natural products C1=C(OC)C(OC)=CC(OC)=C1C1=COC2=C(C=CC(C)(C)O3)C3=C(OC)C=C2C1=O CJWQYWQDLBZGPD-UHFFFAOYSA-N 0.000 claims abstract description 47
- 235000008696 isoflavones Nutrition 0.000 claims abstract description 47
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 claims abstract description 32
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 claims abstract description 32
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 claims abstract description 32
- ZWSNUPOSLDAWJS-QNDFHXLGSA-N 6,7-dihydroxy-3-[4-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]chromen-4-one Chemical compound OC[C@H]1O[C@@H](Oc2ccc(cc2)-c2coc3cc(O)c(O)cc3c2=O)[C@H](O)[C@@H](O)[C@@H]1O ZWSNUPOSLDAWJS-QNDFHXLGSA-N 0.000 claims abstract description 23
- ZCOLJUOHXJRHDI-CMWLGVBASA-N genistein 7-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=C2C(=O)C(C=3C=CC(O)=CC=3)=COC2=C1 ZCOLJUOHXJRHDI-CMWLGVBASA-N 0.000 claims abstract description 19
- ZQSIJRDFPHDXIC-UHFFFAOYSA-N daidzein Chemical compound C1=CC(O)=CC=C1C1=COC2=CC(O)=CC=C2C1=O ZQSIJRDFPHDXIC-UHFFFAOYSA-N 0.000 claims description 35
- 238000000855 fermentation Methods 0.000 claims description 26
- 230000004151 fermentation Effects 0.000 claims description 26
- 235000003687 soy isoflavones Nutrition 0.000 claims description 16
- 235000007240 daidzein Nutrition 0.000 claims description 13
- ZCOLJUOHXJRHDI-FZHKGVQDSA-N Genistein 7-O-glucoside Natural products O([C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)c1cc(O)c2C(=O)C(c3ccc(O)cc3)=COc2c1 ZCOLJUOHXJRHDI-FZHKGVQDSA-N 0.000 claims description 10
- CJPNHKPXZYYCME-UHFFFAOYSA-N Genistin Natural products OCC1OC(Oc2ccc(O)c3OC(=CC(=O)c23)c4ccc(O)cc4)C(O)C(O)C1O CJPNHKPXZYYCME-UHFFFAOYSA-N 0.000 claims description 10
- YCUNGEJJOMKCGZ-UHFFFAOYSA-N Pallidiflorin Natural products C1=CC(OC)=CC=C1C1=COC2=CC=CC(O)=C2C1=O YCUNGEJJOMKCGZ-UHFFFAOYSA-N 0.000 claims description 10
- 235000015097 nutrients Nutrition 0.000 claims description 9
- OZBAVEKZGSOMOJ-MIUGBVLSSA-N glycitin Chemical compound COC1=CC(C(C(C=2C=CC(O)=CC=2)=CO2)=O)=C2C=C1O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O OZBAVEKZGSOMOJ-MIUGBVLSSA-N 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 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 6
- 244000061456 Solanum tuberosum Species 0.000 claims description 6
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 5
- XJTZHGNBKZYODI-UHFFFAOYSA-N Glycitin Natural products OCC1OC(Oc2ccc3OC=C(C(=O)c3c2CO)c4ccc(O)cc4)C(O)C(O)C1O XJTZHGNBKZYODI-UHFFFAOYSA-N 0.000 claims description 4
- 239000002068 microbial inoculum Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000000813 microbial effect Effects 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 238000009629 microbiological culture Methods 0.000 claims 1
- 235000006539 genistein Nutrition 0.000 abstract description 9
- TZBJGXHYKVUXJN-UHFFFAOYSA-N genistein Natural products C1=CC(O)=CC=C1C1=COC2=CC(O)=CC(O)=C2C1=O TZBJGXHYKVUXJN-UHFFFAOYSA-N 0.000 abstract description 9
- 229940045109 genistein Drugs 0.000 abstract description 9
- 238000006243 chemical reaction Methods 0.000 abstract description 8
- 235000008466 glycitein Nutrition 0.000 abstract description 6
- NNUVCMKMNCKPKN-UHFFFAOYSA-N glycitein Natural products COc1c(O)ccc2OC=C(C(=O)c12)c3ccc(O)cc3 NNUVCMKMNCKPKN-UHFFFAOYSA-N 0.000 abstract description 6
- DXYUAIFZCFRPTH-UHFFFAOYSA-N glycitein Chemical compound C1=C(O)C(OC)=CC(C2=O)=C1OC=C2C1=CC=C(O)C=C1 DXYUAIFZCFRPTH-UHFFFAOYSA-N 0.000 abstract description 6
- 244000005700 microbiome Species 0.000 abstract description 4
- 230000003647 oxidation Effects 0.000 abstract description 3
- 238000007254 oxidation reaction Methods 0.000 abstract description 3
- 238000004321 preservation Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 11
- GMTUGPYJRUMVTC-UHFFFAOYSA-N Daidzin Natural products OC(COc1ccc2C(=O)C(=COc2c1)c3ccc(O)cc3)C(O)C(O)C(O)C=O GMTUGPYJRUMVTC-UHFFFAOYSA-N 0.000 description 9
- KYQZWONCHDNPDP-UHFFFAOYSA-N Daidzoside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C2C(=O)C(C=3C=CC(O)=CC=3)=COC2=C1 KYQZWONCHDNPDP-UHFFFAOYSA-N 0.000 description 9
- KYQZWONCHDNPDP-QNDFHXLGSA-N daidzein 7-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C2C(=O)C(C=3C=CC(O)=CC=3)=COC2=C1 KYQZWONCHDNPDP-QNDFHXLGSA-N 0.000 description 9
- 239000001965 potato dextrose agar Substances 0.000 description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 5
- 241000088436 Neurospora sp. Species 0.000 description 5
- 229930182470 glycoside Natural products 0.000 description 5
- 150000002338 glycosides Chemical class 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 241000233866 Fungi Species 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- -1 flavonoid compound Chemical class 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 230000002292 Radical scavenging effect Effects 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000010307 cell transformation Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- OCZVHBZNPVABKX-UHFFFAOYSA-N 1,1-diphenyl-2-(2,4,6-trinitrophenyl)hydrazine;ethanol Chemical compound CCO.[O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1NN(C=1C=CC=CC=1)C1=CC=CC=C1 OCZVHBZNPVABKX-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241001362614 Crassa Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000221960 Neurospora Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000003064 anti-oxidating effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000012084 conversion product Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 230000001076 estrogenic effect Effects 0.000 description 2
- 229930003935 flavonoid Natural products 0.000 description 2
- 235000017173 flavonoids Nutrition 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- SHHLMGCHMMCOOS-UHFFFAOYSA-N 4h-chromen-3-one Chemical compound C1=CC=C2CC(=O)COC2=C1 SHHLMGCHMMCOOS-UHFFFAOYSA-N 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 108091023242 Internal transcribed spacer Proteins 0.000 description 1
- UBORTCNDUKBEOP-UHFFFAOYSA-N L-xanthosine Natural products OC1C(O)C(CO)OC1N1C(NC(=O)NC2=O)=C2N=C1 UBORTCNDUKBEOP-UHFFFAOYSA-N 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000228153 Penicillium citrinum Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000235546 Rhizopus stolonifer Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 235000002597 Solanum melongena Nutrition 0.000 description 1
- UBORTCNDUKBEOP-HAVMAKPUSA-N Xanthosine Natural products O[C@@H]1[C@H](O)[C@H](CO)O[C@H]1N1C(NC(=O)NC2=O)=C2N=C1 UBORTCNDUKBEOP-HAVMAKPUSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003388 anti-hormonal effect Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 235000013527 bean curd Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000003889 chemical engineering Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000002856 computational phylogenetic analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 150000002215 flavonoids Chemical class 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
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000003075 phytoestrogen Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007788 roughening Methods 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 235000013597 soy food Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- UBORTCNDUKBEOP-UUOKFMHZSA-N xanthosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(NC(=O)NC2=O)=C2N=C1 UBORTCNDUKBEOP-UUOKFMHZSA-N 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- 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
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
-
- 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
- C12R2001/645—Fungi ; Processes using fungi
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Mycology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a Neurospora crassa WC2022 strain and application thereof. The invention separates and obtains a strain of mairei (Neurosporasp.) WC2022 which can mediate the conversion of combined soybean isoflavone glycoside to generate free soybean isoflavone aglycone, and the strain is preserved in the microorganism strain preservation center of Guangdong province in the 6 th month of 2022, and the preservation number is GDMCC NO:62523. The strain can completely convert the combined soybean isoflavone glycoside into free soybean isoflavone aglycone within 120 hours, and the contents of the converted soybean aglycone, genistein and glycitein are respectively increased by 23.15 times, 38.09 times and 12.08 times; meanwhile, the oxidation resistance of the converted product is obviously improved. The invention provides excellent strain resources for producing the free soybean isoflavone aglycone.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms. More particularly, it relates to a strain of Neurospora crassa WC2022 and its use.
Background
Soy isoflavones are secondary metabolites of flavonoids produced by plants such as soybean or alfalfa, a typical phytoestrogen, with weak estrogenic activity. The soybean isoflavone can play a role in regulating estrogen-like or anti-hormone bi-directionally, has the activities of resisting oxidation, tumor, bacteria and the like, and is closely related to human health when being eaten deeply. However, the active components of soybean isoflavone mainly exist in the form of glycoside in a combined state, and the bioavailability is extremely low, which restricts the full utilization of the active components.
Researches show that intestinal flora can mediate biological transformation of combined soybean isoflavone glycoside to produce free soybean isoflavone aglycone, remarkably improve bioavailability and promote full play of the physiological activity of soybean isoflavone. In addition to bacteria, fungi and their enzymes can also transform the bound soy isoflavone glycoside to produce free soy isoflavone aglycones, as disclosed in the prior art, the amount of aglycones can be increased by fermenting soy foods with rhizopus stolonifer; genistein and daidzein can be produced by fermenting Penicillium citrinum in potato dextrose broth rich in soy isoflavone soy extract; glycosidases can also be produced using Aspergillus niger or Aspergillus oryzae, which constantly convert the bound soy isoflavone glycoside to a biologically active aglycone form. The fungi can convert and produce the soybean isoflavone aglycone, but the strains capable of converting the soybean isoflavone aglycone are fewer at present, so that more new strain resources capable of mediating the biological conversion of the combined soybean isoflavone glycoside to produce the free soybean isoflavone aglycone are needed to be developed, more efficient and rapid conversion sources and ways are provided for converting the free soybean isoflavone aglycone, and the method is fully utilized in the processing of bean products.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings of the problems and provide a Neurospora crassa WC2022 strain and application thereof.
The first object of the present invention is to provide a Neurospora crassa WC2022 strain.
A second object of the invention is to provide the use of said WC2022 strain.
The third object of the invention is to provide a microbial inoculum for producing free soybean isoflavone aglycone.
The fourth object of the invention is to provide a method for preparing free soybean isoflavone aglycone.
The above object of the present invention is achieved by the following technical scheme:
the invention identifies and obtains a Neurospora crassa (Neurospora sp.) WC2022 strain, and the morphological characteristics of the strain are as follows: the initial mycelium is white mycelium, the mycelium is sparse and extends to the periphery, and then the mycelium is light orange yellow villus; the mycelium is transparent and has branches and partitions; aerial hyphae irregularly grow around to form branch chains at the top; spherical or nearly spherical conidia are produced, which are initially yellowish and later orange-red or red. The nucleotide sequence of the WC2022 strain is shown as SEQ ID NO. 1, and sequence comparison and phylogenetic tree construction are carried out, so that the similarity between the ITS gene sequence of the Neurospora crassa WC2022 strain provided by the invention and Neurospora crassa (GenBank: JN 198494.1) is up to 99.13%. Thus, the WC2022 strain was identified as Neurospora (Neurospora sp.) -Neurospora crassa (Neurospora crassa), which was deposited at the collection of microorganism strains in the cantonese province at month 7 of 2022, accession number: GDMCC NO 62523, deposit address: guangzhou city first middle road No. 100 college No. 59 building 5.
The research of the invention shows that the adoption of WC2022 strain to carry out whole-cell transformation of combined soybean isoflavone glycoside produces free soybean isoflavone aglycone, and the contents of daidzein, genistein and glycitein in the transformation products are respectively increased by 23.15 times, 38.09 times and 12.08 times; the DPPH removing capability of the converted product is stronger, and the oxidation resistance of the soybean isoflavone is obviously improved after the soybean isoflavone is subjected to in-vitro biotransformation by Neurospora crassa.
The invention provides application of a Neurospora crassa WC2022 strain and/or fermentation liquor thereof in converting combined soybean isoflavone glycoside to generate free soybean isoflavone or in preparing a microbial inoculum for converting combined soybean isoflavone glycoside to generate free soybean isoflavone aglycone.
The invention provides a microbial inoculum for producing free soybean isoflavone aglycone, which contains a Neurospora crassa WC2022 strain and/or fermentation liquor thereof.
Preferably, the fermentation broth is a spore suspension.
Preferably, the spore suspension has a concentration of 10 2 ～10 8 Individual spores/mL.
The invention provides a preparation method of free soybean isoflavone aglycone, which comprises the steps of adding a Neurospora crassa WC2022 strain and/or fermentation liquor thereof into a nutrient solution containing soybean isoflavone or purified soybean isoflavone glycoside, fermenting and culturing, collecting fermentation liquor after culturing, and removing hyphae to obtain a conversion product free soybean isoflavone aglycone. Since soybean isoflavone is a flavonoid compound, and a compound group with 3-benzopyrone as a parent nucleus is a mixture containing various substances such as daidzin, genistin, glycitin and the like, free soybean isoflavone aglycone can be produced by directly using soybean isoflavone or purified soybean isoflavone glycoside for fermentation culture.
Preferably, the purified soybean isoflavone glycoside is one or more of daidzin, daidzin or genistin.
Preferably, the formula of the nutrient solution is as follows: adding 20% potato and 2% glucose into distilled water, sterilizing, and adding 0.1-10% soybean isoflavone or purified soybean isoflavone glycoside.
More preferably, the nutrient solution adopts 1000mL of distilled water, 200g of potato, 20g of glucose and sterilization at 121 ℃ for 20min; after cooling, 0.2% soy isoflavones are added.
Preferably, the fermentation culture conditions are: the temperature is 20-40 ℃, the rotating speed is 100-220 rpm, and the fermentation time is 0-144 h.
More preferably, the fermentation time is 120 hours.
The invention has the following beneficial effects:
the Neurospora crassa (Neurospora sp.) WC2022 strain provided by the invention has the capability of converting combined soybean isoflavone glycoside to generate free soybean isoflavone aglycone, and completely converts soybean glycoside, genistin and soybean xanthosine in 120h, wherein the content of the converted soybean aglycone is increased by 23.15 times, the content of genistein is increased by 38.09 times, and the content of glycitein is increased by 12.08 times. Meanwhile, the DPPH free radical clearance of the fermentation broth transformed by the WC2022 strain is higher and is 1.92 times of that of unconverted soybean isoflavone, which shows that the WC2022 strain effectively improves the DPPH free radical clearance of the transformed product after the soybean isoflavone is transformed by whole cells, and has good antioxidation effect. The Neurospora crassa WC2022 strain provided by the invention not only can be used for efficiently converting and producing free soybean isoflavone aglycone, but also has high antioxidant activity and high protease yield, and a novel strain is provided for producing soybean isoflavone.
Drawings
FIG. 1 is a schematic representation of colony morphology, hyphae, and conidia of WC2022 strain on PDA medium;
FIG. 2 is a phylogenetic tree result diagram of the ITS sequence construction of the WC2022 strain;
FIG. 3 is a diagram showing the content change of the glycoside and aglycone substances (daidzin, daidzein, genistin, genistein, daidzein, glycitin) of the converted soybean isoflavone by high performance liquid chromatography;
FIG. 4 is a schematic diagram showing DPPH radical scavenging ability of whole cell transformed soybean isoflavone by WC2022 strain.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The reagents used in the following examples were:
dimethyl sulfoxide DMSO (analytically pure) was purchased from the company of fine chemical engineering, prefecture, the division of the Tianjin city;
soy isoflavones (BR, 40%) were purchased from shanghai source leaf biotechnology limited;
standard substances (more than or equal to 98%) such as daidzin, daidzein, genistin, daidzein, glycitein and genistein are all purchased from Shanghai source leaf biotechnology limited company;
methanol (chromatographic purity), acetonitrile (chromatographic purity) were all purchased from sammer feishier technologies.
The media used in the following examples were:
potato Dextrose Broth (PDB): 200g of potato, 20g of glucose, adding water to a constant volume of 1L, and sterilizing at 121 ℃ for 20min for later use.
PDA medium: after adding 2% agar to PDB, sterilizing at 121deg.C for 20 min.
The formula of the whole cell transformed nutrient solution comprises the following steps: 200g of potato, 20g of glucose, 1000mL of distilled water, natural pH and sterilization at 121 ℃ for 20min; after cooling, 0.2% soy isoflavones are added.
Example 1 isolation and identification of strains
1g of traditional red fungus bean curd in Pingyou county of Guangdong province is weighed, added into 90mL of sterile normal saline, crushed, shaken uniformly, diluted in a gradient manner, coated on a PDA solid medium, cultured at 30 ℃, and separated to obtain 1 strain of fungus named WC2022.
The colony morphology, hypha and conidium of WC2022 strain on PDA culture medium are shown in figure 1, the initial stage of the mycelium in figure 1A is white hypha, the hypha is sparse and extends to the periphery, and the later stage is light orange yellow villus as shown in figure 1B; in FIG. 1C, the hyphae are transparent, branched and segregated; aerial hyphae irregularly grow around to form branch chains at the top; in fig. 1D, spherical or nearly spherical conidia are produced, initially yellowish, followed by reddish orange or red.
And amplifying the strain ITS sequence by adopting a universal primer ITS1/ITS4, and sequencing by adopting a conventional method. The ITS sequence of the strain is shown as SEQ ID NO. 1, the length is about 620bp, the sequence of the WC2022 strain is compared with that of the strain at NCBI, and the phylogenetic tree constructed by the ITS sequence of the WC2022 strain is shown as figure 2, and the phylogenetic tree is found by analysis: WC2022 strain has a similarity of up to 99.13% with Neurospora crassa (GenBank: JN 198494.1). Thus, by combining the morphological characteristics and phylogenetic tree analysis described above, the WC2022 strain was identified as Neurospora (Neurospora sp.) -Neurospora crassa (Neurospora crassa), designated Neurospora crassa (Neurospora sp.) WC2022, which was deposited at the cantonese microorganism strain deposit center at month 7 of 2022, deposit number: GDMCC NO 62523, deposit address: guangzhou city first middle road No. 100 college No. 59 building 5.
EXAMPLE 2 Whole cell transformation of the WC2022 Strain with conjugated Soy isoflavone glycoside to its free aglycone
The Neurospora crassa WC2022 strain isolated in example 1 was used, and the WC2022 strain was cultured in Neurospora crassa WC2022 seed medium (potato 200g, glucose 20g, agar 20g, distilled water 1000mL, pH was natural, and sterilization was performed at 121℃for 20 min) under the following conditions: and (3) carrying out stationary culture on the eggplant in a bottle at a culture temperature of 30 ℃ for 5 days.
And then, washing the solid flat-plate culture medium fully grown with the neurospora crassa spores after the culture by using sterile water containing 0.05% of Tween-80, and lightly scraping the surface of the culture medium by using a pipette tip to obtain the neurospora crassa conidium suspension. Adding the conidium suspension into whole cell transformation nutrient solution, wherein the final concentration of the conidium is as follows: 10 6 Individual spores/mL. Culturing at 30 deg.c and shaking table rotation speed of 150-200 rpm/min for 144 hr.
In whole cell transformation nutrient solution culture, soybean isoflavone transformation products were taken out at regular intervals of 24 hours, and Neurospora crassa hyphae were removed, and filtration was performed using a 0.22 μm filter membrane. After the filtrate is reserved, the content change of the glycoside and aglycone substance (daidzin, daidzein, genistin, genistein, daidzein, glycitin) of the converted soybean isoflavone is detected by high performance liquid chromatography.
Setting parameters of the high performance liquid chromatography detection method: the mobile phase is A: primary aqueous solution, B:100% pure methanol solution, C:100% pure acetonitrile solution; column temperature: 36 ℃; sample injection amount: 10. Mu.L; detection wavelength: 260nm; flow rate: 0.8mL/min. The gradient elution conditions for soy isoflavones are shown in table 1 below.
TABLE 1 gradient elution conditions of Soy isoflavone
As shown in FIG. 3, WC2022 strain can convert the conjugated soybean isoflavone glycoside into free soybean isoflavone aglycone, wherein daidzin in the soybean isoflavone glycoside is converted into daidzein, genistin is converted into genistein, and daidzein is converted into glycitein; in FIG. 3A, daidzin was completely converted at 120h, daidzein content increased gradually with the conversion of daidzin, with a peak at 120h and a 23.15-fold increase in content. In FIG. 3B, genistin was completely converted at 120h, genistein content was gradually increased with the conversion of genistin, the peak reached at 120h, and the content was increased 38.09 times. In FIG. 3C, huang Douhuang glycoside was completely converted at 120h, glycitein content was gradually increased with the conversion of daidzein, the peak was reached at 120h, and the content was increased by 12.08 times.
In conclusion, the MAICHO WC2022 can convert soybean isoflavone glycoside into corresponding aglycone through liquid fermentation, and can be used as a novel microbial resource for producing free soybean isoflavone aglycone through soybean isoflavone conversion.
EXAMPLE 3 DPPH radical scavenging ability test of soybean isoflavone conversion products
The whole cell transformed nutrient solution prepared in example 2 was cultured with sterile water and the fermentation broth was diluted for use. Preparing DPPH-ethanol solution: weighing 20mgDPPH reagent in beaker, dissolving with absolute ethanol, fixing volume in 250mL volumetric flask, and preserving in dark place.
Respectively sucking 2mL of diluted fermentation liquor, adding the diluted fermentation liquor into a test tube, mixing the fermentation liquor and the 2mL of diluted fermentation liquor, standing the mixture at room temperature in the dark for reaction for 30min, measuring the absorbance at 517nm wavelength, and marking the absorbance as Ai. Absolute ethyl alcohol is used to replace the sample to be tested and DPPH-ethanol solution in the above steps respectively, and the absorbance value is measured under the same condition and recorded as A 0 And Ar. Experiments were repeated 3 times. DPPH radical scavenging (K) was calculated according to the following formula.
The calculation formula is as follows: k (%) = [1- (Ai-Ar)/A 0 ]×100
As shown in the experimental result in figure 4, the clearance of DPPH free radicals of the fermentation liquor obtained after the soybean isoflavone is converted by the Neurospora crassa WC2022 is the highest (p is less than 0.05) in 120 hours, and is 1.92 times of that of the unconverted soybean isoflavone, which shows that the DPPH free radical clearance is effectively improved after the soybean isoflavone is converted by the whole cell of the Neurospora crassa WC2022, and the fermentation liquor has good antioxidation effect.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
<110> agricultural university of south China
<120> a Neurospora crassa WC2022 strain and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 620
<212> DNA
<213> Mai cell-roughening WC2022 Strain ITS sequence (SIPOSEQUENCELISTERING 1.0)
<400> 1
ccgagcactt atttcctccg gccttatgat atgcttaagt tcagcgggta ttcctacctg 60
atccgaggtc aaccttagaa atggggggtt ttacggcaag aacccgccgc acgaccatag 120
cgatgtagag ttactacgct cggtgtgact agcgagcccg ccactgattt tgagggaccg 180
cggacagccg cggatcccca acgcaagcag agcttgatgg ttgaaatgac gctcgaacag 240
gcatgctcgc cagaatactg gcgagcgcaa tgtgcgttca aagattcgat gattcactga 300
attctgcaat tcacattact tatcgcattt cgctgcgttc ttcatcgatg ccagaaccaa 360
gagatccgtt gttgaaagtt ttgacttatt taaaagttta ctcagagaga cataaaatat 420
caagagttta gtttcggcac tccggcgggc agcctcccgc gagcgggaga cccgaggatc 480
cgggagggcc cgagggcctt tccggaccgc cagcgccgag gcaaccgtac gggtaagatt 540
cgcgatggtt tgtgggagtt ttgcaactct gtaatgatcc ctccgcaggt cccccttacg 600
gagggatcat tacagagttg 620
Claims (10)
1. Neurospora crassa (L.) KuntzeNeurosporasp.) WC2022 strain, characterized in that it was deposited with the Guangdong province microbiological culture Collection center (GDMCC) on day 7 of 6.2022, with the accession numberGDMCC NO:62523。
2. Use of the strain neurospora crassa WC2022 and/or its fermentation broth according to claim 1 for converting a bound soy isoflavone glycoside to produce free soy isoflavone aglycone.
3. Use of the neurospora crassa WC2022 strain and/or its fermentation broth according to claim 1 for the preparation of a bacterial agent for converting a bound soy isoflavone glycoside to produce free soy isoflavone aglycone.
4. A microbial preparation for producing free soybean isoflavone aglycone, which comprises the Neurospora crassa WC2022 strain and/or a fermentation liquid thereof according to claim 1.
5. The microbial inoculum of claim 4, wherein the fermentation broth is a spore suspension.
6. The microbial preparation of claim 5, wherein said spore suspension has a concentration of 10 2 ～10 8 Individual spores/mL.
7. A process for preparing free soybean isoflavone aglycone, which is characterized in that the strain WC2022 of Neurospora crassa and/or the fermentation liquor thereof is adopted, added into the nutrient solution containing soybean isoflavone or purified soybean isoflavone glycoside, fermented and cultured, and after the culture is finished, the fermentation liquor is collected, hyphae are removed, and the converted product free soybean isoflavone aglycone can be obtained.
8. The method of claim 7, wherein the purified soy isoflavone glycoside is one or more of daidzein, glycitin, or genistin.
9. The method of claim 7, wherein the nutrient solution is formulated as follows: adding 20% potato and 2% glucose into distilled water, sterilizing, and adding 0.1-10% soybean isoflavone or purified soybean isoflavone glycoside.
10. The method of claim 7, wherein the fermentation culture conditions are: the temperature is 20-40 ℃, the rotating speed is 100-220 rpm, and the fermentation time is 48-144 h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210676359.5A CN115029252B (en) | 2022-06-15 | 2022-06-15 | Neurospora crassa WC2022 strain and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210676359.5A CN115029252B (en) | 2022-06-15 | 2022-06-15 | Neurospora crassa WC2022 strain and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115029252A CN115029252A (en) | 2022-09-09 |
| CN115029252B true CN115029252B (en) | 2024-01-05 |
Family
ID=83124824
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210676359.5A Active CN115029252B (en) | 2022-06-15 | 2022-06-15 | Neurospora crassa WC2022 strain and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115029252B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101479380A (en) * | 2006-06-26 | 2009-07-08 | 帝斯曼知识产权资产管理有限公司 | Peptidylarginine deiminase and uses thereof in the production of citrullinated proteins and peptides |
| CN105886561A (en) * | 2014-09-10 | 2016-08-24 | 瑞安市普罗生物科技有限公司 | Neurospora crassa-based microbial conversion method |
-
2022
- 2022-06-15 CN CN202210676359.5A patent/CN115029252B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101479380A (en) * | 2006-06-26 | 2009-07-08 | 帝斯曼知识产权资产管理有限公司 | Peptidylarginine deiminase and uses thereof in the production of citrullinated proteins and peptides |
| CN105886561A (en) * | 2014-09-10 | 2016-08-24 | 瑞安市普罗生物科技有限公司 | Neurospora crassa-based microbial conversion method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115029252A (en) | 2022-09-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102321563B (en) | Amycolatopsis sp. and method for preparing vanillin through whole-cell transformation of Amycolatopsis sp. | |
| CN104342390A (en) | Sinorhizobium meliloti strain and composition and application of sinorhizobium meliloti strain | |
| CN106010980B (en) | A kind of endogenetic fungus Brazil class shell roundlet spore bacterial strain and its application | |
| CN114350722B (en) | Method for preparing genistein | |
| CN103740606A (en) | Streptomyces phytohabitans, method for producing new antibiotics Novonestmycin from Streptomyces phytohabitans, and application of Novonestmycin | |
| CN117106595A (en) | Industrial cannabis endophytic fungus HMY07 capable of producing flavonoid compounds and application thereof | |
| CN115029252B (en) | Neurospora crassa WC2022 strain and application thereof | |
| CN114747423B (en) | Inonotus obliquus culture method and culture product thereof | |
| CN116218690A (en) | Curvularia robusta producing cloth Lei Feide bacteria A and fermentation method thereof | |
| CN105861363A (en) | Eggerthella, S-equol producing engineering bacteria and construction method and application thereof | |
| CN113881602B (en) | High-yield C 21 Steroid bacillus cereus X-32 and application thereof | |
| CN113337432B (en) | Methylophilus for producing pyrroloquinoline quinone and application thereof | |
| JPH05504469A (en) | A83543 collection method | |
| CN102329829B (en) | Method for converting daidzein into 8-hydroxydaidzein by utilizing penicillium | |
| Venkata Dasu et al. | Studies on production of griseofulvin | |
| CN112143681A (en) | Bacillus belgii capable of producing feruloyl esterase and application thereof | |
| CN107354182B (en) | A kind of method that ash green soy bean endogenetic fungus fermentation prepares (R) -4- benzyl -2- oxazolidinone compounds | |
| CN111534553A (en) | Method for biotransformation of dihydrodeoxyartemisinin B by artemisia annua cells and application of dihydrodeoxyartemisinin B | |
| CN104673722B (en) | Oxytolerant Prunella and application thereof in aerobic synthesis of dihydrosoybean isoflavone | |
| CN111534548A (en) | Method for segmented fermentation of flowers and fruits based on saccharomycetes and lactobacillus plantarum | |
| CN107058137A (en) | A kind of wet graceful mould and its method for producing wortmannin | |
| CN110642823A (en) | Pyran derivative and preparation method and application thereof | |
| CN115044628B (en) | Method for producing soybean isoflavone aglycone by whole cell transformation | |
| CN113980821B (en) | Aspergillus niger capable of converting hesperidin and application thereof | |
| CN108948036A (en) | A kind of cochliobolus quinone B derivative and its production bacterial strain and application |
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 |