CN113712207B - Probiotic prebiotic composition for improving gastrointestinal tract immunity and application thereof - Google Patents
Probiotic prebiotic composition for improving gastrointestinal tract immunity and application thereof Download PDFInfo
- Publication number
- CN113712207B CN113712207B CN202010435608.2A CN202010435608A CN113712207B CN 113712207 B CN113712207 B CN 113712207B CN 202010435608 A CN202010435608 A CN 202010435608A CN 113712207 B CN113712207 B CN 113712207B
- Authority
- CN
- China
- Prior art keywords
- lactobacillus paracasei
- cfu
- breast milk
- composition
- milk oligosaccharide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 46
- 210000001035 gastrointestinal tract Anatomy 0.000 title claims abstract description 21
- 230000036039 immunity Effects 0.000 title claims abstract description 16
- 239000006041 probiotic Substances 0.000 title abstract description 35
- 235000018291 probiotics Nutrition 0.000 title abstract description 35
- 235000013406 prebiotics Nutrition 0.000 title abstract description 26
- 230000000529 probiotic effect Effects 0.000 title abstract description 16
- 241000186605 Lactobacillus paracasei Species 0.000 claims abstract description 59
- 210000004251 human milk Anatomy 0.000 claims abstract description 56
- 235000020256 human milk Nutrition 0.000 claims abstract description 56
- 229920001542 oligosaccharide Polymers 0.000 claims abstract description 49
- 150000002482 oligosaccharides Chemical class 0.000 claims abstract description 49
- 235000016709 nutrition Nutrition 0.000 claims abstract description 16
- 238000004321 preservation Methods 0.000 claims abstract description 10
- SNFSYLYCDAVZGP-UHFFFAOYSA-N UNPD26986 Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(OC(O)C(O)C2O)CO)OC(CO)C(O)C1O SNFSYLYCDAVZGP-UHFFFAOYSA-N 0.000 claims abstract description 9
- HWHQUWQCBPAQQH-BWRPKUOHSA-N 2-fucosyllactose Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O HWHQUWQCBPAQQH-BWRPKUOHSA-N 0.000 claims abstract description 7
- 229940062827 2'-fucosyllactose Drugs 0.000 claims abstract description 6
- HWHQUWQCBPAQQH-UHFFFAOYSA-N 2-O-alpha-L-Fucosyl-lactose Natural products OC1C(O)C(O)C(C)OC1OC1C(O)C(O)C(CO)OC1OC(C(O)CO)C(O)C(O)C=O HWHQUWQCBPAQQH-UHFFFAOYSA-N 0.000 claims abstract description 6
- 230000000968 intestinal effect Effects 0.000 claims description 34
- 244000052616 bacterial pathogen Species 0.000 claims description 23
- 101710098275 C-X-C motif chemokine 10 Proteins 0.000 claims description 16
- 108090001007 Interleukin-8 Proteins 0.000 claims description 13
- 235000013305 food Nutrition 0.000 claims description 13
- 230000002757 inflammatory effect Effects 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 8
- 230000001900 immune effect Effects 0.000 claims description 5
- 230000007358 intestinal barrier function Effects 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 235000013361 beverage Nutrition 0.000 claims description 4
- 210000005027 intestinal barrier Anatomy 0.000 claims description 4
- 230000009545 invasion Effects 0.000 claims description 4
- 239000002775 capsule Substances 0.000 claims description 2
- 230000006872 improvement Effects 0.000 claims description 2
- 230000009467 reduction Effects 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 230000002496 gastric effect Effects 0.000 claims 3
- 230000002708 enhancing effect Effects 0.000 claims 2
- 241000186660 Lactobacillus Species 0.000 claims 1
- 235000013365 dairy product Nutrition 0.000 claims 1
- 229940039696 lactobacillus Drugs 0.000 claims 1
- 239000000825 pharmaceutical preparation Substances 0.000 claims 1
- 229940127557 pharmaceutical product Drugs 0.000 claims 1
- 235000013402 health food Nutrition 0.000 abstract description 5
- 210000004027 cell Anatomy 0.000 description 41
- 238000012360 testing method Methods 0.000 description 35
- 238000002474 experimental method Methods 0.000 description 34
- 230000000694 effects Effects 0.000 description 33
- 241000588724 Escherichia coli Species 0.000 description 21
- 230000001717 pathogenic effect Effects 0.000 description 19
- 241000894006 Bacteria Species 0.000 description 16
- 230000012010 growth Effects 0.000 description 16
- 239000000126 substance Substances 0.000 description 16
- 244000052769 pathogen Species 0.000 description 14
- 239000002609 medium Substances 0.000 description 11
- 238000001179 sorption measurement Methods 0.000 description 11
- 208000015181 infectious disease Diseases 0.000 description 10
- 239000013642 negative control Substances 0.000 description 9
- 230000004083 survival effect Effects 0.000 description 8
- 230000007423 decrease Effects 0.000 description 7
- 235000013350 formula milk Nutrition 0.000 description 7
- 229920001817 Agar Polymers 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000004977 Hueckel calculation Methods 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 235000015872 dietary supplement Nutrition 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 238000009629 microbiological culture Methods 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 230000002195 synergetic effect Effects 0.000 description 4
- SNFSYLYCDAVZGP-OLAZETNGSA-N 2'-fucosyllactose Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@@H]2[C@H](OC(O)[C@H](O)[C@H]2O)CO)O[C@H](CO)[C@H](O)[C@@H]1O SNFSYLYCDAVZGP-OLAZETNGSA-N 0.000 description 3
- 230000005353 IP-10 production Effects 0.000 description 3
- 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 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 210000000481 breast Anatomy 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 230000021995 interleukin-8 production Effects 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 241000186000 Bifidobacterium Species 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 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
- 241000476240 Escherichia coli O119 Species 0.000 description 2
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 2
- 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 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 2
- 241000218587 Lactobacillus paracasei subsp. paracasei Species 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000014564 chemokine production Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000016396 cytokine production Effects 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 238000010149 post-hoc-test Methods 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 210000000813 small intestine Anatomy 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 108700012359 toxins Proteins 0.000 description 2
- 239000011787 zinc oxide Substances 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
- 102000001327 Chemokine CCL5 Human genes 0.000 description 1
- 108010055166 Chemokine CCL5 Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010058839 Enteritis infectious Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010061126 Escherichia infection Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101100314144 Mus musculus Tnip1 gene Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 210000000447 Th1 cell Anatomy 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 229930003448 Vitamin K Natural products 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000006472 autoimmune response Effects 0.000 description 1
- 235000008452 baby food Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 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
- 230000036755 cellular response Effects 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 210000003022 colostrum Anatomy 0.000 description 1
- 235000021277 colostrum Nutrition 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 235000020247 cow milk Nutrition 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000002416 diarrheagenic effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000023011 digestive tract development Effects 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
- 239000012153 distilled water Substances 0.000 description 1
- 230000000688 enterotoxigenic effect Effects 0.000 description 1
- 230000004890 epithelial barrier function Effects 0.000 description 1
- 208000020612 escherichia coli infection Diseases 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020774 essential nutrients Nutrition 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 239000005417 food ingredient Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000004153 glucose metabolism Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 210000005026 intestinal epithelial barrier Anatomy 0.000 description 1
- 230000007413 intestinal health Effects 0.000 description 1
- 230000008944 intestinal immunity Effects 0.000 description 1
- 208000002551 irritable bowel syndrome Diseases 0.000 description 1
- 230000006651 lactation Effects 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 210000002540 macrophage Anatomy 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
- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 230000009974 thixotropic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 150000004043 trisaccharides Chemical group 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 235000019168 vitamin K Nutrition 0.000 description 1
- 239000011712 vitamin K Substances 0.000 description 1
- 150000003721 vitamin K derivatives Chemical class 0.000 description 1
- 229940046010 vitamin k Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/165—Paracasei
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention mainly relates to a probiotic prebiotic composition for improving gastrointestinal tract immunity and application thereof, wherein the composition is a nutritional composition consisting of lactobacillus paracasei (Lactobacillus paracasei) and breast milk oligosaccharide (Human Milk Oligosaccharides), the lactobacillus paracasei is lactobacillus paracasei with a preservation number of CGMCC No.15139 or DSM27447 and/or a preservation number of CGMCC No.15077, and the breast milk oligosaccharide comprises 2' -fucosyllactose. The composition of the present invention can effectively improve the immunity of the gastrointestinal tract, and can be added into various health foods and health foods.
Description
Technical Field
The invention mainly relates to a probiotic prebiotic composition capable of improving gastrointestinal tract immunity and application thereof, in particular to a nutritional composition consisting of lactobacillus paracasei (Lactobacillus paracasei) and breast milk oligosaccharide (Human Milk Oligosaccharides, HMOs) 2' -fucosyllactose, which can be added into various health foods and health foods.
Background
In the last thousand years, medical literature has described infants not breast fed with higher disease and mortality rates. The breast milk not only provides needed nutrition for infants, but also provides guarantee for intestinal development and immunity improvement of infants due to active ingredients in the breast milk. Breast-fed infants have a higher relative abundance of beneficial bacteria, particularly bifidobacteria and lactic acid bacteria, in the intestinal flora than formula-fed infants.
The breast milk is transmitted by flora, and active ingredients such as breast milk oligosaccharide and cytokines in the breast milk are added, so that a healthy intestinal flora is established for the newborn. Infants ingest 10 a day by breast milk 7 -10 8 Bacteria, including lactic acid bacteria and bifidobacteria. The bacteria are directly transmitted to the infant through breast milk, part of the bacteria can colonize the intestinal tracts of the infant, and the establishment of intestinal flora in early life is promoted. The establishment of the intestinal flora of infants has a short-term, even lifetime, impact on the development of their intestinal tract, as well as on the health and immune system.
Breast milk oligosaccharides (Human Milk Oligosaccharides, abbreviated as HMOs) are substances in breast milk which are third-enriched in content in addition to lactose and fat. The total content varies at various stages of lactation, about 12-14g/L in mature milk and about 20-24g/L in colostrum. Each breast milk oligosaccharide has a lactose at the reducing end, mostly with a polylactosamine as the structural backbone, and contains fucose, sialic acid, or both at the chain ends. The presence and amount of HMOs are subject to individual differences and are related to the lewis secretory composition of the lactating mother. Because infant formulas are typically cow milk, which is usually free or contains little such oligosaccharide material, HMOs form a gap that infant formulas must cross to more closely approximate the breast milk components.
In the 90 s of the last century, HMO, 2-fucosyllactose (2' -FL) contained in most breast milk was found to be effective in reducing the toxicity of stable toxins in escherichia coli; by 2003, the oligosaccharide was reported to inhibit adhesion and infection of E.campylobacter. Subsequently, three major functions of breast milk oligosaccharides were gradually reported and discovered: (1) inhibiting the attachment and infection of specific pathogens; (2) As a prebiotic, promoting the growth of bacteria in the intestinal symbiotic system; (3) Directly reduces the inflammatory response of mucous membrane under toxic stimulation. The first clinical intervention trial with 2' -FL demonstrated that the addition of this particular ingredient to a low calorie formula was not only safe but also comparable to breast-fed infants in terms of growth rate of the formula-fed infants. 2' -FL is also used as a nutritional supplement for adults, to relieve irritable bowel syndrome or inflammatory bowel disease, or as a prebiotic to maintain intestinal flora balance.
The intestinal flora is an important component of human intestinal microecological system, and has important effects on human health, such as providing essential nutrients, generating vitamin K, assisting digestion process, and promoting angiogenesis and intestinal nerve. Prebiotics and probiotics are regarded as a means of microecological management that improves the health of the body, and can alter, regulate and recombine the intestinal flora that is already present.
At present, in the fields of infant formula powder, complementary food and nutritional supplements, solutions for relieving intestinal discomfort and improving autoimmune ability of infants are needed. Meanwhile, in the fields of children, teenagers and adults over 3 years old, the balance of intestinal flora is also required to be maintained, and the immunity is regulated.
Disclosure of Invention
It is an object of the present invention to provide a nutritional composition that can enhance the immunity of the gastrointestinal tract.
The inventor finds that the combination of lactobacillus paracasei (Lactobacillus paracasei) and breast milk oligosaccharide has a synergistic effect on improving the gastrointestinal tract immunity.
Thus, in one aspect, the present invention provides a nutritional composition consisting of lactobacillus paracasei (Lactobacillus paracasei) and breast milk oligosaccharides.
According to a specific embodiment of the present invention, in the composition of the present invention, the breast milk oligosaccharide is 2' -fucosyllactose.
2' -fucosyllactose (2 ' -Fucosyl lactose,2' -FL or 2-FL) is a trisaccharide structure formed by fucose and lactose, and is generally commercially available as a material prepared by microbial fermentation, and has the same structure as oligosaccharides found in human milk.
According to a specific embodiment of the present invention, the lactobacillus paracasei comprises lactobacillus paracasei with a preservation number of CGMCC No.15139 or DSM27447, and/or lactobacillus paracasei with a preservation number of CGMCC No.15077.
Lactobacillus paracasei with accession number CGMCC No.15139 or DSM27447, also known as lactobacillus paracasei (Lactobacillus paracasei subsp. Paracasei) K56 strain, has been stored in german collection for microorganisms and cell cultures (German Collection of Microorganisms and Cell Cultures) at month 27 of 2013, accession number DSM27447; in addition, lactobacillus paracasei (Lactobacillus paracasei subsp. Paracasei) K56 strain was also preserved in China general microbiological culture Collection center (China General Microbiological Culture Collection Center, CGMCC) at 12 and 29 days of 2017, with a preservation number of CGMCC No.15139.
Lactobacillus paracasei with the preservation number of CGMCC No.15077 is also named as lactobacillus paracasei ET-22 in the invention. The strain is preserved in China general microbiological culture Collection center (CGMCC) (address: national institute of microbiology, national academy of sciences of China, including national academy of sciences of China) at 12 months 18 of 2017: lactobacillus paracasei (Lactobacillus paracasei); the preservation number is CGMCC No.15077.
According to a specific embodiment of the invention, the ratio of Lactobacillus paracasei to breast milk oligosaccharide in the composition according to the invention is 1X 10 3 CFU～1×10 12 CFU:5mg, preferably 1X 10 6 CFU～1×10 10 CFU：5mg。
According to a specific embodiment of the invention, the Lactobacillus paracasei is used in the nutritional composition in an amount of 1X 10 in the composition according to the invention 3 CFU～1×10 12 CFU/day, preferably 1X 10 6 CFU～1×10 11 CFU/day.
According to a specific embodiment of the present invention, the amount of breast milk oligosaccharide used in the nutritional composition is 1mg to 15g per day in the composition of the present invention.
In another aspect, the invention also provides application of the composition in preparing food or medicine with the efficacy of improving gastrointestinal tract immunity. More specifically, the functional component in the food or medicine with the effect of improving the gastrointestinal tract immunity (namely, the component with the effect of improving the gastrointestinal tract immunity) is the composition consisting of lactobacillus paracasei (Lactobacillus paracasei) and breast milk oligosaccharide.
According to a specific embodiment of the invention, the use of the composition of the invention for increasing the immunological competence of the gastrointestinal tract comprises combating the invasion of pathogenic bacteria in the intestinal system, more particularly reducing the adsorption of pathogenic bacteria EPEC 0119 to intestinal cells.
According to a specific embodiment of the invention, the use of the composition of the invention for increasing the immunological competence of the gastrointestinal tract comprises decreasing the release of inflammatory factor IL-8 by intestinal cells.
According to a specific embodiment of the invention, the use of the composition of the invention for increasing the immunological competence of the gastrointestinal tract comprises decreasing the release of inflammatory factor IP-10 by intestinal cells.
According to a specific embodiment of the invention, the use of the composition of the invention for increasing the immunological competence of the gastrointestinal tract comprises protecting the integrity of the intestinal barrier.
According to a specific embodiment of the present invention, the nutritional composition of the present invention may be used for preparing various health foods, and the like. Specifically, the food can be liquid beverage, solid beverage, oral liquid, milk product, tablet or capsule, etc., for example, can be added into infant food (including infant formula powder, supplementary food and nutritional supplement), and nutritional supplement or food for children, teenagers and adults over 3 years old, and has wide application prospect. Preferably, the lactobacillus paracasei CGMCC No.15139 is added into food in an amount of 1×10 3 CFU～1×10 12 CFU/day, more preferably 1X 10 6 CFU～1×10 11 CFU/day. The addition amount of the lactobacillus paracasei CGMCC No.15077 in food is 1×10 3 CFU～1×10 12 CFU/day, more preferably 1X 10 6 CFU～1×10 11 CFU/day. The addition amount of breast milk oligosaccharide in food is 1mg-15 g/day.
The food or medicine comprising the nutritional composition has the function of improving the gastrointestinal tract immunity due to the inclusion of the nutritional composition.
Drawings
FIG. 1 shows the effect of breast milk oligosaccharide 2' -FL on EPEC survival of test pathogens.
FIG. 2 shows the effect of Lactobacillus paracasei K56 and ET-22 strains on the survival of the test pathogenic bacteria EPEC.
FIG. 3 shows the effect of Lactobacillus paracasei K56 in combination with breast milk oligosaccharide 2' -FL on the survival of the test pathogenic bacteria EPEC.
FIG. 4 shows the effect of Lactobacillus paracasei ET-22 in combination with breast milk oligosaccharide 2' -FL on the survival of the test pathogenic bacteria EPEC.
FIG. 5 shows the results of an adhesion experiment of Lactobacillus paracasei K56 in combination with breast milk oligosaccharide 2' -FL on pathogenic bacteria EPEC to intestinal cells Caco-2.
FIG. 6 shows the results of an adhesion experiment of Lactobacillus paracasei ET-22 in combination with breast milk oligosaccharide 2' -FL on pathogenic bacteria EPEC to intestinal cells Caco-2.
FIG. 7 shows the effect of Lactobacillus paracasei K-56 in combination with breast milk oligosaccharide 2' -FL on secretion of inflammatory factor IP-10 by intestinal cells in case of E.coli ETEC.
FIGS. 8A and 8B show the effect of Lactobacillus paracasei ET-22 in combination with breast milk oligosaccharides 2' -FL on secretion of inflammatory factors IL-8 and IP-10 by intestinal cells in the case of E.coli ETEC.
Fig. 9A and 9B show the effect of lactobacillus paracasei K56 and ET-22 in combination with breast milk oligosaccharide 2' -FL formation, respectively, on the intestinal barrier.
Detailed Description
In order to more clearly understand the technical features, objects and advantages of the present invention, the following detailed description of the technical solution of the present invention will be made with reference to specific examples and accompanying drawings, it being understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art.
The inventor proves that the probiotic prebiotic composition has a synergistic effect in regulating gastrointestinal tract immunity through specific experiments.
The experimental methods and test subjects used in each example and control were as follows:
1. experimental preparation procedure
1.1 Medium configuration and storage and configuration of test prebiotics
The medium containing the prebiotics and probiotics was freshly prepared in a sterile environment on the day of each experiment and preheated to 37 ℃. The prebiotics/HMOs tested were stored in a dry, light-protected room temperature environment. The concentration used in the experiment was 5g/L (this concentration was used alone or in combination with probiotics).
1.2 Probiotics culture, growth Curve drawing and Activity identification
The probiotic powder was sent to the laboratory in a freeze-dried state prior to the experiment. Probiotics were inoculated onto MRS-agar plates, individual strain populations were taken for 16S identification and used to prepare glycerol stocks (stored at-80 ℃). The probiotics were removed from glycerol stock and inoculated on MRS plates at 37℃before each experiment, harvested at rest and rinsed with MEM medium before the experiment. Before probiotic testing, probiotic activity and cell number were measured with a fluorescence activated cell sorting system. The probiotic concentration used in the experiment was 1×10 8 CFU/mL (this concentration is used alone or in combination with prebiotics). Lactobacillus paracasei K56 or ET-22 was cultured under anaerobic conditions at 37℃and growth curves were drawn prior to the experiment. The activity of the probiotic strains was tested with a flow cytometer before the probiotic feed was received and before all experiments were started. 16S sequencing was used to determine strain identity. Before the test of specific experiments of probiotics, the activity and the cell number of the probiotics are measured by a fluorescence activated cell sorting system.
Lactobacillus paracasei ET-22:
the lactobacillus paracasei ET-22 of the invention is preserved in China general microbiological culture Collection center (CGMCC) (address: national institute of microbiology, national academy of sciences of China, including North Chen West Lu No.1 and No. 3) of the Korean area of Beijing) in 12 months and is named after classification: lactobacillus paracasei (Lactobacillus paracasei); the preservation number is CGMCC No.15077.
The taxonomic characteristics of the ET-22 strain were confirmed based on the 16S rDNA sequence analysis and the API bacteria identification system analysis results. The method is characterized by comprising the following steps:
morphological features: 1. when the MRS culture solution is used for culturing, the bacterial cells are in a middle-short rod shape, have round two ends, are usually chain-shaped, and occasionally appear in pairs. 2. Gram-positive bacillus does not generate spores, does not have thixotropic enzyme, oxidase and motility, can grow in aerobic and anaerobic environments, has an optimal growth temperature of 37+/-1 ℃, belongs to facultative heterogeneous fermentation strains, and does not generate gas during glucose metabolism.
The fermentation conditions of the ET-22 strain are as follows: MRS liquid medium: peptone, 10.0g; beef extract, 10.0g; yeast extract powder, 5.0g; glucose, 20.0g; dipotassium hydrogen phosphate, 5.0g; 2.0g of diammonium hydrogen citrate; sodium acetate, 5.0g; magnesium sulfate heptahydrate, 0.5g; manganese sulfate tetrahydrate, 0.2g; tween 80,1.0g; 15.0g of agar; distilled water 1000mL. Adjusting the pH value to between 6.2 and 6.4, and sterilizing for 15 minutes at the temperature of 121 ℃.
Lactobacillus paracasei ET-22 is a microaerophilic bacterium, has better growth in a facultative anaerobic environment, produces lactic acid, has acid resistance, can resist an acidic environment with a pH value of 2.5 and a bile salt environment with a concentration of 0.4 percent for 4 hours, has mesophilic bacteria, and has a growth temperature ranging from 15 ℃ to 45 ℃ and an optimal growth temperature of about 37 ℃.
1.3 Caco-2 cell culture
Human colon tumor cell line (Caco-2) was purchased from DSMZ (Donlorek, germany) and cultured in the presence of 5% CO2 at 37℃under a certain humidity. Caco-2 cells at passages 40-44 were used for the experiments. MEM medium was supplemented with 20% (v/v) Fetal Bovine Serum (FBS), 1% non-essential amino acids, 1% Glutamax,1% sodium pyruvate, with or without 1% penicillin-streptomycin solution, and 50ug/mL gentamicin (all available from Invitrogen corporation of Netherlands Radar). Cells were grown to 80% abundance in T75 flasks and harvested by trypsinization.
1.4 cultivation of pathogenic bacteria ETEC and EPEC
The study used two pathogenic bacteria, E.coli ETEC H10407 and E.coli EPEC serotype O119, which are enterotoxigenic, respectively. Both bacteria can be used to simulate small intestine infections in vitro. Both of these are common pathogens causing diarrhea in infants and travelers, especially in developing areas where sanitary conditions are poor. ETEC H10407 is a well-defined model strain commonly used in vitro experiments and has been widely used in other studies to evaluate probiotics and prebiotics for pathogenic adsorption and inflammatory signaling. This strain is also used in animal experiments to develop vaccines. EPEC serotype O119 was isolated from infant faeces and prebiotics have been shown to reduce their adsorption.
ETEC cell line H10407 (ATCC 35401) was cultured with BHI-B medium (Merck, N.Y.). After overnight incubation at 37℃under anaerobic conditions, the pathogen was again incubated prior to infection to reach mid-log phase. Cells were collected by centrifugation, washed and resuspended in PBS prior to the experiment.
EPEC serotype O119 strain was purchased from DSMZ under freeze-drying conditions (DSM 8699). The strain was cultured with BHI-B medium (Merck, N.Y.A.). After overnight incubation at 37℃under anaerobic conditions, the pathogen was again incubated prior to infection to reach mid-log phase. Cells were collected by centrifugation, washed and resuspended in PBS prior to the experiment.
1.5 data analysis
Statistical analysis of each individual test was performed with three replicates (sometimes six replicates are used), if possible. Anti-adhesion data were transformed with 10 logs. Statistical analysis was performed with one-way ANOVA for anti-adhesion data and epidermal signaling data after 10log transformation. Statistical differences from negative control (neg. Control) or from E.coli-stimulated conditions were identified using the Dunnett's posthoc test. P <0.05 was considered to be significantly different. Because Dunnett's posthoc test uses ANOVA MSResidual as a pooled assessment of differences and uses a modified significance value to adjust the number of comparisons, the same result may be significant in one graph and not the other.
2. Specific experimental procedure
2.1 anti-adhesion test
Caco-2 cells were cultured on 24-well plates. On the day of the assay, caco-2 cells were rinsed with pre-warmed PBS. The test substances were added to Caco-2 cells in triplicate replicates. Cells were incubated with the test substance for 1 hour. Pathogenic E.coli was then added at a fold infection (MOI) of 50:1 addition (final concentration 10) 7 CFU/mL), co-cultured with the test substance at 37 ℃ for 1 hour. As a negative control, caco-2 cells were cultured in medium with only pathogenic bacteria. 1mM zinc oxide (ZnO) was used as a positive control as it was reported to reduce pathogenic adsorption. After culturing, caco-2 cells are washed and lysed, and the pathogen is then inoculated onto agar. After overnight incubation on agar plates at 37 ℃, the CFU colonies of the bacteria were counted to measure pathogen adsorption. The number of E.coli colonies in growth was counted and recorded as CFU/mL. Coli (final concentration) 10 in parallel with the anti-adhesion test 7 CFU/mL was added to 1mL of the five combinations tested and co-cultured at 37 ℃ for 1 hour to measure activity. After incubation, E.coli was collected from each sample by centrifugation, resuspended in PBS, and inoculated on agar plates. After overnight incubation on agar plates at 37 ℃, the CFU colonies of the bacteria were counted to measure pathogen adsorption. The number of E.coli colonies in growth was counted and recorded as CFU/mL.
2.2 inflammatory factor Release test
The prebiotics and probiotics have immunoregulatory (promoting or anti-inflammatory) effects, and can increase resistance to infection or promote intestinal health. The immunomodulatory effects of prebiotic probiotics can be measured by measuring cytokine/chemokine production by small intestine epithelial cells in the presence or absence of pro-inflammatory stimuli. The effect of prebiotics and probiotics on chemokine/cytokine production can be screened by stimulating Caco-2 cells with E.coli strains and measuring IL-8 and IP-10 production in the supernatant. IL-8 is important for an urgent autoimmune response and can lead to neutrophil aggregation. IP-10 is important in secondary responses to immunization. It attracts monocytes and macrophages, including Th1 cells, which play an important role in clearing infection. Proinflammatory prebiotics and probiotics may increase IL-8 and/or IP-10 production, while anti-inflammatory prebiotics and probiotics may decrease IL-8 and/or IP-10 production.
Caco-2 cells were plated in 96-well plates to appropriate abundance. At the beginning of the experiment, the cells were rinsed once with medium without antibiotics. The monolayer cells were co-cultured with the test substance at 37℃for 1 hour in a medium without antibiotics, and repeated three times. E.coli cells were stimulated (MOI 200:1). After 1 hour of culture, a monolayer of cells was co-cultured with the pathogen and washed and cultured overnight with a culture medium containing the test substance and 50ug/mL gentamicin. As a blank, only the culture broth was used without E.coli stimulation. Culture medium stimulated with E.coli but without test substance was used as a control for E.coli response. In addition, as a control for Caco-2 cell responses, cells were stimulated with a mixture culture broth containing Rec TNFα (10 ng/mL) and Rec IFNγ (5 ng/mL), both purchased from R & D systems of Abin, england. Supernatants were collected 24 hours of stimulation and stored at-20 ℃. IL-8, IP-10 and RANTES were tested using the Bio-Plex kit (BioRad, calif. USA) according to the manufacturer's instructions.
2.3 intestinal barrier integrity test
The ideal intestinal epithelial barrier function is a prerequisite for protecting the host from pathogenic invasion and/or pathogenic toxins. In this study, barrier integrity in vitro was demonstrated by measuring transepithelial electrical resistance (TEER) of the intestinal cell layer. The food ingredients may have the function of protecting the intestinal barrier function from decline after infection (alleviating decline in TEER after infection). To study the effect of prebiotics and probiotics on infection, TEER was measured before and after escherichia coli infection over time.
Caco-2 cells were seeded into Transwell polycarbonate cell culture inserts with an average pore size of 0.4um and an area of 0.33cm 2 Until fully differentiated (+ -1000 omega). Transepithelial electrical resistance (TEER) was measured using an EVOM2 epidermoid voltmeter purchased from world-precise instruments to measure barrier integrity.
On the day of testing, cells were washed and incubated at 37℃for 1 hour in medium without antibiotics and serum, but with the test substance. Coli was then added to the test material (fold MOI 200:1) and incubated for 6 hours. TEER was measured 1 hour after exposure of the test substance to the pathogen and before addition of the pathogen (t=0) before the start of the experiment (t= -1), and 1 hour, 2 hours, 3 hours, 4 hours and 6 hours after pathogen exposure, respectively. TEER values under the individual conditions after contact with a pathogen are correlated with their TEER values each at t=0 and are expressed as Δteer (Ω·cm2). Negative controls (E.coli alone) and positive controls that were not exposed to the pathogen or test substance are also included in the experimental group. All conditions were repeated three times and some control groups were repeated 6 times.
3. Test of the influence of test substances on the survival rate of pathogenic bacteria EPEC
To verify whether the reduction in pathogenic adsorption is related to pathogenic activity, pathogenic activity was also detected after the prebiotics and probiotic cultures. As shown in fig. 1, 2, 3 and 4, the survival rate of the escherichia coli EPEC 0119 bacteria is not significantly affected after the test substance K-56 or 2' -FL or the combination of the two is added, similar to other test probiotics or prebiotics. Like other probiotics or prebiotics tested, ET-22 or 2' -FL did not significantly affect the survival rate of EPEC 0119 bacteria. While the combination of the two did not significantly reduce the survival rate of EPEC 0119 bacteria.
Example 1: lactobacillus paracasei K-56 and breast milk oligosaccharide 2' -FL combined on adhesion of pathogenic bacteria EPEC in intestinal tract
Results of experiments
The preparation step before the experiment and the specific experimental method are disclosed in the previous paragraph. The test Lactobacillus paracasei K-56 has a normal growth curve.
The protective effect of probiotics and prebiotic compositions against pathogenic bacteria adsorbing to small intestine epithelial cells was investigated by the common diarrheagenic strain (EPEC O119).
As a result, as shown in fig. 5, the adhesion effect of the breast milk oligosaccharide 2' -FL alone on pathogenic bacteria such as escherichia coli O119 on intestinal cells Caco-2 is not significantly different from that of the negative control, while the positive control zinc oxide can significantly reduce the adsorption of pathogenic bacteria on intestinal cells (p < 0.0001). The adhesion of the single probiotics K56 to pathogenic bacteria E.coli O119 to intestinal cells Caco-2 was not significantly different from that of the negative control. But when prebiotic 2' -FL was used as a treatment group test substance with the probiotic, both significantly reduced the adsorption of pathogenic bacteria EPEC 0119 to intestinal cells (p < 0.001). Showing a synergistic effect when the two are formed into a composition.
Example 2: lactobacillus paracasei ET-22 and breast milk oligosaccharide 2' -FL combined to adhere to pathogenic bacteria EPEC in intestinal tract
Experimental results
The preparation step before the experiment and the specific experimental method are disclosed in the previous paragraph. The tested Lactobacillus paracasei ET-22 had a normal growth curve.
As a result, as shown in fig. 6, the adhesion effect of the breast milk oligosaccharide 2' -FL alone on pathogenic bacteria such as escherichia coli O119 on intestinal cells Caco-2 is not significantly different from that of the negative control, while the positive control zinc oxide can significantly reduce the adsorption of pathogenic bacteria on intestinal cells (p < 0.0001). The adhesion of the individual probiotics ET-22 to pathogenic bacteria E.coli O119 to intestinal cells Caco-2 was not significantly different from that of the negative control. But when prebiotic 2' -FL was used as a treatment group test substance with the probiotic, both significantly reduced the adsorption of pathogenic bacteria EPEC 0119 to intestinal cells (p < 0.001). Showing a synergistic effect when the two are formed into a composition.
Example 3: lactobacillus paracasei K-56 and breast milk oligosaccharide 2' -FL combined for secreting inflammatory factors to intestinal cells
Influence of IP-10
The preparation step before the experiment and the specific experimental method are disclosed in the previous paragraph. The test Lactobacillus paracasei K-56 has a normal growth curve.
As a result, referring to FIG. 7, in the case of the E.coli ETEC invasion, the release of IP-10 was not affected by the breast milk oligosaccharide 2'-FL, K56 was able to reduce the release of IP-10, and when the two compositions were added, the compositions were able to change the case where 2' -FL itself had no effect on the release of IP-10, which in turn reduced the release of IP-10. The nutritional composition of K56+2' -FL has a certain regulation effect on the release of inflammatory factors.
Example 4: lactobacillus paracasei ET-22 and breast milk oligosaccharide 2' -FL combined for secreting inflammatory factors to intestinal cells
IL-8 and IP-10 effects
The preparation step before the experiment and the specific experimental method are disclosed in the previous paragraph. The tested Lactobacillus paracasei ET-22 had a normal growth curve.
As a result, referring to FIGS. 8A and 8B, in the case of E.coli ETEC, the human milk oligosaccharide 2'-FL does not affect the release of IL-8, while ET-22 reduces the release of IL-8, and when a combination of the two is added, the combination changes the condition that 2' -FL has no effect on the release of IL-8, thereby reducing the release of IL-8. Breast milk oligosaccharide 2'-FL can slightly reduce the release of IP-10, ET-22 can obviously reduce the release of IP-10, and when the composition of the two can be added, the composition can change the condition that the release of 2' -FL per se is slightly reduced to IP-10, so that the release of IP-10 is greatly reduced. The nutritional composition of ET-22+2' -FL has a certain regulation effect on the release of inflammatory factors.
Example 5: effect of lactobacillus paracasei K56 in combination with breast milk oligosaccharide 2' -FL on transmembrane resistance (TEER)
Results of experiments
The preparation step before the experiment and the specific experimental method are disclosed in the previous paragraph. The test lactobacillus paracasei K56 has a normal growth curve. As shown in fig. 9A, the effect of the test substances on the transmembrane resistance was tested at different time points, the individual breast milk oligosaccharide 2' -FL had no effect on the decrease in transmembrane resistance TEER caused by ETEC, and was close to the negative control group, while the probiotic K56 could have a tendency to raise the value of transmembrane resistance TEER or prevent it from decreasing to some extent at a plurality of time points; and after the breast milk oligosaccharide 2' -FL is added, the effect of improving or preventing the decline is also achieved, and the effect of the breast milk oligosaccharide is easier than that of a blank group.
Example 6: influence of Lactobacillus paracasei ET-22 in combination with Breast milk oligosaccharide 2' -FL on transmembrane resistance (TEER)
Experimental results
The preparation step before the experiment and the specific experimental method are disclosed in the previous paragraph. The tested Lactobacillus paracasei ET-22 had a normal growth curve. As shown in fig. 9B, the effect of the test substances on the transmembrane resistance was tested at different time points, and the individual breast milk oligosaccharide 2' -FL had no effect on the decrease in transmembrane resistance TEER caused by ETEC, which was similar to that of the negative control group, whereas the probiotic ET-22 could have a tendency to raise the value of transmembrane resistance TEER or prevent it from decreasing to some extent at a plurality of time points; and after the addition of the breast milk oligosaccharide 2' -FL, the effect of improving or preventing the decline tends to be enhanced, compared with the effect of the blank group.
Claims (10)
1. A nutritional composition is prepared from Lactobacillus paracaseiLactobacillus paracasei) And breast milk oligosaccharide, wherein the breast milk oligosaccharide is 2' -fucosyllactose,
wherein the lactobacillus paracasei is lactobacillus paracasei with a preservation number of DSM27447 and/or lactobacillus paracasei with a preservation number of CGMCC No. 15077;
wherein the ratio of Lactobacillus paracasei to breast milk oligosaccharide is 1×10 3 CFU~1×10 12 CFU：5mg。
2. The composition of claim 1, wherein the ratio of lactobacillus paracasei to breast milk oligosaccharide is 1 x 10 6 CFU~1×10 10 CFU：5mg。
3. The composition of claim 1, wherein the lactobacillus paracasei is used in the nutritional composition in an amount of 1 x 10 3 CFU~1×10 12 CFU/day.
4. The composition of claim 1, wherein the lactobacillus paracasei is used in the nutritional composition in an amount of 1 x 10 6 CFU~1×10 11 CFU/day.
5. The composition of claim 1, wherein the amount of breast milk oligosaccharide applied in the nutritional composition is 1mg to 15g per day.
6. Use of a composition according to any one of claims 1 to 5 for the preparation of a food or pharmaceutical product having the efficacy of enhancing the immunological competence of the gastrointestinal tract;
wherein the enhancing the gastrointestinal immunity comprises defending the intestinal system against pathogenic bacteria invasion and/or protecting the integrity of the intestinal barrier; or alternatively
The improvement of gastrointestinal immunity comprises reduction of inflammatory factor IL-8 released by intestinal cells; or alternatively
The improving gastrointestinal immunity comprises reducing inflammatory factor IP-10 released by intestinal cells.
7. The use according to claim 6, wherein the food product is a liquid beverage or a solid beverage.
8. The use according to claim 6, wherein the food product is an oral liquid.
9. The use according to claim 6, wherein the food product is a dairy product.
10. The use according to claim 6, wherein the food product is a tablet or capsule.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010435608.2A CN113712207B (en) | 2020-05-21 | 2020-05-21 | Probiotic prebiotic composition for improving gastrointestinal tract immunity and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010435608.2A CN113712207B (en) | 2020-05-21 | 2020-05-21 | Probiotic prebiotic composition for improving gastrointestinal tract immunity and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113712207A CN113712207A (en) | 2021-11-30 |
| CN113712207B true CN113712207B (en) | 2023-06-06 |
Family
ID=78671271
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010435608.2A Active CN113712207B (en) | 2020-05-21 | 2020-05-21 | Probiotic prebiotic composition for improving gastrointestinal tract immunity and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113712207B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117586926B (en) * | 2024-01-18 | 2024-04-16 | 东北农业大学 | Lactobacillus paracasei LP-116, LP-116 metagen composition, and preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107916236A (en) * | 2016-10-05 | 2018-04-17 | 益福生医股份有限公司 | Lactobacillus paracasei subspecies paracasei K56 |
| CN108741083A (en) * | 2010-12-31 | 2018-11-06 | 雅培制药有限公司 | Promote the neutral human milk oligosaccharides of beneficial bacteria growth |
| CN110839702A (en) * | 2018-08-21 | 2020-02-28 | 内蒙古伊利实业集团股份有限公司 | Breast milk oligosaccharide composition for improving abundance of infant intestinal flora and feces smell and application thereof |
| CN110892940A (en) * | 2019-11-20 | 2020-03-20 | 内蒙古伊利实业集团股份有限公司 | New application of lactobacillus paracasei K56 in relieving intestinal inflammation |
| WO2020063515A1 (en) * | 2018-09-30 | 2020-04-02 | 内蒙古伊利实业集团股份有限公司 | Novel application of lactobacillus paracasei k56 capable of regulating balance of gastrointestinal flora |
| CN110964658A (en) * | 2018-09-30 | 2020-04-07 | 内蒙古伊利实业集团股份有限公司 | Lactobacillus paracasei ET-22 with immunoregulation function |
-
2020
- 2020-05-21 CN CN202010435608.2A patent/CN113712207B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108741083A (en) * | 2010-12-31 | 2018-11-06 | 雅培制药有限公司 | Promote the neutral human milk oligosaccharides of beneficial bacteria growth |
| CN107916236A (en) * | 2016-10-05 | 2018-04-17 | 益福生医股份有限公司 | Lactobacillus paracasei subspecies paracasei K56 |
| CN110839702A (en) * | 2018-08-21 | 2020-02-28 | 内蒙古伊利实业集团股份有限公司 | Breast milk oligosaccharide composition for improving abundance of infant intestinal flora and feces smell and application thereof |
| WO2020063515A1 (en) * | 2018-09-30 | 2020-04-02 | 内蒙古伊利实业集团股份有限公司 | Novel application of lactobacillus paracasei k56 capable of regulating balance of gastrointestinal flora |
| CN110964658A (en) * | 2018-09-30 | 2020-04-07 | 内蒙古伊利实业集团股份有限公司 | Lactobacillus paracasei ET-22 with immunoregulation function |
| CN110959865A (en) * | 2018-09-30 | 2020-04-07 | 内蒙古伊利实业集团股份有限公司 | New application of lactobacillus paracasei K56 capable of adjusting gastrointestinal flora balance |
| CN110892940A (en) * | 2019-11-20 | 2020-03-20 | 内蒙古伊利实业集团股份有限公司 | New application of lactobacillus paracasei K56 in relieving intestinal inflammation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113712207A (en) | 2021-11-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112869170B (en) | Probiotics prebiotic nutritional composition capable of improving gastrointestinal tract immunity and application | |
| CN112868800A (en) | Infant formula milk powder containing breast milk oligosaccharide for improving immunity and preparation method thereof | |
| CN112870233A (en) | Composition containing bifidobacterium lactis and breast milk oligosaccharide and application thereof | |
| CN112515170B (en) | Bifidobacterium lactis and prebiotics composition for improving gastrointestinal tract immunity and application thereof | |
| CN112869168B (en) | Probiotics prebiotic composition capable of improving gastrointestinal tract immunity and application thereof | |
| US20200009202A1 (en) | Bacterial composition and its use | |
| EP1007625B1 (en) | Immunity enhancing lactic acid bacteria | |
| US20040110270A1 (en) | Bacterial composition and its use | |
| CN113519849B (en) | Breast milk oligosaccharide for improving intestinal tract resistance to escherichia coli infection and application thereof | |
| AU2021388477A9 (en) | Use of Lactobacillus paracasei ET-22 for enhancing resistance of intestinal tract to bacterial infection and improving intestinal immunity | |
| CN113913346A (en) | Lactobacillus paracasei JN-1 and application thereof | |
| CN114568516A (en) | Composition containing bifidobacterium longum subsp. infantis and breast milk oligosaccharide and application thereof | |
| CN113712207B (en) | Probiotic prebiotic composition for improving gastrointestinal tract immunity and application thereof | |
| CN113679048A (en) | High-activity probiotic freeze-dried powder for adjusting balance of intestinal flora of infants | |
| CN114568520A (en) | Infant formula powder containing bifidobacterium longum subspecies infantis and application thereof | |
| CN114246339A (en) | Composition and application thereof in preparation of product for improving intestinal immunity | |
| CN114634884B (en) | Bifidobacterium longum subspecies GB-1496 and application thereof in improving intestinal bacterial infection resistance and intestinal immunity | |
| CN114287629A (en) | Composition and product for improving intestinal immunity | |
| US11839635B1 (en) | Method against Salmonella typhimurium infection with symbiotic composition | |
| CN116790402B (en) | Bacteroides simplex strain with anti-inflammatory property, culture method and application | |
| WO2024065274A1 (en) | Method against salmonella typhimurium infection with symbiotic composition | |
| CN114468305A (en) | Probiotic composition for relieving adverse reaction caused by excessive exercise and application thereof | |
| CN117917475A (en) | Lactobacillus plantarum P16 for regulating intestinal flora, application, product and method thereof | |
| Geigerová et al. | Selection of prebiotic oligosaccharides suitable for |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |