CN113528381B - An antagonistic escherichia coli O157: h7 pediococcus pentosaceus and application thereof - Google Patents
An antagonistic escherichia coli O157: h7 pediococcus pentosaceus and application thereof Download PDFInfo
- Publication number
- CN113528381B CN113528381B CN202110735107.0A CN202110735107A CN113528381B CN 113528381 B CN113528381 B CN 113528381B CN 202110735107 A CN202110735107 A CN 202110735107A CN 113528381 B CN113528381 B CN 113528381B
- Authority
- CN
- China
- Prior art keywords
- pediococcus pentosaceus
- escherichia coli
- pediococcus
- pentosaceus
- antagonistic
- 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
- 241000191996 Pediococcus pentosaceus Species 0.000 title claims abstract description 101
- 241001646719 Escherichia coli O157:H7 Species 0.000 title claims abstract description 17
- 230000003042 antagnostic effect Effects 0.000 title claims abstract description 10
- -1 pplication Species 0.000 title description 4
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- 239000003814 drug Substances 0.000 claims abstract description 10
- 229940079593 drug Drugs 0.000 claims abstract description 7
- 235000013305 food Nutrition 0.000 claims abstract description 7
- 241001465754 Metazoa Species 0.000 claims abstract description 6
- 235000013373 food additive Nutrition 0.000 claims abstract description 6
- 239000002778 food additive Substances 0.000 claims abstract description 6
- 230000000813 microbial effect Effects 0.000 claims abstract description 5
- 241000588724 Escherichia coli Species 0.000 claims description 23
- 208000015181 infectious disease Diseases 0.000 claims description 14
- 239000003242 anti bacterial agent Substances 0.000 claims description 12
- 210000000813 small intestine Anatomy 0.000 claims description 10
- 230000000694 effects Effects 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 230000002757 inflammatory effect Effects 0.000 claims description 4
- 241000192001 Pediococcus Species 0.000 claims description 3
- 230000002441 reversible effect Effects 0.000 claims description 3
- 230000000845 anti-microbial effect Effects 0.000 claims description 2
- 239000004599 antimicrobial Substances 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 18
- 206010061218 Inflammation Diseases 0.000 abstract description 9
- 230000004054 inflammatory process Effects 0.000 abstract description 9
- 230000006378 damage Effects 0.000 abstract description 8
- 241001333951 Escherichia coli O157 Species 0.000 abstract description 6
- 239000012530 fluid Substances 0.000 abstract description 6
- 244000005700 microbiome Species 0.000 abstract description 6
- 239000006228 supernatant Substances 0.000 abstract description 6
- 238000000855 fermentation Methods 0.000 abstract description 5
- 230000004151 fermentation Effects 0.000 abstract description 5
- 230000002496 gastric effect Effects 0.000 abstract description 4
- 230000001575 pathological effect Effects 0.000 abstract description 4
- 210000004082 barrier epithelial cell Anatomy 0.000 abstract description 3
- 230000004890 epithelial barrier function Effects 0.000 abstract description 3
- 238000001727 in vivo Methods 0.000 abstract description 2
- 208000018522 Gastrointestinal disease Diseases 0.000 abstract 1
- 238000000338 in vitro Methods 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 18
- 230000004083 survival effect Effects 0.000 description 17
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 16
- 230000001580 bacterial effect Effects 0.000 description 10
- 238000012258 culturing Methods 0.000 description 10
- 230000000968 intestinal effect Effects 0.000 description 10
- 238000003752 polymerase chain reaction Methods 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 229940088710 antibiotic agent Drugs 0.000 description 8
- 238000011156 evaluation Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 239000004310 lactic acid Substances 0.000 description 8
- 235000014655 lactic acid Nutrition 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 229920001817 Agar Polymers 0.000 description 7
- 241000186660 Lactobacillus Species 0.000 description 7
- 239000008272 agar Substances 0.000 description 7
- 229940039696 lactobacillus Drugs 0.000 description 7
- 229960001180 norfloxacin Drugs 0.000 description 7
- OGJPXUAPXNRGGI-UHFFFAOYSA-N norfloxacin Chemical compound C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCNCC1 OGJPXUAPXNRGGI-UHFFFAOYSA-N 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 239000011248 coating agent Substances 0.000 description 6
- 238000000576 coating method Methods 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 238000012408 PCR amplification Methods 0.000 description 5
- 210000004051 gastric juice Anatomy 0.000 description 5
- 210000001630 jejunum Anatomy 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 4
- 206010018910 Haemolysis Diseases 0.000 description 4
- 244000052616 bacterial pathogen Species 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 238000003304 gavage Methods 0.000 description 4
- 230000002949 hemolytic effect Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 238000009630 liquid culture Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000006041 probiotic Substances 0.000 description 4
- 230000000529 probiotic effect Effects 0.000 description 4
- 235000018291 probiotics Nutrition 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 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 3
- 238000007400 DNA extraction Methods 0.000 description 3
- 206010059866 Drug resistance Diseases 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101001133081 Homo sapiens Mucin-2 Proteins 0.000 description 3
- 102000003777 Interleukin-1 beta Human genes 0.000 description 3
- 108090000193 Interleukin-1 beta Proteins 0.000 description 3
- 102000004889 Interleukin-6 Human genes 0.000 description 3
- 108090001005 Interleukin-6 Proteins 0.000 description 3
- 102100034263 Mucin-2 Human genes 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 102000003940 Occludin Human genes 0.000 description 3
- 108090000304 Occludin Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 3
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000002354 daily effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000009792 diffusion process Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 210000003405 ileum Anatomy 0.000 description 3
- 210000005027 intestinal barrier Anatomy 0.000 description 3
- 210000005026 intestinal epithelial barrier Anatomy 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 238000004393 prognosis Methods 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000010200 validation analysis Methods 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 108020000946 Bacterial DNA Proteins 0.000 description 2
- 240000002791 Brassica napus Species 0.000 description 2
- 235000011293 Brassica napus Nutrition 0.000 description 2
- 206010016952 Food poisoning Diseases 0.000 description 2
- 208000019331 Foodborne disease Diseases 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 102000003814 Interleukin-10 Human genes 0.000 description 2
- 108090000174 Interleukin-10 Proteins 0.000 description 2
- 108010079723 Shiga Toxin Proteins 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- WKDDRNSBRWANNC-UHFFFAOYSA-N Thienamycin Natural products C1C(SCCN)=C(C(O)=O)N2C(=O)C(C(O)C)C21 WKDDRNSBRWANNC-UHFFFAOYSA-N 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 238000009412 basement excavation Methods 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 2
- 229960002227 clindamycin Drugs 0.000 description 2
- KDLRVYVGXIQJDK-AWPVFWJPSA-N clindamycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 KDLRVYVGXIQJDK-AWPVFWJPSA-N 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000003628 erosive effect Effects 0.000 description 2
- 229960003276 erythromycin Drugs 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 229960002182 imipenem Drugs 0.000 description 2
- ZSKVGTPCRGIANV-ZXFLCMHBSA-N imipenem Chemical compound C1C(SCC\N=C\N)=C(C(O)=O)N2C(=O)[C@H]([C@H](O)C)[C@H]21 ZSKVGTPCRGIANV-ZXFLCMHBSA-N 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 230000007358 intestinal barrier function Effects 0.000 description 2
- 210000004347 intestinal mucosa Anatomy 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 2
- 229960001225 rifampicin Drugs 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 229960002180 tetracycline Drugs 0.000 description 2
- 229930101283 tetracycline Natural products 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 150000003522 tetracyclines Chemical class 0.000 description 2
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 2
- 239000001393 triammonium citrate Substances 0.000 description 2
- 235000011046 triammonium citrate Nutrition 0.000 description 2
- 230000001018 virulence Effects 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 101000939689 Araneus ventricosus U2-aranetoxin-Av1a Proteins 0.000 description 1
- 241000301512 Bacillus cereus ATCC 14579 Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 244000178924 Brassica napobrassica Species 0.000 description 1
- 235000011297 Brassica napobrassica Nutrition 0.000 description 1
- 101000633673 Buthacus arenicola Beta-insect depressant toxin BaIT2 Proteins 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 101000654318 Centruroides noxius Beta-mammal toxin Cn2 Proteins 0.000 description 1
- 101001028695 Chironex fleckeri Toxin CfTX-2 Proteins 0.000 description 1
- 241001135265 Cronobacter sakazakii Species 0.000 description 1
- 206010012741 Diarrhoea haemorrhagic Diseases 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 208000032759 Hemolytic-Uremic Syndrome Diseases 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 241001468155 Lactobacillaceae Species 0.000 description 1
- 241000708064 Listeria monocytogenes ATCC 19117 Species 0.000 description 1
- 241000211181 Manta Species 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241001103617 Pseudomonas aeruginosa ATCC 15442 Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 108010091769 Shiga Toxin 1 Proteins 0.000 description 1
- 108010090763 Shiga Toxin 2 Proteins 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 201000007023 Thrombotic Thrombocytopenic Purpura Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 241000607447 Yersinia enterocolitica Species 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 238000009635 antibiotic susceptibility testing Methods 0.000 description 1
- 230000037429 base substitution Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000011331 genomic analysis Methods 0.000 description 1
- 208000027909 hemorrhagic diarrhea Diseases 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000004673 intestinal mucosal barrier function Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- NFGXHKASABOEEW-LDRANXPESA-N methoprene Chemical compound COC(C)(C)CCCC(C)C\C=C\C(\C)=C\C(=O)OC(C)C NFGXHKASABOEEW-LDRANXPESA-N 0.000 description 1
- 238000012543 microbiological analysis Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 230000004682 mucosal barrier function Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 229960001699 ofloxacin Drugs 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 231100000272 reduced body weight Toxicity 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000009528 severe injury Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
- 229940098232 yersinia enterocolitica Drugs 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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- 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
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- 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
- A23V2400/427—Pentosaceus
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Mycology (AREA)
- Biomedical Technology (AREA)
- Food Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Nutrition Science (AREA)
- Virology (AREA)
- Biophysics (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
Abstract
The invention discloses antagonistic Escherichia coli O157: h7 Pediococcus pentosaceus and its application. The Pediococcus pentosaceus is Pediococcus pentosaceus IM96, is preserved in Guangdong province microorganism strain preservation center at 17 th 5 th 2021, and has a preservation number of GDMCC NO: 61654. The viable bacteria and fermentation supernatant of the strain have good antibacterial activity, and particularly have antibacterial activity on Escherichia coli O157: h7 has strong antagonistic activity in vitro and in vivo. In addition, the pediococcus pentosaceus has good tolerance to gastrointestinal fluids and high safety, and can relieve and improve the gastrointestinal diseases caused by Escherichia coli O157: pathological damage to the gut, inflammation of the gut and damage to the epithelial barrier of the gut caused by H7. The pediococcus pentosaceus can be used for preparing medicines, foods or food additives, animal feeds and microbial preparations with antibacterial activity in the future, and has good application prospects.
Description
The technical field is as follows:
the invention belongs to the technical field of microorganisms, and particularly relates to pediococcus pentosaceus with antibacterial activity and application thereof.
Background art:
enterohemorrhagic escherichia coli (EHEC) is a pathogenic subgroup of shiga toxin-producing escherichia coli (STEC) that can produce shiga toxin 1 and/or 2(Stx1 and Stx2), often containing adhesion genes (eae). Escherichia coli O157H 7 is the most common pathogenic serotype of enterohemorrhagic Escherichia coli. Enterohemorrhagic escherichia coli can cause systemic complications such as hemorrhagic diarrhea, hemorrhagic enteritis, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. Since enterohemorrhagic Escherichia coli was first discovered in 1982 in the United states, contamination of food with enterohemorrhagic Escherichia coli has occurred in many countries of the world, leading to many deaths. In 1996, a pandemic of the world's largest, worldwide, E.coli O157 infection involving tens of thousands of people occurred in Japan. In germany, in 5 months 2011, a large-scale food poisoning event by EHEC O157 was outbreaked, resulting in infection of more than 4000 consumers, out of which 908 had hemolytic uremic symptoms and 40 died. Since the first report in 1986, China has discovered Escherichia coli EHEC O157 in many provinces and cities in sequence: sporadic cases of H7 infection. In addition, enterohemorrhagic escherichia coli is a major cause of morbidity and mortality in children under 5 years of age, particularly in developing countries. In conclusion, the food poisoning caused by enterohemorrhagic escherichia coli is always one of the major public health problems, and simultaneously causes great harm to the life and property safety of people. Currently for entero-hemorrhagic escherichia coli O157: the prevention and control of H7 is mainly by using antibiotics. However, due to the overuse and irregular use of antibiotics in recent years, many resistant microorganisms and super-resistant bacteria appear, which will bring serious threat to human health. Therefore, there is a need for a safe, stable, cost-effective antimicrobial agent that can reduce or even replace the use of antibiotics.
Lactic acid bacteria are recognized as food-grade safe microorganisms worldwide, and some strains have been listed as probiotics by FAO and WHO. A large number of researches show that the probiotic lactic acid bacteria have the functions of antagonizing pathogenic bacteria, regulating immunity, enhancing intestinal barrier and balancing intestinal flora. The lactobacillus can be directly used as a food additive to improve the health of human bodies, and fermentation metabolites of the lactobacillus can be used as an antibacterial agent to inhibit the growth of a large number of food-borne pathogenic bacteria. Therefore, probiotics and their metabolites are of great interest as potential antibacterial agents in the treatment of diseases caused by pathogenic bacteria. Pediococcus pentosaceus (Pediococcus pentosaceus) belongs to the family Lactobacillaceae (Lactobacillus) and Pediococcus (Pediococcus). Pediococcus pentosaceus is a Generally Regarded As Safe (GRAS) species that has been included in the list of edible species by relevant national departments.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a novel polypeptide having antagonistic activity against Escherichia coli O157: h7 active Pediococcus pentosaceus IM96 and application thereof. The strain was deposited at the Guangdong province culture Collection (GDMCC) at 11/5/2021, address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070, accession number: GDMCC No: 61654.
another object of the present invention is to provide a use of Pediococcus pentosaceus IM96 for producing a medicament having an antibacterial function.
Preferably, the drug having an antibacterial function is capable of antagonizing escherichia coli O157: H7.
it is further preferred to alleviate and reduce the risk of infection of the body with e.coli O157: the level of inflammation after H7, and can improve the activity of Escherichia coli O157: h7 infection is a drug for pathological damage caused by small intestine of infected people.
Another object of the present invention is to provide a use of Pediococcus pentosaceus IM96 for producing food or food additives.
Another object of the present invention is to provide the use of Pediococcus pentosaceus IM96 for the production of animal feed.
Another object of the present invention is to provide the use of Pediococcus pentosaceus IM96 for the preparation of a microbial preparation.
Preferably, the microbial preparation is used as an antibacterial agent in the fields of medicines, foods or food additives and animal feeds.
Another objective of the present invention is to provide a specific molecular target of pediococcus pentosaceus IM96 and primers for detecting the specific target, wherein the specific molecular target is shown in SEQ ID No.2, and the primers for detecting the specific target include a forward primer: 5'-ATCGCCAAAGCCAAACGTC-3' and reverse primer: 5'-TTCCTCCTGTCAACTCATAGC-3' are provided.
The invention has the beneficial effects that:
1. the Pediococcus pentosaceus IM96 provided by the invention is obtained by separating and purifying Brassica napobrassica pickled in inner Mongolian Manta, and the food source is safe and reliable. According to an antibiotic susceptibility test, Pediococcus pentosaceus IM96 is not resistant to 7 common antibiotics; meanwhile, the whole genome analysis finds that the strain has no virulence genes and drug resistance genes, so the safety is high.
2. Pediococcus pentosaceus IM96 has good Escherichia coli O157 antagonism: the activity of H7, the diameter of the inhibition zone is as high as 24.67 mm.
3. Pediococcus pentosaceus IM96 has good gastrointestinal fluid resistance. The survival rate in gastric fluid was 80.31%, and in intestinal fluid was 80.21%.
4. Pediococcus pentosaceus IM96 can relieve and reduce the infection of organisms with Escherichia coli O157: the level of inflammation after H7, and can improve the inflammation of Escherichia coli O157: h7 infection causes pathological damage to the small intestine of infected persons.
Pediococcus pentosaceus IM96 was deposited at 11 th 5 th 2021 with the collection of microorganisms of the Guangdong province (GDMCC) at the address: building 5 of the prefecture Zhonglu No. 100 large yard No. 59 in Guangzhou city, the postcode: 510070, accession number: GDMCC No: 61654.
drawings
FIG. 1 shows that Pediococcus pentosaceus IM96 antagonizes E.coli O157: graph of experimental results for H7;
FIG. 2 is a graph showing the results of the survival rate and weight change of mice;
FIG. 3 is a graph showing the results of the morphology of the small intestine of mice;
FIG. 4 is a graph showing the results of the mouse jejunal inflammation indicators;
FIG. 5 is a graph showing the results of the mouse jejunal intestinal epithelial barrier indicators;
FIG. 6 is a diagram of the validation of specific molecular targets of Pediococcus pentosaceus IM 96.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The present invention will be described in detail with reference to the following embodiments in order to better illustrate the objects, technical solutions and advantages of the present invention.
The media referred to in the examples below are as follows
MRS agar plate (g/L): 10.0g/L of peptone, 5.0g/L of beef extract, 4.0g/L of yeast extract powder, 20.0g/L of glucose and 801.0 ml/L, K of tween 2 PO 4 ·3H 2 02.0 g/L, sodium acetate 5.0g/L, and triammonium citrate 2.0g/L, MgSO g 4 ·7H 2 0 0.2g/L、MnSO 4 ·4H 2 00.05 g/L agar 20g/L solvent is water, and its preparation method comprises mixing the above components, and sterilizing. MRS liquid medium was without agar addition.
MRS broth medium (g/L): 10.0g/L of casein digest, 10.0g/L of beef extract, 4.0g/L of yeast extract powder, 2.0g/L of triammonium citrate and 5.0g/L, MgSO of sodium acetate 4 ·7H 2 0 0.2g/L、MnSO 4 ·4H 2 0 0.05g/L、K 2 PO 4 ·3H 2 02.0 g/L, glucose 20.0g/L, Tween 801.0 g/L, and water as solvent, and its preparation method comprises mixing the above components, and sterilizing.
Example 1 isolation and identification of lactic acid bacteria
1. Isolation of lactic acid bacteria
Collecting Brassica napus in Zhoulian of Columbus city of autonomous region of inner Mongolia of China as sample, adding 0.1g Brassica napus sample into 5ml MRS liquid culture medium under aseptic environment, shaking, mixing, performing enrichment culture at 37 deg.C under anaerobic condition for 24h, and sucking 0.5ml bacterial liquid for gradient dilution. Adding physiological saline to obtain 10 -1 To 10 -5 Diluting the gradiental bacterial suspension, and respectively sucking 10 -3 、10 -4 、10 -5 And (3) uniformly smearing three gradiental bacteria suspensions to an MRS agar culture medium by using a coating rod, and then culturing for 48 hours at 37 ℃ under an anaerobic condition. And selecting colonies with typical shapes on the plate to an MRS agar culture medium for streak purification, selecting a single colony after two purifications to inoculate into an MRS liquid culture medium, and culturing at 37 ℃ for 24 hours to collect thalli for extracting thalli DNA.
2. Identification of lactic acid bacteria
Bacterial DNA extraction was performed using a bacterial DNA extraction kit (Mabio, CHINA), followed by PCR amplification using 2 × PCR mix (Dongshengbio, CHINA). The PCR amplification primer adopts a 16SrRNA gene universal primer, and the sequence of an upstream primer is 27F: 5'-AGA GTTTGATCCTGGCTCAG-3', respectively; the sequence of the downstream primer is 1492R: 5'-CTACGGC TACCTTGTTACGA-3' is added. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 35 cycles of 95 ℃ 30s, 56 ℃ 30s and 72 ℃ 1min 30s, and annealing and extending at 72 ℃ for 10 min. The PCR products were recovered by gel cutting and subjected to one-generation sequencing (performed by Jinzhi Biotechnology, Inc., Suzhou). The sequence of the obtained 16SrRNA gene is shown as SEQ ID No. 1. This sequence was aligned with the NCBI database (https:// blast.ncbi.nlm.nih.gov), and as a result, it was suggested that the homology with Pediococcus pentosaceus was highest, so this strain was named Pediococcus pentosaceus (Pediococcus pentosaceus) IM 96.
Pediococcus pentosaceus (Pediococcus pentosaceus) IM96 was deposited at 11/5/2021 with the GDMCC (GDMCC) in the guangdong province collection of microorganisms, address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070, accession number: GDMCC No: 61654.
3. method for activating, culturing and preserving lactic acid bacteria
The pediococcus pentosaceus strain IM96 was removed from the refrigerator at-80 deg.C and activated twice in MRS agar medium. Selecting colonies, inoculating in MRS liquid culture medium, and anaerobically culturing at 37 deg.C for 24-48 h. Adding glycerol into the cultured bacterial liquid, and storing in a refrigerator at-80 deg.C.
EXAMPLE 2 evaluation of antibacterial ability of Pediococcus pentosaceus IM96
1. Preparation of lactic acid bacteria fermentation liquor
A single colony of Pediococcus pentosaceus IM96 was inoculated into MRS broth and cultured anaerobically at 37 ℃ for 48 hours. Centrifuging at 10000r × 4 deg.C for 10min to obtain fermentation supernatant. Filtering the fermentation supernatant by using a 0.22 mu m filter membrane to obtain cell-free supernatant fermented by Pediococcus pentosaceus IM96, and freezing the cell-free supernatant at-80 ℃ for later use.
2. Preparation of indicator bacterium
Escherichia coli O157: h7 ATCC43895, inoculating to LB liquid medium, culturing at 37 deg.C and 200r/min for 6H, adjusting concentration to 1 × 10 8 CFU/mL, antibacterial experiments were performed.
3. Pediococcus pentosaceus IM96 antibacterial ability determination
Taking 200 mu L of the prepared indicator bacterium liquid, uniformly coating the indicator bacterium liquid on a nutrient agar culture medium, placing sterile oxford cups on a flat plate by using sterilized tweezers, and adding 200 mu L of the prepared supernatant into each oxford cup. Meanwhile, MRS medium was used as a blank control group. The plate was placed in a refrigerator at 4 ℃ for uniform diffusion, and then cultured at 37 ℃ for 18 hours, and the diameter of the zone of inhibition was measured. As can be seen from FIG. 1, Pediococcus pentosaceus IM96 has a negative effect on Escherichia coli O157: h7 ATCC43895 has a zone of inhibition of 24.67 + -0.58 mm.
Example 3 evaluation of probiotic Properties of Pediococcus pentosaceus IM96
1. Resistance to Artificial gastric juice test of Pediococcus pentosaceus IM96
Tolerance to artificial gastric juice test: PBS (pH7.4) buffer solution was adjusted to pH 3.0 with 0.1mol/L HCl, pepsin (3g/L) was added, and after dissolution, the mixture was filtered through a 0.22 μm sterile filter membrane and ready to use. Activating the selected strain, inoculating the prepared bacterial liquid of Pediococcus pentosaceus IM96 into 10mL MRS liquid culture medium with the inoculation amount of 2% by volume ratio, standing and culturing for 24h under the anaerobic condition at 37 ℃, performing centrifugation for 10min to collect the bacterial, washing the bacterial for 3 times by PBS (pH7.4), and adjusting the concentration of the bacterial liquid to 10 by PBS 9 CFU/mL, spare. Adding 1mL of PBS resuspended lactobacillus bacterial liquid into 5mL of simulated gastric juice, adding 1.5mL of 0.5% (w/v) NaCl, mixing uniformly, quickly placing into a 37 ℃ incubator, culturing for 0h and 3h, diluting with sterile physiological saline according to a gradient of 1:10, sucking 100 mu L of diluent, uniformly coating the diluent in an MRS solid culture medium by using a disposable coating rod, culturing for 48h at 37 ℃, counting the viable lactobacillus, and calculating the survival rates of 0h and 3h of Pediococcus pentosaceus IM96 under the condition of pH 3.0 according to the following formula.
Survival rate (%). 3h survival number of strain/0 h survival number of strain × 100%
2. Tolerance to Artificial intestinal juice test of Pediococcus pentosaceus IM96
Tolerance to artificial intestinal juice experiment: PBS buffer was adjusted to pH 8.0 with 0.1mol/L NaOH, and 1mg/mL trypsin and 0.3% bovine bile salt were added. Adding 1mL of resuspended Pediococcus pentosaceus IM96 strain (bacterial suspension is prepared to be tolerant to artificial gastric juice) into 5mL of simulated intestinal fluid, quickly and uniformly mixing, putting the mixture into a 37 ℃ incubator, diluting the mixture by sterile physiological saline according to a gradient of 1:10 after 0h and 4h, sucking 100 mu L of diluent, uniformly coating the diluent in an MRS solid culture medium by using a disposable coating rod, culturing the mixture for 48h at 37 ℃, and counting viable bacteria of Pediococcus pentosaceus IM96, wherein the survival rate of different lactic acid bacteria strains for 4h is calculated by the following formula.
Survival rate (%). 4h survival number of strain/0 h survival number of strain × 100%
The result of an artificial gastrointestinal fluid simulation experiment on the screened strain shows that the gastric juice resistant survival rate of Pediococcus pentosaceus IM96 is 80.31 +/-2.88%. The survival rate of the intestinal juice tolerance is 80.21 +/-3.46%.
3. Antibiogram of Pediococcus pentosaceus IM96
Inoculating single colonies of Salmonella typhimurium ATCC 14028, Enterobacter sakazakii ATCC 29544, Yersinia enterocolitica GDMCC60852, Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 19117, Bacillus cereus ATCC 14579 and Bacillus subtilis ATCC 6633 into LB liquid culture medium respectively, culturing at 37 ℃ and 200r/min overnight, and adjusting the concentration to 1 × 10 8 CFU/mL. The antimicrobial spectrum of Pediococcus pentosaceus IM96 was determined by the Oxford cup agar diffusion method. As can be seen from the diameter of the inhibition zone in Table 1, Pediococcus pentosaceus IM96 has good antibacterial activity against the food-borne pathogenic bacteria, which indicates that Pediococcus pentosaceus IM96 is a potential probiotic with good application prospect and broad-spectrum antibacterial activity.
TABLE 1 bacteriostasis spectra of Pediococcus pentosaceus IM96
Example 4 Pediococcus pentosaceus IM96 genome and phenotypic safety evaluation thereof
1. Genome safety evaluation of Pediococcus pentosaceus IM96
Whole genome sequencing was performed on Pediococcus pentosaceus IM96 using the Illumina Nextseq 550 second generation sequencing platform. The extraction method of bacterial genome DNA is the same as that of the previous method. The second generation sequencing was performed by AMT Rapid DNA-Seq Kit for Illumina (CISTRO, CHINA) library and High Output v2.5 Kit (Illumina, USA).
The offline data is subjected to quality control by using Trimmomatic software (v0.39), and then is assembled by using SPAdes software (v 3.13.1). After assembly, the pediococcus pentosaceus genome was subjected to quality control evaluation using Quast software (v5.0.2), and for genomes that were qualified in quality control, the Prokka software (v1.13) was used for annotation. The comparison of VFDB (viral Factor database), ARG-Ant (inflammatory Resistance Gene-ANNOTAT) and CARD (the Comprehensive anti-inflammatory Research database) databases by Abricate software does not find that the bacteria contain virulence genes or drug Resistance genes.
2. Phenotypic safety evaluation of Pediococcus pentosaceus IM96
2.1 evaluation of the drug resistance phenotype of Pediococcus pentosaceus IM96
The sensitivity of Pediococcus pentosaceus IM96 to antibiotics was tested using the paper diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) standards. Seven antibiotics, namely ampicillin, erythromycin, clindamycin, tetracycline, chloramphenicol, imipenem and rifampin, were selected for the test, and the results are shown in Table 2 (note: R is drug-resistant and S is sensitive).
TABLE 2 susceptibility testing of antibiotics
As can be seen from Table 2, Pediococcus pentosaceus IM96 is sensitive to the antibiotics ampicillin, erythromycin, clindamycin, tetracycline, chloramphenicol, imipenem, and rifampicin.
2.2 evaluation of the hemolytic phenotype of Pediococcus pentosaceus IM96
mu.L of Pediococcus pentosaceus IM96 was spotted onto blood plates, respectively, using Staphylococcus aureus ATCC 25923 as control. The results were observed after incubation at 37 ℃ for 48 h. If a clear circular mark is observed around the colony, beta-hemolysis is observed, alpha-hemolysis is observed in brown or green, and gamma-hemolysis is not observed.
The results show that no phenomenon occurs around Pediococcus pentosaceus IM96, and the hemolytic type is gamma-hemolysis. Pediococcus pentosaceus IM96 is not hemolytic.
Example 5 Pediococcus pentosaceus IM96 ameliorates and improves the efficacy of mice infected with escherichia coli O157: pathological injury of intestinal tract, intestinal inflammation and injury of epithelial barrier of intestinal tract caused by H7
1. Animal experiment and design
For the strain with antagonistic Escherichia coli O157: h7 activity, good probiotic properties and safety properties of Pediococcus pentosaceus IM96 was performed in vivo in mice experimental validation. A7 week old SPF rated C57BL/6 mouse from Beijing Huafukang Biotech GmbH. Mice were placed under controlled environmental conditions (temperature 23 ± 3 ℃, relative humidity 50% -60%, light/dark cycle 12/12h, free access to water and food during the experiment). Mice were acclimated starting 1 week prior to the experiment and randomized into 4 groups. The mouse experiment was designed as follows: control group (Control group): intragastrically administering 0.1ml PBS every day, and intragastrically administering 0.1ml PBS 4h later; enterohemorrhagic escherichia coli O157: h7 infection group (EHEC group): intragastric administration of 1X 10 daily 9 cfu E.coli O157: h7 ATCC43895, gavage 0.1ml PBS 4H later; norfloxacin dried group (Norfloxacin group): intragastric administration of 1X 10 daily 9 cful E.coli O157: h7 ATCC43895, gavage 5mg/kg of noro 4H laterA ofloxacin compound; pediococcus pentosaceus IM96 dried group (IM96 group): intragastric administration of 1X 10 daily 9 cful E.coli O157: h7 ATCC43895, gavage 1X 10 after 4H 8 Pediococcus pentosaceus IM 96. Gavage continued for 1 week.
2. Survival rate and weight change of mice
Experimental procedure measurements mice were recorded for survival and weight change, and the results are shown in figure 2. The survival rate of the control group was 100%, whereas E.coli O157: the survival rate of the H7-infected group decreased to 80%. However, after Pediococcus pentosaceus IM96 dry prognosis, the survival rate of the IM96 dry prognosis group is increased to 100%, and the norfloxacin dry prognosis group has the same effect. Coli O157: the H7-infected group had significantly reduced body weight. Infection with escherichia coli O157 with significant remission of Pediococcus pentosaceus pentasaceus im96 and norfloxacin intervention: h7 mice lost weight. Statistical analysis was performed using software SPSS 26.0 using either t-test or one-way analysis of variance. Values are expressed as mean ± standard deviation, with p-values <0.05 considered statistically significant. In addition, p values are expressed as p <0.05, p <0.01, p < 0.001.
3. Morphological observations of jejunum and ileum tissues
At the end of the experiment, the mice were sampled and the jejunum and ileum of the mice were rapidly fixed in 10% neutral formalin. The tissue is dehydrated and transparent, embedded in paraffin and sliced to prepare tissue slices. Morphological observation of large and small intestine tissues was performed by H & E staining. The results are shown in FIG. 3.
As can be seen from FIG. 3, the villi in the small intestine of the control mice are completely structured and regularly arranged, and have no shedding phenomenon. The epithelium of the intestinal mucosa is intact, and the epithelial cell structure is normal without fracture. Coli O157: the H7 infected group has different erosion and shedding of small intestine villi, severe damage to intestinal mucosa, inflammatory cell infiltration and congestion in ileum. Compared with a model group, the norfloxacin and Pediococcus pentosaceus IM96 dry preparation group can relieve inflammatory infiltration of small intestine, relieve shedding of small intestine villi, relieve villi erosion and mucosal barrier damage, and protect small intestine villi structure and intestinal mucosal barrier.
4. Measurement of intestinal inflammation indicators
Preparing jejunum tissue homogenate, and detecting and analyzing the intestinal inflammation indexes and levels of the mice by using IL-1 beta, IL-6, TNF-alpha and IL-10 inflammatory factor Elisa kit. As can be seen from FIG. 4, in comparison with the control group, the ratio of Escherichia coli O157: h7 infection group was determined in e.coli O157: after H7 ATCC43895 is infected, the concentrations of proinflammatory factors IL-1 beta, IL-6 and TNF-alpha are obviously improved, and the successful modeling of the infection model is proved. Compared with the infected group, the proinflammatory factors IL-1 beta, IL-6 and TNF-alpha of the norfloxacin dried group and the Pediococcus pentosaceus IM96 dried group are obviously reduced. In addition, Pediococcus pentosaceus IM96 also significantly increased the production of the anti-inflammatory factor IL-10 compared to the infected group. In conclusion, Pediococcus pentosaceus IM96 has a good function of regulating intestinal inflammation. In particular, p <0.05, p <0.01, p < 0.001.
5. Intestinal epithelial barrier index measurement
Jejunal tissue homogenates were prepared and the content of MUC2, Occludin, ZO-1 in the jejunum of mice was measured for analysis of the epithelial barrier of the intestine of mice according to the protocol of the Elisa test kit. As can be seen from fig. 5, escherichia coli O157: h7 infection group was determined in e.coli O157: after H7 ATCC43895 infection, the concentration of MUC2, Occludin and ZO-1 in jejunum of mice is remarkably reduced, which indicates that the mice are infected by Escherichia coli O157: h7 disrupts the integrity of the intestinal barrier of mice. Compared with an infected group, the content of MUC2, Occludin and ZO-1 in the norfloxacin dried group and the Pediococcus pentosaceus IM96 dried group is remarkably increased. In conclusion, Pediococcus pentosaceus IM96 has a good function of repairing intestinal epithelial barriers. In particular, p <0.05, p <0.01, p < 0.001.
Example 6 excavation and validation of specific molecular targets of Pediococcus pentosaceus IM96
1. Specific molecular target excavation of Pediococcus pentosaceus IM96
Pangenomic analysis was performed on the genomes of Pediococcus pentosaceus IM96 and the other 68 strains of Pediococcus pentosaceus and the other 787 strains of Lactobacillus in the NCBI database using the Prokka (v1.11) and Roary (v3.11.2) software. After obtaining the core genome, genes containing a higher density of base substitutions were identified using Gubbins (v2.4.1) software. The Pediococcus pentosaceus IM96 obtained based on pan-genomic analysis has a specific sequence SEQ ID No.2 different from other Pediococcus pentosaceus and Lactobacillus. Designing a specific PCR amplification primer group according to a specific gene fragment sequence SEQ ID NO.2 of Pediococcus pentosaceus IM96 strain: a forward primer: 5'-ATCGCCAAAGCCAAACGTC-3' and reverse primer: 5'-TTCCTCCTGTCAACTCATAGC-3', primer set sequences are shown in Table 3 below.
TABLE 3 PCR primer set for detecting specific gene fragment sequence
2. Validity verification of identification target of Pediococcus pentosaceus IM96 specific molecule
The effectiveness of the specific molecular target sequence of Pediococcus pentosaceus IM96 was verified by Polymerase Chain Reaction (PCR) and agarose electrophoresis. The detection template is the DNA of bacteria, and the DNA extraction method is the same as the previous method.
The PCR reaction system is configured as follows:
the PCR reaction conditions were as follows:
after the PCR was completed, 5. mu.l of the PCR product was subjected to 1.2% agarose electrophoresis. If Pediococcus pentosaceus IM96 can generate a single specific band at 388bp, and other non-target bacteria can not generate a band at 388bp, the target pair has good efficiency of recognizing Pediococcus pentosaceus IM 96.
The result of PCR amplification product gel is shown in FIG. 6, where M is DL2000DNA standard marker, + is Pediococcus pentosaceus IM96, and C is negative control (the template of the control is DEPC water without genome). 1-230 are non-target pediococcus pentosaceus
As shown in FIG. 6, no specific amplification product was formed in any of the other Pediococcus pentosaceus and other species, except for the specific amplification product formed at 388bp after the primer amplification of the DNA of the target strain Pediococcus pentosaceus IM 96. The result proves that the PCR amplification primer is a specific molecular target of Pediococcus pentosaceus IM 96.
The strains used and the results of the tests are shown in table 4 below; in the table, "+" indicates positive and "-" indicates negative in the test result column.
TABLE 4 detection results of Pediococcus pentosaceus IM96 specific target
Although the preferred embodiments of the present invention have been disclosed, it should be understood that they are not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. Therefore, the protection scope of the present invention should be subject to the content defined by the claims.
Sequence listing
<110> institute of microbiology, academy of sciences of Guangdong province (center for microbiological analysis and detection in Guangdong province)
GUANGDONG HUANKAI BIOTECHNOLOGY Co.,Ltd.
<120> an antagonistic Escherichia coli O157: h7 pediococcus pentosaceus and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1436
<212> DNA
<213> Pediococcus pentosaceus IM96(Pediococcus pentosaceus)
<400> 1
agacggctag ctcctaaaag gttaccccac cggctttggg tgttacaaac tctcatggtg 60
tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg cggcatgctg atccgcgatt 120
actagcgatt ccgacttcgt gtaggcgagt tgcagcctac agtccgaact gagaatggtt 180
ttaagagatt agcttaacct cgcggtttcg cgactcgttg taccatccat tgtagcacgt 240
gtgtagccca ggtcataagg ggcatgatga tttgacgtcg tccccacctt cctccggttt 300
gtcaccggca gtctcactag agtgcccaac ttaatgctgg caactagtaa taagggttgc 360
gctcgttgcg ggacttaacc caacatctca cgacacgagc tgacgacaac catgcaccac 420
ctgtcattct gtccccgaag ggaacctcta atctcttaga ctgtcagaag atgtcaagac 480
ctggtaaggt tcttcgcgta gcttcgaatt aaaccacatg ctccaccgct tgtgcgggcc 540
cccgtcaatt cttttgagtt tcaaccttgc ggtcgtactc cccaggcgga ttacttaatg 600
cgttagctgc agcactgaag ggcggaaacc ctccaacact tagtaatcat cgtttacggc 660
atggactacc agggtatcta atcctgttcg ctacccatgc tttcgagcct cagcgtcagt 720
tgcagaccag acagccgcct tcgccactgg tgttcttcca tatatctacg catttcaccg 780
ctacacatgg agttccactg tcctcttctg cactcaagtc tcccagtttc caatgcactt 840
cttcggttga gccgaaggct ttcacattag acttaaaaga ccgcctgcgc tcgctttacg 900
cccaataaat ccggataacg cttgccacct acgtattacc gcggctgctg gcacgtagtt 960
agccgtggct ttctggttaa ataccgtcac tgggtaaaca gttactctta cccacgttct 1020
tctttaacaa cagagcttta cgagccgaaa cccttcttca ctcacgcggc gttgctccat 1080
cagacttgcg tccattgtgg aagattccct actgctgcct cccgtaggag tctgggccgt 1140
gtctcagtcc caatgtggcc gattaccctc tcaggtcggc tacgtatcac tgccttggtg 1200
agcctttacc tcaccaacta gctaatacgc cgcgggtcca tccagaagtg atagcagagc 1260
catcttttaa aagaaaacca tgcggttttc tctgttatac ggtattagca tctgtttcca 1320
ggtgttatcc cctacttctg ggcaggttac ccacgtgtta ctcacccgtt cgccactcac 1380
ttcgtgttaa aatctcaatc agtacaagta cgtcataatc aattaacgga agttcg 1436
<210> 2
<211> 825
<212> DNA
<213> Pediococcus pentosaceus IM96(Pediococcus pentosaceus)
<400> 2
atgtttaaaa agtgtgaaat ttgtaataaa aagattggat tattttctaa accatttaca 60
actgatactg gtgcaaaagt atgtacctct tgtacatctt atgtagatcc aattttttta 120
actgaaaata ttacaacagc cgaatcaata gctttcgata tggataatga acttggaaat 180
tcttacttag caaaaattgg aaaagagtca ccttctgact tacgtaaaaa agaagaagag 240
gccgctaaac aagaactact tcaaaagcag aaagaagagg ctgaacgaca agaaatttta 300
aaacaagcac aagaaactat cgccaaagcc aaacgtcaaa aaatatatca ttttaaagtt 360
cgaggcgtat ctcactatga tttatcaaaa ttagttagtt atgcaaggaa aaatgactta 420
attttcccct atgatggttt cactgcatca gaaataaagg agatatgccc atacgaaaaa 480
gtttttgaaa cagacttagt gggaacatca ctaactatta gcttaattcc tgaacccgaa 540
aataaatatg atccaaacgc cataaaggta atagcaactc atgaagaaaa acaatttatg 600
ttaggttatg ttccaactga acatactaaa gaagttgcag atattatgca gaagcaaaat 660
gaaaacaaaa ttagaatgca catcagctat gagttgacag gaggaaaata caaaatagct 720
gaggaaaatt ttgatgattt ttcagatgat ccaaagctaa aaattcgtac tggaaaagtt 780
gaatatggtt ttaacataga aatccacgat atgaatattg attaa 825
Claims (10)
1. Pediococcus pentosaceusPediococcus pentosaceus IM96, accession number: GDMCC No: 61654.
2. pediococcus pentosaceus as defined in claim 1PediococcuspentosaceusUse of IM96 in the manufacture of a medicament having antibacterial properties.
3. The use according to claim 2, wherein the antibacterial agent is an agent capable of antagonizing the activity of O157: h7.
4. The use according to claim 3, wherein the drug with antibacterial function is a drug that relieves and reduces the activity of Escherichia coli O157: h7, and can ameliorate the inflammatory level of e.coli O157: h7 infection to the small intestine.
5. Pediococcus pentosaceus as defined in claim 1Pediococcus pentosaceus Use of IM96 in the manufacture of a food product or food additive.
6. Pediococcus pentosaceus according to claim 1PediococcuspentosaceusUse of IM96 in the production of animal feed.
7. Use of pediococcus pentosaceus according to claim 1 for the preparation of a microbial preparation.
8. Use according to claim 7, wherein the microbial preparation is used as an antimicrobial in the field of pharmaceuticals, food additives or animal feed.
9. A pediococcus pentosaceus-specific molecular target according to claim 1, wherein the nucleotide sequence is set forth in SEQ ID No. 2.
10. A primer for identifying pediococcus pentosaceus according to claim 1, comprising a forward primer: 5'-ATCGCCAAAGCCAAACGTC-3' and reverse primer: 5'-TTCCTCCTGTCAACTCATAGC-3' are provided.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110735107.0A CN113528381B (en) | 2021-06-30 | 2021-06-30 | An antagonistic escherichia coli O157: h7 pediococcus pentosaceus and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110735107.0A CN113528381B (en) | 2021-06-30 | 2021-06-30 | An antagonistic escherichia coli O157: h7 pediococcus pentosaceus and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113528381A CN113528381A (en) | 2021-10-22 |
| CN113528381B true CN113528381B (en) | 2022-09-30 |
Family
ID=78097339
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110735107.0A Active CN113528381B (en) | 2021-06-30 | 2021-06-30 | An antagonistic escherichia coli O157: h7 pediococcus pentosaceus and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113528381B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114292794A (en) * | 2022-01-18 | 2022-04-08 | 赵一格 | Pediococcus pentosaceus SD22 and screening method and application thereof |
| CN115747111B (en) * | 2022-11-25 | 2023-12-12 | 四川大学 | Pediococcus pentosaceus and application thereof |
| CN117264850B (en) * | 2023-11-09 | 2024-05-14 | 潍坊君薇生物科技有限责任公司 | Pediococcus pentosaceus SW006 with auxiliary treatment of colpitis and immunity enhancing functions and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136126B (en) * | 2018-08-21 | 2021-04-13 | 云南农业大学 | Pediococcus pentosaceus for preventing diarrhea of weaned piglets |
| CN112375696B (en) * | 2020-06-27 | 2022-04-01 | 石河子大学 | Donkey milk source pediococcus pentosaceus and application thereof |
| CN111793586B (en) * | 2020-08-13 | 2022-05-20 | 石河子大学 | Breast milk source pediococcus pentosaceus and application thereof |
-
2021
- 2021-06-30 CN CN202110735107.0A patent/CN113528381B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN113528381A (en) | 2021-10-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113528381B (en) | An antagonistic escherichia coli O157: h7 pediococcus pentosaceus and application thereof | |
| CN113999797B (en) | Pediococcus acidilactici for improving productivity and immunity level of broiler chickens, and screening method and application thereof | |
| CN114350578B (en) | Lactobacillus plantarum LP1Z for producing lysozyme and efficiently antagonizing multidrug-resistant helicobacter pylori and application thereof | |
| CN116004483B (en) | Lactococcus garvieae for preventing or treating diarrhea and application thereof | |
| CN115851500B (en) | Lactobacillus plantarum and application thereof | |
| CN111281895A (en) | Lactic acid bacteria for treating colitis and application thereof | |
| CN111534452B (en) | Lactobacillus strain with broad-spectrum inhibition of multi-drug-resistance food-borne pathogenic bacteria and application thereof | |
| CN117143767B (en) | Breast milk-derived fermented lactobacillus mucilaginosus MSJK capable of regulating intestinal flora and application thereof | |
| CN115044504A (en) | Enterococcus faecalis YZ-1 and probiotic application thereof | |
| CN112322553B (en) | Clostridium difficile resistant lactococcus lactis and application thereof | |
| CN114317334B (en) | Lactobacillus sake capable of co-aggregating with helicobacter pylori and application thereof | |
| CN113186136B (en) | Multiple drug-resistant helicobacter pylori and application thereof | |
| CN114774315A (en) | Application of lactobacillus rhamnosus strain LRa05 in preparing immunity enhancing and/or eczema relieving product | |
| CN117660253B (en) | Lactobacillus rhamnosus FMBL L23004 CNN, yoghurt starter, yoghurt and application thereof | |
| CN116121110B (en) | Lactococcus lactis YJ0801 with strong antibacterial and anti-inflammatory effects and application thereof | |
| CN114806953B (en) | Lactobacillus gasseri with effect of improving type 1 diabetes | |
| CN113913334B (en) | Enterococcus faecalis EF-ZA1107-06 and application thereof | |
| CN116769655A (en) | Streptococcus salivarius thermophilus JIAN+ and application thereof | |
| CN116622568A (en) | Lactobacillus harbini P1-1 and application thereof | |
| CN116590181A (en) | Lactobacillus paracasei for improving inflammatory reaction caused by microplastic pollution and application thereof | |
| CN111518724B (en) | Lactobacillus gasseri HMV18 and secreted protein and application thereof | |
| CN115838652A (en) | Lactobacillus reuteri LR21 and application thereof | |
| CN113508907A (en) | Application of heat-resistant lactobacillus fermentum in preparation of defecation promoting food or medicine | |
| CN116240151B (en) | Fender-like bacillus and application thereof | |
| CN117305187B (en) | Pediococcus acidilactici for improving intestinal health condition and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |