CN113846035A - 一株缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌及其应用 - Google Patents
一株缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌及其应用 Download PDFInfo
- Publication number
- CN113846035A CN113846035A CN202111268340.9A CN202111268340A CN113846035A CN 113846035 A CN113846035 A CN 113846035A CN 202111268340 A CN202111268340 A CN 202111268340A CN 113846035 A CN113846035 A CN 113846035A
- Authority
- CN
- China
- Prior art keywords
- lactobacillus salivarius
- meningitis
- intestinal
- enteritis
- development
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000186869 Lactobacillus salivarius Species 0.000 title claims abstract description 100
- 208000004232 Enteritis Diseases 0.000 title claims abstract description 33
- 230000023011 digestive tract development Effects 0.000 title claims abstract description 32
- 201000009906 Meningitis Diseases 0.000 title claims abstract description 30
- 230000001737 promoting effect Effects 0.000 title claims abstract description 24
- 241001135265 Cronobacter sakazakii Species 0.000 claims abstract description 45
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- 239000003814 drug Substances 0.000 claims abstract description 11
- 238000011161 development Methods 0.000 claims abstract description 9
- 230000018109 developmental process Effects 0.000 claims abstract description 9
- 235000013305 food Nutrition 0.000 claims abstract description 9
- 238000009629 microbiological culture Methods 0.000 claims abstract description 5
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 24
- 230000000968 intestinal effect Effects 0.000 claims description 19
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 16
- 230000004888 barrier function Effects 0.000 claims description 13
- 210000002220 organoid Anatomy 0.000 claims description 11
- 102000004889 Interleukin-6 Human genes 0.000 claims description 10
- 108090001005 Interleukin-6 Proteins 0.000 claims description 10
- 102000003777 Interleukin-1 beta Human genes 0.000 claims description 9
- 108090000193 Interleukin-1 beta Proteins 0.000 claims description 9
- 101150058357 Muc2 gene Proteins 0.000 claims description 9
- 230000006378 damage Effects 0.000 claims description 9
- 230000002757 inflammatory effect Effects 0.000 claims description 8
- 102000003940 Occludin Human genes 0.000 claims description 6
- 108090000304 Occludin Proteins 0.000 claims description 6
- 238000009630 liquid culture Methods 0.000 claims description 5
- 235000020256 human milk Nutrition 0.000 claims description 4
- 210000004251 human milk Anatomy 0.000 claims description 4
- 210000004347 intestinal mucosa Anatomy 0.000 claims description 4
- 230000002265 prevention Effects 0.000 claims 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 claims description 3
- 206010061218 Inflammation Diseases 0.000 claims description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 3
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims description 3
- 230000004054 inflammatory process Effects 0.000 claims description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 2
- 102000001621 Mucoproteins Human genes 0.000 claims description 2
- 108010093825 Mucoproteins Proteins 0.000 claims description 2
- 102000011154 Tight junction protein ZO-1 Human genes 0.000 claims description 2
- 108050001370 Tight junction protein ZO-1 Proteins 0.000 claims description 2
- 229910052791 calcium Inorganic materials 0.000 claims description 2
- 239000011575 calcium Substances 0.000 claims description 2
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims 2
- 206010027202 Meningitis bacterial Diseases 0.000 claims 2
- 201000009904 bacterial meningitis Diseases 0.000 claims 2
- 230000015572 biosynthetic process Effects 0.000 claims 2
- 208000011140 intestinal infectious disease Diseases 0.000 claims 2
- 210000005027 intestinal barrier Anatomy 0.000 claims 1
- 230000004673 intestinal mucosal barrier function Effects 0.000 claims 1
- 239000006041 probiotic Substances 0.000 abstract description 11
- 235000018291 probiotics Nutrition 0.000 abstract description 11
- 230000000529 probiotic effect Effects 0.000 abstract description 8
- 229940079593 drug Drugs 0.000 abstract description 5
- 241000186606 Lactobacillus gasseri Species 0.000 abstract description 2
- 239000000654 additive Substances 0.000 abstract 1
- 235000013365 dairy product Nutrition 0.000 abstract 1
- 239000002207 metabolite Substances 0.000 abstract 1
- 239000008267 milk Substances 0.000 abstract 1
- 235000013336 milk Nutrition 0.000 abstract 1
- 210000004080 milk Anatomy 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 15
- 210000004379 membrane Anatomy 0.000 description 13
- 239000012528 membrane Substances 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 9
- 108020004999 messenger RNA Proteins 0.000 description 9
- 238000007792 addition Methods 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 244000052616 bacterial pathogen Species 0.000 description 5
- 230000037396 body weight Effects 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 210000000813 small intestine Anatomy 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 102000004162 Claudin-1 Human genes 0.000 description 4
- 108090000600 Claudin-1 Proteins 0.000 description 4
- 241000186660 Lactobacillus Species 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 210000000936 intestine Anatomy 0.000 description 4
- 229940039696 lactobacillus Drugs 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 102000002029 Claudin Human genes 0.000 description 3
- 108050009302 Claudin Proteins 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000003531 protein hydrolysate Substances 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- 102000015728 Mucins Human genes 0.000 description 2
- 108010063954 Mucins Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 210000005252 bulbus oculi Anatomy 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 230000005484 gravity Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102100027211 Albumin Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 208000031729 Bacteremia Diseases 0.000 description 1
- 108010062877 Bacteriocins Proteins 0.000 description 1
- 241000606124 Bacteroides fragilis Species 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 102100035882 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 208000012239 Developmental disease Diseases 0.000 description 1
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 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 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
- 240000001046 Lactobacillus acidophilus Species 0.000 description 1
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 description 1
- 244000199866 Lactobacillus casei Species 0.000 description 1
- 235000013958 Lactobacillus casei Nutrition 0.000 description 1
- 241000186673 Lactobacillus delbrueckii Species 0.000 description 1
- 240000006024 Lactobacillus plantarum Species 0.000 description 1
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 1
- 241000917009 Lactobacillus rhamnosus GG Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 206010058780 Meningitis neonatal Diseases 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 206010051606 Necrotising colitis Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 241000191998 Pediococcus acidilactici Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 235000014897 Streptococcus lactis Nutrition 0.000 description 1
- 244000057717 Streptococcus lactis Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 102000000591 Tight Junction Proteins Human genes 0.000 description 1
- 108010002321 Tight Junction Proteins Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000003266 anti-allergic effect Effects 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 238000002737 cell proliferation kit Methods 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 208000010227 enterocolitis Diseases 0.000 description 1
- 210000001842 enterocyte Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000004153 glucose metabolism Effects 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 238000013427 histology analysis Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 208000037817 intestinal injury Diseases 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 229940039695 lactobacillus acidophilus Drugs 0.000 description 1
- 229940017800 lactobacillus casei Drugs 0.000 description 1
- 229940072205 lactobacillus plantarum Drugs 0.000 description 1
- 229940059406 lactobacillus rhamnosus gg Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 208000004995 necrotizing enterocolitis Diseases 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 201000006195 perinatal necrotizing enterocolitis Diseases 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 230000004580 weight loss Effects 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
- 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
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- 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
- A61P25/00—Drugs for disorders of the nervous system
-
- 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
- A61P31/04—Antibacterial agents
-
- 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
- A23V2400/11—Lactobacillus
- A23V2400/181—Salivarius
-
- 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
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Mycology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Food Science & Technology (AREA)
- Molecular Biology (AREA)
- Nutrition Science (AREA)
- Animal Husbandry (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Physiology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明涉及一株缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌(Lactobacillus gasseri),名称为唾液乳杆菌菌株YL20,保藏编号为CGMCC NO:20590,保藏日期:2020年9月3日,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心。所述菌株具有促进早期肠道发育的功能;此外该菌株还能够缓解阪崎肠杆菌诱导的新生儿肠炎、脑膜炎,增强机体的抵抗能力。该菌株有望应用于新生儿奶制品的添加剂以及婴幼儿乳制品食品的制备,同时其代谢产物还可应用于肠炎药品、益生食品、饲料产品的开发,具有很广的益生前景。
Description
技术领域
本发明主要涉及微生物技术领域,具体涉及一株缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌(Lactobacillus salivarius)YL20。
背景技术
阪崎克罗诺肠杆菌作为一种条件致病菌,能导致严重的新生儿脑膜炎、小肠结肠炎和菌血症,是近几年来受到广泛关注的一种非常重要的影响新生儿健康的食源性致病菌。1岁以下婴儿、出生体质量偏低和未满28d的新生儿被认为是阪崎克罗诺肠杆菌最容易感染的人群,其死亡率高达50%。免疫力低下的成年人食用阪崎克罗诺肠杆菌污染的食物也能导致疾病。尽管在阪崎克罗诺肠杆菌感染的临床治疗中使用抗生素可以使患者康复,但患者往往伴随有严重的神经系统后遗症和发育障碍等症状。
肠道微生物是机体胃肠道中不可缺少的组成部分,依据是否对宿主有利分为有益微生物和致病微生物。大量的研究证明,肠道有益微生物能够与致病菌竞争黏附位点,从而抵御病原菌的入侵。已有研究证实脆弱拟杆菌ZY-312株可对阪崎肠杆菌诱导的坏死性小肠结肠炎起保护作用;植物乳杆菌ATCC 8014与阪崎肠杆菌共孵育后对阪崎肠杆菌有抑制作用;双歧杆菌PRL2010能在胃肠道存活,且能粘附在人类上皮肠细胞单层(Caco2和HT-29)上,从而抑制阪崎肠杆菌的粘附。
唾液乳杆菌(Lactobacillus salivarius)属于乳杆菌科,乳杆菌属,是革兰氏阳性菌,不生成孢子,不具触酶、氧化酶及运动性,能产乳酸,在好氧及厌氧环境均能生长,属于兼性异质发酸性菌株,葡糖代谢时不产生气体。已有研究证实唾液乳杆菌是一种可刺激免疫细胞分泌抗过敏相关细胞激素的益生菌,有良好的益生特性,且具有一定的吸附和免疫调节作用,可用于改善免疫功能,增强机体免疫力;并且定殖在肠道的乳杆菌在生长代谢过程中可分泌抗生素、细菌素等物质,抑制病原菌的生长等多种功效。唾液乳杆菌是我国卫生部批准使用的食品生产加工的菌种之一,其具有良好的耐酸和耐胆碱特性,可在人体和动物肠胃中生存。其作为一种具有极大潜力的益生性乳杆菌,被广泛应用于制成适用于人和动物的益生菌制剂。但有关唾液乳杆菌是否抑制阪崎肠杆菌尚未见报道。
新生儿时期是肠道菌群定植的关键时期,由于新生儿肠道菌群较为单一,因此容易导致免疫力低下等疾病的发生。早期肠道微生物的定植可影响婴幼儿的生长发育,肠道菌群与婴幼儿肠道屏障的建立、神经发育、免疫发育等都密切相关。目前研究已经发现,益生菌干预可以有效地增强肠道的消化吸收能力,促进免疫快速成熟,进而提高机体防疫能力,对于婴幼儿的健康发育具有重要意义。已有研究证实鼠李糖乳杆菌(Lactobacillusrhamnosus)GG的早期定殖能促进小鼠肠道的发育。但唾液乳杆菌对于肠道发育的研究较少。
因此,寻找一种可促进肠道发育且能缓解阪崎肠杆菌对肠道造成损伤的益生菌具有重要的意义。
通过检索并未发现与本发明专利申请相关的专利公开文献。
发明内容
本发明的目的是针对现有技术中存在的不足之处,提供一株缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌及其应用。
为达到以上目的,我们是通过以下技术方案来实现的:
一株缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌,名称为:YL20,分类名称为:唾液乳杆菌Lactobacillus salivarius,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区北辰西路1号院3号,保藏日期:2020年9月3日,保藏号:CGMCC NO:20590。
而且,所述唾液乳杆菌是从健康母亲的母乳中分离获得的;
或者,所述唾液乳杆菌在MRS固体培养基上菌落乳白色,边缘光滑;
或者,所述唾液乳杆菌的基因序列为SEQ ID No.1。
而且,所述唾液乳杆菌能够缓解新生儿肠炎、脑膜炎和促进早期肠道发育;
或者,所述唾液乳杆菌能够促进肠道中黏液层重要黏蛋白2mucin2(Muc2)和紧密连接蛋白occludin(OCLN)、zonula occluden-1(ZO-1)以及闭合蛋白claudin-1(CLDN-1)的表达;
或者所述唾液乳杆菌能够促进小鼠肠道类器官的生长,能够增加小鼠肠道绒毛的长度、降低隐窝的深度;
或者所述唾液乳杆菌能够缓解阪崎肠杆菌所致的肠道粘膜屏障和细胞屏障的损伤,抑制阪崎肠杆菌诱导的炎症因子IL-6、TNF-α、IL-1β的释放。
如上所述的能够缓解新生儿肠炎、脑膜炎和促进早期肠道发育作用的唾液乳杆菌菌株在制备预防和/或治疗肠炎、脑膜炎的药物中的应用。
如上所述的具有缓解肠炎、脑膜炎和促进肠道发育作用的唾液乳杆菌菌株在制备促进肠道发育的药物中的应用。
所述的具有缓解肠炎和促进肠道发育作用的唾液乳杆菌在加工生产制得的食品或饲料产品中的应用。
本发明取得的优点和积极效果如下:
1、本发明唾液乳杆菌YL20具有促进肠道早期发育功能。
2、本发明唾液乳杆菌YL20具有治疗阪崎肠杆菌造成的肠炎和脑膜炎效果,其治肠炎效果显著。
3、本发明唾液乳杆菌YL20具有预防生命早期肠炎的功能。
4、本发明唾液乳杆菌YL20有望用于制备预防和治疗肠道炎症及促进发育的药品及食品或饲料产品,具有非常广泛的应用前景。
5、本发明唾液乳杆菌YL20能够应用在预防与治疗炎症性肠炎、促进动物肠道发育相关的微生态制剂中,为研发预防和治疗结肠炎的益生菌制剂提供理论支持,为研发促进肠道发育的微生态制剂提供理论支持。
附图说明
图1为本发明中唾液乳杆菌YL20的生长曲线图;
图2为本发明中唾液乳杆菌YL20与类器官共培养后EDU染色结果
图3为本发明中唾液乳杆菌YL20与类器官共培养后EDU染色阳性细胞数数据统计结果;
图4为本发明中唾液乳杆菌YL20灌胃小鼠后肠道组织切片HE染色图。
具体实施方式
下面结合具体实施例,对本发明作进一步详述,以下实施例只是描述性的,不是限定性的,不能以此限定本发明的保护范围。
本发明中所使用的原料,如无特殊说明,均为常规的市售产品;本发明中所使用的方法,如无特殊说明,均为本领域的常规方法。
一株能够缓解坂崎肠杆菌诱导的新生儿肠炎和脑膜炎以及促进早期肠道发育作用的唾液乳杆菌,名称为YL20,分类名称为Lactobacillus salivarius,保藏编号为:CGMCCNO:20590,保藏日期:2020年9月22日,北京市朝阳区北辰西路1号院3号,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心。
较优地,所述唾液乳杆菌是从健康母亲的母乳中分离获得的;
或者,所述唾液乳杆菌在MRS固体培养基上菌落乳白色,边缘光滑;
或者,所述唾液乳杆菌的基因序列为SEQ ID No.1。
较优地,所述唾液乳杆菌能够缓解新生儿肠炎、脑膜炎和促进早期肠道发育;
或者,所述唾液乳杆菌能够促进肠道中黏液层重要黏蛋白2mucin2(Muc2)和紧密连接蛋白occludin(OCLN)、zonula occluden-1(Zo-1)以及闭合蛋白claudin-1(CLDN-1)的表达;
或者所述唾液乳杆菌能够促进小鼠肠道类器官的生长,能够增加小鼠肠道绒毛的长度、降低隐窝的深度;
或者所述唾液乳杆菌能够缓解阪崎肠杆菌所致的肠道粘膜屏障和细胞屏障的损伤,抑制阪崎肠杆菌诱导的炎症因子IL-6、TNF-α、IL-1β的释放。
如上所述的能够缓解新生儿肠炎、脑膜炎和促进早期肠道发育作用的唾液乳杆菌菌株在制备预防和/或治疗肠炎、脑膜炎的药物中的应用。
较优地,所述药物为益生菌制剂。
如上所述的具有能够缓解新生儿肠炎、脑膜炎和促进早期肠道发育作用的唾液乳杆菌菌株在制备促进肠道发育的药物中的应用。
较优地,所述药物为微生态制剂;
所述的具有缓解肠炎和促进肠道发育作用的唾液乳杆菌在加工生产制得的食品或饲料产品中的应用。
具体地,相关制备及检测步骤如下:
实施例1:菌株的分离纯化、鉴定
取新鲜健康的母乳1mL,梯度稀释(10-1至10-7),然后取0.2mL均匀涂布于加有碳酸钙的MRS平板上,置于37℃培养箱培养24~48h。挑取出现溶钙圈的菌落反复接种筛选,直至得到均匀的单个菌落,命名为YL20。革兰氏染色镜检:菌株YL20为革兰氏阳性菌株,显微镜下呈短杆状,在MRS平板培养基上生长,可形成表面圆润的小菌落,边缘整齐,有溶钙圈;在MRS液体培养基中呈均匀浑浊生长,久置菌体呈白色沉淀
该菌株与NCBI中GenBank的Lactobacillus salivarius菌种的同源性达99%。结果显示该菌种为唾液乳杆菌,命名为唾液乳杆菌YL20。
实施例2:菌株的理化性质
(1)菌株的形态特征:菌株菌体呈球状;革兰氏染色呈阳性,无芽孢。MRS平板培养基上菌落光滑,呈圆形或近圆形,凸起,平铺,呈白色,不透明。
(2)菌株的生理生化特征:能产生蛋白酶、纤维素酶,能使淀粉水解,能够利用葡萄糖、甘露醇、木糖、蔗糖、L-阿拉伯糖,但不能利用肌醇、乳糖;V-P实验、明胶液化实验和氧化酶实验均呈阳性。
实施例3:菌株的生长曲线
将实施例1中的唾液乳杆菌接入MRS固体培养基上于37″C培养48h。挑取生长状态良好的单菌落接种至MRS液体培养基中进行活化,再以1%的接种量接种于MRS液体培养基中,静置培养24h,每小时取菌悬液测定0D600,并绘制唾液乳杆菌的生长曲线。如图1所示。
实施例4:唾液乳杆菌YL20体外抑菌能力
将保存在-80℃唾液乳杆菌YL20菌种常温下解冻,按2%接种于MRS液体培养基中,经过二次活化,每次培养12h,最终接菌于10mL培养基中。在无菌操作台上,将浓度为109CFU/mL的阪崎肠杆菌菌悬液加入冷却至45″C的MRS固体培养基(灭菌后)中混匀,制备成4mm左右的病原菌琼脂平板将灭菌的牛津杯放置在培养基上,轻轻加压,使其与培养基接触无空隙,待10分钟后,分别向各小管中滴加200uL保存好的实施例1中的唾液乳杆菌,以及勿使其外溢,37C培养36-96h,然后测量抑菌圈直径。每个实验三个重复,取平均值。
结果发现唾液乳杆菌YL20抑制阪崎肠杆菌的抑菌圈达到12.5±0.1mm,比乳酸乳球菌ML2018、乳酸片球菌Hao2018、干酪乳杆菌LH23、嗜酸乳杆菌YL01、德氏乳杆菌KY02等的抑菌圈均大一些,如表1所示,则说明唾液乳杆菌YL20具有一定的体外抑制阪崎肠杆菌的能力。
表1唾液乳杆菌YL20等益生菌体外抑制阪崎肠杆菌的抑菌圈直径
实施例5:唾液乳杆菌YL20体外保护阪崎肠杆菌所致的细胞屏障损伤
(1)紧密连接蛋白Zo-1、Occuldin基因mRNA水平的检测。
HT-29细胞用含10%FCS的DMEM培养基(含1×105U/L青霉素和100mg/L链霉素),在37℃、5%CO2及饱和湿度条件下培养。24h换液,以后每2、3天更换一次培养液。细胞长满后,用2.5g/L胰酶消化后传代。HT-29细胞以104/mL的密度均匀铺种在六孔板中培养两天,经加入108CFU/mL唾液乳杆菌YL20样品处理24h后,Trizol法提取HT-29细胞中RNA,用M-MLV逆转录酶逆转录2μg样品。real-time RCR半定量法检测Zo-1、Occuldin基因的mRNA水平。PCR体系:7.6μL DDW,10μL Mix(2×SYBR Green qPCR Mix),0.4μL ROX(50×ROX ReferenceDye),0.5μL上游引物,0.5μL下游引物,1μL cDNA模板。PCR反应条件:95℃预变性2min,95℃变性10s,60℃退火30s,95℃延伸1min,共40个循环,95℃终止反应15s。引物序列如表2。
表2 real-timeRCR引物
结果相对于阪崎肠杆菌组,加入唾液乳杆菌YL20的治疗组的Zo-1 mRNA水平显著上升,*P<0.01有显著差异;结果相对于阪崎肠杆菌组,加入唾液乳杆菌YL20的治疗组的Occuldin mRNA水平显著上升,*P<0.01有显著性差异。结果显示,加入唾液乳杆菌YL20可以在体外有效保护阪崎肠杆菌所致的细胞屏障损伤。
(2)紧密连接蛋白Zo-1、Occuldin以及闭合蛋白Claudin-1蛋白表达的检测。
配蛋白裂解液(比例:ddH2O 488μL,6×蛋白质裂解液100μL,PMSF 12μL),混合均匀后,放置于冰上备用;用细胞外用PBS缓冲液冲洗6孔板2次。每孔加入100μL配制好的蛋白裂解液,将6孔板在4℃冰箱裂解30min;裂解结束后,将6孔板取出,用细胞刮刀刮下皿底的细胞,收集于EP管中并做标记,沸水煮沸10min,使蛋白彻底变形,于-80℃保存备用;每管中按照1∶6的比例加入6×loading buffer,漩涡震荡混匀后煮沸10min,瞬时离心数秒;将蛋白胶放入电泳槽中,加入电极液,将蛋白样点入胶孔。初始恒压为70V,约1.5h,蛋白压缩之后调整电压为120V,继续跑胶。通过蛋白marker确定蛋白完全分离时间;取出事先裁剪好的NC膜、滤纸(5cm宽*8cm长)放在干转液中,让它们浸泡充分;按照滤纸、NC膜、蛋白胶的顺序放整齐,每一步充分赶走气泡;NC膜靠近阳极碳板、蛋白胶靠近阴极碳板;恒流进行转膜,电流大小为1.5mA/cm2,根据目的蛋白条带的大小确定转膜时间长短,本实验中最大条带Zo-1需转3.5h;转膜之后,将NC膜放入配好的5%脱脂牛奶中,将其放在摇床上室温封闭1h,之后用TBST洗膜3遍;将洗好的NC膜根据蛋白条带的大小进行裁切,放入孵育盒,倒入稀释好的一抗,4℃过夜孵育;所用抗体的稀释比为:Occuldin(1∶100);Claudin-1(1∶100);Zo-1(1∶100);β-actin(1∶5000)。回收一抗,倒入TBST缓冲液,摇床上摇晃10min,倒掉,重新倒入TBST,摇晃10min,如此重复3次;倒入相应的二抗,置于摇床上室温孵育1.5-2h;该步骤全程避光;回收二抗,洗膜3次,每次10min,避光操作;避光条件下将NC膜平铺于Oddysey远红外成像仪中,设置程序扫膜,保存数据。
结果在分别用紧密连接蛋白ZO-1、Occuldin以及闭合蛋白Claudin-1的一抗检测后发现与阪崎肠杆菌的条带相比,加入唾液乳杆菌YL20的治疗组的条带明显加粗,灰度分析可看出比阪崎肠杆菌组上升0.214~0.603不等,说明加入唾液乳杆菌YL20可以在体外有效保护阪崎肠杆菌所致的细胞屏障损伤。
实施例6:唾液乳杆菌YL20体外促进粘蛋白Muc2的表达
HT-29细胞均匀铺种在六孔板中培养两天,经加入108CFU/mL唾液乳杆菌YL20样品处理24h后,ELISA检测Muc2的蛋白水平。结果发现,相对于阪崎肠杆菌组,加入唾液乳杆菌YL20的治疗组Muc2含量从1527.91上升到4040.32±0.78pg/mL,*P<0.001,有显著差异。结果显示,加入唾液乳杆菌YL20可以在体外有效保护阪崎肠杆菌所致的细胞屏障损伤。
实施例7:唾液乳杆菌YL20体外促进类器官的生长
从4周龄的小鼠体内分离小肠隐窝,在特定的培养基中进行培养。即在小鼠胃附近取约20cm的肠段,用预冷的D-PBS反复冲洗肠道内容物并除去小肠上的脂肪。然后将小肠剪成2mm长的片段,置于含有15mL D-PBS的离心管中,反复吹打肠道碎片3次,然后使其重力沉降并弃去上清。如此重复15~20次,直至上清液澄清。重悬组织碎片于25mL的细胞解离试剂中,室温缓慢摇晃15min,然后重力沉降并弃去上清液。加入10mL 0.1%BSA-PBS,上下吹打3次,然后重力沉降。轻轻吸取上清于70μm的细胞筛中进行过滤。4℃,290r/min离心5min,弃去上清。沉淀加入10mL预冷DMEM/F12培养基进行垂悬。显微镜下进行计数,取所需体积离心后,用完全培养基进行垂悬。取预热的48孔细胞培养板,每孔加入50μL垂悬液和基质胶的混合液,于37℃孵育10min。然后沿孔壁加入200μL的完全培养基,置于37℃培养箱中培养,每2天换1次培养基。
(1)EdU检测类器官增殖
1天至两天后类器官形成球形时,加入热灭活唾液乳杆菌YL20(1×104CFU/well),观察其生长状态,3天后用细胞增殖试剂盒(RiboBio,C10310-3)做EDU细胞增殖实验。检测方法按说明书进行。在Zeiss 710激光扫描共聚焦显微镜下观察细胞DNA含量。应用image J软件对各类型器官中EdU+细胞的数量进行分析。如图2、图3所示。
结果发现,EdU阳性细胞数量显著增加,从8±1上升到58±2,*P<0.001,有显著差异。结果说明,唾液乳杆菌YL20体外促进类器官的生长。
(2)检测类器官中Muc2含量
从小肠中分离肠隐窝,在基质胶中培养,大概一天后类器官形成球形时,加入热灭唾液乳杆菌YL20(1×104CFU/well),观察其生长状态,3天后取培养基上清进行ELISA实验。试剂盒采用鼠Muc2 ELISA检测试剂盒(科赛博,CSB-E15065m)。检测方法按说明书进行。
结果发现,唾液乳杆菌YL20可使Muc2蛋白的表达量从973.74±0.031上升到2218.92±0.014,与对照组相比,具有统计学意义(*P<0.05)。
实施例8:唾液乳杆菌YL20体内抑制阪崎肠杆菌
(1)动物和饲养
将购自中国食品药品监督管理实验动物资源研究所的雄性雌性各36只C57BL/6小鼠,一公一母合笼,共四组,每组三个重复,每个重复三只,置于25℃、55%湿度、12h光照/黑暗周期的对照条件下,饲喂标准实验室食品。
(2)实验流程
一周适应期,给与无限制食物和饮用水。适应期后,合笼繁殖,乳鼠出生后第一天开始对三个实验组分别灌胃唾液乳杆菌YL20(108CFU/mL)、阪崎肠杆菌(109CFU/mL)、灌胃唾液乳杆菌YL20(108CFU/mL)及阪崎肠杆菌(109CFU/mL)14天,100μL/鼠/天;对照组灌胃同等体积PBS。在试验期间内所有小鼠饮用纯净水。实验过程中每天记录体重。实验结束,眼球法取血后将其脱臼处死,取肠道及内脏进行后续检测。
(3)体重记录
为了评价唾液乳杆菌YL20对阪崎导致肠损伤的影响,每天记录乳鼠体重。与阪崎肠杆菌组终体重4.29±0.03g相比,唾液乳杆菌YL20组6.47±0.02g明显缓解了小鼠体重的减轻,说明唾液乳杆菌YL20能够体内抑制阪崎肠杆菌。
(4)比较肠道长度
解刨取乳鼠全肠,进行长度测量,结果发现,与阪崎肠杆菌组肠道长度17.03±0.03cm相比,灌喂唾液乳杆菌YL20可显著增加小鼠的肠道长度达到19.47±0.04cm,说明唾液乳杆菌YL20能够体内抑制阪崎肠杆菌。
(5)绒毛长度评价
为了进一步评价唾液乳杆菌YL20对肠道发育的影响,进行乳鼠实验并进行了H&E染色。与对照组对比,唾液乳杆菌YL20组小肠组织学分析显示绒毛更长,密度更大。如图4所示。
(6)炎症因子IL-6、IL-1β和Tnf-α检测
ELISA检测唾液乳杆菌YL20对小鼠血清中相关炎症因子的调节作用。在最后一天小鼠被处死前,眼球法取血,分离出血清,并使用ELISA检测各组小鼠血清中炎症因子IL-6、IL-1β和Tnf-α的水平。灌胃唾液乳杆菌YL20组血清中IL-6、IL-1β和Tnf-α明显低于阪崎肠杆菌组,如表3所示。
表3唾液乳杆菌YL20对小鼠血清炎症因子IL-6、IL-1β和Tnf-α的影响
实时荧光定量PCR的方法测小鼠肠道中IL-6、IL-1β和Tnf-α的mRNA水平影响。引物序列如表4。
表4 real-time PCR引物
结果相对于阪崎肠杆菌组,加入唾液乳杆菌YL20的治疗组的IL-1β mRNA水平显著下降,*P<0.01,有显著差异;IL-6 mRNA水平显著下降,*P<0.05,有显著性差异;Tnf-αmRNA水平显著下降,*P<0.01,有显著差异。结果显示,加入唾液乳杆菌YL20能下调小鼠肠道中由于阪崎肠杆菌造成的IL-6、IL-1β和Tnf-α的mRNA水平的上升,可以在体内有效保护阪崎肠杆菌所致的细胞屏障损伤。
Claims (9)
1.一株缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌(Lactobacillus salivariusYL20),保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,地址:中国微生物菌种保藏管理委员会普通微生物中心,保藏日期:2020年9月3日,保藏号:CGMCC NO:20590。
该菌株革兰氏阳性;在MRS平板培养基上生长,可形成表面圆润的小菌落,边缘整齐,有溶钙圈;在MRS液体培养基中呈均匀浑浊生长,久置菌体呈白色沉淀;最适生长温度35~38℃,适宜pH为5.0~7.0。
2.如权利要求1所述的缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌,其特征在于,所述肠炎、脑膜炎为新生儿肠炎、脑膜炎。
3.如权利要求1所述的缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌,其特征在于,所述肠炎、脑膜炎为细菌性肠炎、脑膜炎。
4.如权利要求3所述的缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌,其特征在于,所述细菌性肠炎、脑膜炎为阪崎肠杆菌诱导的肠炎、脑膜炎。
5.根据权利要求1所述的缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌,其特征在于:所述唾液乳杆菌菌株是从健康母亲的母乳中分离。
6.根据权利要求1和2所述的缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌,其特征在于:所述唾液乳杆菌能够促进肠道黏膜屏障形成、促进肠道发育,抑制阪崎肠杆菌所致的肠炎;
或者所述唾液乳杆菌能够促进肠道黏蛋白Muc2、紧密连接蛋白ZO-1、Occuldin以及闭合蛋白CLDN-1的表达,促进肠道黏膜屏障的形成;
或者所述唾液乳杆菌能够促进小鼠肠道类器官的生长,能够增加小鼠肠道绒毛的长度、降低隐窝的深度;
或者所述唾液乳杆菌能够缓解阪崎肠杆菌所致的肠道粘膜屏障和细胞屏障的损伤,抑制阪崎肠杆菌诱导的炎症因子IL-6、TNF-α、IL-1β的释放。
7.如权利要求1至5任一项所述的具有缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌在制备预防和/或治疗肠炎、脑膜炎的药物中的应用。
8.如权利要求1至5任一项所述的具有缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌在制备促进肠道发育的药物中的应用。
9.根据权利要求1至5任一项所述的具有缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌在加工生产制得的食品或饲料产品中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111268340.9A CN113846035B (zh) | 2021-10-29 | 2021-10-29 | 一株缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111268340.9A CN113846035B (zh) | 2021-10-29 | 2021-10-29 | 一株缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113846035A true CN113846035A (zh) | 2021-12-28 |
CN113846035B CN113846035B (zh) | 2024-01-26 |
Family
ID=78983418
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111268340.9A Active CN113846035B (zh) | 2021-10-29 | 2021-10-29 | 一株缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113846035B (zh) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111849805A (zh) * | 2020-06-17 | 2020-10-30 | 天津科技大学 | 一种具有抗宫颈癌作用的乳酸片球菌及应用 |
CN115725456A (zh) * | 2022-10-11 | 2023-03-03 | 四川大学 | 一种唾液乳杆菌及其应用 |
CN115927045A (zh) * | 2022-07-13 | 2023-04-07 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | 具有降胆固醇、缓解由高脂血症引起肝损伤功能的唾液乳杆菌069及其应用 |
CN115969888A (zh) * | 2022-10-25 | 2023-04-18 | 深圳未知君生物科技有限公司 | 一株唾液联合乳杆菌及其在制备治疗癌症的药物中的应用 |
CN116536225A (zh) * | 2023-07-05 | 2023-08-04 | 美维仕(北京)健康管理有限公司 | 益生菌组合物及其用途 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090214498A1 (en) * | 2005-11-21 | 2009-08-27 | Teagasc- National Diary Products Research Centre | Antimicrobial peptides and bacterial strains that produce them |
US20140286904A1 (en) * | 2011-07-27 | 2014-09-25 | Agriculture And Food Development Authority (Teagasc) | Anitmicrobial peptide produced by intestinal lactobacillus salivarius |
CN107267408A (zh) * | 2017-03-17 | 2017-10-20 | 杨凌职业技术学院 | 一种唾液乳杆菌jm55及其应用 |
CN110144310A (zh) * | 2019-05-20 | 2019-08-20 | 天津科技大学 | 一株具有缓解肠炎和促进肠道发育作用的枯草芽孢杆菌和应用 |
KR102063544B1 (ko) * | 2018-09-12 | 2020-01-09 | (주)성운파마코피아 | 항진균 활성 또는 항균 활성을 갖는 락토바실러스 살리바리우스 swpm101 |
KR102153080B1 (ko) * | 2019-12-11 | 2020-09-07 | 주식회사 에이치이엠 | 신규한 락토바실러스 살리바리우스 hem 1047 균주, 및 상기 균주 또는 이의 배양물을 포함하는 장내 환경 개선용 조성물 |
-
2021
- 2021-10-29 CN CN202111268340.9A patent/CN113846035B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090214498A1 (en) * | 2005-11-21 | 2009-08-27 | Teagasc- National Diary Products Research Centre | Antimicrobial peptides and bacterial strains that produce them |
US20140286904A1 (en) * | 2011-07-27 | 2014-09-25 | Agriculture And Food Development Authority (Teagasc) | Anitmicrobial peptide produced by intestinal lactobacillus salivarius |
CN107267408A (zh) * | 2017-03-17 | 2017-10-20 | 杨凌职业技术学院 | 一种唾液乳杆菌jm55及其应用 |
KR102063544B1 (ko) * | 2018-09-12 | 2020-01-09 | (주)성운파마코피아 | 항진균 활성 또는 항균 활성을 갖는 락토바실러스 살리바리우스 swpm101 |
CN110144310A (zh) * | 2019-05-20 | 2019-08-20 | 天津科技大学 | 一株具有缓解肠炎和促进肠道发育作用的枯草芽孢杆菌和应用 |
KR102153080B1 (ko) * | 2019-12-11 | 2020-09-07 | 주식회사 에이치이엠 | 신규한 락토바실러스 살리바리우스 hem 1047 균주, 및 상기 균주 또는 이의 배양물을 포함하는 장내 환경 개선용 조성물 |
Non-Patent Citations (1)
Title |
---|
王毅超: "肠源益生性抑菌乳酸菌的分离筛选及其抑菌物质的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, no. 9, pages 059 - 146 * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111849805A (zh) * | 2020-06-17 | 2020-10-30 | 天津科技大学 | 一种具有抗宫颈癌作用的乳酸片球菌及应用 |
CN111849805B (zh) * | 2020-06-17 | 2022-04-22 | 天津科技大学 | 一种具有抗宫颈癌作用的乳酸片球菌及应用 |
CN115927045A (zh) * | 2022-07-13 | 2023-04-07 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | 具有降胆固醇、缓解由高脂血症引起肝损伤功能的唾液乳杆菌069及其应用 |
CN115927045B (zh) * | 2022-07-13 | 2023-10-20 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | 具有降胆固醇、缓解由高脂血症引起肝损伤功能的唾液乳杆菌069及其应用 |
CN115725456A (zh) * | 2022-10-11 | 2023-03-03 | 四川大学 | 一种唾液乳杆菌及其应用 |
CN115969888A (zh) * | 2022-10-25 | 2023-04-18 | 深圳未知君生物科技有限公司 | 一株唾液联合乳杆菌及其在制备治疗癌症的药物中的应用 |
CN116536225A (zh) * | 2023-07-05 | 2023-08-04 | 美维仕(北京)健康管理有限公司 | 益生菌组合物及其用途 |
Also Published As
Publication number | Publication date |
---|---|
CN113846035B (zh) | 2024-01-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111484957B (zh) | 动物双歧杆菌乳亚种i797、其分离纯化方法及应用 | |
CN113846035B (zh) | 一株缓解肠炎、脑膜炎和促进肠道发育的唾液乳杆菌及其应用 | |
CN106834187B (zh) | 一种两歧双歧杆菌及其用途 | |
CN110122877B (zh) | 鼠李糖乳杆菌及其用途 | |
CN113604384B (zh) | 一种鼠李糖乳杆菌及其应用 | |
CN111235070A (zh) | 一株母乳婴儿源植物乳杆菌bf_15及其应用 | |
CN112625979B (zh) | 一种对抗幽门螺杆菌的干酪乳杆菌及其应用 | |
CN110144310B (zh) | 一株具有缓解肠炎和促进肠道发育作用的枯草芽孢杆菌和应用 | |
CN116024130A (zh) | 一株降血尿酸发酵乳杆菌a21215及其应用 | |
CN114752529B (zh) | 植物乳杆菌hom3201菌株及其活菌制剂、制备方法和用途 | |
CN116445356B (zh) | 一种调节肠道菌群及增强免疫力的动物双歧杆菌乳亚种ba67及其应用 | |
CN113122466A (zh) | 粪肠球菌及其应用 | |
CN114410547B (zh) | 一株能促进5-htp分泌及缓解抑郁的戊糖乳杆菌lpq1及其应用 | |
CN116286439A (zh) | 一株婴幼儿肠道来源的两歧双歧杆菌及应用 | |
CN109576165B (zh) | 一种贝酵母菌及其应用 | |
CN111743158A (zh) | 一种具有增强免疫功能的益生菌片剂及其制备方法 | |
WO2019174002A1 (zh) | 戊糖片球菌ccfm1012、其发酵食品及其在制备拮抗空肠弯曲杆菌感染药物中的应用 | |
CN115873765A (zh) | 一株类肠膜魏斯氏菌MbWp-1及其产品与应用 | |
CN114806953A (zh) | 一种具有改善1型糖尿病特性的格氏乳杆菌 | |
CN113005066A (zh) | 抗过敏、增加免疫力、降血糖、降脂减肥的复合双歧杆菌制剂及其制备方法 | |
CN116019842B (zh) | 嗜酸乳杆菌la85的抑菌新用途及其在制备缓解eiec腹泻的药物方面的用途 | |
CN114317310B (zh) | 一种抗过敏的婴儿双歧杆菌制剂及其制备方法 | |
CN116200290B (zh) | 一种具有抑制结直肠癌细胞增殖的副干酪乳酪杆菌及其应用 | |
WO2024090414A1 (ja) | 乳酸菌、該乳酸菌由来の自然免疫活性化剤及び該乳酸菌を含有する食品 | |
WO2024090415A1 (ja) | 乳酸菌、該乳酸菌由来の自然免疫活性化剤及び該乳酸菌を含有する食品 |
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 |