CN113215035A - 长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用 - Google Patents
长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用 Download PDFInfo
- Publication number
- CN113215035A CN113215035A CN202110490551.0A CN202110490551A CN113215035A CN 113215035 A CN113215035 A CN 113215035A CN 202110490551 A CN202110490551 A CN 202110490551A CN 113215035 A CN113215035 A CN 113215035A
- Authority
- CN
- China
- Prior art keywords
- bifidobacterium
- solution
- preparation
- bifidobacterium longum
- animalis
- 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
- 241001608472 Bifidobacterium longum Species 0.000 title claims abstract description 61
- 229940009291 bifidobacterium longum Drugs 0.000 title claims abstract description 61
- 241001134770 Bifidobacterium animalis Species 0.000 title claims abstract description 59
- 229940118852 bifidobacterium animalis Drugs 0.000 title claims abstract description 59
- 238000002360 preparation method Methods 0.000 title claims abstract description 36
- 230000003712 anti-aging effect Effects 0.000 title claims abstract description 27
- 241000186000 Bifidobacterium Species 0.000 claims abstract description 61
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000001963 growth medium Substances 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 15
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000011790 ferrous sulphate Substances 0.000 claims abstract description 11
- 235000003891 ferrous sulphate Nutrition 0.000 claims abstract description 11
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims abstract description 11
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims abstract description 11
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000009630 liquid culture Methods 0.000 claims abstract description 8
- 239000008367 deionised water Substances 0.000 claims abstract description 7
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 6
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229960004889 salicylic acid Drugs 0.000 claims abstract description 6
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims abstract description 5
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims abstract description 5
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 5
- -1 potassium ferricyanide Chemical compound 0.000 claims abstract description 5
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 claims abstract description 5
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000002994 raw material Substances 0.000 claims abstract description 4
- 239000000523 sample Substances 0.000 claims description 29
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 24
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 16
- 230000001580 bacterial effect Effects 0.000 claims description 16
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- 229920001817 Agar Polymers 0.000 claims description 14
- 239000008272 agar Substances 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 10
- 238000010521 absorption reaction Methods 0.000 claims description 9
- 239000011521 glass Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 229910052742 iron Inorganic materials 0.000 claims description 8
- 239000004310 lactic acid Substances 0.000 claims description 8
- 235000014655 lactic acid Nutrition 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 239000006041 probiotic Substances 0.000 claims description 8
- 235000018291 probiotics Nutrition 0.000 claims description 8
- 235000013361 beverage Nutrition 0.000 claims description 7
- 210000003608 fece Anatomy 0.000 claims description 7
- 235000013336 milk Nutrition 0.000 claims description 7
- 239000008267 milk Substances 0.000 claims description 7
- 210000004080 milk Anatomy 0.000 claims description 7
- 230000000529 probiotic effect Effects 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- 238000011081 inoculation Methods 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 5
- 235000018102 proteins Nutrition 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 239000013589 supplement Substances 0.000 claims description 5
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 4
- 102000011632 Caseins Human genes 0.000 claims description 4
- 108010076119 Caseins Proteins 0.000 claims description 4
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 claims description 4
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 claims description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 4
- 229930003316 Vitamin D Natural products 0.000 claims description 4
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 claims description 4
- 230000003385 bacteriostatic effect Effects 0.000 claims description 4
- 239000011575 calcium Substances 0.000 claims description 4
- 229910052791 calcium Inorganic materials 0.000 claims description 4
- 239000011574 phosphorus Substances 0.000 claims description 4
- 229910052698 phosphorus Inorganic materials 0.000 claims description 4
- 230000001737 promoting effect Effects 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 235000019166 vitamin D Nutrition 0.000 claims description 4
- 239000011710 vitamin D Substances 0.000 claims description 4
- 150000003710 vitamin D derivatives Chemical class 0.000 claims description 4
- 229940046008 vitamin d Drugs 0.000 claims description 4
- 235000021249 α-casein Nutrition 0.000 claims description 4
- 229940099352 cholate Drugs 0.000 claims description 3
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 claims description 3
- 239000012470 diluted sample Substances 0.000 claims description 3
- 230000002550 fecal effect Effects 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 238000011403 purification operation Methods 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 235000008924 yoghurt drink Nutrition 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 19
- 102000019197 Superoxide Dismutase Human genes 0.000 abstract description 12
- 108010012715 Superoxide dismutase Proteins 0.000 abstract description 12
- 230000032683 aging Effects 0.000 abstract description 11
- 210000002966 serum Anatomy 0.000 abstract description 9
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 abstract description 6
- 150000002632 lipids Chemical class 0.000 abstract description 3
- 102000006587 Glutathione peroxidase Human genes 0.000 abstract description 2
- 108700016172 Glutathione peroxidases Proteins 0.000 abstract description 2
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 abstract description 2
- 208000026139 Memory disease Diseases 0.000 abstract description 2
- 229940118019 malondialdehyde Drugs 0.000 abstract description 2
- 230000003647 oxidation Effects 0.000 abstract description 2
- 238000007254 oxidation reaction Methods 0.000 abstract description 2
- 150000002978 peroxides Chemical class 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 38
- 241000894006 Bacteria Species 0.000 description 17
- 230000000968 intestinal effect Effects 0.000 description 16
- 238000012360 testing method Methods 0.000 description 16
- 210000001035 gastrointestinal tract Anatomy 0.000 description 14
- 241001465754 Metazoa Species 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 11
- 241000699670 Mus sp. Species 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 206010012735 Diarrhoea Diseases 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 244000005700 microbiome Species 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 206010010774 Constipation Diseases 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000003833 bile salt Substances 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 230000000971 hippocampal effect Effects 0.000 description 6
- 230000009286 beneficial effect Effects 0.000 description 5
- 108010005774 beta-Galactosidase Proteins 0.000 description 5
- 238000003304 gavage Methods 0.000 description 5
- 230000036039 immunity Effects 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 229920001542 oligosaccharide Polymers 0.000 description 5
- 150000002482 oligosaccharides Chemical class 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 102100026189 Beta-galactosidase Human genes 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 230000002292 Radical scavenging effect Effects 0.000 description 4
- 244000052616 bacterial pathogen Species 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 208000019902 chronic diarrheal disease Diseases 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 210000002381 plasma Anatomy 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 3
- 108010059881 Lactase Proteins 0.000 description 3
- 241000186660 Lactobacillus Species 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
- 238000004364 calculation method Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000009194 climbing Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000008273 gelatin Substances 0.000 description 3
- 229920000159 gelatin Polymers 0.000 description 3
- 235000019322 gelatine Nutrition 0.000 description 3
- 235000011852 gelatine desserts Nutrition 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 210000004347 intestinal mucosa Anatomy 0.000 description 3
- 229940039696 lactobacillus Drugs 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000186016 Bifidobacterium bifidum Species 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- 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 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 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 2
- 230000003064 anti-oxidating effect Effects 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000000711 cancerogenic effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 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 2
- 238000004043 dyeing Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 229940116108 lactase Drugs 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 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 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- ZFRKQXVRDFCRJG-UHFFFAOYSA-N skatole Chemical compound C1=CC=C2C(C)=CNC2=C1 ZFRKQXVRDFCRJG-UHFFFAOYSA-N 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 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 description 1
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 231100000699 Bacterial toxin Toxicity 0.000 description 1
- 108010062877 Bacteriocins Proteins 0.000 description 1
- 244000177578 Bacterium linens Species 0.000 description 1
- 241000193468 Clostridium perfringens Species 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 102000001390 Fructose-Bisphosphate Aldolase Human genes 0.000 description 1
- 108010068561 Fructose-Bisphosphate Aldolase Proteins 0.000 description 1
- 208000018522 Gastrointestinal disease 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
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 208000005016 Intestinal Neoplasms Diseases 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 244000199866 Lactobacillus casei Species 0.000 description 1
- 235000013958 Lactobacillus casei Nutrition 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 208000003100 Pseudomembranous Enterocolitis Diseases 0.000 description 1
- 241000589157 Rhizobiales Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000607762 Shigella flexneri Species 0.000 description 1
- 241000973497 Siphonognathus argyrophanes Species 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- UQZIYBXSHAGNOE-USOSMYMVSA-N Stachyose 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[C@@H]2[C@@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O2)O1 UQZIYBXSHAGNOE-USOSMYMVSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 102100033220 Xanthine oxidase Human genes 0.000 description 1
- 108010093894 Xanthine oxidase Proteins 0.000 description 1
- 208000023505 abnormal feces Diseases 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 201000009840 acute diarrhea Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- VBIXEXWLHSRNKB-UHFFFAOYSA-N ammonium oxalate Chemical compound [NH4+].[NH4+].[O-]C(=O)C([O-])=O VBIXEXWLHSRNKB-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 230000002790 anti-mutagenic effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000000688 bacterial toxin Substances 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 229940002008 bifidobacterium bifidum Drugs 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000005978 brain dysfunction Effects 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 230000006652 catabolic pathway Effects 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000003920 cognitive function Effects 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000013872 defecation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000002996 emotional effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 206010016766 flatulence Diseases 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002431 foraging effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- FTSSQIKWUOOEGC-RULYVFMPSA-N fructooligosaccharide Chemical compound OC[C@H]1O[C@@](CO)(OC[C@@]2(OC[C@@]3(OC[C@@]4(OC[C@@]5(OC[C@@]6(OC[C@@]7(OC[C@@]8(OC[C@@]9(OC[C@@]%10(OC[C@@]%11(O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@H]%12O)O[C@H](CO)[C@@H](O)[C@@H]%11O)O[C@H](CO)[C@@H](O)[C@@H]%10O)O[C@H](CO)[C@@H](O)[C@@H]9O)O[C@H](CO)[C@@H](O)[C@@H]8O)O[C@H](CO)[C@@H](O)[C@@H]7O)O[C@H](CO)[C@@H](O)[C@@H]6O)O[C@H](CO)[C@@H](O)[C@@H]5O)O[C@H](CO)[C@@H](O)[C@@H]4O)O[C@H](CO)[C@@H](O)[C@@H]3O)O[C@H](CO)[C@@H](O)[C@@H]2O)[C@@H](O)[C@@H]1O FTSSQIKWUOOEGC-RULYVFMPSA-N 0.000 description 1
- 229940107187 fructooligosaccharide Drugs 0.000 description 1
- 235000021255 galacto-oligosaccharides Nutrition 0.000 description 1
- 150000003271 galactooligosaccharides Chemical class 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 210000001320 hippocampus Anatomy 0.000 description 1
- 230000005745 host immune response Effects 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 201000002313 intestinal cancer Diseases 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 230000007413 intestinal health Effects 0.000 description 1
- 244000000074 intestinal pathogen Species 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- DLRVVLDZNNYCBX-RTPHMHGBSA-N isomaltose 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(O)O1 DLRVVLDZNNYCBX-RTPHMHGBSA-N 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 229940017800 lactobacillus casei Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000006386 memory function Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 230000000626 neurodegenerative effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000008621 organismal health Effects 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 description 1
- 229940074386 skatole Drugs 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- UQZIYBXSHAGNOE-XNSRJBNMSA-N stachyose 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[C@@H]3[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O3)O)O2)O)O1 UQZIYBXSHAGNOE-XNSRJBNMSA-N 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- QGVNJRROSLYGKF-UHFFFAOYSA-N thiobarbital Chemical compound CCC1(CC)C(=O)NC(=S)NC1=O QGVNJRROSLYGKF-UHFFFAOYSA-N 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
-
- 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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- 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
- A23V2400/51—Bifidobacterium
- A23V2400/515—Animalis
-
- 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
- A23V2400/51—Bifidobacterium
- A23V2400/533—Longum
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Nutrition Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- General Engineering & Computer Science (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明提供长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,涉及双歧杆菌技术领域,由以下重量份的原料制备而成:MRS液体培养基,去离子水;0.2mmol/LDPPH甲醇溶液;2mmol/L硫酸亚铁溶液,6mmol/L过氧化氢溶液,6mmol/L水杨酸溶液;150mmol/LpH=8.0的Tris‑HCL溶液,1.2mmol/L邻苯三酚溶液;0.4%的硫酸亚铁溶液,1%的VC,0.2mol/L的氢氧化钠溶液,10%的三氯乙酸溶液,0.1%邻二氮菲溶液;pH=6.6的磷酸缓冲液,1%铁氰化钾溶液,0.1%的三氯化铁溶液。本发明,可以缓解D-半乳糖衰老小鼠的记忆障碍,增加了小鼠血清中超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH‑PX)活性,降低了血清中脂质过氧化物丙二醛的浓度,减少细胞的氧化速度,有效提高单个细胞的寿命。
Description
技术领域
本发明涉及双歧杆菌技术领域,尤其涉及长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用。
背景技术
双歧杆菌是一个细菌属名。双歧杆菌属(Bifidobacterium)是一种革兰氏阳性、不运动、细胞呈杆状、一端有时呈分叉状、严格厌氧的细菌属,广泛存在于人和动物的消化道、阴道和口腔等生境中。双歧杆菌属的细菌是人和动物肠道菌群的重要组成成员之一。一些双歧杆菌的菌株可以作为益生菌而用在食品、医药和饲料方面。
衰老几乎是所有生物都会经历的过程,衰老的特征在于随着时间推移,机体细胞、组织、器官功能逐渐衰退,同时伴随着认知和记忆功能减退。随着人口的老龄化,包括神经退行性疾病和神经精神疾病在内的脑功能障碍的发生率预计会迅速增加。寻找可行性的抗衰老药物成为对老龄化问题的研究热点。目前关于衰老的机制主要有氧化应激、细胞凋亡、DNA损伤、免疫、炎性衰老等。
发明内容
本发明的目的是为了解决现有技术中存在的缺点,而提出的长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用。
为了实现上述目的,本发明采用了如下技术方案:长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,由以下重量份的原料制备而成:MRS液体培养基,去离子水;0.2mmol/LDPPH甲醇溶液;2mmol/L硫酸亚铁溶液,6mmol/L 过氧化氢溶液,6mmol/L水杨酸溶液;150mmol/LpH=8.0的Tris-HCL溶液,1.2mmol/L邻苯三酚溶液;0.4%的硫酸亚铁溶液,1%的VC,0.2mol/L的氢氧化钠溶液,10%的三氯乙酸溶液,0.1%邻二氮菲溶液;pH=6.6的磷酸缓冲液,1%铁氰化钾溶液,0.1%的三氯化铁溶液。
优选的,所述MRS液体培养基内部接种有长双歧杆菌和动物双歧杆菌,所述长双歧杆菌和动物双歧杆菌均提取与百岁老人的粪便中分离的耐酸、耐胆盐和抑菌的益生菌株。
优选的,所述长双歧杆菌和动物双歧杆菌的制备方法包括:
步骤一:取定量的百岁老人的粪便加入量筒中,并采用50%比例的甘油进行封存保存;
步骤二:取出一定量的粪便样本至托盘上并通过无菌PBS(1%,w/v)进行稀释,将稀释后的样本涂抹在MRS琼脂平板上;
步骤三:将处理后的MRS琼脂平板放置于无菌室中,并设置室内温度至37℃,培养24小时,并逐步观察MRS琼脂平板上的菌落情况;
步骤四:观察MRS琼脂平板上的菌落状态,使用消毒后的划针对菌落进行划线纯化操作;
步骤五:取上述操作中菌落中的样本并再次进行接种,并对接种后的样本再次进行接种操作,重复接种三次,并提取操作样本中的菌液使其与50%甘油以 1:1的比例搅拌混合,将样本存放在-80℃;
步骤六:即可对样本进行后续操作。
优选的,所述粪便取固体状,并采用玻璃棒捣碎搅拌,将内部的大颗粒状杂物剔除。
优选的,所述长双歧杆菌和动物双歧杆菌每日补充量各为109CFU/mL,可通过喝含双歧杆菌的酸奶或饮料来补充双歧杆菌,也可直接摄入含有双歧杆菌的微生态制剂进行补充。
优选的,所述长双歧杆菌和动物双歧杆菌可进一步制成流体乳品、饮料或益生菌菌片。
优选的,所述双歧杆菌最主要产物包括乳酸、乙酸等,促进铁和维生素D 的吸收并提高磷、铁、钙的利用率;双歧杆菌通过磷蛋白磷酸酶分解α-酪蛋白,促进蛋白吸收。
与现有技术相比,本发明的优点和积极效果在于,
1、本发明中,涉及的长双歧杆菌和动物双歧杆菌来源于百岁老人粪便样本中分离得到的,该菌株经菌种测序分析,其序列如SEQIDNO.1所示,将得到的序列经NCBI核酸序列对比,结果显示菌株为双歧杆菌,命名为长双歧杆菌 BAMA-B05/BAu-B1024和动物双歧杆菌LPL-RH。所述长双歧杆菌和动物双歧杆菌,它可以缓解D-半乳糖衰老小鼠的记忆障碍,增加了小鼠血清中超氧化物歧化酶 (SOD)和谷胱甘肽过氧化物酶(GSH-PX)活性,降低了血清中脂质过氧化物丙二醛的浓度,减少细胞的氧化速度,有效提高单个细胞的寿命。
附图说明
图1为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中长双歧杆菌和动物双歧杆菌耐酸结果;
图2为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中长双歧杆菌和动物双歧杆菌耐胆盐结果;
图3为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中长双歧杆菌和动物双歧杆菌抗氧化结果;
图4为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中长双歧杆菌和动物双歧杆菌抑菌结果;
图5为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中长双歧杆菌和动物双歧杆菌HT-29细胞黏附结果;
图6为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中实验小鼠旷场实验测试结果;
图7为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中实验小鼠爬杆实验测试结果;
图8为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中实验小鼠巴恩斯迷宫实验测试结果;
图9为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中实验小鼠海马中SOD、GSH-Px、MDA测定实验结果;
图10为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中实验小鼠β-半乳糖苷酶染色实验结果。
具体实施方式
为了能够更清楚地理解本发明的上述目的、特征和优点,下面结合附图和实施例对本发明做进一步说明。需要说明的是,在不冲突的情况下,本申请的实施例及实施例中的特征可以相互组合。
在下面的描述中阐述了很多具体细节以便于充分理解本发明,但是,本发明还可以采用不同于在此描述的其他方式来实施,因此,本发明并不限于下面公开说明书的具体实施例的限制。
实施例1:本发明提供了长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,由以下重量份的原料制备而成:MRS液体培养基,去离子水; 0.2mmol/LDPPH甲醇溶液;2mmol/L硫酸亚铁溶液,6mmol/L过氧化氢溶液, 6mmol/L水杨酸溶液;150mmol/LpH=8.0的Tris-HCL溶液,1.2mmol/L邻苯三酚溶液;0.4%的硫酸亚铁溶液,1%的VC,0.2mol/L的氢氧化钠溶液,10%的三氯乙酸溶液,0.1%邻二氮菲溶液;pH=6.6的磷酸缓冲液,1%铁氰化钾溶液,0.1%的三氯化铁溶液。
MRS液体培养基内部接种有长双歧杆菌和动物双歧杆菌,长双歧杆菌和动物双歧杆菌均提取与百岁老人的粪便中分离的耐酸、耐胆盐和抑菌的益生菌株。
长双歧杆菌和动物双歧杆菌的制备方法包括:
步骤一:取定量的百岁老人的粪便加入量筒中,并采用50%比例的甘油进行封存保存;
步骤二:取出一定量的粪便样本至托盘上并通过无菌PBS(1%,w/v)进行稀释,将稀释后的样本涂抹在MRS琼脂平板上;
步骤三:将处理后的MRS琼脂平板放置于无菌室中,并设置室内温度至37℃,培养24小时,并逐步观察MRS琼脂平板上的菌落情况;
步骤四:观察MRS琼脂平板上的菌落状态,使用消毒后的划针对菌落进行划线纯化操作;
步骤五:取上述操作中菌落中的样本并再次进行接种,并对接种后的样本再次进行接种操作,重复接种三次,并提取操作样本中的菌液使其与50%甘油以 1:1的比例搅拌混合,将样本存放在-80℃;
步骤六:即可对样本进行后续操作。
粪便取固体状,并采用玻璃棒捣碎搅拌,将内部的大颗粒状杂物剔除。
长双歧杆菌和动物双歧杆菌每日补充量各为109CFU/mL,可通过喝含双歧杆菌的酸奶或饮料来补充双歧杆菌,也可直接摄入含有双歧杆菌的微生态制剂进行补充。
长双歧杆菌和动物双歧杆菌可进一步制成流体乳品、饮料或益生菌菌片。
双歧杆菌最主要产物包括乳酸、乙酸等,促进铁和维生素D的吸收并提高磷、铁、钙的利用率;双歧杆菌通过磷蛋白磷酸酶分解α-酪蛋白,促进蛋白吸收。
长双歧杆菌和动物双歧杆菌的获得及其鉴定
一、长双歧杆菌和动物双歧杆菌的分离纯化
百岁老人的粪便采用50%的甘油保存,使用无菌PBS(1%,w/v)稀释,涂布于MRS琼脂平板上,37℃静置培养24h后,根据菌落形态,在MRS琼脂平板上反复划线纯化,直到固体培养基上长出纯单一菌落,将分离的乳酸菌连续传代3次,然后以50%甘油与菌液1:1混合保存于-80℃。
二、长双歧杆菌和动物双歧杆菌的鉴定
将挑选的菌落连续培养后,取部分菌液离心,按照天根细菌DNA提取试剂盒说明书提取DNA,然后送于公司,由公司进行16SrDNA序列扩增、纯化,然后测序。
双歧杆菌形态学上主要定义为两种形态,分叉形态定义为Ⅰ型,命名为乳杆菌;杆状则定义为Ⅱ型,命名为副分叉乳杆菌。在肠道内,双歧杆菌多呈直杆状,极少以分叉状、弯杆状的形态呈现。初次分离时,由于相应的培养条件不足,通常呈现为Ⅰ型,菌株形态主要包括不分叉、分叉两种形态,其中,分叉状呈现为V字形、Y字形,但于后续的培养中,分叉状通常呈现为弯曲、杆状。显微镜下菌体呈Y字形、勺状、V字形、弯曲状、棍棒状等,经由一定程度培养后,将转变为Ⅱ型,且长时间、稳定性的遗传。因此,临床上认为Ⅱ型向Ⅰ型的转变是由于对环境、温度等不适应造成的退化,而Ⅰ型向Ⅱ型的转变则是由于可更好地适应环境、温度等而出现的进化。
实施例2:长双歧杆菌和动物双歧杆菌的耐酸实验
从-80℃取出长双歧杆菌和动物双歧杆菌接种于MRS培养基中,37℃培养过夜,再按2%接种至新鲜MRS培养基中,37℃培养24h,用PBS缓冲液梯度稀释101, 103,105倍(取100ul加入到900ul的PBS缓冲液,再从中取10ul加入到990ul 的PBS中,再从中取10ul加入到990ul的PBS中),4-5000rpm离心10min,弃掉上清。加入pH为3、5、7、9的PBS缓冲液,放置4小时,混匀各取10uL涂板, 37℃培养箱培养过夜,活菌计数。结果如图1所示。
实施例2:长双歧杆菌和动物双歧杆菌的耐胆盐实验
从-80℃取出长双歧杆菌和动物双歧杆菌接种于MRS培养基中,37℃培养过夜,再按2%接种分别至0%胆盐、0.1%胆盐、0.3%胆盐和0.5%胆盐的MRS培养基中,37℃培养24h,用PBS缓冲液梯度稀释102,104,106,108倍,混匀各取 10uL涂板,37℃培养箱培养过夜。活菌计数。结果如图2所示。
双歧杆菌是一种重要的肠道有益微生物。双歧杆菌作为一种生理性有益菌,对人体健康具有生物屏障、营养作用、抗肿瘤作用、免疫增强作用、改善胃肠道功能、抗衰老等多种重要的生理功能。
人体肠道中定植着大量的微生物。肠道微生物与人体健康与疾病之间存在着十分密切的关系。根据所知道的肠道微生物对人体健康的影响,肠道微生物可分为有益、无害和有害3大类。
实施例3:长双歧杆菌和动物双歧杆菌的抗氧化实验
溶液配制:MRS液体培养基,去离子水;0.2mmol/LDPPH甲醇溶液;2mmol/L 硫酸亚铁溶液,6mmol/L过氧化氢溶液,6mmol/L水杨酸溶液;150mmol/LpH=8.0 的Tris-HCL溶液,1.2mmol/L邻苯三酚溶液;0.4%的硫酸亚铁溶液,1%的VC, 0.2mol/L的氢氧化钠溶液,10%的三氯乙酸溶液,0.1%邻二氮菲溶液;pH=6.6的磷酸缓冲液,1%铁氰化钾溶液,0.1%的三氯化铁溶液。
灭菌:以上的溶液
活化:各取100uL的双歧杆菌液加到装有5mL的MRS培养基的玻璃试剂管内, 37℃培养箱培养过夜。第二天,离心,取上清液。
①DPPH自由基清除能力的测定
各取2mL的上清液加到玻璃试管内,再加2mL配制好的DPPH甲醇溶液,黑暗下室温反应30分钟,离心取上清。另一根玻璃试管内加入2mL的去离子水和2mL 的DPPH甲醇溶液作为对照。517nm测OD值。记录对照组和实验组数据。
DPPH自由基清除率=[1-A517(样品)/A517(空白)]×100%
②羟基自由基清除能力的测定
各取1mL的细菌上清液加到玻璃试管内,加入1mL的硫酸亚铁溶液,1mL的过氧化氢溶液,1mL的水杨酸溶液,静置30分钟。另一根玻璃管内加入上述溶液,并加入1mL的去离子水。30分钟后510nm处测吸光度。记录对照组和实验组数据。
羟基自由基清除率=[1-A510(样品)/A510(空白)]×100%
③超氧自由基清除能力
取0.5m将培养的不同菌分别作涂片(注意涂片切不可过于浓厚)、干燥、固定。固定时用甲醇固定。
(2)初染:加草酸铵结晶紫1滴,约1min,使用纯水对样本进行清洗。
(3)媒染:加碘液覆盖约1min,使用纯水对样本进行清洗。
(4)脱色:将载玻片上面的水甩净,并衬以白背景,用95%酒精滴洗至流出酒精刚刚不出现紫色时为止,约20-30s,立即用水冲洗。
(5)复染:用番红染1-2min,使用纯水对样本进行清洗。
(6)镜检:待样本干燥后,置油镜下观察,革兰氏阴性菌呈红色,革兰氏阳性菌呈紫色;以分散开的细菌的革兰氏染色反应为准。
在正常情况下,人体内的肠道微生物形成了一个相对平衡的状态。一旦平衡受到破坏,如服用抗生素、放疗、化疗、情绪压抑、身体衰弱、缺乏免疫力等,就会导致肠道菌群失去平衡,某些肠道微生物如产气荚膜梭菌等在肠道中过度增殖并产生氨、胺类、硫化氢、粪臭素、吲哚、亚硝酸盐、细菌毒素等有害物质,从而进一步影响机体的健康。
实施例6:长双歧杆菌和动物双歧杆菌菌株制剂的制备
A.将-80℃保存的长双歧杆菌和动物双歧杆菌菌株接种于10mLMRS培养基, 37℃静置培养16小时;
B.然后按1:100接种于新鲜培养基37℃震荡或静置培养24小时左右;
C.采用大体积离心机离心,离心前将离心筒清洗干净并用75%酒精或紫外消毒,4700rpm/20min;
D.弃上清,PBS重悬后采用50mL离心管,6000rpm/5-10min;
E.弃上清,称重菌泥重量,按1:1加入1%灭菌的海藻酸钠重悬菌泥,再按 1:1体积加入16%灭菌的脱脂牛奶重悬,-20℃冻存后再置于-80℃冻存24小时左右,至样品完全冷冻;
F.置于真空冷冻干燥机,24-48小时,至样本完全冻干成粉末;
G.研钵使用前清洗干净并消毒,将冻干粉与少许红薯粉一起研磨;
H.称取0.01g混合物溶解于1mL无菌PBS梯度稀释后,点板于相应固体培养基中,待菌液晾干后,37℃倒置培养过夜;
I.计数,根据数量进行混合包装,干酪乳杆菌菌株制剂总数不少于5× 109cfu/g。
双歧杆菌和乳酸菌等有益细菌则能抑制人体有害细菌的生长,抵抗病原菌的感染,合成人体需要的维生素,促进人体对矿物质的吸收,产生醋酸、丙酸、丁酸和乳酸等有机酸刺激肠道蠕动,促进排便,防止便秘以及抑制肠道腐败作用、净化肠道环境、分解致癌物质、刺激人体免疫系统,从而提高抗病能力等方面有着重要作用。具体地,双歧杆菌的有益作用包括以下几个方面。
治疗慢性腹泻与抗生素相关性腹泻
双歧杆菌具有调节肠道菌群的作用。通过用双歧杆菌对慢性腹泻患者临床观察研究表明,在服药两周以后,患者大便次数、形状异常,临床症状消失,对总有效率为90.3%,复发率低。许多国内医院已将双歧杆菌制剂作为治疗慢性腹泻的首选药物。此外,双歧杆菌还可以治疗因过量使用抗生素而导致的抗生素相关性腹泻疾病。有人采用双歧杆菌制剂治疗伪膜性肠炎380例,临床总治愈率无明显差异,但临床副作用和复发率均明显降低。双歧杆菌对儿童急慢性腹泻具有很好的治疗作用。
营养作用
双歧杆菌缺少醛酶、葡萄糖,因此其分解代谢途径不同于乳酸菌。双歧杆菌最主要产物主要包括乳酸、乙酸等,可改善机体pH值,促进铁和维生素D的吸收并提高磷、铁、钙的利用率;双歧杆菌可以通过磷蛋白磷酸酶分解α-酪蛋白,促进蛋白吸收。临床上,机体在缺乏乳糖酶的情况下,摄入的乳糖或纯牛奶不能被消化吸收进血液,仍然留在肠道内,肠道细菌就会在发酵分解乳糖的过程中产生大量气体,造成腹泻、胃胀和气胀等症状。动物生产过程中,豆粕为最主要的蛋白来源,但由于寡糖不易消化,其使用受到一定的限制,主要为棉籽糖、水苏糖。而双歧杆菌含有活性较多数细菌高的乳糖酶,这种酶能发酵乳糖产生半乳糖。因此,乳糖酶缺乏者,饮用经双杆菌发酵的乳制品,既可获得乳制品中丰富的营养,又免受胃肠道病痛之苦。
拮抗作用
COLLADO等研究表明,当两歧双歧杆菌(S17)与致病性大肠埃希菌、福氏志贺菌、沙门菌等肠道致病菌共同竞争培养时,对HeLa细胞的黏附能力均明显下降。双歧杆菌除与病原菌争夺营养物质和空间位置外,还可通过其代谢产物,以及产生抗生素、细菌素等阻止病原菌的生长。双歧杆菌可以阻断致病菌和毒素的特异性结合位点,可能是由胞外酶能够降解肠上皮细胞表面多糖来实现的。
通便功能
便秘是指粪便干燥难解或排便次数减少。临床上根据病因可分为功能性便秘和器质性便秘,双歧杆菌对功能性的便秘有明显作用。有研究报道,双歧杆菌在人体肠道定植数量随人年龄和健康状态的变化而改变,母乳喂养儿童肠道中的双歧杆菌可占肠道总菌数的91%,而老年人的肠道菌群中双歧杆菌数量下降较明显,很多老年人口服双歧杆菌微生态制剂可明显改善因缺乏双歧杆菌而引发的便秘。
增强免疫力
双歧杆菌具有调节免疫功能的作用,主要通过对肠道黏膜的刺激,激活肠道黏膜的免疫系统,使其产生抗体、细胞因子,进而更好地提高肠道黏膜的免疫、抗感染能力。
抗肿瘤的作用
双歧杆菌具有抗结肠癌作用,可能是通过影响肠道菌群代谢、提高宿主免疫应答;黏附及降解潜在致癌物,预防肠道癌症;改变肠道菌群;产生抗癌抗诱变物质;提高宿主的免疫应答;影响宿主的生理活动来实现。
抗衰老功能
人体的衰老往往从肠道老化先表现出来。双歧杆菌在维持机体肠道健康和减缓肠道老化方面具有重要作用。VERENA等将含有乳酸双歧杆菌牛乳添加至人体肠道细胞DNA的培养基中,可使肠道细胞避免氧化损伤,具有延缓肠道衰老作用。研究证明,双歧杆菌能明显增加血液中过氧化物歧化酶的含量及其生物活性,有效促进机体内自由基的清除,将体内有害物质毒性降低90%,抑制血浆脂质过氧化反应,延缓机体衰老。
实施例7:长双歧杆菌和动物双歧杆菌对小鼠的抗衰应用
A.实验菌株的制备:分别将长双歧杆菌和动物双歧杆菌按1%(v/v)接种于 MRS培养基,37℃培养过夜,至OD600=0.6,8000rpm离心3min收集菌体,用无菌生理盐水清洗2次,再用含0.1%的明胶生理盐水重悬,调整菌数为1× 109cfu/mL;
B.实验动物及分组治疗:将6周龄的Balb/c雄性小鼠55只(购买自湖南思湖王实验动物有限公司),适应性饲养一周,标记称重分组后开始实验,其中M 组、A组、L组、LA组小鼠腹腔注射D-半乳糖(150mg/kg/d),连续注射12周,同时C组(连续灌胃含0.1%明胶生理盐水治疗,n=11),M组(连续灌胃含0.1%明胶生理盐水治疗,n=11),A组(连续灌胃长双歧杆菌1×109cfu/只/天,n=11), L组(连续灌胃动物双歧杆菌1×109cfu/只/天,n=11),LA组(连续灌胃长双歧杆菌和动物双歧杆菌1×109cfu/只/天,n=11)。12周以后,在进行旷场实验、爬杆实验以及巴恩斯迷宫实验测试后处死小鼠,并收集小鼠脑组织。
C.旷场实验:将每只小鼠置于(90cm×90cm×30cm)暗箱活动室10分钟,使用activitymonitor视频跟踪软件测量小鼠运动的总距离及去到中央区域的时间。为了最大程度地减少气味干扰,每次运行后都要同75%乙醇清洁暗室。结果如图6所示。
D.爬杆实验:老鼠被放置在直径为10毫米高度为55厘米的粗糙木杆顶部。每只老鼠接受下杆动作训练3天(3-5分钟/次/天),只有那些在预试验(通常在试验前2.5h进行)中运动时间<18s的老鼠才能继续进行后续实验。结果如图 7所示。
E.巴恩斯迷宫实验:实验开始前一天,将动物单个从目标洞置于目标箱内适应4min。将动物置于迷宫中央的塑料圆桶(直径20cm,高27cm)内限制活动 5s。移开圆桶,启动计时器,实验者在挡帘后进行观察。动物四肢均进入目标箱,则计为一次逃避(escape),并让动物在箱内停留30s。每一动物一次最多观察4min。在此期间如果动物仍然找不到目标箱,则将动物从迷宫移开,放入目标箱内并停留30s。利用这一间隙清洁迷宫。动物每天训练两次,连续5~6d。从第二次训练开始,每次训练之前将迷宫随机转动一至数个洞的位置,但目标箱始终固定在同一方位。这样做的目的是防止动物依靠气味、而非凭借记忆来确定目标洞的位置。实验记录以下参数:探究任何一个洞的潜伏期、到达目标箱的潜伏期和每只动物的错误次数(一次错误定义为动物把头伸向或探究任何一个非目标洞,包括专注于探究同一个非目标洞)。结果如图8所示。
F.小鼠海马组织SOD活力的测定:
(1)分光光度计预热30min以上,调节波长至560nm,蒸馏水调零;
(2)测定前将试剂一、三和五37℃水浴5min以上;
(3)把试管编号,分别编为测定管、对照管、空白管1和空白管2;试管编号后按下表加入试剂:
充分混匀,37℃水浴30min后,置于1mL玻璃比色皿测定560nm下的吸光度,分别记为A测定、A对照、A空1、A空2,计算△A测定=A测定-A对照,△A 空白=A空1-A空2。
(4)SOD活性计算:
①抑制百分率的计算:抑制百分率=(ΔA空白-ΔA测定)÷ΔA空白×100%
②SOD酶活性单位:在上述黄嘌呤氧化酶藕联反应体系中抑制百分率为50%时,反应体系中的SOD酶活力定义为一个酶活力单位。
③SOD酶活性计算:血清(浆)SOD活性(U/mL)=[抑制百分率÷(1-抑制百分率)×V反总]÷V样×F=11.4×抑制百分率÷(1-抑制百分率)×F。结果如图 9所示。
G.小鼠海马组织MDA的测定:采用硫代巴比妥法检测血浆中MDA的浓度,操作步骤如下:
(1)把试管编号,分别编为标准管、标准空白管、测定管和测定空白管,试管编号后按下表加入试剂:
(2)漩涡混匀器混匀,试管口用保鲜薄膜扎紧,用针头刺一小孔,95℃水浴 40min;
(3)取出后流水冷却,然后4000r/min,离心10min,使沉淀完全;
(4)取上清,吸取上清比色时用移液器吸取上清加入比色皿中,尽量避免倾倒,以免沉淀进入比色皿,影响吸光度;
(5)把比色皿置于532nm处,1cm光径,蒸馏水调零,测各管吸光度值;
(6)计算血浆中MDA的含量:血浆中MDA含量(ng/ml)=(测定管吸光度-测定空白管吸光度)/(标准管吸光度-标准空白管吸光度)×标准品浓度 (10ng/ml)×样品测试前稀释倍数。结果如图9所示。
H.小鼠海马组织GSH-Px的测定:
(1)酶促反应:
(2)血清中GSH—PX酶活力的计算:规定每O.1ml血清在37℃反应5分钟,扣除非酶促反应作用,使反应体系中GSH浓度降低19mol/L为一个酶活力单位;血清GSH-PX活力=(非酶管OD值.酶管OD值)÷(标准管OD值-空白管OD 值)×标准品浓度(20umol/L)×稀释倍数(6)×样本测试前稀释倍数。结果如图9所示。
I.小鼠海马组织β-半乳糖苷酶染色阳性水平的测定行为学实验后处死小鼠,各组解剖并保藏三只小鼠的海马组织。制备海马组织冷冻切片(6μm;-25℃),切片用PBS洗涤两次,在室温下用固定液固定15min,在避光和隔绝CO2情况下,37℃用X-Gal染色液染色24小时,切片用PBS洗涤,孵育后在明视野显微镜下观察。使用ImageJImageProPlus软件进行定量图像分析。结果如图10所示。
序列表
应用方法:促进体内双歧杆菌增殖的方法有两种。第一种办法是喝含双歧杆菌的酸奶或饮料来补充双歧杆菌,也可直接摄入含有双歧杆菌的微生态制剂。此外,还可以摄入含有双歧因子的产品来促进人体自身固有双歧杆菌的生长繁殖。所谓双歧因子,主要是指些功能性的非消化性的低聚糖,这些低聚糖可以选择性地促进双歧杆菌的增殖。具有使双歧杆菌增殖作用的低聚糖主要有异麦芽低聚糖、低聚果糖、低聚半乳糖、大豆低聚糖、棉子糖混合乳蔗糖、低聚木糖等。
以上所述,仅是本发明的较佳实施例而已,并非是对本发明作其它形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例应用于其它领域,但是凡是未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型,仍属于本发明技术方案的保护范围。
Claims (7)
1.长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,其特征在于:由以下重量份的原料制备而成:MRS液体培养基,去离子水;0.2mmol/LDPPH甲醇溶液;2mmol/L硫酸亚铁溶液,6mmol/L过氧化氢溶液,6mmol/L水杨酸溶液;150mmol/LpH=8.0的Tris-HCL溶液,1.2mmol/L邻苯三酚溶液;0.4%的硫酸亚铁溶液,1%的VC,0.2mol/L的氢氧化钠溶液,10%的三氯乙酸溶液,0.1%邻二氮菲溶液;pH=6.6的磷酸缓冲液,1%铁氰化钾溶液,0.1%的三氯化铁溶液。
2.根据权利要求1所述的长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,其特征在于:所述MRS液体培养基内部接种有长双歧杆菌和动物双歧杆菌,所述长双歧杆菌和动物双歧杆菌均提取与百岁老人的粪便中分离的耐酸、耐胆盐和抑菌的益生菌株。
3.根据权利要求1所述的长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,其特征在于:所述长双歧杆菌和动物双歧杆菌的制备方法包括:
步骤一:取定量的百岁老人的粪便加入量筒中,并采用50%比例的甘油进行封存保存;
步骤二:取出一定量的粪便样本至托盘上并通过无菌PBS(1%,w/v)进行稀释,将稀释后的样本涂抹在MRS琼脂平板上;
步骤三:将处理后的MRS琼脂平板放置于无菌室中,并设置室内温度至37℃,培养24小时,并逐步观察MRS琼脂平板上的菌落情况;
步骤四:观察MRS琼脂平板上的菌落状态,使用消毒后的划针对菌落进行划线纯化操作;
步骤五:取上述操作中菌落中的样本并再次进行接种,并对接种后的样本再次进行接种操作,重复接种三次,并提取操作样本中的菌液使其与50%甘油以1:1的比例搅拌混合,将样本存放在-80℃;
步骤六:即可对样本进行后续操作。
4.根据权利要求2所述的长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,其特征在于:所述粪便取固体状,并采用玻璃棒捣碎搅拌,将内部的大颗粒状杂物剔除。
5.根据权利要求2所述的长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,其特征在于:所述长双歧杆菌和动物双歧杆菌每日补充量各为109CFU/mL,可通过喝含双歧杆菌的酸奶或饮料来补充双歧杆菌,也可直接摄入含有双歧杆菌的微生态制剂进行补充。
6.根据权利要求2所述的长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,其特征在于:所述长双歧杆菌和动物双歧杆菌可进一步制成流体乳品、饮料或益生菌菌片。
7.根据权利要求2所述的长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,其特征在于:所述双歧杆菌最主要产物包括乳酸、乙酸等,促进铁和维生素D的吸收并提高磷、铁、钙的利用率;双歧杆菌通过磷蛋白磷酸酶分解α-酪蛋白,促进蛋白吸收。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110490551.0A CN113215035A (zh) | 2021-05-06 | 2021-05-06 | 长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110490551.0A CN113215035A (zh) | 2021-05-06 | 2021-05-06 | 长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113215035A true CN113215035A (zh) | 2021-08-06 |
Family
ID=77091330
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110490551.0A Pending CN113215035A (zh) | 2021-05-06 | 2021-05-06 | 长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113215035A (zh) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111778196A (zh) * | 2020-08-03 | 2020-10-16 | 江西善行生物科技有限公司 | 一种抗衰老动物双歧杆菌及其应用 |
CN111826322A (zh) * | 2020-08-03 | 2020-10-27 | 江西善行生物科技有限公司 | 一种长双歧杆菌及其在抗衰老中的应用 |
CN114437959A (zh) * | 2021-11-29 | 2022-05-06 | 内蒙古普泽动保生物技术有限公司 | 一种动物双歧杆菌及其在抗氧化和提升免疫中的应用 |
CN117106672A (zh) * | 2023-10-16 | 2023-11-24 | 微康益生菌(苏州)股份有限公司 | 一种改善衰老相关的认知障碍的短双歧杆菌及其应用 |
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103275893A (zh) * | 2013-05-06 | 2013-09-04 | 哈尔滨美华生物技术股份有限公司 | 长双歧杆菌和动物双歧杆菌的复合菌剂及其制备方法 |
CN103589658A (zh) * | 2013-07-05 | 2014-02-19 | 中国农业大学 | 一株产细菌素双歧杆菌及其在抑制酸奶后酸化中的应用 |
CN110373368A (zh) * | 2019-08-29 | 2019-10-25 | 卓源健康科技有限公司 | 一种长双歧杆菌菌株zj1及其应用 |
CN110438042A (zh) * | 2019-07-23 | 2019-11-12 | 广东益可维健康科技有限公司 | 一株长双歧杆菌及其应用 |
CN111705025A (zh) * | 2020-08-03 | 2020-09-25 | 江西善行生物科技有限公司 | 一种干酪乳杆菌的制备及其在抗衰老方面的应用 |
CN111778196A (zh) * | 2020-08-03 | 2020-10-16 | 江西善行生物科技有限公司 | 一种抗衰老动物双歧杆菌及其应用 |
CN111826322A (zh) * | 2020-08-03 | 2020-10-27 | 江西善行生物科技有限公司 | 一种长双歧杆菌及其在抗衰老中的应用 |
CN111876356A (zh) * | 2020-08-03 | 2020-11-03 | 江西善行生物科技有限公司 | 一种具有抗衰老功能的发酵乳杆菌及其应用 |
CN114515298A (zh) * | 2022-03-22 | 2022-05-20 | 哈尔滨美华生物技术股份有限公司 | 用于防治骨质疏松、提高骨密度的动物双歧杆菌及其应用 |
-
2021
- 2021-05-06 CN CN202110490551.0A patent/CN113215035A/zh active Pending
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103275893A (zh) * | 2013-05-06 | 2013-09-04 | 哈尔滨美华生物技术股份有限公司 | 长双歧杆菌和动物双歧杆菌的复合菌剂及其制备方法 |
CN103589658A (zh) * | 2013-07-05 | 2014-02-19 | 中国农业大学 | 一株产细菌素双歧杆菌及其在抑制酸奶后酸化中的应用 |
CN110438042A (zh) * | 2019-07-23 | 2019-11-12 | 广东益可维健康科技有限公司 | 一株长双歧杆菌及其应用 |
CN110373368A (zh) * | 2019-08-29 | 2019-10-25 | 卓源健康科技有限公司 | 一种长双歧杆菌菌株zj1及其应用 |
CN111705025A (zh) * | 2020-08-03 | 2020-09-25 | 江西善行生物科技有限公司 | 一种干酪乳杆菌的制备及其在抗衰老方面的应用 |
CN111778196A (zh) * | 2020-08-03 | 2020-10-16 | 江西善行生物科技有限公司 | 一种抗衰老动物双歧杆菌及其应用 |
CN111826322A (zh) * | 2020-08-03 | 2020-10-27 | 江西善行生物科技有限公司 | 一种长双歧杆菌及其在抗衰老中的应用 |
CN111876356A (zh) * | 2020-08-03 | 2020-11-03 | 江西善行生物科技有限公司 | 一种具有抗衰老功能的发酵乳杆菌及其应用 |
CN114515298A (zh) * | 2022-03-22 | 2022-05-20 | 哈尔滨美华生物技术股份有限公司 | 用于防治骨质疏松、提高骨密度的动物双歧杆菌及其应用 |
Non-Patent Citations (4)
Title |
---|
CHAOFEI XIA等: "Anti-aging effect of the combination of Bifidobacterium longum and B.", 《JOURNAL OF FUNCTIONAL FOODS》 * |
李平兰: "长寿老人源双歧杆菌优良菌株的筛选及生理功能研究", 《万方》 * |
王光慈主编: "《食品营养学》", 31 May 2001, 中国农业出版社 * |
罗满林主编: "《兽医生物制品学》", 28 February 2019, 中国农业出版社 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111778196A (zh) * | 2020-08-03 | 2020-10-16 | 江西善行生物科技有限公司 | 一种抗衰老动物双歧杆菌及其应用 |
CN111826322A (zh) * | 2020-08-03 | 2020-10-27 | 江西善行生物科技有限公司 | 一种长双歧杆菌及其在抗衰老中的应用 |
CN114437959A (zh) * | 2021-11-29 | 2022-05-06 | 内蒙古普泽动保生物技术有限公司 | 一种动物双歧杆菌及其在抗氧化和提升免疫中的应用 |
CN114437959B (zh) * | 2021-11-29 | 2022-11-18 | 内蒙古普泽动保生物技术有限公司 | 一种动物双歧杆菌及其在抗氧化和提升免疫中的应用 |
CN117106672A (zh) * | 2023-10-16 | 2023-11-24 | 微康益生菌(苏州)股份有限公司 | 一种改善衰老相关的认知障碍的短双歧杆菌及其应用 |
CN117106672B (zh) * | 2023-10-16 | 2024-01-30 | 微康益生菌(苏州)股份有限公司 | 一种改善衰老相关的认知障碍的短双歧杆菌及其应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1005353B1 (en) | Pharmaceutical preparation comprising lactobacillus casei rhamnosus | |
CN113215035A (zh) | 长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用 | |
KR0123456B1 (ko) | 프로바이오틱 균 | |
Gopal et al. | Effects of the consumption of Bifidobacterium lactis HN019 (DR10TM) and galacto-oligosaccharides on the microflora of the gastrointestinal tract in human subjects | |
ES2435065T3 (es) | Cepas de lactobacilos probióticos para mejorar la salud vaginal | |
EP2179028B1 (en) | A novel strain of bifidobacterium and active peptides against rotavirus infections | |
CN110144304B (zh) | 干酪乳杆菌菌株及其应用 | |
CN100378214C (zh) | 益生细菌:发酵乳杆菌 | |
CN113234640B (zh) | 一株长双歧杆菌mf-269及其应用 | |
KR19980703102A (ko) | 상피 흡착성 락토바실러스 | |
MX2007001863A (es) | Lactobacillus rhamnosus con actividad reductora de la grasa corporal y alimentos que los contienen. | |
US11911423B2 (en) | Bifidobacterium lactis BL-99 and application thereof | |
CN112625979B (zh) | 一种对抗幽门螺杆菌的干酪乳杆菌及其应用 | |
CN113293113A (zh) | 一株长双歧杆菌mi-186及其应用 | |
Sato et al. | Intestinal distribution and intraluminal localization of orally administered Clostridium butyricum in rats | |
Kolida et al. | Gastrointestinal microflora: probiotics | |
US9055763B2 (en) | Probiotics for use in relieving symptoms associated with gastronitestinal disorders | |
CN111686134A (zh) | 一种益生菌组合物及其制备方法和用途 | |
RU2303058C2 (ru) | Средство для лечения кишечных инфекций, осложненных дисбактериозом "биобаланс-к" | |
Kesen et al. | Beneficial characteristics and evaluation criteria of probiotics | |
CN113337440B (zh) | 一株唾液乳杆菌mg-587及其应用 | |
WO2018112740A1 (zh) | 一种加氏乳杆菌及其培养方法和应用 | |
CN113549567B (zh) | 一株具有促排便功能的鼠李糖乳杆菌nsl0401及其应用 | |
CN114933992B (zh) | 一种长双歧杆菌及其复合制剂在缓解溃疡性结肠炎中的应用 | |
RU2584600C2 (ru) | ШТАММ L.bulgaricus, СПОСОБНЫЙ ИНГИБИРОВАТЬ АДГЕЗИЮ ШТАММОВ Н.pylori К ЭПИТЕЛИАЛЬНЫМ КЛЕТКАМ |
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 | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210806 |