CN114958649B - Pediococcus acidilactici and application thereof in degradation of ochratoxin A - Google Patents
Pediococcus acidilactici and application thereof in degradation of ochratoxin A Download PDFInfo
- Publication number
- CN114958649B CN114958649B CN202210380740.7A CN202210380740A CN114958649B CN 114958649 B CN114958649 B CN 114958649B CN 202210380740 A CN202210380740 A CN 202210380740A CN 114958649 B CN114958649 B CN 114958649B
- Authority
- CN
- China
- Prior art keywords
- pediococcus acidilactici
- njb421
- ota
- mice
- ochratoxin
- 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
- 241000191998 Pediococcus acidilactici Species 0.000 title claims abstract description 171
- 230000015556 catabolic process Effects 0.000 title claims abstract description 28
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 28
- RWQKHEORZBHNRI-BMIGLBTASA-N ochratoxin A Chemical compound C([C@H](NC(=O)C1=CC(Cl)=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 RWQKHEORZBHNRI-BMIGLBTASA-N 0.000 title claims abstract description 20
- VYLQGYLYRQKMFU-UHFFFAOYSA-N Ochratoxin A Natural products CC1Cc2c(Cl)cc(CNC(Cc3ccccc3)C(=O)O)cc2C(=O)O1 VYLQGYLYRQKMFU-UHFFFAOYSA-N 0.000 title claims abstract description 15
- DAEYIVCTQUFNTM-UHFFFAOYSA-N ochratoxin B Natural products OC1=C2C(=O)OC(C)CC2=CC=C1C(=O)NC(C(O)=O)CC1=CC=CC=C1 DAEYIVCTQUFNTM-UHFFFAOYSA-N 0.000 title claims abstract description 15
- 238000004321 preservation Methods 0.000 claims abstract description 10
- 206010067125 Liver injury Diseases 0.000 claims abstract description 7
- 206010061481 Renal injury Diseases 0.000 claims abstract description 7
- 239000000654 additive Substances 0.000 claims abstract description 5
- 230000001580 bacterial effect Effects 0.000 claims description 64
- 239000007788 liquid Substances 0.000 claims description 50
- 238000000034 method Methods 0.000 claims description 43
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 claims description 42
- 239000003795 chemical substances by application Substances 0.000 claims description 25
- 241000894006 Bacteria Species 0.000 claims description 24
- 229960000956 coumarin Drugs 0.000 claims description 21
- 235000001671 coumarin Nutrition 0.000 claims description 21
- 238000012258 culturing Methods 0.000 claims description 20
- 230000000813 microbial effect Effects 0.000 claims description 20
- 230000000593 degrading effect Effects 0.000 claims description 17
- 230000006378 damage Effects 0.000 claims description 16
- 239000003963 antioxidant agent Substances 0.000 claims description 10
- 230000003078 antioxidant effect Effects 0.000 claims description 10
- 230000003213 activating effect Effects 0.000 claims description 7
- 208000027418 Wounds and injury Diseases 0.000 claims description 6
- 208000014674 injury Diseases 0.000 claims description 6
- 208000037806 kidney injury Diseases 0.000 claims description 6
- 229930183344 ochratoxin Natural products 0.000 claims description 6
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 5
- 231100000753 hepatic injury Toxicity 0.000 claims description 5
- 230000008816 organ damage Effects 0.000 claims description 5
- 208000024891 symptom Diseases 0.000 claims description 5
- 230000003247 decreasing effect Effects 0.000 claims description 4
- 244000005700 microbiome Species 0.000 claims description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 235000012631 food intake Nutrition 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims 2
- 241000192001 Pediococcus Species 0.000 claims 1
- 238000003745 diagnosis Methods 0.000 claims 1
- 238000002405 diagnostic procedure Methods 0.000 claims 1
- 238000002560 therapeutic procedure Methods 0.000 claims 1
- 241000699670 Mus sp. Species 0.000 abstract description 86
- 210000004185 liver Anatomy 0.000 abstract description 37
- 230000012010 growth Effects 0.000 abstract description 17
- 239000003833 bile salt Substances 0.000 abstract description 14
- 102000004142 Trypsin Human genes 0.000 abstract description 13
- 108090000631 Trypsin Proteins 0.000 abstract description 13
- 239000012588 trypsin Substances 0.000 abstract description 13
- 238000012216 screening Methods 0.000 abstract description 9
- 230000000968 intestinal effect Effects 0.000 abstract description 8
- 230000002378 acidificating effect Effects 0.000 abstract description 7
- 230000003647 oxidation Effects 0.000 abstract description 7
- 238000007254 oxidation reaction Methods 0.000 abstract description 7
- 239000003053 toxin Substances 0.000 abstract description 7
- 231100000765 toxin Toxicity 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 4
- 208000037817 intestinal injury Diseases 0.000 abstract description 4
- 239000001963 growth medium Substances 0.000 description 45
- 230000000694 effects Effects 0.000 description 37
- 238000012360 testing method Methods 0.000 description 32
- 210000003734 kidney Anatomy 0.000 description 30
- 108020004414 DNA Proteins 0.000 description 26
- 210000003405 ileum Anatomy 0.000 description 23
- 230000002829 reductive effect Effects 0.000 description 23
- 239000000243 solution Substances 0.000 description 23
- 238000009630 liquid culture Methods 0.000 description 21
- 210000001519 tissue Anatomy 0.000 description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- 239000002609 medium Substances 0.000 description 16
- 241001465754 Metazoa Species 0.000 description 15
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 14
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 14
- 229940118019 malondialdehyde Drugs 0.000 description 14
- 210000000056 organ Anatomy 0.000 description 14
- 210000002966 serum Anatomy 0.000 description 13
- 239000007787 solid Substances 0.000 description 13
- 238000011144 upstream manufacturing Methods 0.000 description 13
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 12
- 108010082126 Alanine transaminase Proteins 0.000 description 12
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 11
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 11
- 231100000678 Mycotoxin Toxicity 0.000 description 11
- 239000011521 glass Substances 0.000 description 11
- 239000002636 mycotoxin Substances 0.000 description 11
- 239000006041 probiotic Substances 0.000 description 11
- 235000018291 probiotics Nutrition 0.000 description 11
- 108090001005 Interleukin-6 Proteins 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 230000002496 gastric effect Effects 0.000 description 10
- 108020004999 messenger RNA Proteins 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 210000000813 small intestine Anatomy 0.000 description 9
- 230000001954 sterilising effect Effects 0.000 description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 8
- 108010065472 Vimentin Proteins 0.000 description 8
- 102100035071 Vimentin Human genes 0.000 description 8
- 230000037396 body weight Effects 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- 239000000843 powder Substances 0.000 description 8
- 238000010186 staining Methods 0.000 description 8
- 210000005048 vimentin Anatomy 0.000 description 8
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 7
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 7
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 7
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 7
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 7
- 229940109239 creatinine Drugs 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 102000004162 Claudin-1 Human genes 0.000 description 6
- 108090000600 Claudin-1 Proteins 0.000 description 6
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 102000003777 Interleukin-1 beta Human genes 0.000 description 6
- 108090000193 Interleukin-1 beta Proteins 0.000 description 6
- 102000003940 Occludin Human genes 0.000 description 6
- 108090000304 Occludin Proteins 0.000 description 6
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 6
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 6
- 238000001784 detoxification Methods 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 235000021050 feed intake Nutrition 0.000 description 6
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 6
- IQZZFVDIZRWADY-UHFFFAOYSA-N isocoumarin Chemical compound C1=CC=C2C(=O)OC=CC2=C1 IQZZFVDIZRWADY-UHFFFAOYSA-N 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 238000004806 packaging method and process Methods 0.000 description 6
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 6
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 229940041514 candida albicans extract Drugs 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 5
- 230000003902 lesion Effects 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 239000012138 yeast extract Substances 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000003794 Gram staining Methods 0.000 description 4
- 102000004889 Interleukin-6 Human genes 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 4
- 235000019341 magnesium sulphate Nutrition 0.000 description 4
- 231100000915 pathological change Toxicity 0.000 description 4
- 230000036285 pathological change Effects 0.000 description 4
- 230000000770 proinflammatory effect Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 3
- 108010035532 Collagen Proteins 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 241000192125 Firmicutes Species 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
- 206010061218 Inflammation Diseases 0.000 description 3
- 241000186660 Lactobacillus Species 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 3
- 235000015278 beef Nutrition 0.000 description 3
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 229920001436 collagen Polymers 0.000 description 3
- 230000002354 daily effect Effects 0.000 description 3
- 230000008021 deposition Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 210000004969 inflammatory cell Anatomy 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 230000007358 intestinal barrier function Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 229940039696 lactobacillus Drugs 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 229940099596 manganese sulfate Drugs 0.000 description 3
- 239000011702 manganese sulphate Substances 0.000 description 3
- 235000007079 manganese sulphate Nutrition 0.000 description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000003340 mental effect Effects 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 230000007170 pathology Effects 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 231100000572 poisoning Toxicity 0.000 description 3
- 230000000607 poisoning effect Effects 0.000 description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 3
- 229920000053 polysorbate 80 Polymers 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 239000001632 sodium acetate Substances 0.000 description 3
- 235000017281 sodium acetate Nutrition 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 230000003068 static effect Effects 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 241000228212 Aspergillus Species 0.000 description 2
- 241000194108 Bacillus licheniformis Species 0.000 description 2
- 206010070545 Bacterial translocation Diseases 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- 108010002352 Interleukin-1 Proteins 0.000 description 2
- 102000000589 Interleukin-1 Human genes 0.000 description 2
- 206010023421 Kidney fibrosis Diseases 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- BPZZWRPHVVDAPT-UHFFFAOYSA-N Ochratoxin-A-ethylester Natural products C=1C(Cl)=C2CC(C)OC(=O)C2=C(O)C=1C(=O)NC(C(=O)OCC)CC1=CC=CC=C1 BPZZWRPHVVDAPT-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 208000031481 Pathologic Constriction Diseases 0.000 description 2
- 241000228143 Penicillium Species 0.000 description 2
- 108010060059 Sarcosine Oxidase Proteins 0.000 description 2
- 102000008118 Sarcosine oxidase Human genes 0.000 description 2
- 108010046334 Urease Proteins 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000002115 aflatoxin B1 Substances 0.000 description 2
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 description 2
- 229930020125 aflatoxin-B1 Natural products 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 239000003674 animal food additive Substances 0.000 description 2
- 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 2
- 230000007375 bacterial translocation Effects 0.000 description 2
- 239000003855 balanced salt solution Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000005252 bulbus oculi Anatomy 0.000 description 2
- 101150004928 bun gene Proteins 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 235000013330 chicken meat Nutrition 0.000 description 2
- 208000019425 cirrhosis of liver Diseases 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000003111 delayed effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 210000000416 exudates and transudate Anatomy 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 210000003736 gastrointestinal content Anatomy 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 231100000767 hemotoxin Toxicity 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 239000002068 microbial inoculum Substances 0.000 description 2
- 230000002906 microbiologic effect Effects 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- BPZZWRPHVVDAPT-PXAZEXFGSA-N ochratoxin C Chemical compound C([C@@H](C(=O)OCC)NC(=O)C=1C(=C2C(=O)O[C@H](C)CC2=C(Cl)C=1)O)C1=CC=CC=C1 BPZZWRPHVVDAPT-PXAZEXFGSA-N 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 230000036262 stenosis Effects 0.000 description 2
- 208000037804 stenosis Diseases 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 230000004580 weight loss Effects 0.000 description 2
- UJOYFRCOTPUKAK-MRVPVSSYSA-N (R)-3-ammonio-3-phenylpropanoate Chemical compound OC(=O)C[C@@H](N)C1=CC=CC=C1 UJOYFRCOTPUKAK-MRVPVSSYSA-N 0.000 description 1
- GOLORTLGFDVFDW-UHFFFAOYSA-N 3-(1h-benzimidazol-2-yl)-7-(diethylamino)chromen-2-one Chemical compound C1=CC=C2NC(C3=CC4=CC=C(C=C4OC3=O)N(CC)CC)=NC2=C1 GOLORTLGFDVFDW-UHFFFAOYSA-N 0.000 description 1
- AXDJCCTWPBKUKL-UHFFFAOYSA-N 4-[(4-aminophenyl)-(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]aniline;hydron;chloride Chemical compound Cl.C1=CC(=N)C(C)=CC1=C(C=1C=CC(N)=CC=1)C1=CC=C(N)C=C1 AXDJCCTWPBKUKL-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 206010000234 Abortion spontaneous Diseases 0.000 description 1
- 229930195730 Aflatoxin Natural products 0.000 description 1
- 241000272525 Anas platyrhynchos Species 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 208000005156 Dehydration Diseases 0.000 description 1
- 208000020401 Depressive disease Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 241000186394 Eubacterium Species 0.000 description 1
- 241000186399 Holdemanella biformis Species 0.000 description 1
- 206010022678 Intestinal infections Diseases 0.000 description 1
- 206010061298 Mucosal haemorrhage Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 241000286209 Phasianidae Species 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 206010062237 Renal impairment Diseases 0.000 description 1
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000555745 Sciuridae Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000007059 acute toxicity Effects 0.000 description 1
- 231100000403 acute toxicity Toxicity 0.000 description 1
- 239000005409 aflatoxin Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 229940093761 bile salts Drugs 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 229940099352 cholate Drugs 0.000 description 1
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 1
- 230000007665 chronic toxicity Effects 0.000 description 1
- 231100000160 chronic toxicity Toxicity 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000003235 crystal violet staining Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000005485 electric heating Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000003008 fumonisin Substances 0.000 description 1
- 230000001434 glomerular Effects 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000008236 heating water Substances 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000008946 inflammatory intestinal reaction Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 210000005027 intestinal barrier Anatomy 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 230000005977 kidney dysfunction Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000005976 liver dysfunction Effects 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 201000003102 mental depression Diseases 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 208000015994 miscarriage Diseases 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000007826 nucleic acid assay Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- DAEYIVCTQUFNTM-ABAIWWIYSA-N ochratoxin B Chemical compound C([C@H](NC(=O)C1=CC=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 DAEYIVCTQUFNTM-ABAIWWIYSA-N 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000013021 overheating Methods 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 201000001474 proteinuria Diseases 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 231100000272 reduced body weight Toxicity 0.000 description 1
- 238000007430 reference method Methods 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000013424 sirius red staining Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 208000000995 spontaneous abortion Diseases 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000033 toxigenic Toxicity 0.000 description 1
- 230000001551 toxigenic effect Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 125000001813 vomitoxin group Chemical group 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- C—CHEMISTRY; METALLURGY
- 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
-
- 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
-
- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/41—Pediococcus
- A23V2400/413—Acidilactici
-
- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Nutrition Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Communicable Diseases (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Oncology (AREA)
- Epidemiology (AREA)
- Gastroenterology & Hepatology (AREA)
- General Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses Pediococcus acidilactici and application thereof in degradation of ochratoxin A. According to the invention, through screening a natural sample, a Pediococcus acidilactici (Pediococcus acidilactici) NJB421 with good degradation capability on ochratoxin A is obtained, and the preservation number is CGMCC No.23554. The degradation rate of the pediococcus acidilactici NJB421 disclosed by the invention on OTA reaches 48.53% after 2mg/L OTA toxin is treated for 48 hours at the temperature of 37 ℃ at the pH of 6.8-7.0; and has good growth characteristics and better tolerance to 46 ℃ and acidic environment with pH=3.0, 0.3 percent of bile salt and 1.4 percent of trypsin. The Pediococcus acidilactici NJB421 disclosed by the invention is used for feeding mice for 21 days, is safe and harmless to the mice, can relieve intestinal, liver and kidney injuries caused by OTA, and improves the oxidation resistance of the mice, so that the Pediococcus acidilactici NJB421 can be widely applied to feeds, medicines and related additives.
Description
Technical Field
The invention relates to the technical field of microorganisms and feed additives, in particular to pediococcus acidilactici and application thereof in degradation of ochratoxin A.
Background
Mycotoxins are structurally diverse secondary metabolites produced by fungi parasitic thereon, and are produced during the processing, transportation and storage of foods and feeds, and are widely found in foods, animal feeds and their raw materials. According to the statistics of the grain and agricultural organization of the united nations, about 25% of grain crops in the whole world are polluted by mycotoxins, and the health of human beings and animals is seriously threatened.
Ochratoxins are a group of toxic metabolites produced by certain species of the Aspergillus and Penicillium genera (Aspergillus) and Penicillium, a combination of L-beta-phenylalanine and isocoumarin, which are a further class of mycotoxins identified as being highly toxic following aflatoxin B1. Respectively referred to as Ochratoxin a (OTA), ochratoxin B (OTB) and Ochratoxin C (OTC) in their order of discovery, with the OTA having the longest biological half-life and the greatest toxicity. OTA mainly pollutes crops such as grains, peanuts, vegetables and the like. OTA-containing feeds are ingested by animals which cause accumulation of OTA in the body, causing acute and chronic toxicity to the animals. People are endangered by feeding crops and animal tissues contaminated with OTA. The existing research finds that the damage of OTA is mainly expressed as follows: (1) reducing feed intake and production performance; (2) reducing the immunity and the antioxidation capability of the organism; (3) causing kidney lesions and necrosis; (4) visual mucosal hemorrhage, (5) enteritis, dehydration and diuresis, with proteinuria and diabetes; (6) pregnant female animals bleed from the uterine mucosa and miscarriage occurs. Hamilton was the first report of Hamilton equals 1982 on large scale Haematotoxin poisoning of turkeys, after which there were reports of Haematotoxin poisoning of a large number of animals in the United states, canada, european countries and China, causing serious economic losses to the animal industry and bringing potential harm to human health.
At present, in actual production, some measures are taken to prevent and control mycotoxin pollution, and a certain effect is achieved. The method for detoxication of crop mycotoxins mainly comprises three methods, namely physical, chemical and biological detoxication methods. The physical detoxification method mainly removes mycotoxin in the feed by means of drying, adsorption and the like, but has higher investment cost of manpower and material resources, influences the appearance of the feed product and has common effect. The chemical detoxification method converts mycotoxins into other substances through chemical reaction or extracts mycotoxins by using chemical reagents, so as to achieve the purpose of detoxification, but at the same time, the nutritional characteristics of the feed are destroyed. The biological detoxification method, which is a method for biologically degrading mycotoxins to remove toxicity by using certain enzymes or microorganisms capable of producing toxin degrading enzymes, has been recently developed. Compared with physical and chemical detoxification methods, the biological detoxification method has the advantages of no addition of harmful chemical substances, mild implementation conditions, no loss of nutritive value, simple and convenient operation and the like, and is an important research direction of mycotoxin degradation at present.
Probiotics are "living organisms of microorganisms that can act on the health of a host when ingested in a certain amount", and mainly include lactic acid bacteria, bifidobacteria, bacillus, photosynthetic bacteria, yeasts, and the like. Research shows that probiotics can prevent intestinal infection by competitive exclusion of pathogens and strengthen intestinal barrier function, maintain intestinal flora balance and improve immune function. In recent years, researches report that probiotics have the effect of degrading mycotoxins, such as bacillus subtilis can degrade fumonisins, and lactobacillus can degrade aflatoxins or vomitoxins. At present, reports of degrading ochratoxin A by probiotics are also reported, for example, researchers separate a strain of bacillus licheniformis from Thailand bean paste, degradation rates of the degradation aflatoxin B1 and the ochratoxin A are 74% and 92.5%, and the degradation rates can inhibit the growth of toxigenic fungi, but the animal safety of the bacillus licheniformis is not evaluated in the study, and the safety of the strain is unknown. Researchers separate a strain of eubacterium bifidus (Eubacterium biforme) with degradation capability to OTA from intestinal contents of pigs, and the degradation rate is more than 75%, however, the related researches on the strain are less at present, and the strain lacks enough theoretical support in clinical application and cannot be applied to production. Among the strains with OTA degradation capability, only a few strains with animal safety evaluation are known, the safety of the strains is unknown, and the protection effect of the strains in animal bodies is not studied, so that the application of the strains in actual production is limited. Therefore, the method is extremely important for screening and researching safe and efficient OTA degrading bacteria.
Disclosure of Invention
In order to overcome at least one problem in the prior art, the invention screens and separates a pediococcus acidilactici strain capable of degrading ochratoxin A (OTA) by collecting a large number of natural samples, and the safety and the effectiveness of the pediococcus acidilactici strain are verified by animal experiments.
In theory, OTA is a derivative composed of isocoumarin, phenylalanine and different structural groups (mainly methyl and ethyl), strains capable of degrading coumarin and phenylalanine can degrade OTA with high probability, and various researches show that coumarin and isocoumarin are available as substitutes of OTA to screen OTA degrading bacteria. Compared with OTA, coumarin is safer, cheaper and less in environmental pollution, so that when a large amount of screening work is needed in the early stage, coumarin is selected to replace OTA for primary screening. According to the invention, a large number of natural samples are collected, diluted and added into a basic culture medium with coumarin as a unique carbon source, and strains capable of degrading coumarin as a carbon source for growth are screened out. Based on coumarin pre-experiments, morphological, physiological and biochemical and molecular identification are carried out, probiotics are selected from strains obtained through preliminary screening, and then the probiotics capable of degrading the OTA are screened by using the OTA, specifically, the MRS culture medium added with a certain concentration of the OTA is adopted for secondary screening, so that the probiotics capable of degrading the OTA, namely Pediococcus acidilactici, are screened from buffalo feces, and the probiotics are named as NJB421. The invention also carries out biological characteristic research and safety evaluation on the strain, and further feeds the strain to mice under OTA treatment, which can effectively reduce organ damage caused by OTA and improve the antioxidant capacity of the mice.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides Pediococcus acidilactici, the 16srDNA sequence of which is shown in SEQ ID No. 1.
Further, the pediococcus acidilactici is named as NJB421, is classified and named as Pediococcus acidilactici, has a preservation number of CGMCC No.23554, has a preservation date of 2021, 10 and 09 days, and has a preservation unit of China general microbiological culture Collection center (CGMCC), and a preservation unit address of China institute of microbiological culture Collection, national institute of sciences No. 3, national institute of sciences 1, north-west road, guangxi, beijing, city.
Further, the microbiological characteristics of the pediococcus acidilactici NJB421 are: gram positive bacteria, catalase test negative, spherical cell morphology, single colony size of about 1-3 mm on MRS culture medium, white color, opacity, smooth colony surface, slightly upward bulge and neat edge; pediococcus acidilactici NJB421 has a viable count of more than 10 after 2 hours of treatment at 57 DEG C 7 CFU/mL; pediococcus acidilactici NJB421 can grow in an acidic environment with the pH value of more than 2.0, and has strong bile salt resistance. Furthermore, the pediococcus acidilactici NJB421 has good growth characteristics, is better tolerant to 46 ℃ and acidic environment with pH=3.0, 0.3% bile salt and 1.4% trypsin, and is safe and harmless to mice after being fed to mice for 21 days.
Further, the pediococcus acidilactici NJB421 has the ability to degrade coumarin and OTA. In a specific embodiment, the degradation rate of Pediococcus acidilactici NJB421 to OTA after 2mg/L of OTA toxin is treated for 24 hours, 48 hours and 72 hours at the pH of 6.8-7.0 and the temperature of 37 ℃ is 21.58 percent, 48.53 percent and 48.52 percent respectively; furthermore, the pediococcus acidilactici NJB421 can relieve intestinal, liver and kidney damage caused by OTA and improve the oxidation resistance of mice.
In a second aspect the present invention provides a microbial agent comprising pediococcus acidilactici as described in any one of the first aspects of the invention; or is obtained by culturing the Pediococcus acidilactici.
Further, the viable bacteria concentration of the microbial agent is 2×10 8 ～2×10 10 CFU/mL. Preferably 1X 10 9 ～5×10 9 CFU/mL, more preferably 2X 10 9 CFU/mL. In practical use, the microbial agent may be diluted to a predetermined concentration for use.
In a third aspect, the invention provides the use of a pediococcus acidilactici according to any one of the first aspects of the invention or a microbial agent according to any one of the second aspects of the invention for degrading coumarin and/or ochratoxin a.
Further, the application includes: the pediococcus acidilactici or the microbial agent is prepared into a product for relieving symptoms caused by ochratoxin A.
Further, the product comprises feed, medicines and additives.
Further, the symptoms include: organ damage, decreased food consumption, and decreased antioxidant capacity, wherein the organ damage comprises: intestinal tract injury, liver injury, and kidney injury.
In a specific embodiment, the perfused Pediococcus acidilactici NJB421 has no abnormality in the body weight, organ index, serum alanine Aminotransferase (ALT), glutamic-oxaloacetic Aminotransferase (AST), alkaline phosphatase (ALP) activity, urea nitrogen (BUN), creatinine (CREA), total Protein (TP) content, total antioxidant capacity (T-AOC) of liver, kidney and ileum tissues and Malondialdehyde (MDA) content of the mice, and has no damage to the liver, kidney and ileum, which indicates that Pediococcus acidilactici NJB421 is safe and harmless.
In a specific embodiment, the infusion of Pediococcus acidilactici NJB421 improves changes in the criteria of reduced mouse body weight, reduced organ index, elevated serum Cr, BUN, AST and ALT levels, significant pathological changes in the kidney, liver and intestine, elevated levels of transcription of kidney and liver α -SMA, vimentin and TGF- β, reduced levels of transcription of ileum ZO-1, occludin and Claudin-1, elevated levels of transcription of kidney, liver and intestinal pro-inflammatory cytokines IL-6, IL-1 β and TNF- α and MDA levels, and reduced T-AOC caused by OTA.
In a fourth aspect the present invention provides a method of degrading coumarin and/or ochratoxin a comprising the steps of: activating and culturing the bacterial liquid of the Pediococcus acidilactici in the first aspect of the invention to obtain a microbial agent, and applying the microbial agent to an object to be treated in a preset dosage to degrade coumarin and/or ochratoxin A.
Further, the specific preparation steps of the microbial agent comprise: pediococcus acidilactici NJB421 stored in an ultralow temperature refrigerator at-80 ℃ is quickly thawed and then inoculated on a slant activation culture medium (namely MRS solid culture medium) and cultured for 48 hours at 37 ℃; after being activated for 3 times continuously, the mixture is inoculated into 1L of MRS liquid culture medium, and is cultured for 24 hours at 37 ℃ to obtain the pediococcus acidilactici bacterial agent; and (3) adjusting CFU of the microbial agent to obtain the microbial agent.
Further, the viable bacteria concentration of the microbial agent is 2×10 8 ～2×10 10 CFU/mL. Preferably 1X 10 9 ～1×10 10 CFU/mL, more preferably 2X 10 9 CFU/mL. In practical use, the microbial agent may be diluted to a predetermined concentration for use.
Further, the MRS liquid culture medium comprises the following components: 10.0g of peptone, 10.0g of beef extract powder, 5.0g of yeast extract powder, 20.0g of glucose, 2.0g of dipotassium hydrogen phosphate, 2.0g of diammonium hydrogen citrate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 1.0g of tween 80 and 1000mL of distilled water, wherein the pH value is 6.8+/-0.2, sterilizing for 30min at 115 ℃ by high-pressure steam, and packaging for later use.
Further, the degradation rate of ochratoxin A is 5-50%, preferably 20-50%. In a specific embodiment, pediococcus acidilactici NJB421 has 21.58%, 48.53% and 48.52% degradation of OTA after treatment with 2mg/L OTA toxin at 37℃for 24h, 48h and 72h, respectively.
Further, the object to be treated is an animal including, but not limited to: pig, chicken, duck, mouse, etc.
Further, in a specific embodiment, the object to be treated is a mouse, and the microbial agent is in a dosage of 10 8 ～5×10 8 CFU/dose, the administration frequency is 14-28 days of continuous drenching. Preferably, the microbial agent is used in a dose of 2X 10 8 CFU/dose, the frequency of administration was 21 days of continuous drenching. The above conditions are applicable to tests for safety and mitigation of OTA damage in embodiments of the present invention.
Compared with the prior art, the invention has the following beneficial effects by adopting the technical scheme:
the invention discovers that Pediococcus acidilactici NJB421 has the capability of degrading ochratoxin A for the first time. The pediococcus acidilactici NJB421 has good growth characteristics, the in vitro method identification is carried out on the beneficial effects of the pediococcus acidilactici NJB421, the pediococcus acidilactici NJB421 has good tolerance to 46 ℃, the acidic environment with the pH value of 3.0, 0.3% bile salt and 1.4% trypsin, the degradation rate of the pediococcus acidilactici NJB421 to OTA reaches 48.53% after 2mg/L OTA toxin is treated for 48 hours at the pH value of 6.8-7.0 and the temperature of 37 ℃, and the result shows that the pediococcus acidilactici NJB421 can resist acid and bile salt and the internal environment of the gastrointestinal tract, and has the potential of probiotics. The invention also carries out in vivo tests on the protection effect of the pediococcus acidilactici NJB421 on the OTA-induced mouse organism injury, and after the pediococcus acidilactici NJB421 is used for feeding mice for 21 days, the results show that the pediococcus acidilactici NJB421 is safe and harmless to the mice, has a certain protection effect on the OTA-induced mouse organism injury, can relieve the intestinal, liver and kidney injury caused by the OTA, improves the oxidation resistance of the mice, and can be widely applied to feeds, medicines and related additives.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention. In the drawings:
FIG. 1 is a colony morphology of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 in one embodiment of the invention.
FIG. 2 is a gram stain of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 in one embodiment of the invention.
FIG. 3 is a tree of pediococcus acidilactici (Pediococcus acidilactici) NJB421 according to one embodiment of the invention.
Fig. 4 is a schematic diagram of in vitro degradation rate of OTA by pediococcus acidilactici (Pediococcus acidilactici) NJB421 according to an embodiment of the present invention.
FIG. 5 is a graph showing the growth of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 in one embodiment of the invention.
FIG. 6 is a linear regression equation of the relationship between the OD600 value of the Pediococcus acidilactici (Pediococcus acidilactici) NJB421 strain solution and the number of viable bacteria in an embodiment of the invention.
FIG. 7 is a schematic diagram showing the results of a heat resistance test of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 in one embodiment of the present invention.
FIG. 8 is a graph showing the results of an acid resistance test of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 in one embodiment of the present invention.
FIG. 9 is a graph showing the results of a cholate test of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 in one embodiment of the present invention.
FIG. 10 is a graph showing the results of trypsin resistance test of Pediococcus acidilactici (Pediococcus acidilactici i) NJB421 in one embodiment of the invention.
FIG. 11 is a graph showing the effect of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 on body weight and feed intake in normal mice according to one embodiment of the present invention.
FIG. 12 is a graph showing the effect of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 on organ index and small intestine length in normal mice according to one embodiment of the present invention.
FIG. 13 is a graph showing the results of an NJB421 shift assay for Pediococcus acidilactici (Pediococcus acidilactici) in one embodiment of the present invention.
FIG. 14 is a graph showing the effect of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 on serum biochemical indicators of normal mice in an embodiment of the invention.
FIG. 15 is a graph showing the effect of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 on the antioxidant capacity of normal mice in an embodiment of the present invention.
FIG. 16 is a graph showing the effect of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 on pathological changes in normal mice in one embodiment of the invention.
FIG. 17 is a graph showing the effect of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 on the weight and feed intake of OTA treated mice in accordance with one embodiment of the present invention.
FIG. 18 is a graph showing the effect of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 on organ index and small intestine length of OTA treated mice in accordance with one embodiment of the present invention.
FIG. 19 is a graph showing the effect of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 on the renal pathology of OTA treated mice and serum creatinine and urea nitrogen levels in an embodiment of the present invention.
FIG. 20 is a graph showing the effect of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 on kidney fibrosis index and proinflammatory cytokine mRNA levels in OTA treated mice according to one embodiment of the invention.
FIG. 21 is a graph showing the effect of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 on liver pathology and serum AST and ALT levels in OTA treated mice according to one embodiment of the invention.
FIG. 22 is a graph showing the effect of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 on liver fibrosis index and proinflammatory cytokine mRNA levels in OTA treated mice according to one embodiment of the invention.
FIG. 23 is a graph showing the effect of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 on intestinal pathology, intestinal barrier function and proinflammatory cytokine mRNA levels in OTA treated mice according to one embodiment of the invention.
FIG. 24 is a graph showing the effect of Pediococcus acidilactici (Pediococcus acidilactici) NJB421 on the antioxidant capacity of OTA treated mice in accordance with one embodiment of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental procedures, which are not specified in the following examples, are generally determined according to national standards. The experimental materials not shown in the examples below are all commercially available. The equipment used in each step in the following examples is conventional equipment. If the corresponding national standard does not exist, the method is carried out according to the general international standard, the conventional condition or the condition recommended by the manufacturer. Unless otherwise indicated, all parts are parts by weight and all percentages are percentages by mass. Unless defined or otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any method and material similar or equivalent to those described may be used in the methods of the present invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The invention is further described below with reference to the drawings and specific examples, which are not intended to be limiting.
In the following examples, the following media and related reagents were used:
LB solid medium: 10.0g of tryptone, 5.0g of yeast extract powder, 10.0g of sodium chloride, 20.0g of agar, 1000mL of distilled water, pH of 7.0+/-0.1, sterilizing for 15min at 121 ℃ by high-pressure steam, and packaging for later use.
LB liquid medium: 10.0g of tryptone, 5.0g of yeast extract powder, 10.0g of sodium chloride, 1000mL of distilled water, pH of 7.0+/-0.1, sterilizing for 15min at 121 ℃ by high-pressure steam, and packaging for later use.
MRS solid medium: 10.0g of peptone, 10.0g of beef extract powder, 5.0g of yeast extract powder, 20.0g of glucose, 2.0g of dipotassium hydrogen phosphate, 2.0g of diammonium hydrogen citrate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 20.0g of agar, 1.0g of tween 80, 1000mL of distilled water, pH of 6.8+/-0.2, and sterilizing for 30min at 115 ℃ by high-pressure steam, and packaging for later use.
MRS liquid medium: 10.0g of peptone, 10.0g of beef extract powder, 5.0g of yeast extract powder, 20.0g of glucose, 2.0g of dipotassium hydrogen phosphate, 2.0g of diammonium hydrogen citrate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 1.0g of tween 80 and 1000mL of distilled water, wherein the pH value is 6.8+/-0.2, sterilizing for 30min at 115 ℃ by high-pressure steam, and packaging for later use.
Auxotroph solid medium with coumarin as unique carbon source: 2.0g of ammonium sulfate, 0.2g of magnesium sulfate, 0.01g of calcium chloride, 0.001g of ferrous sulfate, 1.5g of disodium hydrogen phosphate, 1.5g of monopotassium phosphate, 1.7g of coumarin, 20.0g of agar, 1000mL of distilled water, pH of 7.0+/-0.1, sterilizing by high-pressure steam at 121 ℃ for 15min, and packaging for later use.
Example 1 isolation and identification of Pediococcus acidilactici (Pediococcus acidilactici) NJB421
The pediococcus acidilactici with degradation OTA is obtained by the following method:
(1) Isolation of Strain NJB421
1) Isolation culture of strains
And respectively collecting fresh feces of cattle, sheep and dogs, intestinal contents of chickens, moldy soil, and samples such as yoghurt, lactobacillus beverage and the like for screening. The method is described in the "Berger's bacteria identification Manual", and is specifically performed as follows: according to the sample and sterile physiological saline as 1:10 (specifically, weighing 0.5g of solid sample or taking 0.5mL of liquid sample by a pipette, adding the liquid sample into 5mL of sterilized normal saline, fully vibrating and uniformly mixing, respectively taking 0.1mL of suspension and 0.9mL of sterilized normal saline, uniformly mixing in a sterile EP tube, fully mixing 0.1mL of suspension and 0.9mL of sterilized normal saline, repeating for a plurality of times), diluting the sample for a plurality of times by a ten-fold ratio, and diluting the sample for 10 times 5 、10 6 、10 7 Taking 0.1mL of the suspension, uniformly coating the suspension on the surface of LB solid medium, marking, and inversely culturing in a constant temperature incubator at 37 ℃ for 48 hours. Observing colony morphology on LB solid medium, respectively selecting bacterial single colonies with different morphologies and sizes from the surface of medium with moderate dilution, respectively inoculating into 5mL LB liquid medium, marking, and standing in a constant temperature incubator at 37 ℃ for 48 hours. Observing whether the bacterial culture solution is turbid, taking 0.01mL of the culture solution after fully and uniformly mixing the bacterial culture solution, dividing and marking the surface of the LB solid culture medium by an inoculating loop, marking the LB solid culture medium, and inversely culturing the LB solid culture medium in a constant-temperature incubator at 37 ℃ for 48 hours. The bacterial colony morphology on the surface of the culture medium is observed, a plurality of single colonies (for example, 2-3) with regular morphology are selected from each culture medium, and are respectively inoculated into 5mL of LB liquid culture medium, marked and subjected to static culture in a constant temperature incubator at 37 ℃ for 48 hours. The streaking and single colony inoculation steps are repeated at least three times until the single colony on the surface of the LB solid medium has the same shape and size and is the same bacteria, which indicates that the strain purification is completed.
Taking 0.1mL of the purified bacterial culture solution, uniformly coating the bacterial culture solution on the surface of an auxotroph solid culture medium which takes coumarin as a unique carbon source by using a sterilizing glass coater, marking, and inversely culturing in a constant-temperature incubator at 37 ℃ for 1-7 days. The bacterial colony growth condition on the surface of each culture medium is observed daily, and recorded, if no bacterial colony grows or the bacterial colony number is extremely small after 7 days, the bacterial strain cannot degrade coumarin or has extremely poor coumarin degrading capability, and the bacterial colony is discarded. Selecting a strain with more colony growth and faster growth speed on the surface of the culture medium, inoculating the strain into 5mL of LB liquid culture medium, marking, and performing stationary culture in a constant temperature incubator at 37 ℃ for 48 hours for subsequent experiments.
And then 100 microliters of the strain culture solution screened above is coated on an MRS solid culture medium, marking is carried out, the culture is carried out for 24-48 hours at 37 ℃, 15-20 single colonies with different appearances are randomly selected from each plate, and the strain of the purified degradable coumarin is obtained by streaking and purifying at least three times on the MRS plate. Colony morphology was observed and recorded.
2) Gram staining of strains
A clean glass slide is taken, 0.01mL of bacterial culture solution is dripped on the clean glass slide, and the bacterial culture solution is spread on the glass slide as much as possible, so that the smear is prevented from being too thick. The back of the glass slide is quickly passed through the outer flame of the alcohol lamp for a plurality of times, and the smear is fixed (the glass slide is prevented from overheating, and the glass slide is not scalded). Dripping crystal violet staining solution to completely cover the area where the bacterial solution is located, staining for 1min, washing with water, and naturally airing; dripping iodine solution to completely cover the area of the bacteria solution, mordant dyeing for 1min, washing with water, and naturally airing; dropwise adding alkaline fuchsin ethanol solution 50S, washing with water, and naturally airing; and (3) dripping safranin staining solution on the glass slide to completely cover the area where the bacterial solution is located, staining for 1min, washing with water, and naturally airing. When the cells were positive in purple, the cells were negative in red, as observed on a common optical microscope. And (3) after the coumarin degrading bacteria obtained by the primary screening are subjected to gram staining and a catalase test, selecting gram positive bacteria.
3) 16S DNA sequence homology analysis
And carrying out trace biochemical identification on the screened gram-positive bacteria, and primarily screening strains which are possibly probiotics. The total DNA of the strain was extracted with a bacterial genomic DNA extraction kit from Beijing Soxhaust technologies Co., ltd, and then the DNA concentration was measured and PCR amplification was performed. Then, 5. Mu.l of each PCR product was subjected to agarose gel electrophoresis to verify whether the PCR was successful. And sending the single clear and bright strain amplified product to a biological engineering (Shanghai) stock company for sequencing, carrying out similarity comparison on the sequencing 16SrDNA sequence splicing result in an NCBI GenBank database and related sequences, carrying out multiple comparison by using Blast program, determining the species of the strain, and constructing a strain phylogenetic tree by adopting Mega X software (MEGA 5.0 software) in an adjacent method.
The PCR system was 25. Mu.l (12.5. Mu.l Master MIX, 1.25. Mu.l upstream primer, 1.25. Mu.l downstream primer, appropriate amount of DNA template, double distilled water to 25. Mu.l), PCR amplification parameters: pre-denaturation, 94 ℃,4min,1 cycle; denaturation, 94 ℃,10s,34 cycles; annealing at 55 ℃,20s,34 cycles; extending at 72 ℃, for 30s,34 cycles; terminal extension, 72 ℃,5min,1 cycle. Upstream primer using bacterial 16srDNA amplification primer 27F:5-AGAGTTTGATCCTGGCTCAG-3 (SEQ ID No. 2) and a downstream primer 1492R:5-GGTTACCTTTGTTACGACTT-3 (SEQ ID No. 3).
(2) In vitro degradation effect of strain NJB421 on ochratoxin A
Selecting strains with sequencing comparison results of lactobacillus, respectively inoculating the strains to an MRS liquid culture medium, culturing for 48 hours in an anaerobic environment at 37 ℃ to a growth log phase, regulating the OD600 value of each bacterial culture solution to about 0.8 by using a spectrophotometer to ensure that the concentration of bacterial solutions is the same, respectively taking 0.5mL of each bacterial culture solution, adding the bacterial culture solutions into a liquid culture medium containing 2 mug/mL of OTA, uniformly mixing, repeating each group of three strains, standing and culturing for 72 hours at 37 ℃, measuring the OTA content in the culture solution by using an OTA enzyme-linked immunosorbent assay kit every 24 hours, and calculating the degradation rate of the strain on the OTA by taking the non-inoculated OTA-containing liquid culture medium as a reference.
(3) Results of the implementation
1) Morphological identification results
The morphology of single colonies of strain NJB421 in the logarithmic growth phase and stable in colony size on the auxotroph medium was described, including the size, color, transparency, colony surface state and colony edge state of the colonies. The single colony obtained is about 1-2 mm in size, white in color, opaque, smooth in surface and neat in edge (see figure 1). And then, after gram staining is carried out on the separated and screened strain NJB421, the bacterial morphology is observed by adopting an optical microscope. Gram staining was positive, the cell morphology was round, no more than 2 μm in diameter, and no chain arrangement was seen (see FIG. 2).
2) Molecular biological characterization results
And (3) carrying out similarity comparison on the sequencing result and related sequences in a GenBank database, and then constructing a phylogenetic tree by using MEGA X software, wherein the result shows that the strain NJB421 and the pediococcus acidilactici (Pediococcus acidilactici) belong to the same development branch (see figure 3).
3) Degradation ability of strain NJB421 on OTA
Pediococcus acidilactici NJB421 was inoculated into a culture medium containing OTA for culturing for 24 hours, 48 hours and 72 hours, and the ability of the Pediococcus acidilactici NJB421 to degrade OTA was measured, and the results are shown in FIG. 4. The results show that: the concentration of OTA is respectively reduced from 2.00g/mL to 1.57g/mL, 1.03g/mL and 1.03g/mL, and the degradation rates of Pediococcus acidilactici NJB421 on OTA are respectively 21.58%, 48.53% and 48.52%. The results showed that the degradation capacity of the NJB421 for OTA peaked at 48h with time and that the OTA was not re-released into the medium after 48 h.
The Pediococcus acidilactici (Pediococcus acidilactici) NJB421 is preserved with the preservation number of CGMCC No.23554, the preservation date of 2021, 10 month and 09 days, the preservation unit of China general microbiological culture Collection center (CGMCC) and the preservation unit address of China academy of China microbiological study, national institute No. 3, national institute of sciences of China, the Beijing area, chaoyang area.
Example 2 determination of growth Curve of Pediococcus acidilactici NJB421
Activating bacterial liquid of Pediococcus acidilactici NJB421, inoculating the bacterial liquid into MRS liquid culture medium according to an inoculum size of 5%, standing and culturing for 48 hours in an anaerobic environment at 37 ℃, absorbing 4mL of bacterial liquid which is uniformly mixed by vibration every 3 hours, placing the bacterial liquid into a glass light absorption dish, culturing for 24 hours, absorbing 4mL of bacterial liquid every 6 hours, placing the bacterial liquid into the glass light absorption dish, measuring an OD600 value by using a spectrophotometer, zeroing by using an equal volume of MRS liquid culture medium, and repeating the steps. The abscissa is the culture time (hours) of the NJB421, the ordinate is the average value corresponding to the OD600, and the growth curve of the NJB421 is drawn. The growth curve shows that the Pediococcus acidilactici NJB421 is cultured for 48 hours, and the strain can intuitively and clearly show the delayed phase, the logarithmic phase and the stationary phase, wherein the delayed phase is 0-3 hours, the logarithmic phase is 3-30 hours and the stationary phase is 30-48 hours (see figure 5).
Activating Pediococcus acidilactici NJB421, inoculating in an MRS liquid culture medium at a ratio of 5%, standing for culturing at 37 ℃ for 48 hours, selecting 5 time points in the culturing time, absorbing 4mL of the bacterial liquid which is uniformly mixed by vibration, placing in a glass light absorption dish, measuring the OD600 value by using a spectrophotometer, zeroing by using an equal volume of MRS liquid culture medium, repeating for three times, and taking an average value. Meanwhile, 1mL of bacterial liquid which is evenly mixed by vibration is sucked at each time point and placed in an EP tube, ten times of dilution is carried out by using sterile physiological saline, 100 mu L of dilution is evenly coated on an MRS flat plate, standing culture is carried out for 48 hours at 37 ℃, flat plates with the colony number in the range of 30-300 are selected, colony counting is carried out by using a flat plate direct counting method, the steps of repeating for three times, taking an average value, and the viable count (CFU/mL) is calculated. And drawing a linear regression equation of the relation between the OD600 value of the NJB421 bacterial liquid and the number of the viable bacteria by taking the OD600 as an abscissa and the number of the corresponding viable bacteria as an ordinate (see FIG. 6).
EXAMPLE 3 biological Property study of Pediococcus acidilactici NJB421
(1) Test of Heat resistance
In order to understand the tolerance of Pediococcus acidilactici NJB421 to different high temperatures, the processing conditions are convenient to optimize in the future, and the Pediococcus acidilactici NJB421 is subjected to a high temperature resistance test, and the steps are briefly described as follows:
the water temperature of the electric heating water bath kettle is regulated to 37, 40, 43 and 46 ℃, and the temperature is measured for 5 minutes by using a thermometer to ensure that the water temperature is kept constant. The bacterial liquid of Pediococcus acidilactici NJB421 is activated and inoculated into MRS liquid culture medium according to the inoculum size of 5 percent, and the Pediococcus acidilactici NJB421 is subjected to static culture for 48 hours under the anaerobic environment at 37 ℃ to rejuvenate the Pediococcus acidilactici. Taking 5mL of bacterial liquid in 4 sterilization EP pipes, respectively carrying out water bath treatment at 37, 40, 43 and 46 ℃ for 10, 20 and 30min, taking the bacterial liquid treated in the water bath at 37 ℃ as a control, measuring the OD600 value by using a spectrophotometer, carrying out standard curve calculation of the OD600 value and the viable count, converting the OD600 into the viable count, and repeating for three times.
Experimental results (see fig. 7) found that: after the reaction is carried out for 30 minutes at 40, 43 and 46 ℃, the number of viable bacteria in the Pediococcus acidilactici NJB421 bacterial liquid is not different from that of the Pediococcus acidilactici NJB421 bacterial liquid after the reaction is carried out for 30 minutes at 37 ℃, and the number can still reach 10 7 CFU/mL above, demonstrated that Pediococcus acidilactici N421 had better tolerance to 40, 43, 46 ℃.
(2) Acid resistance test
In order to verify the resistance of Pediococcus acidilactici NJB421 to acidic environments at different pH values, an acid resistance test was performed on Pediococcus acidilactici NJB421, the procedure of which is briefly described below:
activating the bacterial liquid of Pediococcus acidilactici NJB421, inoculating the bacterial liquid into an MRS liquid culture medium according to an inoculum size of 5%, standing and culturing for 48 hours in an anaerobic environment at 37 ℃, and shaking and mixing uniformly. 1mL of the bacterial liquid was inoculated into 20mL of MRS liquid medium with different pH values (pH 2.0, 3.0, 4.0, 5.0 and 6.0), and 1mL of the bacterial liquid was inoculated into MRS liquid medium (pH=6.8) as a control. And (3) standing and culturing for 3 hours in an anaerobic environment at 37 ℃, sucking 4mL of the uniformly mixed bacterial liquid every 1 hour, measuring an OD600 value by using a spectrophotometer, carrying out standard curve calculation of the OD600 value and the number of viable bacteria, converting the OD600 into the number of viable bacteria, and repeating for three times.
Experimental results (see fig. 8) found that: compared with the control group, after the Pediococcus acidilactici NJB421 is cultured for 1 hour, no obvious difference exists in the number of viable bacteria in the culture medium with each pH value; after 2 hours, the concentration of the bacterial liquid in the culture medium with PH=2.0 is extremely obviously reduced (P is less than 0.01); after 3 hours, the concentration of bacterial liquid in the culture medium at ph=2.0 was extremely reduced (P < 0.01), the concentration of bacterial liquid in the culture medium at ph=3.0 was significantly reduced (P < 0.05), and a significant increase in the concentration of bacterial liquid in the culture medium at ph=3.0 was found compared to that in the culture medium at 0 hours, indicating that pediococcus acidilactici NJB421 can withstand the acidic environment at ph=3.0.
(3) Test for bile salt resistance
To verify the resistance of Pediococcus acidilactici NJB421 to bile salts at different concentrations, the Pediococcus acidilactici NJB421 was subjected to a bile salt resistance test, the procedure of which is briefly described below:
the bacterial liquid of Pediococcus acidilactici NJB421 is activated and inoculated into MRS liquid culture medium according to the inoculum size of 5 percent for culturing for 48 hours, after shaking and mixing, 1mL of bacterial liquid is respectively inoculated into 20mL of MRS liquid culture medium with different bile salt concentrations (0.1 percent, 0.3 percent and 0.5 percent), and 1mL of bacterial liquid is simultaneously inoculated into 20mL of MRS liquid culture medium (pig bile salt is not added) to serve as a control. And (3) standing and culturing for 3 hours in an anaerobic environment at 37 ℃, sucking 4mL of culture solution every 1 hour, measuring an OD600 value by using a spectrophotometer, carrying out standard curve calculation of the OD600 value and the number of viable bacteria, converting the OD600 into the number of viable bacteria, and repeating for three times.
Experimental results (see fig. 9) found that: after 1, 2 and 3 hours of culture, the concentration of bacteria liquid in the MRS liquid culture medium with 0.1%, 0.2% and 0.3% of the concentration of the bile salt of Pediococcus acidilactici NJB421 is not obviously different from that of the MRS liquid culture medium without adding the bile salt, which indicates that the Pediococcus acidilactici NJB421 can tolerate the concentration of 0.3% of the bile salt.
(4) Trypsin resistance test
To verify the resistance of Pediococcus acidilactici NJB421 to trypsin at different concentrations, a trypsin resistance test was performed on Pediococcus acidilactici NJB421, the procedure of which is briefly described below:
Activating bacterial liquid of Pediococcus acidilactici NJB421, inoculating the bacterial liquid into an MRS liquid culture medium according to an inoculum size of 5% for culturing for 48 hours, shaking and uniformly mixing, taking 1mL of bacterial liquid, respectively inoculating the bacterial liquid into 20mL of MRS liquid culture mediums with different trypsin concentrations (1.0%, 1.2% and 1.4%), simultaneously inoculating 1mL of bacterial liquid into 20mL of MRS liquid culture medium (without trypsin added) for comparison, standing and culturing for 3 hours in an anaerobic environment at 37 ℃, sucking 4mL of the culture liquid every 1 hour, measuring an OD600 value by using a spectrophotometer, carrying out standard curve calculation of the OD600 value and the number of viable bacteria, converting the OD600 into the number of viable bacteria, and repeating for three times.
Experimental results (see fig. 10) found that: after 1, 2 and 3 hours of incubation, there was no significant difference in the concentration of the bacterial solutions in the MRS liquid medium with 1.0%, 1.2% and 1.4% trypsin concentration of pediococcus acidilactici NJB421 compared to the MRS liquid medium without trypsin addition, indicating that pediococcus acidilactici NJB421 can tolerate 1.4% trypsin concentration.
Example 4 evaluation of safety of Pediococcus acidilactici NJB421
In this example, normal mice were used as the subjects, and the safety of Pediococcus acidilactici was evaluated by the feeding method.
(1) Production of Pediococcus acidilactici formulations
The Pediococcus acidilactici NJB421 stored in an ultralow temperature refrigerator at the temperature of minus 80 ℃ is quickly thawed and then inoculated on an inclined plane activation culture medium, and is cultured for 32 hours at the temperature of 37 ℃; after being activated for 3 times continuously, the mixture is inoculated into 1L of MRS liquid culture medium, and is cultured for 24 hours at 37 ℃ to obtain the pediococcus acidilactici bacterial agent; post-adjusting CFU of the microbial inoculum to 2×10 9 /mL，Is used for gastric lavage mice.
(2) Implementation of animals and groupings
12 SPF grade C57BL/6 mice of 18-20g were selected and randomly divided into 2 groups of 6 mice each. Group A is control group filled with sterile physiological saline; group B is Pediococcus acidilactici NJB421, 2×10 of the preparation is taken orally 8 The CFU/bacterial liquid is infused once every day at nine am for 21 days. The laboratory mouse room controls the constant temperature and humidity, natural illumination, the mice eat and drink water freely, and the squirrel cage is cleaned every 7 days. In the experimental process, the state of the mice is observed and recorded every day, and the survival condition, the clinical abnormal symptoms and the like exist.
(3) Sample collection and measurement
On day 21 of the formal test, the mice were fasted for 24 hours, weighed and then the eyeballs were bled, left at room temperature for 30 minutes, centrifuged at 3000r/min at 4℃for 10 minutes, the serum was collected, sub-packaged in 1.5mL centrifuge tubes, and stored at-80℃for measuring the Total Protein (TP), aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), alkaline phosphatase (ALP), creatinine (Cr) and urea nitrogen (BUN) contents in the serum.
After blood collection, the mice are sacrificed, the liver, the kidney, the ileum, the heart and the spleen are dissected and separated, the obvious lesions are observed and whether the obvious lesions exist or not is observed, the organ indexes (the organ indexes are expressed by the weight of the organs and are multiplied by 100 percent) are weighed and calculated, then the liver, the kidney and the ileum tissues are longitudinally cut into small blocks with the size of 0.5cm multiplied by 0.5cm, and immediately the small blocks are placed into neutral 4 percent formaldehyde fixing solution for fixing and preserving, so that the histopathological section is manufactured; the remaining tissue was rapidly separated and washed with cold balanced salt solution, then frozen in liquid nitrogen, and stored at-70℃for detection of total antioxidant capacity (T-AOC) and Malondialdehyde (MDA) content.
(4) Measurement index and method
1) AST, ALT, ALP and TP Activity assay
AST, ALT, ALP and TP kits were purchased from the institute of biological engineering, constructed in Nanjing, and tested according to instructions.
2) Analysis of Cr and BUN content
Cr was analyzed by sarcosine oxidase method and BUN was analyzed by urease method. The kit is purchased from Nanjing's institute of biological engineering.
3) Analysis of T-AOC and MDA content
The T-AOC was analyzed by ABTS method and the MDA content by TBA method. The kit is purchased from Nanjing's institute of biological engineering.
4) Translocation assay of Pediococcus acidilactici NJB421
The kidneys, liver, ileum were removed aseptically to organize with saline 1:1 (m/v) is added with proper amount of sterilized normal saline, and after grinding by a tissue homogenizer, 100 mu L of homogenate of each tissue is respectively taken, and the homogenate is coated on the surface of an MRS agar plate containing 1% (w/v) coumarin by a sterilization coater, and is subjected to static culture for 48 hours under an anaerobic environment at 37 ℃ to observe whether colony growth exists.
5) Histopathological examination
Kidneys, livers and ileums immersed in the universal tissue fixative were sent to the Nanjing sciences, inc. for conventional paraffin embedding and staining, wherein kidneys were stained with HE and MASSON, livers were stained with HE and sirius red, and ileums were stained with HE.
(5) Results of the implementation
1) Influence of Pediococcus acidilactici NJB421 on the growth status of mice
The mice have no diarrhea, death and other conditions in the test process, and the mental condition is good. After 21 days of continuous gastric lavage, the mice in the test group normally increase in weight and have no obvious abnormality in feed intake compared with the mice in the control group (see FIG. 11); none of the mice in the test group had significant differences in kidney, liver, heart and spleen index and small intestine length (see figure 12). The bacterial translocation test shows that the NJB421 does not grow on the culture medium of the kidney and the liver, and grows on the culture medium of the small intestine in a large quantity, which indicates that the Pediococcus acidilactici NJB421 only colonizes the intestinal tract in the mouse body, and the bacterial translocation phenomenon does not occur (see figure 13).
2) Influence of Pediococcus acidilactici NJB421 on mouse serum biochemical indicators
After 21 days of continuous gastric lavage with the NJB421 bacterial liquid, the TP, ALT, AST, ALP, BUN and CREA contents of the test and control groups were within the standard range, and there was no significant difference between the test and control groups (see fig. 14).
3) Influence of Pediococcus acidilactici NJB421 on the antioxidant index of mice
After 21 days of continuous gastric lavage with NJB421, no significant differences in the T-AOC and MDA contents of kidney, liver, ileum tissues were observed in the mice of the test group compared to the control group (see FIG. 15).
4) Influence of Pediococcus acidilactici NJB421 on pathological changes in mouse organs
The kidney tissue sections of this experiment were stained with hematoxylin-eosin staining (HE staining) and MASSON, liver tissue sections were stained with hematoxylin-eosin staining (HE staining) and sirius red, and ileum tissue sections were stained with hematoxylin-eosin staining (HE staining). The observation results by the optical microscope are shown in the following FIG. 16: compared with a control group, the kidney, liver and ileum tissues of the Pediococcus acidilactici NJB421 experimental group have no obvious pathological changes, which indicates that the Pediococcus acidilactici NJB421 has no damage to the kidney, liver and ileum of mice.
Example 5 use of Pediococcus acidilactici NJB421
In the embodiment, pediococcus acidilactici NJB421 is applied to mice treated by OTA, and the actual application effect of the mice is verified.
(1) Production of pediococcus acidilactici bacterial agent
Rapidly thawing Pediococcus acidilactici N421 stored in a refrigerator with ultralow temperature of-80 ℃, inoculating on a slant activating culture medium, and culturing for 32h at 37 ℃; after being activated for 3 times continuously, the mixture is inoculated into 1L of MRS liquid culture medium, and is cultured for 24 hours at 37 ℃ to obtain the pediococcus acidilactici bacterial agent; post-adjusting CFU of the microbial inoculum to 2×10 9 /mL, used in lavage mice.
(2) Application of Pediococcus acidilactici NJB421 in OTA poisoning
1) Implementation of animals and groupings
18-20g of SPF grade C57BL/6 mice were selected, the initial body weight was weighed, and the mice were randomly divided into 4 treatment groups of 6 mice each according to the body weight. Group A is a control group; group B is OTA group; group C is NJB 421. Group A and group B were filled with sterile physiological saline on days 1-14, and group C was filled with NJB421 (2×10) 8 CFU/bacterial liquid amount alone), group B and group C were filled with physiological saline and probiotics after 14 consecutive daysBased on (1), OTA of 0.8mg/kg body weight was infused for 7 consecutive days, respectively, and the test lasted 21 days.
2) Daily ration and feeding management
The test mice are pre-fed with standardized mice for 3 days with long-term maintenance of pellet feed so as to adapt to feeding conditions and remove unhealthy mice with abnormal actions, wood chips are crushed on a cage bottom pad and replaced twice a week, daily water is purified water, free feeding and drinking water are carried out, and indoor temperature is 25+/-2 ℃ and illumination time is 12 hours.
3) Sample collection and measurement
On day 21 of the formal test, the mice were fasted for 24 hours, weighed and then the eyeballs were bled, allowed to stand at room temperature for 30 minutes, centrifuged at 3000r/min at 4℃for 10 minutes, serum was collected, sub-packaged in 1.5mL centrifuge tubes, and stored at-80℃for AST, ALT, cr and BUN content.
After blood collection, mice are sacrificed, livers, kidneys and ileums are dissected and separated, obvious lesions are observed and observed, and organ indexes (the organ indexes are expressed by organ weight/body weight multiplied by 100%) are calculated by weighing, then each tissue is longitudinally cut into small blocks with the size of about 0.5cm multiplied by 0.5cm, and immediately placed into neutral 4% formaldehyde fixing solution for fixing and preserving, so that the tissue pathological section detection is manufactured. The remaining tissues were rapidly separated and washed with cold balanced salt solution, then frozen in liquid nitrogen, and stored at-70℃for detection of T-AOC, MDA content, IL-6, IL-1β, TNF- α, ZO-1, occludin, claudin-1, α -SMA, vimentin and TGF- β mRNA levels.
4) Measurement index and method
(1) AST and ALT Activity assays
AST was assayed by the microplate method and ALT activity by the Leidese method. The kit is purchased from Nanjing's institute of biological engineering.
(2) Analysis of Cr and BUN content
Cr was analyzed by sarcosine oxidase method and BUN was analyzed by urease method. The kit is purchased from Nanjing's institute of biological engineering.
(3) Analysis of T-AOC and MDA content
The T-AOC was analyzed by ABTS method and the MDA content by TBA method. The kit is purchased from Nanjing's institute of biological engineering.
(4) Histopathological examination
Kidneys, livers and ileums immersed in the universal tissue fixative were sent to the Nanjing sciences, inc. for conventional paraffin embedding and staining, wherein kidneys were stained with HE and MASSON, livers were stained with HE and sirius red, and ileums were stained with HE.
(5) Determination of Gene mRNA levels by relative fluorescent quantitative PCR
The sequences of 9 target genes and 1 reference gene were designed with Primer (PREMIER Biosoft International, USA) and the Primer sequences are shown in Table 1 below (SEQ ID No.4 to SEQ ID No.23 in sequence), and the primers were synthesized by Invitrogen. Extraction of total RNA from tissues was performed using TRIZOL kit (TaKaRa, china) according to the protocol, and finally the quality of the extracted RNA was measured by 1.5% agarose gel electrophoresis and protein nucleic acid assay. The relevant gene mRNA levels were amplified and data analyzed relative to quantification using SYBR Green I dye, reference method on a ABI Prism Step One Plus detection system (Applied Biosystems, USA) fluorescent quantitative PCR instrument. The relative mRNA levels of the related genes were analyzed and compared by the delta Ct method using beta-action as the reference gene, and the results were expressed as 2-delta Ct.
TABLE 1 fluorescent quantitative PCR primer sequences
5) Results of the implementation
(1) Influence of Pediococcus acidilactici NJB421 on the growth status of mice
The results of the measurements of body weight and feeding of mice during the test are shown in FIG. 17. The results show that each group of mice has good mental status, normal weight gain, feeding, faecal and physical characteristics on days 1-14. From day 15, the gastric lavage OTA is started, and compared with a blank control group, the mental depression, the weight loss and the Mao Zaluan of the mice in the OTA group and the NJB421+OTA group appear, and the mental condition and the weight loss of the mice in the NJB421+OTA group are improved after 3 days of gastric lavage bacteria liquid. At the expiration of the test on day 21, mice in the OTA group and the NJB421+ota group had reduced body weight and feed intake compared to the control group; the NJB421 improved the weight and feed intake reduction in mice caused by OTA compared to the OTA group.
(2) Influence of Pediococcus acidilactici NJB421 on OTA-induced changes in organ index in mice
The mice were examined by dissection without obvious macroscopic lesions in each organ. The kidneys, livers and small intestines of the mice were weighed and the indices of the organs were calculated, and the results are shown in fig. 18: compared with the control group, the index of kidneys, livers and small intestines of the OTA group is obviously reduced, the small intestines are atrophic, and the length is obviously shortened. Compared with the OTA group, the kidney index of the NJB421+OTA group is remarkably increased (P is less than 0.05), the liver and small intestine indexes are increased, and the small intestine length is increased.
(3) Effect of Pediococcus acidilactici NJB421 on OTA-induced kidney injury in mice
The effect of Pediococcus acidilactici NJB421 on OTA-induced kidney injury in mice is shown in FIGS. 19-20. The results of kidney HE staining and MASSON staining showed that the mice in the OTA group had glomerular boundary unclear, flocculent exudates in the tubular, lumen stenosis, presence of large inflammatory cell infiltrates, large collagen fiber deposition, flocculent exudates in the tubular of mice in the NJB421+ota group, lumen stenosis, small collagen fiber deposition (fig. 19). Compared with the control group, the serum creatinine content of the mice in the OTA group is obviously increased, the urea nitrogen content is obviously increased (figure 19), and the relative expression amounts of the kidney alpha-SMA, vimentin, TGF-beta and IL-6, IL-1 beta and TNF-alpha are obviously increased (figure 20). Compared with the OTA group, the serum urea nitrogen content of mice in the NJB421+OTA group is obviously reduced, the creatinine content is reduced (figure 19), the mRNA expression quantity of alpha-SMA, vimentin, TGF-beta, IL-6 and TNF-alpha in the kidney is extremely obviously reduced, and the expression quantity of IL-1 beta is obviously reduced (figure 20). Test results show that continuous 7 days of gastric lavage of 0.8 mg/kg.dw OTA can cause kidney dysfunction, kidney fibrosis injury and serious inflammatory reaction of mice, and the injection of Pediococcus acidilactici NJB421 has a certain protection effect on the kidney injury of the mice caused by the OTA.
(4) Effect of Pediococcus acidilactici NJB421 on OTA-induced liver injury in mice
The effect of Pediococcus acidilactici NJB421 on OTA-induced liver injury in mice is shown in FIGS. 21-22. Liver HE staining and sirius red staining results showed that OTA group liver chordae disorder, liver lobule structure and liver cell boundary were not clear, inflammatory cell infiltration, and there was significant collagen fiber deposition, and that the NJB421+ota group liver chordae arrangement was more regular, part of liver lobule structure and liver cell boundary were not clear, and a small amount of inflammatory cell infiltration (fig. 21). Compared with the control group, the activity of serum AST and ALT of the mice in the OTA group is obviously increased (figure 21), and the relative expression amounts of liver alpha-SMA, vimentin, TGF-beta and IL-6, IL-1 beta and TNF-alpha are obviously increased (figure 22). Compared with the OTA group, the serum ALT activity of mice in the NJB421+OTA group is obviously reduced, the AST activity is reduced (figure 21), the mRNA expression quantity of alpha-SMA, vimentin, TGF-beta and TNF-alpha in the liver is obviously reduced, and the expression quantity of IL-6 and IL-1 beta is obviously reduced (figure 22). Test results show that continuous 7-day gastric lavage of 0.8 mg/kg.dw OTA can cause liver dysfunction, liver fibrosis injury and serious inflammatory reaction of mice, and the injection of Pediococcus acidilactici NJB421 has a certain protection effect on the liver injury of mice caused by OTA.
(5) Influence of Pediococcus acidilactici NJB421 on intestinal injury in mice
The effect of Pediococcus acidilactici NJB421 on OTA-induced intestinal injury in mice is shown in FIG. 23. Ileum HE staining results showed severe cleavage and dissolution of ileal villi in the OTA group mice, and damage of intestinal wall, with little cleavage of ileal villi in the NJB421+ OTA group mice (fig. 23). Compared with the control group, the relative expression level of ZO-1 and Occludin, claudin-1 in ileum tissues of the OTA group is extremely remarkably reduced, and the relative expression level of IL-6, IL-1β and TNF- α is extremely remarkably increased (FIG. 23). Compared with the OTA group, the relative expression quantity of Occludin, claudin-1 of the ileum tissue of the mice in the NJB421+OTA group is obviously increased, and the mRNA expression quantity of IL-6, IL-1 beta and TNF-alpha is extremely obviously reduced. Test results show that continuous 7 days of gastric lavage of 0.8 mg/kg.dw OTA can cause damage of intestinal mucosa, dysfunction of intestinal barrier and inflammatory reaction of mice, and the injection of Pediococcus acidilactici NJB421 has a certain protection effect on the damage of the intestinal tract of the mice caused by OTA.
(6) Influence of Pediococcus acidilactici NJB421 on antioxidant capacity of mouse organism
The effect of Pediococcus acidilactici NJB421 on the antioxidant capacity of the mice is shown in FIG. 24. Compared with a control group, the T-AOC of the kidney and the ileum of the OTA group is obviously reduced, and the T-AOC of the liver is extremely obviously reduced; the MDA content of kidneys, livers and ileum is extremely obviously increased. Compared with the OTA group, the ileum NJB421+OTA group T-AOC is extremely obviously increased, the renal MDA content is obviously reduced, and the liver and ileum MDA content is extremely obviously reduced. The results show that the continuous 7-day gastric lavage of 0.8 mg/kg.dw OTA can reduce the oxidation resistance of the kidneys, livers and ileums of mice, and the reduction of the oxidation resistance caused by the OTA can be relieved by the perfusion of Pediococcus acidilactici NJB421, so that the oxidation resistance of the mice is enhanced to a certain extent.
From the above examples, the Pediococcus acidilactici NJB421 of the present invention has good OTA toxin degradation capability, good growth characteristics, and good tolerance to 46 ℃, acidic environment with pH=3.0, 0.3% bile salt, and 1.4% trypsin; the compound feed additive is safe and harmless to mice, can relieve intestinal, liver and kidney damage caused by OTA, improves the oxidation resistance of the mice, and can be widely applied to feeds, medicines and related additives.
The foregoing description is only illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the scope of the invention, and it will be appreciated by those skilled in the art that equivalent substitutions and obvious variations may be made using the description and illustrations of the invention, and are intended to be included within the scope of the invention.
Sequence listing
<110> Nanjing agricultural university
<120> Pediococcus acidilactici and application thereof in degradation of ochratoxin A
<160> 23
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1482
<212> DNA
<213> 16srDNA sequence of Strain NJB421 (Artificial Sequence)
<400> 1
ccccggtgcc gggtgctata catgcagtcg aacgaacttc cgttaattga ttatgacgtg 60
cttgcactga atgagatttt aacacgaagt gagtggcgga cgggtgagta acacgtgggt 120
aacctgccca gaagcagggg ataacacctg gaaacagatg ctaataccgt ataacagaga 180
aaaccgcctg gttttctttt aaaagatggc tctgctatca cttctggatg gacccgcggc 240
gcattagctg gttggtgagg taacggctca ccaaggcgat gatgcgtagc cgacctgaga 300
gggtaatcgg ccacattggg actgagacac ggcccagact cctacgggag gcagcagtag 360
ggaatcttcc acaatggacg caagtctgat ggagcaacgc cgcgtgagtg aagaagggtt 420
tcggctcgta aagctctgtt gttaaagaag aacgtgggtg agagtaactg ttcacccagt 480
gacggtattt aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 540
gtggcaagcg ttatccggat ttattgggcg taaagcgagc gcaggcggtc ttttaagtct 600
aatgtgaaag ccttcggctc aaccgaagaa gtgcattgga aactgggaga cttgagtgca 660
gaagaggaca gtggaactcc atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc 720
agtggcgaag gcggctgtct ggtctgtaac tgacgctgag gctcgaaagc atgggtagcg 780
aacaggatta gataccctgg tagtccatgc cgtaaacgat gattactaag tgttggaggg 840
tttccgccct tcagtgctgc agctaacgca ttaagtaatc cgcctgggga gtacgaccgc 900
aaggttgaaa ctcaaaagaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960
ttcgaagcta cgcgaagaac cttaccaggt cttgacatct tctgccaacc taagagatta 1020
ggcgttccct tcggggacag aatgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg 1080
agatgttggg ttaagtcccg caacgagcgc aacccttatt actagttgcc agcattcagt 1140
tgggcactct agtgagactg ccggtgacaa accggaggaa ggtggggacg acgtcaaatc 1200
atcatgcccc ttatgacctg ggctacacac gtgctacaat ggatggtaca acgagttgcg 1260
aaaccgcgag gtttagctaa tctcttaaaa ccattctcag ttcggactgt aggctgcaac 1320
tcgcctacac gaagtcggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt 1380
tcccgggcct tgtacacacc gcccgtcaca ccatgagagt ttgtaacacc caaagccggt 1440
ggggtaacct tttaggagct agccgtctaa ggtgacagat ta 1482
<210> 2
<211> 20
<212> DNA
<213> upstream primer 27F (Artificial Sequence)
<400> 2
<210> 3
<211> 20
<212> DNA
<213> downstream primer 1492R (Artificial Sequence)
<400> 3
<210> 4
<211> 20
<212> DNA
<213> GAPDH upstream primer (Artificial Sequence)
<400> 4
<210> 5
<211> 22
<212> DNA
<213> GAPDH downstream primer (Artificial Sequence)
<400> 5
tataggtggt ttcgtggatg cc 22
<210> 6
<211> 23
<212> DNA
<213> IL-6 upstream primer (Artificial Sequence)
<400> 6
gaggatacca ctcccaacag acc 23
<210> 7
<211> 24
<212> DNA
<213> IL-6 downstream primer (Artificial Sequence)
<400> 7
aagtgcatca tcgttgttca taca 24
<210> 8
<211> 23
<212> DNA
<213> IL-1. Beta. Upstream primer (Artificial Sequence)
<400> 8
tcgcagcagc acatcaacaa gag 23
<210> 9
<211> 24
<212> DNA
<213> IL-1. Beta. Downstream primer (Artificial Sequence)
<400> 9
tgctcatgtc ctcatcctgg aagg 24
<210> 10
<211> 25
<212> DNA
<213> TNF-alpha upstream primer (Artificial Sequence)
<400> 10
catcttctca aaattcgagt gacaa 25
<210> 11
<211> 23
<212> DNA
<213> TNF-alpha downstream primer (Artificial Sequence)
<400> 11
tgggagtaga caaggtacaa ccc 23
<210> 12
<211> 21
<212> DNA
<213> ZO-1 upstream primer (Artificial Sequence)
<400> 12
acctctgcag caataaagca g 21
<210> 13
<211> 20
<212> DNA
<213> ZO-1 downstream primer (Artificial Sequence)
<400> 13
<210> 14
<211> 23
<212> DNA
<213> Occludin upstream primer (Artificial Sequence)
<400> 14
ttgaaagtcc acctccttac aga 23
<210> 15
<211> 22
<212> DNA
<213> Occludin downstream primer (Artificial Sequence)
<400> 15
ccggataaaa agagtacgct gg 22
<210> 16
<211> 20
<212> DNA
<213> Claudin-1 upstream primer (Artificial Sequence)
<400> 16
<210> 17
<211> 21
<212> DNA
<213> Claudin-1 downstream primer (Artificial Sequence)
<400> 17
gacaggagca ggaaagtagg a 21
<210> 18
<211> 22
<212> DNA
<213> alpha-SMA-1 upstream primer (Artificial Sequence)
<400> 18
gctgacagag gcaccactga ac 22
<210> 19
<211> 24
<212> DNA
<213> alpha-SMA-1 downstream primer (Artificial Sequence)
<400> 19
agtcacacca tctccagagt ccag 24
<210> 20
<211> 23
<212> DNA
<213> Vimentin upstream primer (Artificial Sequence)
<400> 20
actagccgca gcctctattc ctc 23
<210> 21
<211> 24
<212> DNA
<213> Vimentin downstream primer (Artificial Sequence)
<400> 21
gaagtccacc gagtcttgaa gcag 24
<210> 22
<211> 24
<212> DNA
<213> TGF-beta upstream primer (Artificial Sequence)
<400> 22
gcaacaattc ctggcgttac cttg 24
<210> 23
<211> 21
<212> DNA
<213> TGF-beta downstream primer (Artificial Sequence)
<400> 23
cagccactgc cgtacaactc c 21
Claims (8)
1. Pediococcus acidilacticiPediococcus acidilactici) The method is characterized in that the pediococcus acidilactici is named as NJB421, and the preservation number of the pediococcus acidilactici is CGMCC No. 23554.
2. A microbial agent comprising pediococcus acidilactici according to claim 1; or from Pediococcus acidilactici according to claim 1.
3. A microbial agent according to claim 2, wherein the micro-organisms areThe living bacteria concentration of the biological bacterial agent is 2 multiplied by 10 8 ~ 2×10 10 CFU/mL。
4. Use of pediococcus acidilactici according to claim 1, or of the microbial agent according to claim 2 or 3, for the degradation of coumarin and/or ochratoxin a, characterized in that the use is a non-disease diagnostic or therapeutic method.
5. The use according to claim 4, characterized in that the use is: the pediococcus acidilactici or the microbial agent is prepared into a product for relieving symptoms caused by ochratoxin A.
6. The use according to claim 5, wherein the product comprises feed and additives; the symptoms include: organ damage, decreased food consumption, and decreased antioxidant capacity, wherein the organ damage comprises: intestinal tract injury, liver injury, and kidney injury.
7. A method for degrading coumarin and/or ochratoxin a, comprising the steps of: activating and culturing the bacterial liquid of Pediococcus acidilactici according to claim 1 to obtain a microbial agent, and applying the microbial agent to a subject to be treated in a predetermined dosage to degrade coumarin and/or ochratoxin A, wherein the method is a non-disease diagnosis or treatment method.
8. The method of claim 7, wherein the degradation rate of ochratoxin a is 20-50%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210380740.7A CN114958649B (en) | 2022-04-08 | 2022-04-08 | Pediococcus acidilactici and application thereof in degradation of ochratoxin A |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210380740.7A CN114958649B (en) | 2022-04-08 | 2022-04-08 | Pediococcus acidilactici and application thereof in degradation of ochratoxin A |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114958649A CN114958649A (en) | 2022-08-30 |
| CN114958649B true CN114958649B (en) | 2023-05-09 |
Family
ID=82978163
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210380740.7A Active CN114958649B (en) | 2022-04-08 | 2022-04-08 | Pediococcus acidilactici and application thereof in degradation of ochratoxin A |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114958649B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911123A (en) * | 2015-05-19 | 2015-09-16 | 孙军 | Pediococcus acidilactici P3-4, screening and identifying method thereof and application of pediococcus acidilactici P3-4 in degradation of erythromycin and roxithromycin |
| CN108251322A (en) * | 2016-12-29 | 2018-07-06 | 中粮营养健康研究院有限公司 | Microbial inoculum, feed or additive and the method for removing mycotoxin |
-
2022
- 2022-04-08 CN CN202210380740.7A patent/CN114958649B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911123A (en) * | 2015-05-19 | 2015-09-16 | 孙军 | Pediococcus acidilactici P3-4, screening and identifying method thereof and application of pediococcus acidilactici P3-4 in degradation of erythromycin and roxithromycin |
| CN108251322A (en) * | 2016-12-29 | 2018-07-06 | 中粮营养健康研究院有限公司 | Microbial inoculum, feed or additive and the method for removing mycotoxin |
Non-Patent Citations (3)
| Title |
|---|
| Elimination of ochratoxin A by lactic acid bacteria strains isolated from chickens and their probiotic characteristics;Marta Kupryś-Caruk 等;《Biotechnology and Food Science》;第113-124页 * |
| Isolation, identifiation and safety evaluation of OTA-detoxifiation strain Pediococcus acidilactici NJB421 and its effects on OTA-induced toxicity in mice;Jiangyu Tang 等;《Food and Chemical Toxicology》;第1-11页 * |
| 具有降解真菌毒素乳酸菌的筛选、鉴定及其降解机理研究;张乐;《中国优秀硕士学位论文全文数据库农业科技辑》;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114958649A (en) | 2022-08-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102226157B (en) | Lactobacillus fermentum and application thereof | |
| WO2023087944A1 (en) | Pediococcus acidilactici for improving production performance and immune level of broilers, and screening method therefor and use thereof | |
| CN114085789B (en) | Pediococcus pentosaceus MA.WTPQJ01 and application thereof | |
| CN114806978B (en) | Lactobacillus johnsonii SXDT-23 and application thereof | |
| CN111304119A (en) | Feeding bacillus subtilis for degrading fumonisins and application thereof | |
| CN115992059B (en) | Lactobacillus johnsonii for producing feruloyl esterase and application thereof in relieving ulcerative colitis | |
| CN116555112A (en) | Probiotics for corrosion prevention and fresh keeping and composition thereof | |
| CN110079482B (en) | Bacillus amyloliquefaciens for feed and application thereof | |
| CN113797232B (en) | Composition with insulin resistance relieving function and application thereof | |
| CN117448213B (en) | Lactobacillus plantarum for inhibiting clostridium perfringens and its progeny and application | |
| CN112239739B (en) | Lactobacillus plantarum capable of relieving ETEC (enterotoxigenic enterobacteria) induced diarrhea and application thereof | |
| CN112592870A (en) | Bacillus coagulans G4, fermentation liquor thereof, preparation method and application | |
| CN114958649B (en) | Pediococcus acidilactici and application thereof in degradation of ochratoxin A | |
| CN115337327B (en) | Preparation method and application of probiotic preparation with lipid-lowering, anti-inflammatory and antioxidant functions | |
| CN107260796B (en) | Application of thyme compound plant essential oil in preventing and treating necrotic enteritis of chicken | |
| CN114480206B (en) | High-temperature-resistant enterococcus faecalis and preparation method and application thereof | |
| CN110452843A (en) | A kind of feeding bacillus subtilis and its application | |
| CN114015598B (en) | Pediococcus acidilactici separated from Tibetan mushroom and application of pediococcus acidilactici in prevention and treatment of rotavirus infection | |
| CN114507628A (en) | Staphylococcus equinus FS310 and application thereof | |
| CN109536404B (en) | Bacillus pumilus for feed and application thereof | |
| CN109161501B (en) | Feeding bacillus licheniformis and application thereof | |
| US20110044969A1 (en) | Antimicrobial Agents for the Treatment of Campylobacter Species in the Crop of a Bird | |
| Zhang et al. | Characterization of Lactobacillus rhamnosus GG and its effects on improving the fermentation quality of silages as a novel inoculant | |
| CN107375413B (en) | Application of nutmeg essential oil and basil essential oil in preventing and treating necrotic enteritis of chicken | |
| CN116162557B (en) | A strain of Saccharomyces cerevisiae, chinese medicinal microecological preparation for preventing and treating ruminant diarrhea, and its preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |