CN115287203A - 一种高效降解氨基甲酸乙酯的红酵母及其应用 - Google Patents
一种高效降解氨基甲酸乙酯的红酵母及其应用 Download PDFInfo
- Publication number
- CN115287203A CN115287203A CN202210985886.4A CN202210985886A CN115287203A CN 115287203 A CN115287203 A CN 115287203A CN 202210985886 A CN202210985886 A CN 202210985886A CN 115287203 A CN115287203 A CN 115287203A
- Authority
- CN
- China
- Prior art keywords
- xsy01
- rhodosporidium toruloides
- food
- fermentation
- strain
- 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.)
- Granted
Links
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 title claims abstract description 155
- 230000000593 degrading effect Effects 0.000 title claims abstract description 28
- 241000223252 Rhodotorula Species 0.000 title abstract description 10
- 238000000855 fermentation Methods 0.000 claims abstract description 52
- 230000004151 fermentation Effects 0.000 claims abstract description 52
- 230000015556 catabolic process Effects 0.000 claims abstract description 30
- 238000006731 degradation reaction Methods 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 27
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 23
- 235000021107 fermented food Nutrition 0.000 claims abstract description 20
- 235000013305 food Nutrition 0.000 claims abstract description 20
- 235000021419 vinegar Nutrition 0.000 claims abstract description 20
- 239000000052 vinegar Substances 0.000 claims abstract description 20
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 235000013334 alcoholic beverage Nutrition 0.000 claims abstract description 9
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 235000013555 soy sauce Nutrition 0.000 claims abstract description 8
- 230000004913 activation Effects 0.000 claims abstract description 7
- 241000221523 Rhodotorula toruloides Species 0.000 claims description 57
- 239000007788 liquid Substances 0.000 claims description 27
- 239000001963 growth medium Substances 0.000 claims description 22
- 238000011081 inoculation Methods 0.000 claims description 10
- 238000009630 liquid culture Methods 0.000 claims description 10
- 235000014101 wine Nutrition 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 6
- 230000000813 microbial effect Effects 0.000 claims description 5
- 235000013618 yogurt Nutrition 0.000 claims description 5
- 238000009629 microbiological culture Methods 0.000 claims description 4
- 235000020095 red wine Nutrition 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 150000003673 urethanes Chemical class 0.000 claims 1
- 235000020097 white wine Nutrition 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 abstract description 25
- 230000000694 effects Effects 0.000 abstract description 11
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 238000012216 screening Methods 0.000 abstract description 6
- 230000008569 process Effects 0.000 abstract description 2
- 235000021001 fermented dairy product Nutrition 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 33
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 22
- 206010018910 Haemolysis Diseases 0.000 description 19
- 239000001064 degrader Substances 0.000 description 17
- 239000000047 product Substances 0.000 description 17
- 238000011534 incubation Methods 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 230000008588 hemolysis Effects 0.000 description 14
- 239000002609 medium Substances 0.000 description 14
- 239000007787 solid Substances 0.000 description 13
- 239000000725 suspension Substances 0.000 description 12
- 239000012530 fluid Substances 0.000 description 11
- 230000000968 intestinal effect Effects 0.000 description 11
- 239000008380 degradant Substances 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- 239000013592 cell lysate Substances 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000000576 coating method Methods 0.000 description 9
- 229940088598 enzyme Drugs 0.000 description 9
- 230000004083 survival effect Effects 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 239000013641 positive control Substances 0.000 description 8
- 239000006041 probiotic Substances 0.000 description 8
- 235000018291 probiotics Nutrition 0.000 description 8
- 229920001817 Agar Polymers 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 7
- 239000008272 agar Substances 0.000 description 7
- 230000003115 biocidal effect Effects 0.000 description 7
- 239000012295 chemical reaction liquid Substances 0.000 description 7
- 239000011248 coating agent Substances 0.000 description 7
- 238000012258 culturing Methods 0.000 description 7
- 235000019441 ethanol Nutrition 0.000 description 7
- 210000004051 gastric juice Anatomy 0.000 description 7
- 230000002949 hemolytic effect Effects 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 6
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 6
- 108090000604 Hydrolases Proteins 0.000 description 6
- 102000004157 Hydrolases Human genes 0.000 description 6
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical class OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 6
- 238000007865 diluting Methods 0.000 description 6
- 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 6
- 239000000796 flavoring agent Substances 0.000 description 6
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 6
- 210000001035 gastrointestinal tract Anatomy 0.000 description 6
- 210000001822 immobilized cell Anatomy 0.000 description 6
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 5
- 241000191967 Staphylococcus aureus Species 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 5
- 239000000661 sodium alginate Substances 0.000 description 5
- 235000010413 sodium alginate Nutrition 0.000 description 5
- 229940005550 sodium alginate Drugs 0.000 description 5
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 4
- 229920001661 Chitosan Polymers 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 241001494479 Pecora Species 0.000 description 4
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 4
- 229960002079 calcium pantothenate Drugs 0.000 description 4
- 239000012228 culture supernatant Substances 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000002496 gastric effect Effects 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 229960003512 nicotinic acid Drugs 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 230000000529 probiotic effect Effects 0.000 description 4
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 4
- 230000002000 scavenging effect Effects 0.000 description 4
- 239000007222 ypd medium Substances 0.000 description 4
- FFTVPQUHLQBXQZ-KVUCHLLUSA-N (4s,4as,5ar,12ar)-4,7-bis(dimethylamino)-1,10,11,12a-tetrahydroxy-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1C2=C(N(C)C)C=CC(O)=C2C(O)=C2[C@@H]1C[C@H]1[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]1(O)C2=O FFTVPQUHLQBXQZ-KVUCHLLUSA-N 0.000 description 3
- 206010007269 Carcinogenicity Diseases 0.000 description 3
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 3
- 229930182566 Gentamicin Natural products 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- SSISHJJTAXXQAX-ZETCQYMHSA-N L-ergothioneine Chemical compound C[N+](C)(C)[C@H](C([O-])=O)CC1=CNC(=S)N1 SSISHJJTAXXQAX-ZETCQYMHSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 102000057297 Pepsin A Human genes 0.000 description 3
- 108090000284 Pepsin A Proteins 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 244000062793 Sorghum vulgare Species 0.000 description 3
- 239000004098 Tetracycline Substances 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 108010059993 Vancomycin Proteins 0.000 description 3
- 229960004821 amikacin Drugs 0.000 description 3
- LKCWBDHBTVXHDL-RMDFUYIESA-N amikacin Chemical compound O([C@@H]1[C@@H](N)C[C@H]([C@@H]([C@H]1O)O[C@@H]1[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O1)O)NC(=O)[C@@H](O)CCN)[C@H]1O[C@H](CN)[C@@H](O)[C@H](O)[C@H]1O LKCWBDHBTVXHDL-RMDFUYIESA-N 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 231100000260 carcinogenicity Toxicity 0.000 description 3
- 230000007670 carcinogenicity Effects 0.000 description 3
- 229960001139 cefazolin Drugs 0.000 description 3
- MLYYVTUWGNIJIB-BXKDBHETSA-N cefazolin Chemical compound S1C(C)=NN=C1SCC1=C(C(O)=O)N2C(=O)[C@@H](NC(=O)CN3N=NN=C3)[C@H]2SC1 MLYYVTUWGNIJIB-BXKDBHETSA-N 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 235000013339 cereals Nutrition 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 229940093497 ergothioneine Drugs 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 229960003276 erythromycin Drugs 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 229960002518 gentamicin Drugs 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 235000019713 millet Nutrition 0.000 description 3
- 229960004023 minocycline Drugs 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 229940111202 pepsin Drugs 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 239000008055 phosphate buffer solution Substances 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- 229960002180 tetracycline Drugs 0.000 description 3
- 229930101283 tetracycline Natural products 0.000 description 3
- 235000019364 tetracycline Nutrition 0.000 description 3
- 150000003522 tetracyclines Chemical class 0.000 description 3
- 229960003165 vancomycin Drugs 0.000 description 3
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 3
- MYPYJXKWCTUITO-LYRMYLQWSA-O vancomycin(1+) Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C([O-])=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)[NH2+]C)[C@H]1C[C@](C)([NH3+])[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-O 0.000 description 3
- 238000009423 ventilation Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000194108 Bacillus licheniformis Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 2
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 2
- 241000228143 Penicillium Species 0.000 description 2
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 108010046334 Urease Proteins 0.000 description 2
- 229930003537 Vitamin B3 Natural products 0.000 description 2
- 229930003571 Vitamin B5 Natural products 0.000 description 2
- LXNHXLLTXMVWPM-UHFFFAOYSA-N Vitamin B6 Natural products CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 2
- 229930003756 Vitamin B7 Natural products 0.000 description 2
- 229930003761 Vitamin B9 Natural products 0.000 description 2
- 229960004050 aminobenzoic acid Drugs 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 239000003833 bile salt Substances 0.000 description 2
- 238000006065 biodegradation reaction Methods 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- 238000013124 brewing process Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 244000309464 bull Species 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000000711 cancerogenic effect Effects 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 231100000357 carcinogen Toxicity 0.000 description 2
- 239000003183 carcinogenic agent Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 229940106164 cephalexin Drugs 0.000 description 2
- ZAIPMKNFIOOWCQ-UEKVPHQBSA-N cephalexin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@@H]3N(C2=O)C(=C(CS3)C)C(O)=O)=CC=CC=C1 ZAIPMKNFIOOWCQ-UEKVPHQBSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229910000365 copper sulfate Inorganic materials 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 235000019152 folic acid Nutrition 0.000 description 2
- 239000011724 folic acid Substances 0.000 description 2
- 229960000304 folic acid Drugs 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 229960000367 inositol Drugs 0.000 description 2
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 229940099596 manganese sulfate Drugs 0.000 description 2
- 239000011702 manganese sulphate Substances 0.000 description 2
- 235000007079 manganese sulphate Nutrition 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 244000005700 microbiome Species 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
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 235000001968 nicotinic acid Nutrition 0.000 description 2
- 239000011664 nicotinic acid Substances 0.000 description 2
- DFPAKSUCGFBDDF-UHFFFAOYSA-N nicotinic acid amide Natural products NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 229940056360 penicillin g Drugs 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 2
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 description 2
- 239000011764 pyridoxine hydrochloride Substances 0.000 description 2
- 229960004172 pyridoxine hydrochloride Drugs 0.000 description 2
- 235000015067 sauces Nutrition 0.000 description 2
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000011684 sodium molybdate Substances 0.000 description 2
- 235000015393 sodium molybdate Nutrition 0.000 description 2
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- 235000019160 vitamin B3 Nutrition 0.000 description 2
- 239000011708 vitamin B3 Substances 0.000 description 2
- 235000009492 vitamin B5 Nutrition 0.000 description 2
- 239000011675 vitamin B5 Substances 0.000 description 2
- 235000019158 vitamin B6 Nutrition 0.000 description 2
- 239000011726 vitamin B6 Substances 0.000 description 2
- 235000011912 vitamin B7 Nutrition 0.000 description 2
- 239000011735 vitamin B7 Substances 0.000 description 2
- 235000019159 vitamin B9 Nutrition 0.000 description 2
- 239000011727 vitamin B9 Substances 0.000 description 2
- 229940011671 vitamin b6 Drugs 0.000 description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 description 2
- 229960001763 zinc sulfate Drugs 0.000 description 2
- 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 1
- -1 ABTS free radical Chemical class 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241001302654 Escherichia coli Nissle 1917 Species 0.000 description 1
- 108010006464 Hemolysin Proteins Proteins 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 241001134775 Lysinibacillus fusiformis Species 0.000 description 1
- 241000193386 Lysinibacillus sphaericus Species 0.000 description 1
- 108010061951 Methemoglobin Proteins 0.000 description 1
- 241000191936 Micrococcus sp. Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 241000588777 Providencia rettgeri Species 0.000 description 1
- 241000158504 Rhodococcus hoagii Species 0.000 description 1
- 241000223253 Rhodotorula glutinis Species 0.000 description 1
- 241000223254 Rhodotorula mucilaginosa Species 0.000 description 1
- 102000004495 STAT3 Transcription Factor Human genes 0.000 description 1
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 108010039416 Urethanase Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000006518 acidic stress Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003444 anaesthetic effect Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000009635 antibiotic susceptibility testing Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 235000013527 bean curd Nutrition 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 229940093761 bile salts Drugs 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- FFQKYPRQEYGKAF-UHFFFAOYSA-N carbamoyl phosphate Chemical compound NC(=O)OP(O)(O)=O FFQKYPRQEYGKAF-UHFFFAOYSA-N 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 229960002173 citrulline Drugs 0.000 description 1
- 235000013477 citrulline Nutrition 0.000 description 1
- 239000007382 columbia agar Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 231100001261 hazardous Toxicity 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 239000003228 hemolysin Substances 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 235000021109 kimchi Nutrition 0.000 description 1
- 239000008297 liquid dosage form Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000031864 metaphase Effects 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012599 radical scavenging assay Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12H—PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES
- C12H1/00—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages
- C12H1/12—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages without precipitation
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C7/00—Other dairy technology
- A23C7/04—Removing unwanted substances other than lactose or milk proteins from milk
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/127—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L23/00—Soups; Sauces; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/50—Soya sauce
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12J—VINEGAR; PREPARATION OR PURIFICATION THEREOF
- C12J1/00—Vinegar; Preparation or purification thereof
-
- 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
- C12N1/16—Yeasts; Culture media therefor
-
- 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
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast 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
- C12N1/20—Bacteria; Culture media therefor
-
- 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
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Polymers & Plastics (AREA)
- Mycology (AREA)
- Biomedical Technology (AREA)
- Nutrition Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Molecular Biology (AREA)
- Dispersion Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Soy Sauces And Products Related Thereto (AREA)
Abstract
本发明公开了一种高效降解氨基甲酸乙酯的红酵母及其应用,属于食品生物技术领域。本发明提供一种高效降解氨基甲酸乙酯(EC)的红酵母DL‑XSY01,保藏编号:CGMCC No.23534。所述红酵母DL‑XSY01经筛选、鉴定、活化、发酵和包埋过程,获得EC降解制剂。本发明所获得的菌株DL‑XSY01经包埋后制成降解剂,可以用于发酵食品中EC的去除;本发明进一步使用菌株DL‑XSY01的降解剂去除不同食品体系中的EC,具有去除EC效果明显,生产成本低,使用方便、已于从食品体系中移除等特点,广泛适用于酒类饮料、发酵乳制品、酱油、醋等发酵食品中。
Description
技术领域
本发明涉及一种高效降解氨基甲酸乙酯的红酵母及其应用,属于食品生物技术领域。
背景技术
氨基甲酸乙酯(Ethyl carbamate,EC)是一种具有遗传毒性和较强致癌性的代谢产物,广泛存在于酒精饮料和发酵食品(如腐乳、酱油、乳酪、食醋、泡菜等)中。
近年来,世界各国学者先后在蒸馏酒、黄酒和葡萄酒中发现EC的存在,其中蒸馏酒和黄酒中的EC含量较高,葡萄酒中的EC含量低。EC具有抑菌和抗肿瘤活性,在20世纪40年代初期,EC被作为麻醉药物使用;1943年,有学者发现,EC具有致癌性,但是并未引起足够重视;在1974年,国际癌症研究机构将EC列为2B类致癌物质,并于2007年被升级为2A类致癌物。同时,EC被证明具有多位点致癌性,可以导致肺癌、淋巴癌、肝癌和皮肤癌等。在动物模型的实验中,转录因子STAT3、NF-kB和细胞外信号蛋白激酶ERK被证明参与EC诱导的肿瘤的发展。另一方面,EC也被证明可以通过诱导氧化应激反应、降低解毒能力、消耗能量、破坏膜结构完整性、破坏DNA和蛋白质等方式,诱导人体肝癌细胞HepG2死亡。目前,根据FAO/WHO食品添加剂联合委员会的数据,人体通过酿造酒摄入EC的平均含量为65ng·kg-1·d-1,远高于其他发酵食品五倍之多。
EC主要以尿素、瓜氨酸、氨甲酰磷酸等为前体,与乙醇反应产生。EC的控制方法主要包括限制前体和消减EC两大类。限制前体多使用脲酶消解尿素,但是脲酶特异性单一,需考虑前体类型及不同体系适用性,实用性受到限制,而消减EC适用范围广,较为常用,此方法一般通过EC水解酶以及产酶微生物作用,实现将EC水解为乙醇、氨和二氧化碳的过程。
但是,目前现有的EC降解菌株资源库还不能满足食品中EC生物降解的实际需求,分离并公开报道的EC降解菌株较少,而且已报道的大部分EC降解菌株为非食品来源甚至是条件性致病菌株,在一定程度上限制了菌株直接在发酵食品中EC去除的应用。
1991年,Kobashi等(Chem Pharm Bull,1991,39(12):3303-3306)从小鼠胃肠道中筛选到一株地衣芽孢杆菌Bacillus licheniformis sp.,但由于其EC水解酶对EC的亲和力较弱而无法投入实际应用;1994年Kobashi等(Biol Pharm Bull,1994,17(6):773-778)又分离到一株B.licheniformis sp,只表征了其EC降解酶酶学特征,但至今并无该菌株实际应用报道。1997年,Mohapatra(Lett Appl Microbiol,1997,25(6):393-396.)从海洋生物海绵中分离到一株Micrococcus sp.,从中分离的酸性EC水解酶具有较高的乙醇耐受能力,但至今并无该菌株实际应用报道。2006年,Yukie等人(Appl Microbiol Biotechnol,.2006,70(4):422-429)从土壤样品中分离到一株Rhodococcus equi,解析了其EC水解酶的序列,但该酶对EC的特异性并不强。2014年,李京京等(食品与生物技术学报,2014,33(12):1239-1245.)从从小鼠胃中筛选出一株EC降解菌株Lysinibacillus fusiformis,从其纯化的EC水解酶具有消除酱油中EC的潜力。2014年,卜攀攀等(生物工程学报,2014,30(3):404-411)从小鼠胃部获得了一株EC降解菌株肺炎克雷伯氏菌Klebsiella pneumonia,从其分离到了耐盐的EC水解酶,但由于该酶不能耐受酸胁迫,在环境pH小于6时,酶活显著下降,应用受到局限。2017年,刘等从Providencia rettgeri JN-B815中获得氨基甲酸乙酯水解酶,pH4-7范围内残余酶活达80%,乙醇浓度为35%时残余酶活达40%。此酶可在一定程度下耐受乙醇和酸性环境,但目前没有报道其实际应用能力。田亚平(Appl Biochem Biotechnol,2013:1-11)等从小鼠胃肠道中筛选分离得到一株EC降解菌株变幻青霉菌Penicilliumvariabile,从中分离EC水解酶,发现该酶能够有效的降解白酒中的EC。
而从白酒酿造过程分离获得的菌株,报道的有:徐岩等(J Agric Food Chem,2018,66(6):1583-1590.)从白酒发酵过程中分离到的Lysinibacillus sphaericus MT3,吴群(Appl Biochem Biotechnol,2013:1-13.)等从中国白酒酿造过程中分离得到胶红酵母菌株。发酵食品来源的EC降解菌株资源库亟待扩充。
因此,本发明对发酵食品中可降解EC的微生物进行筛选、分离、鉴定和应用表征,可以扩充发酵食品来源的EC降解菌株资源库,为降低发酵食品中EC提供有效手段,为保障发酵食品安全提供技术支持。
发明内容
针对现有降解菌株较少且多数非食品来源、EC降解菌剂对EC降解效果不佳的问题,本发明目的在于提供一株分离自发酵食品的酵母菌株DL-XSY01,以及用该菌株制备的降解剂。
所述圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23534,保藏日期为2021年10月08日。
所述圆红冬孢酵母DL-XSY01分离采集自山东小米醋醋厂的醋醅中,将测序结果进行Blastn分析,发现该菌与Rhodosporidium toruloides和Rhodotorula mucilaginosa的同源性最高,均达到99%。经形态学和26s rRNA鉴定,该菌株DL-XSY01为Rhodosporidiumtoruloides,命名为圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01。
所述圆红冬孢酵母DL-XSY01在YPD固体平板中生长良好,28℃培养48h,形成湿润、圆形、突起、光滑、浅粉色菌落;镜检发现菌体呈椭圆型长棒状,在4℃静置2d后呈现橘红色。最适生长温度28℃,最适pH5~6。
本发明提供了一株圆红冬孢酵母DL-XSY01或其发酵液在制备可降解氨基甲酸乙酯的产品中的应用。
在本发明的一种实施方式中,所述产品为化学品或微生物菌剂。
在本发明的一种实施方式中,所述微生物菌剂的制备方法为:
(1)将上述圆红冬孢酵母DL-XSY01接种于YPD液体培养基中进行活化,在25~32℃、150~250rpm恒温振荡培养24~48h,获得种子液;
(2)将所述种子液按10%-20%接种量接种于发酵培养基中,在25~32℃、通气量0.6~1.0m3/min,搅拌速率150~250rpm条件下,发酵至菌体数量3×108~1.8×109CFU/mL,得到圆红冬孢酵母DL-XSY01发酵产物;
(3)将步骤(2)制备得到的所述圆红冬孢酵母DL-XSY01发酵产物进行包埋,制成微生物菌剂。
在本发明的一种实施方式中,所述圆红冬孢酵母DL-XSY01在产品中的添加量至少为:3×108CFU/mL。
在本发明的一种实施方式中,所述化学品包括但不限于氨基甲酸乙酯降解剂、氨基甲酸乙酯吸附剂、氨基甲酸乙酯抑制剂、氨基甲酸乙酯分解剂,氨基甲酸乙酯降解生物菌种、氨基甲酸乙酯降解微生物菌剂。
本发明还提供了一种降解食品中氨基甲酸乙酯的方法,在食品的制备过程中,添加所述圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01或其发酵液进行氨基甲酸乙酯的降解,降解后除菌,制备得到食品。
在本发明的一种实施方式中,所述食品包括但不限于发酵食品、酒精饮品。
在本发明的一种实施方式中,所述食品为白酒、黄酒、红酒、酸奶、醋、酱油。
本发明还提供了一种氨基甲酸乙酯降解剂,其特征在于,所述降解剂是按照下述方法制备得到的:
(1)将上述圆红冬孢酵母DL-XSY01接种于YPD液体培养基中进行活化,在25~32℃、150~250rpm恒温振荡培养24~48h,获得种子液;
(2)将所述种子液按10%-20%接种量接种于发酵培养基中,在25~32℃、通气量0.6~1.0m3/min,搅拌速率150~250rpm条件下,发酵至菌体数量3×108~1.8×109CFU/mL,得到圆红冬孢酵母DL-XSY01发酵产物;
(3)将步骤(2)制备得到的所述圆红冬孢酵母DL-XSY01发酵产物进行包埋,制成氨基甲酸乙酯降解剂。
在本发明的一种实施方式中,所述发酵培养基为:YPD液体培养基(蛋白胨20.0g,酵母粉10.0g,葡萄糖20.0g,蒸馏水补至1L,调pH至7.0,高压灭菌20min)。
在本发明的一种实施方式中,所述包埋的方法为:
S1.将海藻酸钠(4%)与含菌发酵液、细胞裂解液和菌体悬液以1:1的比例混合;
S2.用10mL注射器吸取并以2-10滴/秒的速度缓慢滴入固化液(0.6%CaCl2的饱和硼酸溶液)中,固化时间约5h;
S3.将固定化细胞用生理盐水冲洗3-4次,置于壳聚糖(2%)溶液中覆膜40min;
S4.再次用0.8%生理盐水冲洗3-4次,沥干水分,4℃下保存备用。
在本发明的一种实施方式中,所述小球湿润状态下粒径为3-5mm。
在本发明的一种实施方式中,步骤(2)中所述圆红冬孢酵母DL-XSY01发酵产物包括:圆红冬孢酵母DL-XSY01含菌发酵液、圆红冬孢酵母DL-XSY01发酵液上清、圆红冬孢酵母DL-XSY01菌体悬液、圆红冬孢酵母DL-XSY01细胞裂解液。
在本发明的一种实施方式中,所述圆红冬孢酵母DL-XSY01含菌发酵液由所述圆红冬孢酵母DL-XSY01发酵产物经干燥,用去离子水回调至原体积的1/20制成;
所述圆红冬孢酵母DL-XSY01菌体悬液由所述圆红冬孢酵母DL-XSY01发酵产物在4000g转速下离心10min并收集并收集菌体沉淀物,稀释至浓度20OD600得到;
所述圆红冬孢酵母DL-XSY01细胞裂解液由所述圆红冬孢酵母DL-XSY01菌体悬液经过细胞破壁得到。
在本发明的一种实施方式中,所述干燥方法包括冷冻真空干燥。
在本发明的一种实施方式中,步骤(3)中所述进行包埋后制备得到固定化小球,方法包括以下步骤:
S1.将海藻酸钠(4%)与含菌发酵液、细胞裂解液和菌体悬液以1:1的比例混合;
S2.用10mL注射器吸取并以2-10滴/秒的速度缓慢滴入固化液(0.6%CaCl2的饱和硼酸溶液)中,固化时间约5h;
S3.将固定化细胞用生理盐水冲洗3-4次,置于壳聚糖(2%)溶液中覆膜40min;
S4.再次用0.8%生理盐水冲洗3-4次,沥干水分,4℃下保存备用。
在本发明的一种实施方式中,所述小球湿润状态下粒径为3-5mm。
本发明还提供了上述圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01在氨基甲酸乙酯降解中的应用。
有益效果
(1)本发明从小米醋醋醅中筛选获得一株EC高效降解菌株DL-XSY01,该菌株可以利用EC作为唯一碳源生长,可有效降解EC,降解剂对EC的降解率最高可达76%,具有良好的EC生物降解效果。
(2)由于本发明所获的的菌株DL-XSY01分离自发酵食品,该菌株及其制成的EC降解剂可用于发酵食品中EC的去除,白酒中EC解率达到76%,本发明提供方法具有生产成本低、使用方便、易于从发酵产品体系中移除等特点,可以有效提高酒精饮料、酸奶、酱油和醋等发酵食品的品质和安全性。
(3)安全性评价表明:菌株DL-XSY01是安全的。抗生素敏感性中,菌株对抗生素头孢氨苄中度敏感,对10种抗生素头孢唑林、红霉素、庆大霉素、四环素、阿米卡星氨苄西林、链霉素、万古霉素、米诺环素和青霉素G敏感;溶血性实验中,DL-XSY01在羊血平板上28℃培养时未表现出溶血(γ-溶血)。然而,金黄色葡萄球菌ATCC 25923显示出β-溶血作用,阳性对照成立。此外大肠杆菌Nissle 1917显示α-溶血。因此,DL-XSY01被认为是对人体健康无危害的安全的生物体。
(4)益生性评价表明:菌株DL-XSY01在人工模拟胃液中和人工模拟肠液中有良好的存活率;对DPPH和ABTS自由基具有较好清除能力,清除率分别可达90.02%和93.67%。
生物材料保藏
一株圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01,分类命名为圆红冬孢酵母Rhodosporidium toruloides,已于2021年10月08日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23534,保藏地址为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所。
附图说明
图1为本发明圆红冬孢酵母DL-XSY01的显微照片。
图2为本发明圆红冬孢酵母DL-XSY01以EC为碳源时的筛选图片。
具体实施方式
下述实施例仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,本发明主要阐述菌株以及基于所述菌株的应用思想,实施方式中简单参数的替换不能一一在实施例中赘述,其它的任何改变未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,应被视为等效的置换方式,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,都应涵盖在本发明的保护范围之内
以下结合说明书附图和具体实施例进一步说明本发明。除非特殊说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备;除非特别说明,以下实施例所用试剂和材料均为市购。
本发明中使用的各种培养基均采用常规方法配制,实施例中涉及的分子生物学操作如未注明具体试验条件和方法,均参照SambrookJ等主编,科学出版社,2002,分子克隆实验指南(第三版);或参照产品说明书。
下述实施例中所涉及的培养基的配制如下:
YPD液体培养基:蛋白胨20.0g,酵母粉10.0g,葡萄糖20.0g,蒸馏水补至1L,调pH至7.0,高压灭菌20min。
YPD固体培养基:蛋白胨20.0g,酵母粉10.0g,葡萄糖20.0g,15g琼脂,蒸馏水补至1L,调pH至7.0,高压灭菌20min,然后倒平板。
YNB液体培养基:硫酸铵5000mg,肌醇2mg,烟酸(维生素B3)0.4mg,盐酸硫胺素(维生素B1)0.4mg,硫酸铜0.04mg,磷酸二氢钾1000mg,硼酸0.5mg,盐酸吡哆醇(维生素B6)0.4mg,泛酸钙(维生素B5)0.4mg,对氨基苯甲酸0.2mg,硫酸镁500mg,硫酸锰0.4mg,硫酸锌0.4mg,氯化铁0.2mg,核黄素(维生素B2)0.2mg,氯化钙100mg,碘化钾0.1mg,钼酸钠0.2mg,生物素(维生素B7、H)0.002mg,叶酸(维生素B9)0.002mg,氯化钠100mg,蒸馏水补至1L,滤头除菌。
YNB固培养基:硫酸铵5000mg,肌醇2mg,烟酸(维生素B3)0.4mg,盐酸硫胺素(维生素B1)0.4mg,硫酸铜0.04mg,磷酸二氢钾1000mg,硼酸0.5mg,盐酸吡哆醇(维生素B6)0.4mg,泛酸钙(维生素B5)0.4mg,对氨基苯甲酸0.2mg,硫酸镁500mg,硫酸锰0.4mg,硫酸锌0.4mg,氯化铁0.2mg,核黄素(维生素B2)0.2mg,氯化钙100mg,碘化钾0.1mg,钼酸钠0.2mg,生物素(维生素B7、H)0.002mg,叶酸(维生素B9)0.002mg,氯化钠100mg,15g琼脂,蒸馏水补至1L,滤头除菌。
下述实施例中所涉及的EC含量测定方法如下:
采用国标方法GB5009.223-2014测定培养上清液中EC的残留量,同时设不接菌的YPD培养基作对照,每个处理3个重复。该方法的具体步骤:2mL样品上样于CleanertEC氨基甲酸乙酯专用柱上,静置10min,用10mL正己烷淋洗,真空泵抽干,用10mL 5%乙酸乙酯-乙醚溶液以1mL/min流速洗脱,收集洗脱液。接收的洗脱液在室温下氮吹至0.5mL,用甲醇定容至1mL,向其中加入0.8g无水硫酸钠(100℃,24h),10000rpm离心,上清使用0.22μm有机滤膜过滤,流出液供气质检测(约0.5mL),供GC/MS分析。
GC-MS分析条件如下:
毛细管色谱柱:DB-INNOWAX,30m×0.25mm(内径)×0.25μm(膜厚);进样口温度:220℃;柱温:初温50℃,保持1min,然后以8℃/min升至180℃,程序运行完成后,240℃后运行5min;载气:氦气,纯度≥99.999%,流量1.0mL/min;离子源:EI;电离能量:70eV;离子源温度:230℃;四级杆温度:150℃;传输线温度:250℃;溶剂延迟:11min;进样方式:不分流进样;进样量:1μL;检测方式:选择离子检测(SIM);EC选择检测离子(m/z):44、62、74、89,定量离子62。
根据峰面积和标准曲线即可计算氨基甲酸乙酯的浓度。
降解率计算公式:
实施例1:圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01的分离制备
1、菌株的分离和纯化:
(1)样品:采集自山东小米醋醋场的醋醅。
(2)分离筛选方法采用先进行抗氧化富集,再进行EC降解功能验证:
取醋醅1g,加入到10mL含有2mM过氧化氢的YPD液体培养基中,并将上述样品移入均质袋,拍打30min,无菌条件下,移取均质袋中的部分液体至50mL离心管中,在30℃,200rpm条件下培养48h。由于在强氧化环境中菌体的生长会受到抑制甚至不生长,而麦角硫因具有强抗氧化性,因此能够产生较高水平麦角硫因的菌株会在培养基中正常生长,而不能产生麦角硫因或产量较低的菌株则会被筛选压力淘汰。待培养基浑浊后,稀释涂布于YPD固体培养基上,液体吸收后置于30℃倒置培养48h,挑单菌落进行YPD固体培养基上平板划线,连续纯化三代,挑取单菌落进行镜检,确定未污染后,进行菌株冻存保藏,菌株命名为DL-XSY01。
S2(EC降解功能验证):菌株DL-XSY01加入含有5g/L EC的10mL YNB液体培养基,在30℃,200rpm条件下培养24h,500rpm离心5min,取酵母沉淀稀释后均匀涂布在含有10g/L的EC为唯一碳源的YNB固体平板上,在28℃倒置培养72h,菌落形成。发现菌株DL-XSY01能以EC为唯一碳源进行良好生长,说明菌株DL-XSY01具有EC降解功能。
如图1所示,为本发明圆红冬孢酵母DL-XSY01以EC为唯一碳源时的生长图片。
2、菌株DL-XSY01的鉴定:
(1)提取菌株DL-XSY01基因组进行26s rRNA PCR鉴定,基因组提取方法按照《精编分子生物学实验指南》玻璃珠法进行提取。
PCR条件突下:
扩增体系为:2×Taq Master Mix 25μL、引物D1 2μL、引物D2 2μL、灭菌水19μL、模板2μL。PCR反应条件为:95℃预变性3min;95℃变性15S;50℃退火15S;72℃延伸1min;72℃15min;30个循环;4℃无穷。
PCR结束后进行琼脂糖凝胶(1.0%)电泳检测酵母菌样品PCR产物,跑出条带且条带明亮的为PCR扩增成功的基因组,成功的基因组即可送测序。引物序列D1:GCATATCAATAAGCGGAGGAAAAG,D2:GGTCCGTGTTTCAAGACGG。
所述菌株DL-XSY01的26s rRNA序列如下:
TTTACGGCATTCCCTAGTAGCGGCGAGCGAAGCGGGAAGAGCTCAAATTTATAATCTGGCACCTTCGGTGTCCGAGTTGTAATCTCTAGAAATGTTTTCCGCGCTGGACCGCACACAAGTCTGTTGGAATACAGCGGCATAGTGGTGAGACCCCCGTATATGGTGCGGACGCCCAGCGCTTTGTGATACATTTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCAAATTGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACCGTGAGGGAAAGATGAAAAGCACTTTGGAAAGAGAGTTAACAGTACGTGAAATTGTTGGAAGGGAAACGCTTGAAGTCAGACTTGCTTGCCGAGCAATCGGTTTGCAGGCCAGCATCAGTTTTCCGGGATGGATAATGGTAGAGAGAAGGTAGCAGTTTCGGCTGTGTTATAGCTCTCTGCTGGATACATCTTGGGGGACTGAGGAACGCAGTGTGCCTTTGGCGGGGGTTTCGACCTCTTCACACTTAGGATGCTGGTGGAATGGCTTTAAACGACCCGTCTTGAAACACGGACCCAAA。
将上述测序结果进行Blastn分析,发现该菌与Rhodosporidium toruloides和Rhodotorula mucilaginosa的同源性最高,均达到99%。经形态学和26s rRNA鉴定,该菌株DL-XSY01为Rhodosporidium toruloides,命名为圆红冬孢酵母(Rhodosporidiumtoruloides)DL-XSY01。
(2)菌株DL-XSY01在YPD固体平板中生长良好,28℃培养24-48h,如图2所示,形成湿润、圆形、突起、光滑、浅粉色菌落;镜检发现菌体呈椭圆型长棒状,在4℃静置2d后呈现橘红色。最适生长温度28℃,最适pH5~6。
经形态学和26s rRNA鉴定,该EC降解菌株DL-XSY01暂定为Rhodosporidiumtoruloides。并已于2021年10月08日保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,保藏编号:CGMCC No.23534。保藏地址:中国北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。
实施例2:圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01菌株EC降解制剂的制备
具体步骤如下:
(1)将圆红冬孢酵母DL-XSY01菌株接种于YPD液体培养基中进行活化,在25-30℃、150-250rpm恒温振荡培养24h,获得种子液。
(2)将种子液按10%接种量接种于含有YPD发酵培养基的生产发酵罐(装液量70%),通气量0.6~1.0m3/min,搅拌速率为300rpm,培养温度控制在28℃,培养时间为48~96h。发酵结束后得到菌体数量为3×108~1.8×109CFU/mL的DL-XSY01培养液。
发酵结束后,无菌条件下收集发酵液,直接用包装瓶分装成液体剂型。
(3)发酵完成后,将步骤(2)收集得到DL-XSY01培养液经冷冻真空干燥,利用去离子水回调体积至原体积的1/20,得到DL-XSY01发酵液;
(4)发酵完成后,将步骤(2)收集得到DL-XSY01培养液在4000g转速下离心10min收集DL-XSY01菌体沉淀物;
(5)将步骤(4)得到的菌体沉淀物用稀释剂PBS缓冲液稀释后,得到菌株DL-XSY01菌体悬液,其中菌浓为:OD 600=20;
(6)将步骤(5)得到的菌株DL-XSY01菌体悬液经过细胞破壁得到DL-XSY01细胞裂解液;
(7)分别将步骤(3)制备得到的DL-XSY01含菌发酵液、步骤(5)制备得到的DL-XSY01菌体悬液和步骤(6)制备得到的DL-XSY01细胞裂解液,经海藻酸钠包埋,制成相应的固定化小球,具体步骤如下:
将海藻酸钠(4%)分别与步骤(3)制备得到的DL-XSY01含菌发酵液、步骤(5)制备得到的DL-XSY01菌体悬液和步骤(6)制备得到的DL-XSY01细胞裂解液以体积比为1:1的比例混合,用10mL注射器吸取并以2-10滴/秒的速度缓慢滴入固化液(0.6%CaCl2的饱和硼酸溶液)中,固化时间约5h;制备得到固定化细胞;
将固定化细胞用生理盐水冲洗3-4次,置于壳聚糖(2%)溶液中覆膜40min。再次用0.8%生理盐水冲洗3-4次,沥干水分,4℃下保存备用,制备得到EC降解剂。
分别将步骤(3)制备得到的DL-XSY01含菌发酵液、步骤(5)制备得到的DL-XSY01菌体悬液和步骤(6)制备得到的DL-XSY01细胞裂解液所制备的包埋制剂分别为EC降解剂1、EC降解剂2、EC降解剂3。
(8)分别检测EC降解剂1、EC降解剂2、EC降解剂3中的DL-XSY01的菌浓,分别为:OD600=10、OD600=20和OD600=0。
实施例3:圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01制备得到的EC降解剂在白酒中EC降解的应用
具体步骤如下:
分别将实施例2制备得到的EC降解剂1、EC降解剂2、EC降解剂3,各20mL,分别处理含有EC的白酒样品,具体如下:
分别向60mL 45度白酒中添加EC降解剂1、EC降解剂2和EC降解剂3各20mL,分别在250mL发酵瓶中,28℃,100rpm反应5d。
对照组:向60mL 45度白酒中添加20mL YPD培养基,在250mL发酵瓶中,28℃,100rpm反应5d。
反应结束后分别吸取上清液2mL,过0.2μm滤膜,分别测定培养上清液中EC的残留量,每个处理3个重复,结果如表1所示。
表1:不同EC降解剂的EC降解率
结果显示:EC降解剂1、EC降解剂2和EC降解剂3均可以降低白酒中的EC浓度,可以将EC浓度由230.3737μg/L分别降低至86.9693μg/L、100.0902μg/L和55.3112μg/L,降解率分别为62.24%,56.56%和76.00%。
可见,EC降解剂3对于白酒中EC的降解效果最好。
实施例4:圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01制备得到的EC降解剂3在发酵食品中添加EC的降解实验
下述实施例中以EC降解剂3为例,证明本申请的菌株能够降解各种发酵食品及酒精饮品中的EC,下述采用的样品,均是市售产品。
下述实施例中所涉及的EC降解剂3的处理方法为:将圆红冬孢酵母DL-XSY01菌株接种于YPD液体培养基中进行活化,在25-30℃、150-250rpm恒温振荡培养24h,获得种子液。将种子液按10%接种量接种于含有YPD发酵培养基的生产发酵罐(装液量70%),通气量0.6~1.0m3/min,搅拌速率为300rpm,培养温度控制在28℃,培养时间为48~96h。发酵结束后得到菌体数量为3×108~1.8×109CFU/mL的DL-XSY01培养液。发酵完成后,收集得到DL-XSY01培养液在4000g转速下离心10min收集DL-XSY01菌体沉淀物;用稀释剂PBS缓冲液稀释后,得到菌株DL-XSY01菌体悬液,其中菌浓为:OD600=20;将菌体破壁得到DL-XSY01细胞裂解液,随后,与4%海藻酸钠以体积比为1:1的比例混合,用10mL注射器吸取并以2-10滴/秒的速度缓慢滴入固化液(0.6%CaCl2的饱和硼酸溶液)中,固化时间约5h;制备得到固定化细胞;将固定化细胞用生理盐水冲洗3-4次,置于壳聚糖(2%)溶液中覆膜40min。再次用0.8%生理盐水冲洗3-4次,沥干水分,4℃下保存备用。
1、制备红酒EC反应液:
红酒(12度)60mL;EC:3.0ppm;EC降解剂3:20mL,在250mL锥形瓶中温育,28℃,100rpm,5天。
2、制备酸奶EC反应液:
酸奶60mL;EC:3.0ppm;EC降解剂3:20mL,在250mL锥形瓶中温育,28℃,100rpm,5天。
3、制备醋EC反应液:
醋60mL;EC:3.0ppm;EC降解剂3:20mL,在250mL锥形瓶中温育,28℃,100rpm,5天。
4、制备酱油EC反应液:
酱油60mL;EC:3.0ppm;EC降解剂3:20mL,在250mL锥形瓶中温育,28℃,100rpm,5天。
5、制备酱EC反应液
酱30g,加入30mL去离子水,EC:3.0ppm;EC降解剂3:20mL,在250mL锥形瓶中温育,28℃,100rpm,5天。
分别将上述所制得的发酵食品EC反应液,反应后吸取上清液2mL,过0.2μm滤膜,测定培养上清液中EC的残留量,同时设不接菌的YPD培养基作对照,每个处理3个重复,结果如表2所示。
表2:反应5天EC浓度
结果显示,DL-XSY01发酵产品及其EC降解剂对EC有很好的降解作用,发酵食品中乙醇含量、盐等成分对DL-XSY01的EC降解活性影响不大。
实施例5:圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01的应用
下述实施例中以EC降解剂3为例,证明本申请的菌株能够降解实际酒精饮品中的EC,下述采用的样品,均是市售产品。
下述实施例中所涉及的EC降解剂3的处理方法同实施例4。
1、制备芝麻香型白酒EC反应液:
芝麻香型白酒:(53度)60mL;EC降解剂3:20mL,在250mL锥形瓶中温育,28℃,100rpm,2天。
2、制备浓香型白酒EC反应液:
浓香型白酒:(45度)60mL;EC降解剂3:20mL,在250mL锥形瓶中温育,28℃,100rpm,2天。
3、制备酱香型白酒EC反应液
酱香型白酒:(42度)60mL;EC降解剂3:20mL,在250mL锥形瓶中温育,28℃,100rpm,2天。
4、制备清香型白酒EC反应液
清香型白酒:(38度)60mL;EC降解剂3:20mL,在250mL锥形瓶中温育,28℃,100rpm,2天。
5、制备黄酒EC反应液:
黄酒(15度)60mL;EC降解剂3:20mL,在250mL锥形瓶中温育,28℃,100rpm,2天。
分别将上述所制得的发酵食品EC反应液,反应后吸取上清液2mL,过0.2μm滤膜,测定培养上清液中EC的残留量,同时设不接菌的YPD培养基作对照,每个处理3个重复,结果如表3所示。
表3:反应2天的实际酒精饮品中EC浓度
结果显示,DL-XSY01发酵产品及其EC降解剂对实际发酵样品中EC有很好的降解作用,对酒精饮品中EC的降解率可以达到70%左右。
实施例6:圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01在发酵过程中降低EC含量的的应用
在下述实施例中,使用DL-XSY01处理发酵过程中的醋醅,证明本申请的菌株能够在发酵过程中降低EC的产生。
下述实施例中所涉及的DL-XSY01制备方法为:将圆红冬孢酵母DL-XSY01菌株接种于YPD液体培养基中进行活化,在25-30℃、150-250rpm恒温振荡培养24h,获得种子液;将种子液按10%接种量接种于含有YPD发酵培养基的生产发酵罐(装液量70%),通气量0.6~1.0m3/min,搅拌速率为300rpm,培养温度控制在28℃,培养时间为48~96h。发酵结束后得到菌体数量为3×108~1.8×109CFU/mL的DL-XSY01培养液。发酵完成后,收集得到DL-XSY01培养液经冷冻真空干燥,获得干燥菌体。
取5g冻干获得的菌体,加入100g醋醅中进行发酵20天,获得接菌发酵的白醋。另外直接发酵100g醋醅20天,获得未接菌发酵的白醋。
分别将上述的白醋吸取2mL,得顶EC残留量,每组样品设置3个重复,结果如表4所示。
表4:接菌发酵前后白醋中EC含量
结果显示,添加冻干后的DL-XSY01菌体,可以降低发酵过程中的EC浓度。
实施例7:圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01菌株的性质
具体步骤如下:
1、DL-XSY01菌株安全性评价
(1)抗生素敏感性实验
使用纸片扩散法对圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01的抗生素敏感性谱进行表征。
在YPD-琼脂固体平板上挑取DL-XSY01单菌落,转移至YPD液体培养基培养,获得菌液(约1×108cfu/mL);
取200μL DL-XSY01菌液涂布于YPD-琼脂固体平板中,分别缓慢放置庆大霉素(10μg)、链霉素(10μg)、红霉素(15μg)、四环素(30μg)、头孢氨苄(30μg)、万古霉素(30μg)、头孢唑林(30μg)、氨苄西林(10μg)、青霉素(10μg)、米诺环素(30μg)及阿米卡星(30μg)11种抗生素药敏纸片,每个纸片的间距不小于24mm;
将上述放有纸片的培养基,在28℃静置培养24h后测量并统计抑菌圈直径,分析其耐药性抗性R(≤14毫米),中期I(14-20毫米)或敏感S(≥20毫米),每组设置三个重复,DL-XSY01的抗生素敏感性如表5所示。
表5:DL-XSY01的抗生素敏感性
结果显示,菌株对抗生素头孢氨苄中度敏感,对10种抗生素头孢唑林、红霉素、庆大霉素、四环素、阿米卡星氨苄西林、链霉素、万古霉素、米诺环素和青霉素G敏感。因此,根据CLSI指南,这些结果证实DL-XSY01是安全的。
(2)溶血性实验
溶血性可分为三种,其中α溶血:又称草绿色溶血,菌落周围培养基出现1-2mm的草绿色环,为高铁血红蛋白所致,α溶血环中的红细胞未完全溶解,可形成α溶血环的细菌如甲型溶血性链球菌、肺炎链球菌;β溶血是在固体平板上培养时,菌落周围形成的宽大(2-4mm)、界限分明、完全透明的溶血环,β溶血环中的红细胞完全溶解,是菌体产生的溶血素使红细胞完全溶解所致,又称完全溶血,可形成β溶血环的包括乙型溶血性链球菌、金黄色葡萄球菌等;γ溶血:又称不溶血,在菌落周围无溶血环。
具体步骤如下:
培养基配置:哥伦比亚琼脂+5%脱纤维羊血。
在YPD-琼脂固体平板上挑取DL-XSY01单菌落,转移至YPD液体培养基培养,获得菌液(约1×108cfu/mL);
取菌液划线于含有5%脱纤维羊血的哥伦比亚平板中,28℃培养24h,之后观察是否有透明圈。金黄色葡萄球菌ATCC 25923作为阳性对照,每组设置三个重复。
结果显示,在此项溶血试验中,金黄色葡萄球菌ATCC 25923被用作阳性对照。DL-XSY01在羊血平板上28℃培养时未表现出溶血(γ-溶血)。然而,金黄色葡萄球菌ATCC25923显示出β-溶血作用,阳性对照成立。此外大肠杆菌Nissle 1917显示α-溶血。因此,DL-XSY01被认为是对人体健康无危害的安全的生物体。
2、DL-XSY01菌株益生性评价
(1)人工模拟胃液实验
益生菌要定植于胃肠道发挥益生作用,首先要对消化道环境具有一定的耐受性,对胃液的耐受性是其中重要筛选标准。
配制模拟胃液:稀盐酸16.4mL,加水约900mL,胃蛋白酶10g,均匀混合后加水定容至1000mL。分别调节pH为2.5,用0.22μm滤膜过滤除菌,4℃保存。
模拟胃液耐受性实验:
在YPD-琼脂固体平板上挑取DL-XSY01单菌落,转移至YPD液体培养基培养,获得菌液(约1×108cfu/mL),取5mL菌液于10000rpm,4℃离心5min收集菌体,用pH 7.4的无菌磷酸盐缓冲液洗涤2次,重复如上操作。
将菌体重新悬浮于5mL的pH 2.5的人工胃液中。采用平板涂布法计算在pH 2.5的人工模拟胃液中孵育0h和3h的活菌数量,每组设置三个重复。
存活率(%)=logCFU(N1)/logCFU(N0)*100%。
其中,N0表示在人工模拟胃液中孵育0h的活菌数,N1表示在人工模拟胃液中孵育3h后的活菌数。
同时,以大肠杆菌Nissle 1917作为阳性对照。
结果显示,在人工模拟胃液条件(含有0.3%胃蛋白酶,pH值为2.5)培养3h以后,DL-XSY01的存活率为86.73%,而阳性对照组大肠杆菌Nissle 1917的存活率为86.14%。结果表明,与大肠杆菌Nissle 1917相比,DL-XSY01在人工模拟胃液中有良好的存活率。
(2)人工模拟肠液实验
益生菌要定植于胃肠道发挥益生作用,首先要对消化道环境具有一定的耐受性,对肠液的耐受性是其中重要标准。
配置模拟肠液:用灭菌后的磷酸盐缓冲液(pH 7.4)将胰蛋白酶配制成浓度为1mg/mL溶液,在加入0.3%的牛胆盐,并用1mol/L氢氧化钠调节pH值至7.4,而后经0.22μm微孔滤膜过滤除菌后备用。
模拟肠液耐受性实验:
在YPD-琼脂固体平板上挑取DL-XSY01单菌落,转移至YPD液体培养基培养,获得菌液(约1×108cfu/mL);
取5mL菌液于10000rpm,4℃离心5min收集菌体,用pH 7.4的无菌磷酸盐缓冲液洗涤2次,重复如上操作。将菌体重新悬浮于5mL的pH 7.4的人工模拟肠液中。采用平板涂布法计算在pH 7.4人工模拟肠液中0h和4h的活菌数量,每组设置三个重复。
存活率(%)=logCFU(N1)/logCFU(N0)*100%
其中,N0代表在人工模拟肠液中孵育0h的活菌数,N1代表在人工模拟肠液中孵育3h后的活菌数。
同时,以大肠杆菌Nissle 1917作为阳性对照。
结果显示,在人工模拟肠液条件(含有0.3%胰蛋白酶和0.3%牛胆盐)培养4h以后,DL-XSY01的存活率为85.92%,而阳性对照组大肠杆菌Nissle 1917的存活率为84.64%。结果表明,与大肠杆菌Nissle 1917相比,DL-XSY01在人工模拟肠液中有良好的存活率。
(3)抗氧化实验
DPPH自由基清除率试验:
采用0.0078g DPPH,用无水乙醇溶解,定容至100ml,配制得到0.2mmol/L DPPH,避光放置,现配现用。
按照上述步骤(1)制备得到菌浓为108CFU/mL的DL-XSY01菌液;
将上述108CFU/mL的DL-XSY01菌液按照体积比为1:1的比例与100%乙醇DPPH溶液(0.2mM)混合,在25℃的黑暗中培养30min。
单独使用DL-XSY01菌液和100%乙醇作为空白,而DPPH乙醇溶液作为对照。在2330×g(4120rpm)下离心10分钟后收集上清液。在517nm处测量吸光度一式三份。
ABTS自由基清除率试验:
将ABTS(14mM)和过硫酸钾(5mM)溶解在0.1M磷酸钾缓冲液(pH 7.4)中,以1:1的比例混合,并在25℃下反应12~16h。
将100μL菌株DL-BJ01(108CFU/mL)添加到900μL ABTS溶液中,并在25℃下黑暗中培养15min。离心(14000g,1分钟)后,在734nm处测量上清液的吸光度。
结果显示,DL-XSY01的DPPH清除活性为90.02%,ABTS清除活性为93.67%,说明菌株DL-XSY01具有很好的抗氧化活性。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.一株圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23534,保藏日期为2021年10月08日。
2.权利要求1所述的圆红冬孢酵母DL-XSY01或其发酵液在制备可降解氨基甲酸乙酯的产品中的应用。
3.如权利要求2所述的应用,其特征在于,所述产品为化学品或微生物菌剂。
4.如权利要求3所述的应用,其特征在于,所述圆红冬孢酵母DL-XSY01在产品中的添加量至少为:3×108CFU/mL。
5.如权利要求2~4任一所述的应用,其特征在于,所述化学品包括但不限于氨基甲酸乙酯降解剂。
6.一种降解食品中氨基甲酸乙酯的方法,其特征在于,在食品的制备过程中,添加权利要求1所述的圆红冬孢酵母DL-XSY01或其发酵液进行氨基甲酸乙酯的降解,降解后除菌,制备得到食品。
7.如权利要求6所述的方法,其特征在于,所述食品包括但不限于发酵食品、酒精饮品。
8.如权利要求7所述的方法,其特征在于,所述食品为白酒、黄酒、红酒、酸奶、醋、酱油。
9.一种氨基甲酸乙酯降解剂,其特征在于,所述降解剂是按照下述方法制备得到的:
(1)将权利要求1所述的圆红冬孢酵母DL-XSY01接种于YPD液体培养基中进行活化,在25~32℃、150~250rpm恒温振荡培养24~48h,获得种子液;
(2)将所述种子液按10%-20%接种量接种于发酵培养基中,在25~32℃、通气量0.6~1.0m3/min,搅拌速率150~250rpm条件下,发酵至菌体数量3×108~1.8×109CFU/mL,得到圆红冬孢酵母DL-XSY01发酵产物;
(3)将步骤(2)制备得到的所述圆红冬孢酵母DL-XSY01发酵产物进行包埋,制成氨基甲酸乙酯降解剂。
10.权利要求1所述的圆红冬孢酵母DL-XSY01在氨基甲酸乙酯降解中的应用。
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210985886.4A CN115287203B (zh) | 2022-08-16 | 2022-08-16 | 一种高效降解氨基甲酸乙酯的红酵母及其应用 |
PCT/CN2023/102849 WO2024037192A1 (zh) | 2022-08-16 | 2023-06-27 | 一种高效降解氨基甲酸乙酯的红酵母及其应用 |
US18/589,595 US20240225056A1 (en) | 2022-08-16 | 2024-02-28 | Rhodotorula Capable of Efficiently Degrading Ethyl Carbamate and Application Thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210985886.4A CN115287203B (zh) | 2022-08-16 | 2022-08-16 | 一种高效降解氨基甲酸乙酯的红酵母及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN115287203A true CN115287203A (zh) | 2022-11-04 |
CN115287203B CN115287203B (zh) | 2023-05-26 |
Family
ID=83830212
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210985886.4A Active CN115287203B (zh) | 2022-08-16 | 2022-08-16 | 一种高效降解氨基甲酸乙酯的红酵母及其应用 |
Country Status (3)
Country | Link |
---|---|
US (1) | US20240225056A1 (zh) |
CN (1) | CN115287203B (zh) |
WO (1) | WO2024037192A1 (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115322912A (zh) * | 2022-08-16 | 2022-11-11 | 大连工业大学 | 麦角硫因高产菌株及其筛选方法和应用 |
WO2024037192A1 (zh) * | 2022-08-16 | 2024-02-22 | 大连工业大学 | 一种高效降解氨基甲酸乙酯的红酵母及其应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5000966A (en) * | 1987-12-11 | 1991-03-19 | Takeda Chemical Industries, Ltd. | Quality improvement of alcoholic liquors by enzymatic decomposing of ethyl carbamate |
CA1310925C (en) * | 1987-12-11 | 1992-12-01 | Yasuhiro Sumino | Quality improvement of alcoholic liquors |
CN102492633A (zh) * | 2011-12-02 | 2012-06-13 | 江南大学 | 具有降解氨基甲酸乙酯功能的胶红酵母及其在酒类及食品中的应用 |
CN113337417A (zh) * | 2021-03-30 | 2021-09-03 | 大连工业大学 | 一种高效降解氨基甲酸乙酯的农杆菌及其应用 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113174342B (zh) * | 2021-03-30 | 2023-07-07 | 大连工业大学 | 高效降解氨基甲酸乙酯的菌株及其应用 |
CN115287203B (zh) * | 2022-08-16 | 2023-05-26 | 大连工业大学 | 一种高效降解氨基甲酸乙酯的红酵母及其应用 |
CN115322912B (zh) * | 2022-08-16 | 2023-09-19 | 大连工业大学 | 麦角硫因高产菌株及其筛选方法和应用 |
-
2022
- 2022-08-16 CN CN202210985886.4A patent/CN115287203B/zh active Active
-
2023
- 2023-06-27 WO PCT/CN2023/102849 patent/WO2024037192A1/zh unknown
-
2024
- 2024-02-28 US US18/589,595 patent/US20240225056A1/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5000966A (en) * | 1987-12-11 | 1991-03-19 | Takeda Chemical Industries, Ltd. | Quality improvement of alcoholic liquors by enzymatic decomposing of ethyl carbamate |
CA1310925C (en) * | 1987-12-11 | 1992-12-01 | Yasuhiro Sumino | Quality improvement of alcoholic liquors |
CN102492633A (zh) * | 2011-12-02 | 2012-06-13 | 江南大学 | 具有降解氨基甲酸乙酯功能的胶红酵母及其在酒类及食品中的应用 |
CN113337417A (zh) * | 2021-03-30 | 2021-09-03 | 大连工业大学 | 一种高效降解氨基甲酸乙酯的农杆菌及其应用 |
Non-Patent Citations (3)
Title |
---|
EILEEN ABT 等: ""Occurrence of Ethyl Carbamate in Foods and Beverages: Review of the Formation Mechanisms, Advances in Analytical Methods, and Mitigation Strategies"" * |
姚晓瑞宁;高飞飞;王斌;肖婧;史学伟;: "产氨基甲酸乙酯降解酶酵母菌的选育及产酶条件优化" * |
张群;: "酒中安全性指标氨基甲酸乙酯的研究" * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115322912A (zh) * | 2022-08-16 | 2022-11-11 | 大连工业大学 | 麦角硫因高产菌株及其筛选方法和应用 |
CN115322912B (zh) * | 2022-08-16 | 2023-09-19 | 大连工业大学 | 麦角硫因高产菌株及其筛选方法和应用 |
WO2024037192A1 (zh) * | 2022-08-16 | 2024-02-22 | 大连工业大学 | 一种高效降解氨基甲酸乙酯的红酵母及其应用 |
Also Published As
Publication number | Publication date |
---|---|
WO2024037192A1 (zh) | 2024-02-22 |
US20240225056A1 (en) | 2024-07-11 |
CN115287203B (zh) | 2023-05-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN115287203B (zh) | 一种高效降解氨基甲酸乙酯的红酵母及其应用 | |
CN111849810B (zh) | 拮抗幽门螺旋杆菌的乳酸菌zjuids03及其应用 | |
CN112410269B (zh) | 一株枯草芽孢杆菌及其在降解玉米赤霉烯酮中的应用 | |
CN111808765B (zh) | 一株高效降解呕吐毒素的枯草芽孢杆菌及其应用 | |
CN107201322A (zh) | 用于降解黄曲霉毒素b1的枯草芽孢杆菌及其应用 | |
CN115895934B (zh) | 一种同时降解呕吐毒素和玉米赤霉烯酮的贝莱斯芽孢杆菌及应用 | |
CN111826298A (zh) | 一株高效降解玉米赤霉烯酮的凝结芽孢杆菌及其应用 | |
CN116445321A (zh) | 降核苷降血尿酸罗伊氏乳杆菌a21160及其应用 | |
CN115851500A (zh) | 一株植物乳杆菌及其应用 | |
CN111778178B (zh) | 海洋灰平链霉菌hn60在抗细菌方面的应用 | |
CN111088181B (zh) | 短双歧杆菌菌株bk55及在抑制艰难梭菌中的应用 | |
CN113061550B (zh) | 一株乳杆菌新菌株z6及其在食品中的应用 | |
CN113174342B (zh) | 高效降解氨基甲酸乙酯的菌株及其应用 | |
CN113337417B (zh) | 一种高效降解氨基甲酸乙酯的农杆菌及其应用 | |
CN113930367B (zh) | 一株具有降胆固醇性能的乳酸菌及其应用 | |
CN113046276B (zh) | 一种母乳源鼠李糖乳杆菌及其应用 | |
CN112195119B (zh) | 一种能降解生物胺且耐盐的植物乳杆菌及其应用 | |
CN110241052B (zh) | 一株高产叶酸植物乳杆菌gslp-7及其应用 | |
CN116396883B (zh) | 一株耐高温高效降解玉米赤霉烯酮和呕吐毒素的枯草芽孢杆菌及其菌剂 | |
CN110713958A (zh) | 一株酪丁酸梭菌及其应用 | |
CN115838652B (zh) | 一种罗伊氏黏液乳杆菌lr21及其应用 | |
CN115466693B (zh) | 一株斯塔贝基斯鲁梅利杆菌cy2菌株及其应用 | |
CN118374387A (zh) | 一种降解嘌呤核苷酸的短乳杆菌及其应用 | |
CN108576577B (zh) | 快速降解河豚毒素的方法及药剂 | |
CN106967630B (zh) | 一种发酵乳杆菌及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |