CN116762959A - Lactobacillus paracasei microcapsule and preparation method and application thereof - Google Patents
Lactobacillus paracasei microcapsule and preparation method and application thereof Download PDFInfo
- Publication number
- CN116762959A CN116762959A CN202210236610.6A CN202210236610A CN116762959A CN 116762959 A CN116762959 A CN 116762959A CN 202210236610 A CN202210236610 A CN 202210236610A CN 116762959 A CN116762959 A CN 116762959A
- Authority
- CN
- China
- Prior art keywords
- lactobacillus paracasei
- microcapsule
- paracasei
- intestinal
- vhprobi
- 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.)
- Pending
Links
- 241000186605 Lactobacillus paracasei Species 0.000 title claims abstract description 85
- 239000003094 microcapsule Substances 0.000 title claims abstract description 57
- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- 230000000968 intestinal effect Effects 0.000 claims abstract description 87
- 208000026278 immune system disease Diseases 0.000 claims abstract description 49
- 238000004321 preservation Methods 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 16
- 239000000725 suspension Substances 0.000 claims description 15
- 230000000694 effects Effects 0.000 claims description 14
- 239000011259 mixed solution Substances 0.000 claims description 13
- 239000000463 material Substances 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 10
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 8
- 239000004375 Dextrin Substances 0.000 claims description 8
- 229920001353 Dextrin Polymers 0.000 claims description 8
- 108010010803 Gelatin Proteins 0.000 claims description 8
- 229920002472 Starch Polymers 0.000 claims description 8
- 235000019425 dextrin Nutrition 0.000 claims description 8
- 239000008273 gelatin Substances 0.000 claims description 8
- 229920000159 gelatin Polymers 0.000 claims description 8
- 235000019322 gelatine Nutrition 0.000 claims description 8
- 235000011852 gelatine desserts Nutrition 0.000 claims description 8
- 239000000661 sodium alginate Substances 0.000 claims description 8
- 235000010413 sodium alginate Nutrition 0.000 claims description 8
- 229940005550 sodium alginate Drugs 0.000 claims description 8
- 239000008107 starch Substances 0.000 claims description 8
- 235000019698 starch Nutrition 0.000 claims description 8
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- 238000007711 solidification Methods 0.000 claims description 6
- 230000008023 solidification Effects 0.000 claims description 6
- 238000001179 sorption measurement Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 235000013376 functional food Nutrition 0.000 claims description 5
- 239000011162 core material Substances 0.000 claims description 4
- 241000193830 Bacillus <bacterium> Species 0.000 abstract description 29
- 235000013351 cheese Nutrition 0.000 abstract description 29
- 208000024891 symptom Diseases 0.000 abstract description 20
- 241000894006 Bacteria Species 0.000 abstract description 19
- 235000011389 fruit/vegetable juice Nutrition 0.000 abstract description 16
- 210000004051 gastric juice Anatomy 0.000 abstract description 15
- 238000012216 screening Methods 0.000 abstract description 10
- 235000013305 food Nutrition 0.000 abstract description 8
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 230000036541 health Effects 0.000 abstract description 4
- 244000005700 microbiome Species 0.000 abstract description 4
- 102000004127 Cytokines Human genes 0.000 abstract description 3
- 108090000695 Cytokines Proteins 0.000 abstract description 3
- 230000000770 proinflammatory effect Effects 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 description 43
- 241000699670 Mus sp. Species 0.000 description 36
- 239000006041 probiotic Substances 0.000 description 34
- 235000018291 probiotics Nutrition 0.000 description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 28
- 230000000529 probiotic effect Effects 0.000 description 26
- 210000004027 cell Anatomy 0.000 description 25
- 238000000855 fermentation Methods 0.000 description 24
- 230000004151 fermentation Effects 0.000 description 24
- 239000007788 liquid Substances 0.000 description 24
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 23
- 239000001963 growth medium Substances 0.000 description 23
- 239000000243 solution Substances 0.000 description 22
- 108010058846 Ovalbumin Proteins 0.000 description 20
- 229940092253 ovalbumin Drugs 0.000 description 20
- 241001465754 Metazoa Species 0.000 description 18
- 241000894007 species Species 0.000 description 15
- 238000012360 testing method Methods 0.000 description 14
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 230000001965 increasing effect Effects 0.000 description 12
- 210000004877 mucosa Anatomy 0.000 description 12
- 235000012000 cholesterol Nutrition 0.000 description 11
- 238000012258 culturing Methods 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 11
- 229940039696 lactobacillus Drugs 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 102100037850 Interferon gamma Human genes 0.000 description 10
- 108010074328 Interferon-gamma Proteins 0.000 description 10
- 108090001005 Interleukin-6 Proteins 0.000 description 10
- 102000004889 Interleukin-6 Human genes 0.000 description 10
- 241000186660 Lactobacillus Species 0.000 description 10
- 239000001888 Peptone Substances 0.000 description 10
- 108010080698 Peptones Proteins 0.000 description 10
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 10
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 10
- 238000009630 liquid culture Methods 0.000 description 10
- 235000019319 peptone Nutrition 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000011081 inoculation Methods 0.000 description 9
- 239000002504 physiological saline solution Substances 0.000 description 9
- 102000003814 Interleukin-10 Human genes 0.000 description 8
- 108090000174 Interleukin-10 Proteins 0.000 description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 8
- 206010070834 Sensitisation Diseases 0.000 description 8
- 230000002757 inflammatory effect Effects 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 230000008313 sensitization Effects 0.000 description 8
- 206010020751 Hypersensitivity Diseases 0.000 description 7
- 241001052560 Thallis Species 0.000 description 7
- 208000026935 allergic disease Diseases 0.000 description 7
- 239000003242 anti bacterial agent Substances 0.000 description 7
- 229940088710 antibiotic agent Drugs 0.000 description 7
- 229940041514 candida albicans extract Drugs 0.000 description 7
- -1 citric acid diamine Chemical class 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 239000012138 yeast extract Substances 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 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 6
- 108010002616 Interleukin-5 Proteins 0.000 description 6
- 102000000743 Interleukin-5 Human genes 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 210000001035 gastrointestinal tract Anatomy 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 230000036039 immunity Effects 0.000 description 6
- 210000001630 jejunum Anatomy 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 5
- 241000606125 Bacteroides Species 0.000 description 5
- 102000057297 Pepsin A Human genes 0.000 description 5
- 108090000284 Pepsin A Proteins 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 230000007815 allergy Effects 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 229940111202 pepsin Drugs 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 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 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 238000011725 BALB/c mouse Methods 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- 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 4
- 208000000260 Warts Diseases 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 4
- 229910052782 aluminium Inorganic materials 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 4
- 210000002443 helper t lymphocyte Anatomy 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 4
- 235000019341 magnesium sulphate Nutrition 0.000 description 4
- 229940099596 manganese sulfate Drugs 0.000 description 4
- 239000011702 manganese sulphate Substances 0.000 description 4
- 235000007079 manganese sulphate Nutrition 0.000 description 4
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 4
- 201000010153 skin papilloma Diseases 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- 206010003694 Atrophy Diseases 0.000 description 3
- 206010012735 Diarrhoea Diseases 0.000 description 3
- 235000010469 Glycine max Nutrition 0.000 description 3
- 244000068988 Glycine max Species 0.000 description 3
- 244000199866 Lactobacillus casei Species 0.000 description 3
- 235000013958 Lactobacillus casei Nutrition 0.000 description 3
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 239000004098 Tetracycline Substances 0.000 description 3
- 230000037444 atrophy Effects 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 210000002919 epithelial cell Anatomy 0.000 description 3
- 229960003276 erythromycin Drugs 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 210000003608 fece Anatomy 0.000 description 3
- 230000002496 gastric effect Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 229940017800 lactobacillus casei Drugs 0.000 description 3
- 230000003859 lipid peroxidation Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 229960002180 tetracycline Drugs 0.000 description 3
- 229930101283 tetracycline Natural products 0.000 description 3
- 235000019364 tetracycline Nutrition 0.000 description 3
- 150000003522 tetracyclines Chemical class 0.000 description 3
- 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 2
- 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
- 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 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-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
- 238000007400 DNA extraction Methods 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
- 108010065805 Interleukin-12 Proteins 0.000 description 2
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 2
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 2
- 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 2
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 239000005662 Paraffin oil Substances 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 230000000172 allergic effect Effects 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- 208000010668 atopic eczema Diseases 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- 235000015278 beef Nutrition 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
- 210000000601 blood cell Anatomy 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Natural products OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000007760 free radical scavenging Effects 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 239000003228 hemolysin Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000006058 immune tolerance Effects 0.000 description 2
- 210000004347 intestinal mucosa Anatomy 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 2
- 235000020778 linoleic acid Nutrition 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 210000004400 mucous membrane Anatomy 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 238000005502 peroxidation Methods 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000003860 storage Methods 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
- 235000000346 sugar Nutrition 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 1
- XUCIJNAGGSZNQT-JHSLDZJXSA-N (R)-amygdalin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O[C@@H](C#N)C=2C=CC=CC=2)O1 XUCIJNAGGSZNQT-JHSLDZJXSA-N 0.000 description 1
- OCZVHBZNPVABKX-UHFFFAOYSA-N 1,1-diphenyl-2-(2,4,6-trinitrophenyl)hydrazine;ethanol Chemical compound CCO.[O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1NN(C=1C=CC=CC=1)C1=CC=CC=C1 OCZVHBZNPVABKX-UHFFFAOYSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- 241001019659 Acremonium <Plectosphaerellaceae> Species 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 241000234282 Allium Species 0.000 description 1
- 108091093088 Amplicon Proteins 0.000 description 1
- 238000009631 Broth culture Methods 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose 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)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 1
- QWIZNVHXZXRPDR-UHFFFAOYSA-N D-melezitose Natural products O1C(CO)C(O)C(O)C(O)C1OC1C(O)C(CO)OC1(CO)OC1OC(CO)C(O)C(O)C1O QWIZNVHXZXRPDR-UHFFFAOYSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- 206010012442 Dermatitis contact Diseases 0.000 description 1
- 208000027244 Dysbiosis Diseases 0.000 description 1
- 206010013954 Dysphoria Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 208000004262 Food Hypersensitivity Diseases 0.000 description 1
- 206010016946 Food allergy Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 108010006464 Hemolysin Proteins Proteins 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 240000001929 Lactobacillus brevis Species 0.000 description 1
- 235000013957 Lactobacillus brevis Nutrition 0.000 description 1
- 241000186673 Lactobacillus delbrueckii Species 0.000 description 1
- 241000186604 Lactobacillus reuteri Species 0.000 description 1
- 201000010538 Lactose Intolerance Diseases 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 230000002292 Radical scavenging effect Effects 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- NGFMICBWJRZIBI-JZRPKSSGSA-N Salicin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O1)c1c(CO)cccc1 NGFMICBWJRZIBI-JZRPKSSGSA-N 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241001591005 Siga Species 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 210000004241 Th2 cell Anatomy 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 229930003471 Vitamin B2 Natural products 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- NGFMICBWJRZIBI-UHFFFAOYSA-N alpha-salicin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UHFFFAOYSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 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 1
- 230000003321 amplification Effects 0.000 description 1
- 229940089837 amygdalin Drugs 0.000 description 1
- YZLOSXFCSIDECK-UHFFFAOYSA-N amygdalin Natural products OCC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC(C#N)c3ccccc3 YZLOSXFCSIDECK-UHFFFAOYSA-N 0.000 description 1
- 230000003266 anti-allergic effect Effects 0.000 description 1
- 239000000043 antiallergic agent Substances 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 229940125715 antihistaminic agent Drugs 0.000 description 1
- 239000000739 antihistaminic agent Substances 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- DLRVVLDZNNYCBX-ZZFZYMBESA-N beta-melibiose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)O1 DLRVVLDZNNYCBX-ZZFZYMBESA-N 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960002227 clindamycin Drugs 0.000 description 1
- 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 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000002288 cocrystallisation Methods 0.000 description 1
- 230000000112 colonic effect Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 208000010247 contact dermatitis Diseases 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 229940068840 d-biotin Drugs 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000023011 digestive tract development Effects 0.000 description 1
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000007140 dysbiosis Effects 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- YGHHWSRCTPQFFC-UHFFFAOYSA-N eucalyptosin A Natural products OC1C(O)C(O)C(CO)OC1OC1C(OC(C#N)C=2C=CC=CC=2)OC(CO)C(O)C1O YGHHWSRCTPQFFC-UHFFFAOYSA-N 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000020932 food allergy Nutrition 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 210000004211 gastric acid Anatomy 0.000 description 1
- 238000003304 gavage Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 230000009931 harmful effect Effects 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
- 230000008938 immune dysregulation Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229940001882 lactobacillus reuteri Drugs 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- OQTQHQORDRKHFW-UHFFFAOYSA-L manganese(2+);sulfate;heptahydrate Chemical compound O.O.O.O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O OQTQHQORDRKHFW-UHFFFAOYSA-L 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- QWIZNVHXZXRPDR-WSCXOGSTSA-N melezitose Chemical compound O([C@@]1(O[C@@H]([C@H]([C@@H]1O[C@@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O)CO)CO)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O QWIZNVHXZXRPDR-WSCXOGSTSA-N 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000005142 microbroth dilution method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000008807 pathological lesion Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose 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[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 238000002407 reforming Methods 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 206010039083 rhinitis Diseases 0.000 description 1
- 229960002477 riboflavin Drugs 0.000 description 1
- NGFMICBWJRZIBI-UJPOAAIJSA-N salicin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UJPOAAIJSA-N 0.000 description 1
- 229940120668 salicin Drugs 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- MSXHSNHNTORCAW-GGLLEASOSA-M sodium;(2s,3s,4s,5r,6s)-3,4,5,6-tetrahydroxyoxane-2-carboxylate Chemical compound [Na+].O[C@H]1O[C@H](C([O-])=O)[C@@H](O)[C@H](O)[C@H]1O MSXHSNHNTORCAW-GGLLEASOSA-M 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-O vancomycin(1+) Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C([O-])=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)[NH2+]C)[C@H]1C[C@](C)([NH3+])[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-O 0.000 description 1
- 239000011716 vitamin B2 Substances 0.000 description 1
- 235000019164 vitamin B2 Nutrition 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of functional microorganism screening and application, and particularly provides a lactobacillus paracasei microcapsule and a preparation method and application thereof. The lactobacillus paracasei microcapsule comprises a protein with a preservation number of CCTCC No: the novel cheese bacillus paracasei VHProbi F22 strain of M2019941 has high viable bacteria content and good tolerance to artificial gastric juice and artificial intestinal juice, can reduce the level of proinflammatory cytokines, lighten the symptom of intestinal immune disorder, and can be widely applied to the fields of foods, health products and the like.
Description
Technical Field
The invention belongs to the technical field of application of probiotics, and particularly relates to a lactobacillus paracasei microcapsule, a preparation method and application thereof.
Background
During the last decades, the allergic phenomena of the human body have assumed a state of dramatic growth, influenced by western lifestyles or industrialized lifestyles. Examples of diseases caused by allergic reactions include rhinitis, asthma, food allergy, atopic dermatitis, contact dermatitis, etc. There is increasing evidence that intestinal flora is an important environmental factor affecting immune tolerance.
The altered order of intestinal flora structure establishment results in altered differentiation of helper T cells, imbalance of helper T cells type i (Th 1 cells) and helper T cells type ii (Th 2 cells) in the immune system of the body, and in particular, a balance of helper T cells tends to Th2 cells. For example, in infants, the use of broad-spectrum antibiotics can lead to altered intestinal flora and dysbiosis, and increased susceptibility to allergy. In fact, epidemiological studies in recent years have shown that environmental factors affecting colonisation of the intestinal flora have a certain association with allergy risk.
At present, the allergic diseases mainly take antiallergic drugs and antihistamines as the main treatment, but the allergic diseases cannot be fundamentally solved for patients with weak immunity, such as lactose intolerance caused by imperfect intestinal development of infants; in addition, histamine can cause adverse reactions such as reduced attention, slow response and the like of human bodies. Therefore, it is of great importance to find more rational methods for treating allergies. In recent years, the treatment of allergic diseases by probiotics has received increasing attention. Clinical studies have shown that probiotics can induce organisms to produce immune responses and immune tolerance, and the ability of regulating immunity can be improved by presenting antigens through gastrointestinal mucosa. The probiotics can increase IFN-gamma, IL-2 and IL-12, promote T cell differentiation to Th1, reduce IL-4 and reduce T cell differentiation to Th2, regulate Th1 and Th2 immune balance and further reach antiallergic effect.
There have been some studies in the prior art on probiotics to regulate allergic reactions. For example, chinese patent publication No. CN 111560330A discloses a strain of Lactobacillus casei which can regulate immunity, regulate M1/M2 type macrophage balance, increase the number of Treg cells, and improve colonitis symptoms and cervical cancer resistance of mice; chinese patent publication No. CN 102373173B discloses a Lactobacillus casei strain which can induce macrophages to produce IL-12 and IL-6 and TNF-alpha and has the function of enhancing immunoregulation; the Chinese patent publication No. CN 101139557B discloses a Lactobacillus casei strain which can influence the T lymphocyte subgroup of the peripheral blood of a mouse, the serum IgG level and the intestinal mucosa SigA level of the mouse, and enhance the cellular immunity, the humoral immunity and the intestinal mucosa local immunity functions of the mouse. The screening of probiotics with the function of regulating immunity and relieving symptoms of intestinal immune disorder has very important application value.
Disclosure of Invention
The invention provides a lactobacillus paracasei microcapsule, a preparation method and application thereof, and aims to solve the problems in the prior art. The provided lactobacillus paracasei microcapsule has high viable bacteria content and good tolerance to artificial gastric juice and artificial intestinal juice, can reduce the level of proinflammatory cytokines, lighten the symptom of intestinal immune disorder, and can be widely applied to the fields of foods, health products and the like.
The invention first provides a lactobacillus paracasei microcapsule, wherein the core material of the microcapsule comprises lactobacillus paracasei.
The cheese bacillus paracasei is named as cheese bacillus paracasei VHProbi F22 (Lacticaseibacillus paracasei VHProbi F), and is preserved in China center for type culture collection (CCTCC No: m2019941.
The microcapsule also comprises starch in the core material.
The embedding material used in the outer layer of the microcapsule comprises sodium alginate, gelatin and dextrin.
The microcapsule is prepared by mixing porous starch 2-5% (W/W) with Lactobacillus paracasei bacterial suspension, adjusting pH to 6.0, and oscillating at 25-35deg.C for 30min; after full adsorption, the following outer layer embedding materials are added in sequence according to the weight percentage: 1-3% of sodium alginate, 1-5% of gelatin and 1-5% of dextrin, and uniformly stirring; dropping 2-6% (W/W) calcium chloride solution until solidification of the mixed solution occurs; freeze drying to obtain the microcapsule of Lactobacillus paracasei.
Further preferably, the microcapsule is prepared by the following method: mixing 3% porous starch with Lactobacillus paracasei suspension, adjusting pH to 6.0, and oscillating at 25-35deg.C for 30min; after full adsorption, the following outer layer embedding materials are added in sequence according to the weight percentage: 1.5% of sodium alginate, 2.5% of gelatin and 3.5% of dextrin, and uniformly stirring; dropping 6% (W/W) calcium chloride solution until solidification of the mixed solution occurs; freeze drying to obtain the microcapsule of Lactobacillus paracasei.
The invention also provides application of the lactobacillus paracasei microcapsule in regulating intestinal immune disorder.
The invention also provides application of the lactobacillus paracasei microcapsule in preparing functional food with the effect of regulating intestinal immune disorder.
The invention also provides a functional food comprising the lactobacillus paracasei microcapsule.
The cheese bacillus paracasei VHProbi F22 provided by the invention has strong tolerance to artificial intestinal juice; the VHProbi F22 strain does not produce hemolysin, does not dissolve blood cells, is sensitive to common antibiotics such as erythromycin and tetracycline, and has good biological safety; can tolerate higher salinity with a maximum salt tolerance concentration of 6%.
The strain has strong oxidation resistance, and can effectively degrade cholesterol; in addition, the hydrophobicity of the cell surface of the strain is more than 70%, and the strain has certain adhesion capability.
The cheese bacillus paracasei VHProbi F22 has better effect of regulating intestinal immune disorder. The use of the provided cheese bacillus paracasei VHProbi F22 can effectively relieve intestinal immune disorder symptoms.
The use of the Lactobacillus paracasei VHProbi F22 can effectively reduce IL-5, IL-6, IFN-gamma, TNF-alpha and OVA-IgE in serum of an intestinal immune disorder model mouse, and the effect of a probiotic pretreatment group is better than that of a post-treatment group.
From the HE staining results of the jejunum tissues of the mice, after being treated by the Lactobacillus paracasei VHProbi F22, the jejunum mucosa villus swelling degree can be reduced, the phenomenon of epithelial cell shedding can be reduced, the mucosa lamina propria atrophy degree can be reduced, jejunum mucosa villus has better integrity, and the effect of the probiotic pretreatment group is better than that of the post-treatment group.
The use of the Lactobacillus paracasei VHProbi F22 can reduce the wart micro-bacteria in the intestinal tract, increase the bacteroides, lead the probiotic bacteria to belong to the bacteroides in most cases, lead the intestinal flora structure of the allergic mouse model to be more similar to the normal intestinal tract, and lead the intestinal flora diversity to be increased.
The cheese-bacterium paracasei VHProbi F22 provided by the invention has no toxic or harmful effect on organisms, can be added into food, can be prepared into functional food for relieving intestinal immune disorder, and has wide application prospect.
The microcapsule of the cheese bacillus paracasei provided by the invention has high viable bacteria content, has strong tolerance to artificial gastric juice and intestinal juice, has simple preparation process, low embedding material cost and low industrialization cost, can effectively improve the stability of the cheese bacillus paracasei VHProbi F22 in the storage, processing and transportation processes, effectively improves the actual use effect of the cheese bacillus paracasei VHProbi F22, and can be widely applied to the fields of foods, health products and the like.
Drawings
FIG. 1 is a photograph of a cultured colony;
FIG. 2 is a protein fingerprint;
FIG. 3 is a chart of Riboprinter fingerprints;
FIG. 4 is a RAPD fingerprint pattern;
FIG. 5 is a map of rep-PCR fingerprints;
fig. 6 is a graph of symptoms score and wet stool frequency for intestinal immune disorder, where P <0.05 compared to intestinal immune disorder group;
FIG. 7 is a graph showing the results of HE staining of jejunum in each group of mice;
fig. 8 is a graph of cytokine levels for each group, where P <0.05 compared to the group of intestinal immune disorders;
FIG. 9 is a plot of species diversity dilution;
FIG. 10 is a graph of LAD values of species abundance for groups O1 and O2 with statistical differences;
FIG. 11 is a column cumulative plot of the relative abundance of intestinal flora in each group of mice.
Detailed Description
The screening method of the present invention is not limited to the examples, but known screening methods can be used to achieve the screening purpose, and the screening description of the examples is only illustrative of the present invention and is not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The invention will be further illustrated with reference to specific examples.
Example 1 isolation screening of Lactobacillus paracasei VHProbi F22
1. Lactobacillus primary screening
Preparing MRS (Man Rogosa Sharpe) agar medium: 1000mL of purified water, 10g of peptone, 10g of beef extract, 5.0g of yeast extract, 5g of sodium acetate, 5g of glucose, 2g of monopotassium phosphate, 1.0mL of Tween 80, 2.0g of citric acid diamine, 20g of calcium carbonate, 0.58g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate heptahydrate, 15g of agar, pH adjustment of 6.2-6.5 and high-pressure sterilization at 121 ℃ for 15min.
Taking 1g of cheese from the pasture herdsman fermentation of the inner Mongolian tin Lin Guole allium, diluting with sterile normal saline, putting into a sterile sample bag, and beating and mixing uniformly with a homogenizer; and (3) taking 100 mu L of the mixed solution, carrying out gradient dilution, coating the mixed solution on an MRS agar medium, and then culturing at 37 ℃ for 48 hours, and carrying out microscopic examination on a single colony after the plate grows. Based on the microscopic examination, the applicant screened 11 potential Lactobacillus strains designated NL-1, NL-2, NL-3, NL-4, NL-5, NL-6, NL-7, NL-8, NL-9, NL-10, NL-11, respectively.
2. Lactobacillus re-screening
Preparing 1L of MRS liquid culture medium, sterilizing at 115 ℃ for 30min, cooling the culture medium, adding 3.2g of pig mucosa pepsin, shaking to dissolve, and placing in a 37 ℃ water bath shaking table for 1h to obtain the acid-resistant culture medium.
The 11 strains of lactic acid bacteria NL-1, NL-2, NL-3, NL-4, NL-5, NL-6, NL-7, NL-8, NL-9, NL-10 and NL-11 obtained by screening were inoculated into the acid resistant medium in an amount of 6%, and subjected to stationary culture at 37℃for 48 hours, and the fermentation broth was counted.
The results show that the logarithmic values of the viable bacteria amounts in the 11 strains of lactobacillus fermentation broth are 7.19, 8.22, 7.16, 7.52, 5.83, 5.61, 7.85, 6.74, 7.32, 7.08 and 8.58Log CFU/mL respectively, the viable bacteria amount is the largest after the NL-11 strain is subjected to the acid-resistant culture medium rescreening, and the logarithmic value of the bacterial amount is as high as 8.58Log CFU/mL. Therefore, the NL-11 strain screened by the method has the highest acid resistance.
Example 2 identification of strains
2.1 colony morphology identification
NL-11 strain was inoculated on MRS agar medium, and after 48h of culture at 37 ℃, NL-11 single colony was seen to be rough and grey white, and was long rod-shaped or short rod-shaped under a microscope, both ends were flush, and NL-11 single colony and photo under an optical microscope were shown in FIG. 1.
2.2 identification of physiological and Biochemical Properties
The inoculum preparation in this example was as follows: under the aseptic condition, a proper amount of fresh NL-11 bacterial liquid is taken, centrifuged for 5min at 5000rpm/min, washed for 2 times by PBS buffer, and then the bacterial cells are diluted by 50 times after being weighted by the same volume of PBS buffer, so as to be used as an inoculation liquid.
1. Salinity tolerance test
Under aseptic conditions, 190. Mu.L of BSM liquid medium with salt concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% was added to the 96-well plate, respectively, 3 replicates of each salt concentration, and then 10. Mu.L of inoculum was added thereto, and the wells without inoculation were used as controls. 50. Mu.L of autoclaved paraffin oil was added to each well to prevent evaporation of water during the culture. Culturing at 37deg.C, and observing whether the culture medium becomes turbid.
The results showed that NL-11 strain grew at 1% -6% salt concentration and did not grow at 7% -8% salt concentration, with a maximum tolerant salt concentration of 6%.
2. Catalase experiment
A fresh bacterial solution was taken, dropped onto a clean glass slide, and then a drop of 3% hydrogen peroxide solution was added dropwise thereto, and no bubbles were observed to be generated by the NL-11 strain, which was a negative reaction.
3. Carbon source metabolism test
The basal medium formulation used in this example is as follows:
1.5g of peptone; 0.6g of yeast extract; tween 80.1 g; 0.5mL of saline solution; 18mg of phenol red; distilled water 100mL; pH 7.4.+ -. 0.2. Salt solution components: mgSO (MgSO) 4 ·7H 2 O 11.5g，MnSO 4 ·4H 2 O2.8 g, distilled water 100mL.
A10 g/100mL solution of sugar, alcohol and glycoside carbohydrate was prepared and filtered with a 0.22 μm sterile filter. Under aseptic conditions, 20. Mu.L of sterilized carbohydrate solution, 4 per carbohydrate, was added to the 96-well plate, then 170. Mu.L of sterilized phenol red-containing basal medium was added, and 10. Mu.L of inoculum was added, without inoculating the reaction well as a control. 50. Mu.L of liquid paraffin was added to each well to prevent evaporation of water during the culture. Anaerobic culture at 37deg.C, and observing color change of culture medium with phenol red as indicator. The specific results are shown in Table 1.
TABLE 1 carbon source metabolism results of NL-11 Strain
| Cellobiose | Melibiose | Raffinose | Mannitol (mannitol) | Amygdalin | Sucrose | Galactose |
| + | - | - | + | + | + | + |
| Lactose and lactose | Maltose | Mannose | Salicin | Trehalose | Arabinose (Arabic sugar) | Gluconic acid sodium salt |
| + | + | + | - | + | - | + |
| Melezitose | Ribose | Sorbitol | Xylose | Rhamnose (rhamnose) | / | / |
| + | + | + | - | - | / | / |
Note that: a "+" positive response; "-" negative reaction.
2.3 MALDI-TOF-MS detection of full protein expression of strains
Inoculating fresh bacterial liquid into MRS liquid culture medium according to 0.1% inoculum size, culturing at 37deg.C and 150rpm for 48 hr, collecting bacterial cells, washing with sterile water for 4 times, and air drying surface water. And then a small amount of fresh thalli is uniformly coated on a target plate in a film form, 1 mu L of lysate is added to cover the sample, after the sample is dried, 1 mu L of matrix solution is added to cover the sample, after the sample is dried, the sample target is put into a mass spectrometer for identification. The co-crystallization film formed by the sample and the matrix is irradiated by laser to ionize the protein in the sample, the ions are accelerated to fly through a flight pipeline under the action of an electric field of 10 KV to 20KV, and the molecular weight of the protein is detected according to different flight time reaching a detector. Protein fingerprint is obtained by utilizing Autofms 1000 analysis software Autof Analyzer v1.0, and the main ion peaks of the NL-11 strain are as follows: m/z9397.500, 7482.800, 6867.117, 5893.371, 5303.893, 4452.130, 4699.426, the results are shown in FIG. 2.
2.4 molecular biological characterization
2.4.1 16s rDNA Gene sequence analysis
1. Genomic DNA extraction
Reference was made to the Tiangen bacterial genomic DNA extraction kit (catalog number: DP 302).
2. 16s rDNA Gene amplification
1) Primer sequence:
27F：AGAGTTTGATCCTGGCTCA；
1492R：GGTTACCTTGTTACGACTT。
2) Reaction system (50. Mu.L)
Table 2:16s rDNA PCR amplification System Table
3) Electrophoresis verifies that the PCR product meets the requirement when the nucleic acid electrophoresis result is about 1500 bp.
4) Sequencing of PCR products
The 16s rDNA sequence of the NL-11 strain SEQ ID NO. 1 was obtained by sequencing and the sequences were aligned in NCBI database to preliminarily determine that the NL-11 strain was Lactobacillus paracasei.
catgcggcgtgctatacatgcagtcgaacgagttctcgttgatgatcggtgcttgcaccgagattcaacatggaacgagtggcggacgggtgagtaacacgtgggtaacctgcccttaagtgggggataacatttggaaacagatgctaataccgcatagatccaagaaccgcatggttcttggctgaaagatggcgtaagctatcgcttttggatggacccgcggcgtattagctagttggtgaggtaatggctcaccaaggcgatgatacgtagccgaactgagaggttgatcggccacattgggactgagacacggcccaaactcctacgggaggcagcagtagggaatcttccacaatggacgcaagtctgatggagcaacgccgcgtgagtgaagaaggctttcgggtcgtaaaactctgttgttggagaagaatggtcggcagagtaactgttgtcggcgtgacggtatccaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttatccggatttattgggcgtaaagcgagcgcaggcggttttttaagtctgatgtgaaagccctcggcttaaccgaggaagcgcatcggaaactgggaaacttgagtgcagaagaggacagtggaactccatgtgtagcggtgaaatgcgtagatatatggaagaacaccagtggcgaaagcggctgtctggtctgtaactgacgctgaggctcgaaagcatgggtagcgaacaggattagataccctggtagtccatgccgtaaacgatgaatgctaggtgttggagggtttccgcccttcagtgccgcagctaacgcattaagcattccgcctggggagtacgaccgcaaggttgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcttttgatcacctgagagatcaggtttccccttcgggggcaaaatgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttatgactagttgccagcatttagttgggcactctagtaagactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggatggtacaacgagttgcgagaccgcgaggtcaagctaatctcttaaagccattctcagttcggactgtaggctgcaactcgcctacacgaagtcggaatcgctagtaatcgcggatcagcacgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgagagtttgtaacacccgaagccggtggcgtaaccctttagggagcgagccgtctaaggtgacaagg。
2.4.2 Riboprinter fingerprint
The purified single colony is dipped from an agar culture medium flat plate by a fungus taking rod, the single colony is placed into a sample tube with buffer solution, the single colony is stirred by a hand-held stirrer to be suspended in the buffer solution, then the sample holder is placed into a heater for inactivation and then placed into a Riboprinter system, and after DNA preparation, film transfer, imaging detection and data processing, the Riboprinter fingerprint of the strain NL-11 is obtained (figure 3).
2.4.3 RAPD and rep-PCR fingerprint identification
1. RAPD fingerprint identification
1) Primer sequence: m13 (5'-GAGGGTGGCGGTTCT-3');
2) RAPD reaction system
TABLE 3 RAPD reaction System
3) Electrophoresis
1.5% agarose gel plates were prepared, DL2000 DNA markers were used as a result control, 100V electrophoresis was performed for 80min at a constant pressure, and finally the electropherograms were detected using a gel imaging system. RAPD finger prints of NL-11 strain are shown in FIG. 4.
2. rep-PCR fingerprint
1) rep-PCR primer
CTACGGCAAGGCGACGCTGACG。
2) reaction system of rep-PCR
TABLE 4 rep-PCR reaction System
3) Electrophoresis
DL2000 DNA Marker served as a result control. Detecting the amplification result by 100V voltage and 80min electrophoresis time. The rep-PCR fingerprint of NL-11 strain is shown in FIG. 5.
In summary, colony morphology and physiological and biochemical characteristic results of NL-11 strain were uploaded to the website http:// www.tgw1916.net/bacteria_log_desktop. Html, and aligned in combination with the results published in the literature De Clerck E, et al systems and applied microbiology,2004,27 (1) 50. From the results of the molecular biology identification, it can be concluded that the NL-11 strain is a new strain of Lactobacillus paracasei, designated as Lactobacillus paracasei VHProbi F22.
Example 3 test of resistance of Lactobacillus paracasei VHProbi F22 to artificial gastric juice and artificial intestinal juice
3.1 preparation of artificial gastric juice
5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl are weighed respectively, 1000mL of distilled water is added, pH is adjusted to 3.0 by dilute hydrochloric acid, and then sterilization is carried out for 20min at 115 ℃. Then 3.2g of pig mucosa pepsin is added before use, the pig mucosa pepsin is uniformly shaken and dissolved, and the mixture is placed in a water bath shaker at 37 ℃ for warm water bath for 1 hour so as to simulate the temperature of a human body.
3.2 preparation of artificial intestinal juice
Respectively weighing peptone 5g, yeast extract 2.5g, glucose 1g, KH 2 PO 4 6.8g and 3.0g of ox gall salt, 77mL of 0.2mol/L NaOH solution is added, the volume is fixed to 1000mL, the pH is regulated to 6.8+/-0.1 by dilute hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized for 20min at 115 ℃. Then adding 1g of pancreatin before use, shaking to dissolve, and placing in a water bath shaker at 37 ℃ for warm water bath for 1h to simulate the temperature of human body.
3.2 test methods
2mL of fresh bacterial liquid is taken, the bacterial liquid is collected by centrifugation at 5000rpm/min for 5min, the bacterial liquid is washed 3 times by physiological saline, and then 2mL of physiological saline is used as inoculation liquid for resuspension. 1mL of the inoculation liquid is taken and added into 24mL of artificial intestinal juice, and the mixture is placed on a water bath shaking table (200 rpm/min) at 37 ℃ for 3 hours, 1mL of sample is taken, and the viable bacteria amount is detected.
The viable bacteria counting method is used for measuring the bacterial amount according to national standard GB 4789.35-2016-lactobacillus test for food microorganism test, and the viable bacteria amount (Log CFU/mL) of the strain after artificial intestinal juice digestion is shown in Table 5.
TABLE 5 viable count after digestion with Artificial gastric juice and Artificial intestinal juice
As can be seen from Table 5, the viable bacteria amount of the lactobacillus VHProbi F22 screened by the present invention was reduced by about 1.2Log CFU/mL after being digested by artificial gastric juice and artificial intestinal juice. Therefore, the strain has strong tolerance to artificial gastric juice and artificial intestinal juice.
Example 4 antibiotic resistance experiment with Lactobacillus paracasei VHProbi F22
1) Antibiotic formulation
Ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline and vancomycin are prepared into stock solution of 2048 mug/mL, and the stock solution is preserved at-20 ℃ for standby. When in use, the stock solution is serially diluted by 2 times by using BSM liquid culture medium to form use solution, and the gradient dilution concentration is 1-1024 mu g/mL and total 11 gradients.
2) Preparation of inoculum
Taking a proper amount of fresh bacterial liquid (24 h,37 ℃ for culture), centrifuging at 5000rpm for 5min, washing once with sterile physiological saline, and diluting 50 times after re-suspending bacterial cells with the same volume of physiological saline to obtain an inoculation liquid.
3) Determination of minimum inhibitory concentration MIC value of antibiotics for Lactobacillus paracasei VHProbi F22 by micro broth dilution method
an MRS liquid culture medium without antibiotics is added to the 1 st column of the 96-well plate as a negative control, 190 mu L of MRS liquid culture medium with antibiotics with different concentrations is sequentially added to the 2 nd to 12 th columns, 10 mu L of the inoculation liquid is inoculated respectively, 3 parallel wells are made, and 1 well of the non-added bacteria liquid is used as a blank.
b. 50. Mu.L of paraffin oil was added to cover the water and prevent evaporation.
c. The 96-well plate was incubated at 37℃for 24 hours, then removed, and OD was measured 600 Values, MIC values of antibiotics against strains were counted with 24h results, and specific results are shown in table 6.
TABLE 6 antibiotic MIC values for Lactobacillus paracasei VHProbi F22
MIC units μg/mL
From the results shown in Table 6, the Lactobacillus paracasei VHProbi F22 provided by the invention is sensitive to common antibiotics such as erythromycin and tetracycline, and has good biological safety.
EXAMPLE 5 measurement of antioxidant function of Lactobacillus paracasei VHProbi F22
1. Determination of DPPH free radical scavenging ability of Strain
1) Preparation of PBS bacterial suspension
Single colony with excellent growth state is inoculated into 3mL of MRS liquid culture medium, and is cultured for 24h at 37 ℃, the culture solution is taken as an inoculating solution, and is inoculated into 50mL of MRS liquid culture medium according to the inoculating amount of 2 percent, and the culture solution of the strain is obtained by standing and culturing for 24 h. After 1mL of bacterial liquid is sucked up and bacterial cells are collected, the bacterial cells are washed by 1mL of buffer solution for 2 times, and then 2mL of buffer solution is added to resuspend the bacterial cells for standby.
2) Determination of DPPH free radical scavenging ability of Strain
1mL of PBS bacterial suspension of the strain to be detected is taken, 1mL of 0.4mM DPPH free radical solution is added, after uniform mixing, the mixture is placed at room temperature for shading reaction for 30min, then the absorbance A sample of the sample at the wavelength of 517nm is measured, and the sample is measured for 3 times of parallelism. The control samples were zeroed with equal volumes of PBS and DPPH ethanol mixed solution and with equal volumes of PBS and ethanol mixed solution. The clearance is calculated according to the following formula: clearance% = [1- (a) Sample of -A Blank space )/A Control ]×100％。
The results are shown in Table 7 using the Lactobacillus paracasei IMC-4 strain as a positive control.
TABLE 7 DPPH radical scavenging Rate
As can be seen from the data in Table 7, the Lactobacillus paracasei VHProbi F22 provided by the invention can effectively remove DPPH free radicals, and the removal rate is 54.57% which is obviously higher than that of the Lactobacillus paracasei IMC-4 strain.
2. Identification of strains by anti-lipid peroxidation experiments
1) Preparation of culture and fermentation supernatant, cell and intracellular extract of lactic acid bacteria: culturing lactobacillus in MRS liquid culture medium at 37deg.C for 24 hr, transferring for 3 generations, centrifuging at 6000rpm/min at 4deg.C for 10min, and collecting supernatant to obtain fermentation supernatant. The collected cells were centrifuged at 6000r/min for 10min with PBS buffer (pH 7.4), and washed 3 times. The bacterial cells were resuspended in PBS buffer to a bacterial cell concentration of 1.0X10 9 cells/mL to obtain a bacterial suspension.
2) Preparation of linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water.
3) To 0.5mL of PBS solution (pH 7.4) was added 1mL of linoleic acid emulsion, 1mL of LFASO 4 (1%) and 0.5mL of sample are added, water bath is carried out at 37 ℃ for 1.5 hours, 0.2mL of TCA (4%) and 2mL of TBA (0.8%) are added into the mixed solution, water bath is carried out at 100 ℃ for 30 minutes, rapid cooling is carried out, centrifugation is carried out at 4000rpm/min for 15 minutes, and absorbance of the supernatant liquid is collected and measured at 532nm to obtain A; the control group was A0 with 0.5mL distilled water instead of the sample. Inhibition rate/% = (A0-a)/a0×100%
Note that: a is absorbance of a sample group; a0 is absorbance of the control group. The results are shown in Table 8 using Lactobacillus paracasei IMC-4 as a positive control.
TABLE 8 inhibition of lipid peroxidation
From the data in Table 8, it can be seen that the anti-lipid peroxidation inhibition rate of the supernatant of the Lactobacillus paracasei VHProbi F22 provided by the invention is 50.64% which is lower than that of the Lactobacillus paracasei IMC-4 strain; the inhibition rate of the bacterial body to lipid peroxidation is 51.66 percent, which is higher than that of the strain IMC-4 of the cheese bacillus paracasei.
Example 6 in vitro cholesterol degradation experiments with Lactobacillus paracasei VHProbi F22
1. Preparing a cholesterol micelle solution: 1g of cholesterol was accurately weighed, dissolved in absolute ethanol, and fixed to 100mL, and sterilized by filtration through a 0.22 μm microporous filter under aseptic conditions.
2. 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 1.0mL of tween 80, 5.0g of sodium acetate, 0.1g of magnesium sulfate, 0.05 g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate, 1g of bile salt and 1000mL of distilled water, adjusting the pH value to 7.3, sterilizing at 115 ℃ for 30min, and then adding a cholesterol solution to ensure that the final concentration of cholesterol is 0.1%.
Inoculating fresh bacterial liquid according to 0.1% of inoculation amount, standing at 37 ℃ for 48 hours, taking 0.2mL of bacterial liquid, adding 1.8mL of absolute ethyl alcohol, uniformly mixing, standing for 10 minutes, centrifuging at 3000 r for 5 minutes, and taking supernatant for measuring cholesterol content. Cholesterol measurement method according to GB/T5009.128-2003 < measurement of cholesterol in food >.
The results show that: the degradation rate of the cheese bacillus paracasei VHProbi F22 provided by the invention on cholesterol reaches 34.55%.
EXAMPLE 7 Lactobacillus paracasei VHProbi F22 cell surface hydrophobicity test
1. Preparing a bacterial liquid to be tested: the purified bacterial colony of the cheese bacillus paracasei VHProbi F22 is picked and inoculated into a newly prepared MRS liquid culture medium, and the culture is carried out for 24 to 48 hours at 37 ℃. Inoculating 1% (V/V) of the strain into MRS liquid culture medium, continuously culturing at 37deg.C for 24-48 hr, centrifuging at 6000 Xg for 10min, collecting thallus, washing with sterile physiological saline for 2 times, and sterilizing with 0.1MKNO 3 The bacterial cells were resuspended in 1mL of the solution and used as the bacterial liquid to be tested.
2. Surface hydrophobicity determination: mu.L of the above bacterial suspension was pipetted into 2450. Mu.L of 0.1M KNO 3 And record OD 600 Is A 0 1.5ml of the bacterial suspension and 500. Mu.L of xylene were takenAfter mixing, the mixture was allowed to stand at room temperature for 10 minutes (a two-phase system was formed at this time). Vortex oscillating the two-phase system for 2min, standing for 20min, and reforming into water phase and organic phase. The aqueous phase (not to the organic phase) was carefully aspirated and absorbance A was measured at 600nm 1 . Cell Hydrophobicity = (a) according to the formula hydropathicity = (a) 0 -A 1 )/A 1 X% calculation, measurement of the average of three experiments.
The results show that: the hydrophobicity of the surface of the cheese bacillus paracasei VHProbi F22 cell provided by the invention is 70.61%.
Example 8 use of Lactobacillus paracasei VHProbi F22 for alleviating symptoms of intestinal immune disorders in mice
1.1 Experimental materials
1.1.1 laboratory animals
BALB/c mice SPF grade, 30 females, 4-6 weeks, body weight 19-25 g. License number SCXK (robust) 20140007 was produced by the experimental animal breeding limited company, punyue, atanan.
Animal quarantine and identification: after all animals arrive, the adaptation period is at least one week, quarantine observation is carried out, activities, diet and other performances of the animals are observed, the animals need to be checked to be qualified before the test, and the qualified animal can be used for the test. After the animals are qualified for quarantine, a single animal number is assigned to each animal, and the animal tail is marked. The quarantine observation period cage card is marked with a thematic number, an animal number, a cage number, sex, animal receiving date and thematic responsible person; after grouping, the thematic numbers, animal numbers, cage numbers, sexes, groups, test start-stop dates and thematic responsible persons are marked on the cage cards.
Environmental conditions for experimental animal feeding management: the room temperature is 20-26 ℃, the daily temperature difference is less than or equal to 4 ℃, the relative humidity is 40-70%, and the light and shade alternation time is 12/12h. Animals were kept in standard mouse cages, 6 per cage.
Animal feed, drinking water: can be ingested and drunk freely. The feed is SPF-class mice growth breeding feed, which is provided by Jinan Pengyue laboratory animal breeding Limited (lot number: 20190905). The drinking water is city tap water sterilized at high temperature.
1.1.2 reagent consumable
Ovalbumin (OVA) (lot number: S12016): shanghai Yuan Ye Biotech Co., ltd;
d-biotin (lot number: S13004): shanghai Yuan Ye Biotech Co., ltd;
vitamin B2 (lot number: S13020): shanghai Yuan Ye Biotech Co., ltd;
aluminum adjuvant (lot number: UL 292268): simer Feier technology (China);
IL-5 (lot number: E20200605-20187B) kit: shanghai enzyme-linked biotechnology limited company;
IL-6 (lot number: E20200605-20188B) kit: shanghai enzyme-linked biotechnology limited company;
IL-10 (lot number: E20200601-201662B) kit: shanghai enzyme-linked biotechnology limited company;
IFN-gamma (lot number: E20200602-20140B) kit: shanghai enzyme-linked biotechnology limited company;
TNF-alpha cytokine (lot number: E20200607-20852B) kit: shanghai enzyme-linked biotechnology limited company;
OVA-specific IgE (lot number: E20200604-20508B) kit: shanghai enzyme-linked biotechnology limited company;
eosin (lot number: 20181204): beijing Soy Bao technology Co., ltd;
hematoxylin (lot number: 20181204): beijing Soy Bao technology Co., ltd.
1.2 Experimental methods
1.2.1 preparation of bacterial liquid
Anaerobic culturing single colony on MRS plate at 37 deg.C for 24-48 hr, picking single colony, amplifying culturing in MRS broth culture medium for 16 hr, collecting bacterial liquid and regulating its concentration to 10 9 CFU/mL bacterial suspension.
1.2.2 grouping
SPF-class female BALB/c mice of 4-6 weeks old are randomly divided into a blank group, an intestinal immune disorder group, a probiotic pretreatment group and a probiotic post-treatment group after being adaptively fed for 3 days, wherein 6 mice are used in each group, and the probiotic pretreatment group and the post-treatment group are subjected to gastric lavage according to 0.2mL/10 g.
1.2.3 modeling and probiotic intervention scheme
The probiotic pretreatment group mice were given probiotic bacteria by gavage in advance before the beginning of molding, and were given continuously for 10 days, and the other groups were not treated. Starting modeling after 10 days, 3 groups of mice except for a blank group were injected with 200 μl of allergen solution (OVA: aluminum adjuvant: pbs=1:1:1 mixed solution) intraperitoneally on days 0, 14, 100 μg OVA per mouse, and the blank group was injected with a PBS/aluminum adjuvant mixed solution (PBS: aluminum adjuvant=2:1); starting from day 28, a blank control group was primed with 200 μl of PBS solution every three days, and an intestinal immune dysregulation group and 2 probiotic groups were primed with 50mg OVA every three days, and priming was co-activated 6 times; the probiotic pretreatment was started from 3 days after the adaptive feeding to the day before the sacrifice, and the probiotic post-treatment group was fed with the bacterial liquid from the day 0 basal sensitization to the day before the sacrifice.
1.3 index observations
1.3.1 symptoms of intestinal immune disorder in mice
The mice were observed for symptoms of intestinal immune disorder 1h after each gastric lavage with OVA and scored, and the scoring details are shown in table 9.
Table 9 mice intestinal immune disorder symptom score table
1.3.2IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha content and OVA-specific IgE detection
24h after the last administration of mice, eyeballs are bled, serum is prepared and stored at-80 ℃, and the ELISA is used for detecting the IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha cytokine content and OVA specific IgE level in the serum of each group of mice.
1.3.3 histopathological examination
After mice were sacrificed, jejunal tissues were harvested, paraformaldehyde fixed, sampled, dehydrated, paraffin embedded, sectioned, and HE stained for histopathological changes.
1.4 statistical processing method for data
All experimental data are expressed as mean ± standard deviation "± SD", data statistics and plots are performed using Microsoft EXCEL, and comparisons between two sets of data are determined to be significant differences using t-test, with P < 0.05.
1.5 experimental results
1.5.1 symptoms of intestinal immune disorder in mice
Compared with a blank control group, the immune disorder symptoms and the wet stool frequency of the mice in the intestinal immune disorder group are obviously increased, and the immune disorder and diarrhea symptoms are increased along with the increase of sensitization times; at the end of the test period, the allergy symptoms of the intestinal immune disorder group are highest in score, the diarrhea of the mice is serious and dysphoria is caused, the allergy symptoms of the probiotic pretreatment group and the probiotic post-treatment group are reduced in score, the diarrhea symptoms are lighter, and the intestinal immune disorder symptoms are improved. The symptom scores and the stool frequency of each group are shown in fig. 6.
1.5.2 histopathological examination
Compared with intestinal immune disorder groups, the probiotics treatment can reduce the villus swelling degree of jejunum mucosa, the epithelial cell shedding phenomenon is reduced, the integrity is better, the atrophy degree of the mucosa lamina propria is reduced, and the effect of the pretreatment group is better than that of the post-treatment group. Typical pathological lesions are shown in FIG. 7.
1.5.3IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha content and OVA-specific IgE detection
Serum levels of IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha cytokines and OVA-specific IgE were measured in each group of mice using the Elisa method. The results show that compared with a blank control group, the specific antibody of the mice in the intestinal immune disorder group and inflammatory factors are obviously increased, which proves that the construction of the intestinal immune disorder model caused by OVA is successful. Compared with the intestinal tract immune disorder group, the serum of the mice in the probiotic post-treatment group has reduced IL-5, IL-6, IFN-gamma and TNF-alpha cell inflammatory factors, increased IL-10 cell inflammatory factors, reduced OVA specific IgE and significant difference (P < 0.05); the serum of mice in the probiotic pretreatment group has reduced IL-5, IL-6, IFN-gamma, TNF-alpha cell inflammatory factors, increased IL-10 cell inflammatory factors, reduced OVA specific IgE, and significant difference (P < 0.05). The comparison of L-5, IL-6, IL-10, IFN-gamma, TNF-alpha cytokine levels and OVA-specific IgE in the sera of each group of mice is shown in FIG. 8.
In conclusion, compared with the intestinal tract immune disorder group, the cell inflammatory factor and OVA specific antibody after the probiotic treatment are reduced and have obvious difference (P is less than 0.05), and the effect of the probiotic pretreatment group is better than that of the probiotic post-treatment group; compared with mice with intestinal immune disorder groups, the probiotic treatment can reduce the central axis atrophy degree of jejunum mucosa villus, reduce the gap between central axis connective tissue and surface epithelium, reduce the phenomenon of epithelial cell shedding and reduce the pathological change degree.
Example 9 use of Lactobacillus paracasei VHProbi F22 for modulation of intestinal flora in mice
1.1 Experimental methods
The same animal experiment protocol as in application effect example 1 was used. And (3) collecting mouse faeces before OVA sensitization and after experiments of an intestinal immune disorder group and a probiotic pretreatment group, analyzing an intestinal flora structure, taking an collected faeces sample, sending the faeces sample to a norstanding grain source for sequencing, and analyzing community diversity and abundance changes of flora microorganisms by adopting an amplicon sequencing technology, wherein the community diversity and abundance changes comprise a richness index, a diversity index and the like.
1.2 experimental results
On the basis of obtaining 97% similarity OTU, alpha diversity of all samples is analyzed to construct a corresponding dilution curve, as shown in fig. 9, when the curve tends to be gentle, the sequencing quantity is reasonable, most microorganisms can be detected, the sequencing depth can be basically covered on all the species in the samples, the species abundance of the samples (Y2) after the end of the probiotic pretreatment group is higher than that of the samples (O1) before the sensitization of the intestinal immune disorder group and the samples (Y1) before the sensitization of the probiotic pretreatment group, the species abundance of the samples (O1) before the sensitization of the intestinal immune disorder group is higher than that of the samples (O2) after the end of the intestinal immune disorder group, so that the species abundance of intestinal flora is reduced after the symptoms of the intestinal immune disorder caused by OVA sensitization, and the species abundance of the intestinal flora of the intestinal immune disorder mice can be improved by adopting the interference of the Lactobacillus paracasei VHProbi F22, so that the species abundance of the intestinal flora tends to be recovered.
Species with a greater than set point (default set 4), i.e., species with statistical differences between groups, are shown in the LDA value distribution histogram, which shows species with significant differences in abundance among the different groups, and the length of the histogram represents the magnitude of the effect of the different species (i.e., LDA Score). As shown in FIG. 10, in O1, the abundances of bacillus, lactobacillus, enterobacter, lactobacillus reuteri, lactobacillus brevis, lactobacillus delbrueckii and the like are all remarkable; in O2, the wart micro-bacteria, acremonium, erysipelotorich and turicibacillus and the like are all remarkably high in abundance, which indicates that the use of intestinal immune disorder can cause the decrease of the abundance of lactobacillus in intestinal flora.
Comparing the two groups of O1/O2 data, the relative abundance of the firmicutes of the intestinal immune disorder group is reduced, and the relative abundance of the bacteroides and the wart microzyme is increased; comparing the two groups of data of O1/Y1, the pretreatment of the pre-probiotics reduces the relative abundance of the phylum of the firmicutes and increases the relative abundance of the flora of the bacteroides in the intestinal tract of the mice; comparing the two groups of O2/Y2 data, the relative abundance of the probiotics group is reduced compared with that of the wart microzyme of the intestinal immune disorder group after the experiment is finished, and the relative abundance of the bacteroides is increased; the results show that the intestinal flora structure of mice after intestinal immune disorder is better towards that of normal mice after intervention of the Lactobacillus paracasei VHProbi F22, and the relative abundance of each group species is shown in FIG. 11.
After the OVA sensitization causes intestinal immune disorder symptoms, the species abundance of intestinal flora is reduced, the species abundance of the intestinal flora of an intestinal immune disorder mouse can be improved by adopting the intervention of the cheese bacillus Paramygdalis VHProbi F22, so that the intestinal flora tends to be recovered, the abundance of lactobacillus in the intestinal flora can be improved, and the intestinal flora structure tends to be more beneficial to the intestinal flora of a healthy mouse.
In conclusion, the cheese bacillus paracasei VHProbi F22 provided by the invention has strong tolerance to simulated artificial intestinal gastric juice, which lays a foundation for the probiotic strains to successfully pass through the gastrointestinal tract to perform the probiotic function by colonic colonisation. The haemolytic test proves that the cheese bacillus VHProbi F22 does not produce haemolysin, does not dissolve blood cells and has good biological safety. Meanwhile, the cheese bacillus paracasei VHProbi F22 can remove DPPH free radicals, inhibit lipid peroxidation, has a certain antioxidant function activity, can degrade cholesterol, and has the probiotic property of reducing serum cholesterol. Animal experiments prove that the Lactobacillus paracasei VHProbi F22 can improve inflammatory response of a model mouse with intestinal immune disorder, can inhibit TH2 type immune response while enhancing TH1 type cell immune response, reduce inflammatory state of an organism and enhance immunity, and has potential application value in relieving inflammation of intestinal immune disorder.
EXAMPLE 10 Lactobacillus paracasei microcapsules and method of preparing the same
1. Preparation of Lactobacillus paracasei fermentation broth
Under the aseptic condition, inoculating the seed solution of the cheese bacillus paracasei VHProbi F22 into a fermentation culture medium in a volume ratio of 3-10%, wherein the fermentation culture medium comprises the following components in percentage by volume: 50-70 g/L of sucrose, 20-30 g/L of yeast powder, 5-25 g/L of soybean peptone, 1.5-2 g/L of magnesium sulfate and 0.08-0.12 g/L of manganese sulfate; culturing at 37 deg.c for 18-24 hr at 50-100 rpm, ventilating amount of 0.3-1L/min and tank pressure of 0.05-0.08 MPa. After fermentation, obtaining a fermentation broth of the Lactobacillus paracasei VHProbi F22, wherein the viable count of the Lactobacillus paracasei is 10 9 -10 10 CFU/mL。
2. Preparation of Lactobacillus paracasei microcapsules
Centrifuging the fermentation broth of the cheese bacillus paracasei VHProbi F22 at 4 ℃ and 5000r/min for 10min, collecting thalli, washing with sterile physiological saline for 2 times, and re-suspending the thalli to prepare a bacterial suspension.
Mixing 5% (W/W) porous starch with the bacterial suspension, adjusting pH to 6.0, and oscillating at 25-35deg.C for 30min; after full adsorption, the following outer layer embedding materials are added in sequence according to the weight percentage: 3% of sodium alginate, 5% of gelatin and 5% of dextrin, and uniformly stirring; dropping 2% (W/W) calcium chloride solution until solidification of the mixed solution occurs; freeze drying to obtain the microcapsule of Lactobacillus paracasei. The microcapsule viable count of the cheese bacillus paracasei VHProbi F22 is 1.5X10 9 CFU/mL。
EXAMPLE 11 Lactobacillus paracasei microcapsules and method of preparing the same
1. Preparation of Lactobacillus paracasei fermentation broth
Under the aseptic condition, inoculating the seed solution of the cheese bacillus paracasei VHProbi F22 into a fermentation culture medium in a volume ratio of 3-10%, wherein the fermentation culture medium comprises the following components in percentage by volume: 50-70 g/L of sucrose, 20-30 g/L of yeast powder, 5-25 g/L of soybean peptone, 1.5-2 g/L of magnesium sulfate and 0.08-0.12 g/L of manganese sulfate; culturing at 37 deg.c for 18-24 hr at 50-100 rpm, ventilating amount of 0.3-1L/min and tank pressure of 0.05-0.08 MPa. After fermentation, obtaining a fermentation broth of the Lactobacillus paracasei VHProbi F22, wherein the viable count of the Lactobacillus paracasei is 10 9 -10 10 CFU/mL。
2. Preparation of Lactobacillus paracasei microcapsules
Centrifuging the fermentation broth of the cheese bacillus paracasei VHProbi F22 at 4 ℃ and 5000r/min for 10min, collecting thalli, washing with sterile physiological saline for 2 times, and re-suspending the thalli to prepare a bacterial suspension.
Mixing porous starch 2% (W/W) with the bacterial suspension, adjusting pH to 6.0, and oscillating at 25-35deg.C for 30min; after full adsorption, the following outer layer embedding materials are added in sequence according to the weight percentage: 1% sodium alginate, 1% gelatin and 1% dextrin, and uniformly stirring; dropping 4% (W/W) calcium chloride solution until solidification of the mixed solution occurs; freeze drying to obtain the microcapsule of Lactobacillus paracasei. The microcapsule viable count of the cheese bacillus paracasei VHProbi F22 is 4 multiplied by 10 9 CFU/mL。
EXAMPLE 12 Lactobacillus paracasei microcapsules and method for preparing the same
1. Preparation of Lactobacillus paracasei fermentation broth
Under the aseptic condition, inoculating the seed solution of the cheese bacillus paracasei VHProbi F22 into a fermentation culture medium in a volume ratio of 3-10%, wherein the fermentation culture medium comprises the following components in percentage by volume: 50-70 g/L of sucrose, 20-30 g/L of yeast powder, 5-25 g/L of soybean peptone, 1.5-2 g/L of magnesium sulfate and 0.08-0.12 g/L of manganese sulfate; culturing at 37 deg.c for 18-24 hr at 50-100 rpm, ventilating amount of 0.3-1L/min and tank pressure of 0.05-0.08 MPa. After fermentation, obtaining a fermentation broth of the cheese bacillus paracasei VHProbi F22, wherein the fermentation broth comprises the auxiliary componentsThe viable count of the cheese bacillus was 10 9 -10 10 CFU/mL。
2. Preparation of Lactobacillus paracasei microcapsules
Centrifuging the fermentation broth of the cheese bacillus paracasei VHProbi F22 at 4 ℃ and 5000r/min for 10min, collecting thalli, washing with sterile physiological saline for 2 times, and re-suspending the thalli to prepare a bacterial suspension.
Mixing 3% porous starch with the bacterial suspension, adjusting pH to 6.0, controlling temperature to 25-35deg.C, and oscillating for 30min; after full adsorption, the following outer layer embedding materials are added in sequence according to the weight percentage: 1.5% of sodium alginate, 2.5% of gelatin and 3.5% of dextrin, and uniformly stirring; dropping 6% (W/W) calcium chloride solution until solidification of the mixed solution occurs; freeze drying to obtain the microcapsule of Lactobacillus paracasei. The microcapsule viable count of the cheese bacillus paracasei VHProbi F22 is 1 multiplied by 10 10 CFU/mL；
EXAMPLE 13 detection of Performance of Lactobacillus paracasei microcapsules
1. Stress resistance detection
1.1 preparation of Artificial gastric juice
5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl are weighed respectively, 1000mL of distilled water is added, pH is adjusted to 3.0 by dilute hydrochloric acid, and then sterilization is carried out for 20min at 115 ℃. Then 3.2g of pig mucosa pepsin is added before use, the pig mucosa pepsin is uniformly shaken and dissolved, and the mixture is placed in a water bath shaker at 37 ℃ for warm water bath for 1 hour so as to simulate the temperature of a human body.
1.2 preparation of Artificial intestinal juice
Respectively weighing peptone 5g, yeast extract 2.5g, glucose 1g, KH 2 PO 4 6.8g and 3.0g of ox gall salt, 77mL of 0.2mol/L NaOH solution is added, the volume is fixed to 1000mL, the pH is regulated to 6.8+/-0.1 by dilute hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized for 20min at 115 ℃. Then adding 1g of pancreatin before use, shaking to dissolve, and placing in a water bath shaker at 37 ℃ for warm water bath for 1h to simulate the temperature of human body.
1.3 test methods
2g of each of the Lactobacillus paracasei microcapsules prepared in examples 10 to 12 was resuspended in 2mL of physiological saline and used as an inoculation liquid. 1mL of inoculation liquid is taken and added into 9mL of artificial gastric juice which is in advance heated in water bath for 1h, the mixture is placed in a water bath shaking table at 37 ℃ to oscillate for 2h at the rotating speed of 200rpm/min, 1mL is sampled at 0h and 2h respectively, and the viable bacteria amount is detected. Then 1mL of the artificial gastric juice after 2h digestion is taken and added into 24mL of the artificial intestinal juice, the mixture is placed in a water bath shaking table (200 rpm/min) at 37 ℃ for 3h, 1mL of the sample is taken, and the viable bacteria amount is detected. Viable bacteria counting method the bacterial load was measured according to national standard GB 4789.35-2016-lactobacillus test for food microbiological examination, and the viable bacteria load (Log CFU/mL) of the microcapsules prepared in examples 10-12 after digestion with artificial gastric juice and artificial intestinal juice is shown in Table 10.
TABLE 10 Effect of Lactobacillus paracasei microcapsules on tolerance to Artificial gastric juice and artificial intestinal juice
From the results shown in Table 10, the Lactobacillus paracasei microcapsule provided by the invention can still maintain higher viable bacteria after being digested by artificial gastric juice and artificial intestinal juice, thereby indicating that the Lactobacillus paracasei VHProbi F22 microcapsule has strong gastric acid resistance and choline resistance.
The applicant feeds the lactobacillus paracasei microcapsule provided by the invention into a female BALB/c mouse of 4-6 weeks in advance for 10 days, then carries out an intestinal immune disorder modeling test on the mouse, and observes indexes such as intestinal immune disorder of the mouse. The results show that the levels of mouse cell inflammatory factors and OVA specific antibodies of the pre-perfused lactobacillus paracasei microcapsules are significantly reduced and the degree of jejunal pathology in mice is reduced compared to the negative group of mice.
After the Lactobacillus paracasei microcapsule provided by the invention is fed to female BALB/c mice of 4-6 weeks in advance for 10 days, intestinal immune disorder modeling test is carried out on the mice, and the intestinal flora change condition is detected. The results show that mice infused with Lactobacillus paracasei microcapsules have increased abundance of Lactobacillus species in the intestinal flora and intestinal flora structures of mice with intestinal immune disorders are more prone to intestinal flora structures in healthy mice than in mice of the negative group.
The preparation process of the lactobacillus paracasei microcapsule provided by the invention is simple, the embedding material is cheap, the industrialization cost is low, the stability of the lactobacillus paracasei VHProbi F22 in the storage, processing and transportation processes can be effectively improved, the actual use effect of the lactobacillus paracasei VHProbi F22 is effectively improved, and the microcapsule can be widely applied to the fields of foods, health products and the like.
Sequence listing
<110> Qingdao blue biological Co., ltd
QINGDAO VLAND BIOTECH GROUP Co.,Ltd.
<120> a Lactobacillus paracasei microcapsule, and preparation method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1468
<212> DNA
<213> Lactobacillus paracasei (Lacticaseibacillus paracasei)
<400> 1
catgcggcgt gctatacatg cagtcgaacg agttctcgtt gatgatcggt gcttgcaccg 60
agattcaaca tggaacgagt ggcggacggg tgagtaacac gtgggtaacc tgcccttaag 120
tgggggataa catttggaaa cagatgctaa taccgcatag atccaagaac cgcatggttc 180
ttggctgaaa gatggcgtaa gctatcgctt ttggatggac ccgcggcgta ttagctagtt 240
ggtgaggtaa tggctcacca aggcgatgat acgtagccga actgagaggt tgatcggcca 300
cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga atcttccaca 360
atggacgcaa gtctgatgga gcaacgccgc gtgagtgaag aaggctttcg ggtcgtaaaa 420
ctctgttgtt ggagaagaat ggtcggcaga gtaactgttg tcggcgtgac ggtatccaac 480
cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta 540
tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat gtgaaagccc 600
tcggcttaac cgaggaagcg catcggaaac tgggaaactt gagtgcagaa gaggacagtg 660
gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt ggcgaaagcg 720
gctgtctggt ctgtaactga cgctgaggct cgaaagcatg ggtagcgaac aggattagat 780
accctggtag tccatgccgt aaacgatgaa tgctaggtgt tggagggttt ccgcccttca 840
gtgccgcagc taacgcatta agcattccgc ctggggagta cgaccgcaag gttgaaactc 900
aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc 960
gaagaacctt accaggtctt gacatctttt gatcacctga gagatcaggt ttccccttcg 1020
ggggcaaaat gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta 1080
agtcccgcaa cgagcgcaac ccttatgact agttgccagc atttagttgg gcactctagt 1140
aagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta 1200
tgacctgggc tacacacgtg ctacaatgga tggtacaacg agttgcgaga ccgcgaggtc 1260
aagctaatct cttaaagcca ttctcagttc ggactgtagg ctgcaactcg cctacacgaa 1320
gtcggaatcg ctagtaatcg cggatcagca cgccgcggtg aatacgttcc cgggccttgt 1380
acacaccgcc cgtcacacca tgagagtttg taacacccga agccggtggc gtaacccttt 1440
agggagcgag ccgtctaagg tgacaagg 1468
Claims (8)
1. The lactobacillus paracasei microcapsule is characterized in that the core material of the lactobacillus paracasei microcapsule comprises lactobacillus paracasei.
2. The lactobacillus paracasei microcapsule of claim 1, wherein the lactobacillus paracasei has a preservation number of CCTCC No: m2019941.
3. The lactobacillus paracasei microcapsule of claim 2, wherein the core material of the microcapsule comprises starch.
4. A lactobacillus paracasei microcapsule according to claim 2 or 3, wherein the encapsulating material used in the outer layer of the microcapsule comprises sodium alginate, gelatin and dextrin.
5. A lactobacillus paracasei microcapsule according to any of claims 1 to 4, wherein the microcapsule is prepared by the following method: mixing 3% porous starch with Lactobacillus paracasei suspension, adjusting pH to 6.0, and oscillating at 25-35deg.C for 30min; after full adsorption, the following outer layer embedding materials are added in sequence according to the weight percentage: 1.5% of sodium alginate, 2.5% of gelatin and 3.5% of dextrin, and uniformly stirring; dropping 6% (W/W) calcium chloride solution until solidification of the mixed solution occurs; freeze drying to obtain the microcapsule of Lactobacillus paracasei.
6. Use of a lactobacillus paracasei microcapsule according to any of claims 1 to 5 for modulating an intestinal immune disorder.
7. Use of a lactobacillus paracasei microcapsule according to any of claims 1 to 5 for the preparation of a functional food having the effect of modulating intestinal immune disorders.
8. A functional food comprising the lactobacillus paracasei microcapsule according to any of claims 1 to 5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210236610.6A CN116762959A (en) | 2022-03-11 | 2022-03-11 | Lactobacillus paracasei microcapsule and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210236610.6A CN116762959A (en) | 2022-03-11 | 2022-03-11 | Lactobacillus paracasei microcapsule and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116762959A true CN116762959A (en) | 2023-09-19 |
Family
ID=87990184
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210236610.6A Pending CN116762959A (en) | 2022-03-11 | 2022-03-11 | Lactobacillus paracasei microcapsule and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116762959A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117586930A (en) * | 2024-01-19 | 2024-02-23 | 北京市农林科学院 | Microcapsule material for degrading patulin as well as preparation method and application thereof |
-
2022
- 2022-03-11 CN CN202210236610.6A patent/CN116762959A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117586930A (en) * | 2024-01-19 | 2024-02-23 | 北京市农林科学院 | Microcapsule material for degrading patulin as well as preparation method and application thereof |
| CN117586930B (en) * | 2024-01-19 | 2024-04-16 | 北京市农林科学院 | Microcapsule material for degrading patulin as well as preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN116769629A (en) | Lactobacillus paracasei with intestinal immune disorder symptom regulating function and application thereof | |
| CN104894021A (en) | Lactobacillus paracasei strain and application thereof | |
| CN114621888B (en) | Lactobacillus paracasei with intestinal immune disorder symptom regulating function and application thereof | |
| CN110157645B (en) | Lactobacillus salivarius Y4 and application thereof | |
| KR20180021777A (en) | Lactobacillus laminosis bacteria for the treatment of bacterial vaginosis | |
| CN114752529B (en) | Lactobacillus plantarum HOM3201 strain and viable bacteria preparation, preparation method and application thereof | |
| CN114717157A (en) | Lactobacillus paracasei for preventing streptococcus infection of infants and application thereof | |
| CN116515666A (en) | Lactobacillus helveticus with acne treatment effect and application thereof | |
| CN117327608A (en) | Lactobacillus rhamnosus strain and application thereof | |
| CN116762959A (en) | Lactobacillus paracasei microcapsule and preparation method and application thereof | |
| CN114717132B (en) | Bifidobacterium lactis with constipation symptom preventing and relieving function and application thereof | |
| CN114717220B (en) | Lactobacillus reuteri microcapsule and preparation method thereof | |
| CN113528367A (en) | Bacillus coagulans with functions of preventing diarrhea and degrading cholesterol | |
| CN116445356B (en) | Bifidobacterium animalis subspecies BA67 for regulating intestinal flora and enhancing immunity and application thereof | |
| CN114703107B (en) | Lactobacillus paracasei and application thereof in preventing streptococcus infection of infants | |
| CN114717127B (en) | Lactobacillus reuteri for preventing and relieving allergic asthma symptoms and application thereof | |
| CN116875480A (en) | Lactobacillus rhamnosus with antagonism to helicobacter pylori | |
| CN114717219B (en) | Lactobacillus reuteri preparation with antioxidant and cholesterol reducing functions | |
| CN117480244A (en) | Novel lactobacillus fermentum ATG-V5 strain or composition for enhancing immunity comprising said strain | |
| CN113528368A (en) | Bacillus coagulans preparation and preparation method thereof | |
| CN114717133B (en) | Bacillus subtilis with bone health enhancing effect and application thereof | |
| CN114703079B (en) | Lactobacillus paracasei and application thereof in relieving skin injury | |
| CN116590181B (en) | Lactobacillus paracasei for improving inflammatory reaction caused by microplastic pollution and application thereof | |
| CN117925452A (en) | Lactobacillus plantarum for increasing abundance of intestinal Akk bacteria and application thereof | |
| CN117467586B (en) | Lactobacillus rhamnosus with effect of delaying chronic nephrosis process |
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 |