CN110432332B - 青春双歧杆菌ccfm1062、其发酵食品及菌剂制备方法 - Google Patents
青春双歧杆菌ccfm1062、其发酵食品及菌剂制备方法 Download PDFInfo
- Publication number
- CN110432332B CN110432332B CN201910770725.1A CN201910770725A CN110432332B CN 110432332 B CN110432332 B CN 110432332B CN 201910770725 A CN201910770725 A CN 201910770725A CN 110432332 B CN110432332 B CN 110432332B
- Authority
- CN
- China
- Prior art keywords
- ccfm1062
- bifidobacterium adolescentis
- liver
- strain
- mice
- 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
- 241000186018 Bifidobacterium adolescentis Species 0.000 title claims abstract description 124
- 239000002068 microbial inoculum Substances 0.000 title claims abstract description 21
- 235000021107 fermented food Nutrition 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 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 claims abstract description 17
- 239000008103 glucose Substances 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 29
- 239000001963 growth medium Substances 0.000 claims description 21
- 241000758789 Juglans Species 0.000 claims description 19
- 235000009496 Juglans regia Nutrition 0.000 claims description 19
- 235000020234 walnut Nutrition 0.000 claims description 19
- 230000001580 bacterial effect Effects 0.000 claims description 15
- 239000003223 protective agent Substances 0.000 claims description 14
- 244000288157 Passiflora edulis Species 0.000 claims description 13
- 235000000370 Passiflora edulis Nutrition 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 11
- 238000004108 freeze drying Methods 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 11
- 238000007710 freezing Methods 0.000 claims description 8
- 230000008014 freezing Effects 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 239000000725 suspension Substances 0.000 claims description 8
- 235000011925 Passiflora alata Nutrition 0.000 claims description 7
- 235000011922 Passiflora incarnata Nutrition 0.000 claims description 7
- 235000013750 Passiflora mixta Nutrition 0.000 claims description 7
- 235000013731 Passiflora van volxemii Nutrition 0.000 claims description 7
- 239000003513 alkali Substances 0.000 claims description 6
- 235000013361 beverage Nutrition 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 6
- 239000008055 phosphate buffer solution Substances 0.000 claims description 6
- 238000004537 pulping Methods 0.000 claims description 6
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 5
- 241000186000 Bifidobacterium Species 0.000 claims description 5
- 239000005913 Maltodextrin Substances 0.000 claims description 5
- 229920002774 Maltodextrin Polymers 0.000 claims description 5
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 5
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 5
- 229940035034 maltodextrin Drugs 0.000 claims description 5
- 238000011160 research Methods 0.000 claims description 5
- 235000020183 skimmed milk Nutrition 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 4
- 235000015140 cultured milk Nutrition 0.000 claims description 4
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 claims description 3
- 244000174681 Michelia champaca Species 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 235000015278 beef Nutrition 0.000 claims description 3
- 238000009835 boiling Methods 0.000 claims description 3
- 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 claims description 3
- 229960001305 cysteine hydrochloride Drugs 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 238000000855 fermentation Methods 0.000 claims description 3
- 230000004151 fermentation Effects 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 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 claims description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 3
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 230000001502 supplementing effect Effects 0.000 claims description 3
- 239000012137 tryptone Substances 0.000 claims description 3
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 3
- 239000000811 xylitol Substances 0.000 claims description 3
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 claims description 3
- 229960002675 xylitol Drugs 0.000 claims description 3
- 235000010447 xylitol Nutrition 0.000 claims description 3
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 2
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 2
- 235000013365 dairy product Nutrition 0.000 claims description 2
- 235000012055 fruits and vegetables Nutrition 0.000 claims description 2
- 230000000813 microbial effect Effects 0.000 claims 5
- 238000004321 preservation Methods 0.000 claims 2
- 239000002994 raw material Substances 0.000 claims 1
- 210000004185 liver Anatomy 0.000 abstract description 52
- 210000004369 blood Anatomy 0.000 abstract description 27
- 239000008280 blood Substances 0.000 abstract description 27
- 210000002966 serum Anatomy 0.000 abstract description 20
- SNGREZUHAYWORS-UHFFFAOYSA-N perfluorooctanoic acid Chemical compound OC(=O)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)F SNGREZUHAYWORS-UHFFFAOYSA-N 0.000 abstract description 19
- 208000001072 type 2 diabetes mellitus Diseases 0.000 abstract description 18
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 abstract description 17
- 108010082126 Alanine transaminase Proteins 0.000 abstract description 17
- 102000019197 Superoxide Dismutase Human genes 0.000 abstract description 14
- 108010012715 Superoxide dismutase Proteins 0.000 abstract description 14
- 108010028554 LDL Cholesterol Proteins 0.000 abstract description 13
- 238000001179 sorption measurement Methods 0.000 abstract description 11
- 229930182843 D-Lactic acid Natural products 0.000 abstract description 10
- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 abstract description 10
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 abstract description 10
- 229940022769 d- lactic acid Drugs 0.000 abstract description 10
- 206010022489 Insulin Resistance Diseases 0.000 abstract description 9
- 208000004930 Fatty Liver Diseases 0.000 abstract description 8
- 230000003870 intestinal permeability Effects 0.000 abstract description 6
- 210000005228 liver tissue Anatomy 0.000 abstract description 6
- 206010061218 Inflammation Diseases 0.000 abstract description 5
- 235000012000 cholesterol Nutrition 0.000 abstract description 5
- 230000004054 inflammatory process Effects 0.000 abstract description 5
- 210000002381 plasma Anatomy 0.000 abstract description 5
- 230000000451 tissue damage Effects 0.000 abstract description 4
- 231100000827 tissue damage Toxicity 0.000 abstract description 4
- 235000009200 high fat diet Nutrition 0.000 abstract description 3
- 230000003902 lesion Effects 0.000 abstract description 3
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 abstract description 3
- 230000002159 abnormal effect Effects 0.000 abstract description 2
- 238000010521 absorption reaction Methods 0.000 abstract description 2
- 210000004923 pancreatic tissue Anatomy 0.000 abstract description 2
- 108090000340 Transaminases Proteins 0.000 abstract 1
- 102000014898 transaminase activity proteins Human genes 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 57
- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 description 49
- 239000000243 solution Substances 0.000 description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 25
- 230000000694 effects Effects 0.000 description 25
- 241000699666 Mus <mouse, genus> Species 0.000 description 21
- YASAKCUCGLMORW-UHFFFAOYSA-N Rosiglitazone Chemical compound C=1C=CC=NC=1N(C)CCOC(C=C1)=CC=C1CC1SC(=O)NC1=O YASAKCUCGLMORW-UHFFFAOYSA-N 0.000 description 18
- 238000002474 experimental method Methods 0.000 description 15
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 13
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 13
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 description 13
- 102100036761 Protein TMED8 Human genes 0.000 description 13
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- 150000003626 triacylglycerols Chemical class 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 9
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical group CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 9
- 210000001035 gastrointestinal tract Anatomy 0.000 description 9
- 210000005229 liver cell Anatomy 0.000 description 9
- 230000002829 reductive effect Effects 0.000 description 9
- 229960004586 rosiglitazone Drugs 0.000 description 9
- RYMZZMVNJRMUDD-UHFFFAOYSA-N SJ000286063 Natural products C12C(OC(=O)C(C)(C)CC)CC(C)C=C2C=CC(C)C1CCC1CC(O)CC(=O)O1 RYMZZMVNJRMUDD-UHFFFAOYSA-N 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- RYMZZMVNJRMUDD-HGQWONQESA-N simvastatin Chemical compound C([C@H]1[C@@H](C)C=CC2=C[C@H](C)C[C@@H]([C@H]12)OC(=O)C(C)(C)CC)C[C@@H]1C[C@@H](O)CC(=O)O1 RYMZZMVNJRMUDD-HGQWONQESA-N 0.000 description 8
- 229960002855 simvastatin Drugs 0.000 description 8
- 206010019708 Hepatic steatosis Diseases 0.000 description 7
- 101000588302 Homo sapiens Nuclear factor erythroid 2-related factor 2 Proteins 0.000 description 7
- 102000003777 Interleukin-1 beta Human genes 0.000 description 7
- 108090000193 Interleukin-1 beta Proteins 0.000 description 7
- 208000010706 fatty liver disease Diseases 0.000 description 7
- 239000003925 fat Substances 0.000 description 6
- 230000002496 gastric effect Effects 0.000 description 6
- 230000000968 intestinal effect Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 231100000240 steatosis hepatitis Toxicity 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 208000008589 Obesity Diseases 0.000 description 5
- 241000160321 Parabacteroides Species 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 206010012601 diabetes mellitus Diseases 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 206010015037 epilepsy Diseases 0.000 description 5
- 239000004310 lactic acid Substances 0.000 description 5
- 235000014655 lactic acid Nutrition 0.000 description 5
- 235000020824 obesity Nutrition 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 239000008096 xylene Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 206010067125 Liver injury Diseases 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000007935 neutral effect Effects 0.000 description 4
- 239000012188 paraffin wax Substances 0.000 description 4
- 229920002866 paraformaldehyde Polymers 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 238000004807 desolvation Methods 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 231100000753 hepatic injury Toxicity 0.000 description 3
- 239000007928 intraperitoneal injection Substances 0.000 description 3
- 238000013227 male C57BL/6J mice Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 229960001412 pentobarbital Drugs 0.000 description 3
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 3
- 230000010412 perfusion Effects 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000001993 wax Substances 0.000 description 3
- 206010002091 Anaesthesia Diseases 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 240000008067 Cucumis sativus Species 0.000 description 2
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241000186660 Lactobacillus Species 0.000 description 2
- 102100031701 Nuclear factor erythroid 2-related factor 2 Human genes 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 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 2
- -1 amine hydrochloride Chemical class 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 210000005027 intestinal barrier Anatomy 0.000 description 2
- 230000007358 intestinal barrier function Effects 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 229940039696 lactobacillus Drugs 0.000 description 2
- 208000019423 liver disease Diseases 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- 238000012335 pathological evaluation Methods 0.000 description 2
- 235000015206 pear juice Nutrition 0.000 description 2
- 239000006041 probiotic Substances 0.000 description 2
- 235000018291 probiotics Nutrition 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- LOGFVTREOLYCPF-KXNHARMFSA-N (2s,3r)-2-[[(2r)-1-[(2s)-2,6-diaminohexanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxybutanoic acid Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-KXNHARMFSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- GSXOAOHZAIYLCY-UHFFFAOYSA-N D-F6P Natural products OCC(=O)C(O)C(O)C(O)COP(O)(O)=O GSXOAOHZAIYLCY-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 108010052764 Fructose-6-phosphate phosphoketolase Proteins 0.000 description 1
- 102000006587 Glutathione peroxidase Human genes 0.000 description 1
- 108700016172 Glutathione peroxidases Proteins 0.000 description 1
- 208000017170 Lipid metabolism disease Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- BGWGXPAPYGQALX-ARQDHWQXSA-N beta-D-fructofuranose 6-phosphate Chemical compound OC[C@@]1(O)O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O BGWGXPAPYGQALX-ARQDHWQXSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 108091092328 cellular RNA Proteins 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000816 effect on animals Effects 0.000 description 1
- 230000009982 effect on human Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- DZGCGKFAPXFTNM-UHFFFAOYSA-N ethanol;hydron;chloride Chemical compound Cl.CCO DZGCGKFAPXFTNM-UHFFFAOYSA-N 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 244000005709 gut microbiome Species 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 208000037817 intestinal injury Diseases 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 208000018191 liver inflammation Diseases 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000021590 normal diet Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011775 sodium fluoride Substances 0.000 description 1
- 235000013024 sodium fluoride Nutrition 0.000 description 1
- AGDSCTQQXMDDCV-UHFFFAOYSA-M sodium;2-iodoacetate Chemical compound [Na+].[O-]C(=O)CI AGDSCTQQXMDDCV-UHFFFAOYSA-M 0.000 description 1
- JBJWASZNUJCEKT-UHFFFAOYSA-M sodium;hydroxide;hydrate Chemical compound O.[OH-].[Na+] JBJWASZNUJCEKT-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 238000001946 ultra-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Images
Classifications
-
- 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
- A23C11/00—Milk substitutes, e.g. coffee whitener compositions
- A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
- A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
- A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
- A23C11/106—Addition of, or treatment with, microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N1/205—Bacterial isolates
-
- 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
- A23V2400/513—Adolescentes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nutrition Science (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Botany (AREA)
- Agronomy & Crop Science (AREA)
- Mycology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了青春双歧杆菌CCFM1062、其发酵食品及菌剂制备方法,青春双歧杆菌CCFM1062能够显著降低血清中低密度脂蛋白胆固醇、谷丙转氨酶、谷草转氨酶的水平;显著改善高脂饮食诱导的血糖升高且改善胰岛素抵抗;显著降低肝脏中总胆固醇、甘油三酯的水平,减少肝脏脂肪变性、肝脏小叶炎症以及肝脏气球样病变;能够显著增加肝脏中超氧化物歧化酶水平;显著降低血浆中D‑乳酸的水平,改善肠道通透性增加;显著改善二型糖尿病造成的空腹血糖异常;显著改善二型糖尿病造成的胰腺和肝脏组织损伤;此外青春双歧杆菌CCFM1062对全氟辛酸有较强的吸附能力,减少全氟辛酸在体内吸收。
Description
技术领域
本发明属于微生物技术领域,具体涉及青春双歧杆菌CCFM1062、其发酵食品及菌剂制备方法。
背景技术
肠道是一个高度复杂的生态系统,细菌种类繁多且相互作用,肠道菌群的组成影响宿主对外源化合物和病原体的敏感性,而宿主的生理状态影响肠道微生物菌群的组成。肝脏是和肠道联系最为密切的器官,肠道吸收的营养物质和脂肪随着血液进入肝脏循环,以供给全身能量所需。大量研究表明,非酒精性脂肪性肝病(non-alcoholic fatty liverdisease,NAFLD)的形成跟肠道菌群失调有着密切的联系,肠道菌群失调能够促使肠道屏障破坏从而促进肠道通透性增加。随着肠道通透性的增加,肠道中的细菌所产的内毒素以及肠道损伤的产物更容易随血液进入肝脏对肝细胞造成刺激以致损伤,使NAFLD进一步恶化为更为严重的肝脏疾病,因而调节肠道菌群、保护肠道屏障是保护肝脏的重要方式。有研究表明,职业暴露于PFOA的工人的因脂肪肝死亡风险增加,因此日常生活中的PFOA暴露,是人类肝脏疾病发病的潜在风险。
益生菌被认为是一种无毒、无害,对人体健康具有一定促进作用的微生物。大量研究结果表明,多种益生菌对动物疾病具有显著的改善作用。
发明内容
本部分的目的在于概述本发明的实施例的一些方面以及简要介绍一些较佳实施例。在本部分以及本申请的说明书摘要和发明名称中可能会做些简化或省略以避免使本部分、说明书摘要和发明名称的目的模糊,而这种简化或省略不能用于限制本发明的范围。
鉴于上述的技术缺陷,提出了本发明。
因此,作为本发明其中一个方面,本发明克服现有技术中存在的不足,提供青春双歧杆菌CCFM1062,其保藏编号为GDMCC No:60707。
作为本发明的另一个方面,本发明克服现有技术中存在的不足,提供制备青春双歧杆菌CCFM1062菌剂的方法。
为解决上述技术问题,本发明提供了如下技术方案:制备青春双歧杆菌CCFM1062菌剂的方法,其包括,菌株培养基的制备;菌株保护剂的制备;接种培养、冻干;所述菌株培养基,为改良MRS培养基,其配方包括胰蛋白胨8~14g、牛肉浸膏8~12g、酵母粉4~7g、葡萄糖15~25g、醋酸钠4~6g、柠檬酸氢二铵1.5~2.5g、磷酸氢二钾1~3g、七水硫酸镁0.4~0.6g、吐温-80 0.8~1.2mL、一水合硫酸锰0.2~0.3g、半胱氨酸盐酸盐0.4~0.6g,水1000mL;调整其pH为6.5±0.2。
作为本发明所述的制备青春双歧杆菌CCFM1062菌剂的方法的一种优选方案:所述菌株保护剂,其配方为100g/L~150g/L脱脂奶粉、100g/L~150g/L麦芽糊精、140g/L~160g/L海藻糖、余量为水,冻干,得到冻干保护剂。
作为本发明所述的制备青春双歧杆菌CCFM1062菌剂的方法的一种优选方案:所述菌株保护剂,其配方为120g/L脱脂奶粉、120g/L麦芽糊精、150g/L海藻糖、余量为水,冻干,得到冻干保护剂。
作为本发明所述的制备青春双歧杆菌CCFM1062菌剂的方法的一种优选方案:所述接种培养、冻干,包括,青春双歧杆菌CCFM1062以5%的接种量接种到在119~123℃条件下灭菌15~25min后的所述菌株培养基中,在温度35~39℃厌氧条件下培养24~48h,用pH为6.8~7.2磷酸盐缓冲液清洗2~4次,用所述菌株保护剂重悬,使菌浓达到1010CFU/mL;接着,让该菌株重悬液在温度37℃厌氧条件下预培养50~70min,再在-15~-20℃预冻8~14h,之后真空冷冻干燥。
作为本发明所述的制备青春双歧杆菌CCFM1062菌剂的方法的一种优选方案:青春双歧杆菌CCFM1062以5%的接种量接种到在121℃条件下灭菌20min后的培养基中,在温度37℃厌氧条件下培养24h,用pH为6.8磷酸盐缓冲液清洗2~4次,用所述的保护剂重悬,使菌浓达到1010CFU/mL;接着,让该悬浮液在温度37℃厌氧条件下预培养60min,再在-15℃预冻12h,之后真空冷冻干燥。
作为本发明其中一个方面,本发明克服现有技术中存在的不足,提供一种发酵食品。
为解决上述技术问题,本发明提供了如下技术方案:一种发酵食品,其中:使用所述的青春双歧杆菌CCFM1062菌剂进行发酵,所述菌剂含有大于106CFU/g的活性青春双歧杆菌CCFM1062。
作为本发明所述的发酵食品的一种优选方案:所述发酵食品包括乳制品、豆制品与果蔬制品。
作为本发明所述的发酵食品的一种优选方案:所述发酵食品,包括西番莲核桃发酵乳饮料。
作为本发明所述的发酵食品的一种优选方案:所述西番莲核桃发酵乳饮料,其制备方法为,核桃仁用0.5%的NaOH水溶液煮沸5min,将碱液去除,去除核桃仁表面的种皮,用清水洗涤去除核桃仁表面残留的碱液,按照料水比1:8打浆,西番莲果实洗净后打浆,核桃仁浆液与西番莲浆液以9:1的比例混合后过滤,均质,补加2%木糖醇,115℃,灭菌20min,将所述青春双歧杆菌CCFM1062菌剂以109CFU/m L接种到西番莲核桃仁的混合物中,37℃恒温培养箱中发酵12h。
本发明的有益效果:本发明提供的青春双歧杆菌CCFM1062能够显著降低血清中低密度脂蛋白胆固醇(LDL-C)、谷丙转氨酶(ALT)、谷草转氨酶(AST)的水平;显著改善高脂饮食诱导的血糖升高且改善胰岛素抵抗;显著降低肝脏中总胆固醇(TC)、甘油三酯(TG)的水平,显著降低肝脏中IL-1β的水平同时减少肝脏脂肪变性、肝脏小叶炎症以及肝脏气球样病变;能够显著增加肝脏中超氧化物歧化酶(SOD)水平;显著降低血浆中D-乳酸(D-LA)的水平,改善肠道通透性增加;显著改善二型糖尿病造成的空腹血糖异常;显著改善二型糖尿病造成的胰腺和肝脏组织损伤;此外青春双歧杆菌CCFM1062对全氟辛酸(PFOA)有较强的吸附能力,减少PFOA在体内吸收,具有缓解PFOA毒性的能力;显著提高NAFLD肠道中Parabacteroides属的丰度,提高肠道中有益菌群的比例,预防和减少非酒精性脂肪性肝病、全氟辛酸毒性、肥胖、二型糖尿病以及癫痫等疾病发生的药物组合物与发酵食品,具有非常广泛的应用前景。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其它的附图。其中:
图1是青春双歧杆菌CCFM1062的菌落形态;
图2是青春双歧杆菌CCFM1062对NAFLD小鼠肠道中Parabacteroides属等特征性菌群的影响;
图3是青春双歧杆菌CCFM1062对NAFLD小鼠血清低密度脂蛋白胆固醇(LDL-C)水平的影响;
图4是青春双歧杆菌CCFM1062对NAFLD小鼠血清谷丙转氨酶(ALT)的影响;
图5是青春双歧杆菌CCFM1062对NAFLD小鼠血清谷草转氨酶(AST)水平的影响;
图6是青春双歧杆菌CCFM1062对NAFLD小鼠空腹血糖的影响;
图7是青春双歧杆菌CCFM1062对NAFLD小鼠的胰岛素抵抗的影响;
图8是青春双歧杆菌CCFM1062对NAFLD小鼠血浆中D-乳酸(D-LA)的影响
图9是青春双歧杆菌CCFM1062对NAFLD小鼠肝脏中总胆固醇(TC)的影响;
图10是青春双歧杆菌CCFM1062对NAFLD小鼠肝脏甘油三酯(TG)的影响
图11是青春双歧杆菌CCFM1062对NAFLD小鼠肝脏超氧化物歧化酶(SOD)的影响
图12是青春双歧杆菌CCFM1062对NAFLD小鼠肝脏炎症的影响;
图13是青春双岐杆菌CCFM1062对NAFLD小鼠肝脏组织病理的影响;
图14是青春双岐杆菌CCFM1062对脂肪肝细胞Nrf2基因表达的影响;
图15是青春双歧杆菌CCFM1062对PFOA的吸附能力;
图16是青春双歧杆菌CCFM1062对二型糖尿病小鼠空腹血糖的影响;
图17是青春双歧杆菌CCFM1062对二型糖尿病小鼠胰腺组织病理的影响;
图18是青春双歧杆菌CCFM1062对二型糖尿病小鼠肝脏组织病理的影响;
注:a,b,c表示不同字母所代表的组别都存在显著性差异(P<0.05)。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合具体实施例对本发明的具体实施方式做详细的说明。
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其他不同于在此描述的其它方式来实施,本领域技术人员可以在不违背本发明内涵的情况下做类似推广,因此本发明不受下面公开的具体实施例的限制。
其次,此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例,也不是单独的或选择性的与其他实施例互相排斥的实施例。
青春双歧杆菌CCFM1062(Bifidobacterium adoltescentis),于2019年06月28日保藏于广东省微生物菌种保藏中心,地址为广州市先烈中路100号大院59号楼5楼,广东省微生物研究所,保藏编号为GDMCC No:60707。
青春双歧杆菌CCFM1062的特性:
(1)菌体特征:呈革兰氏染色阳性,不形成孢子,不运动的细菌;
(2)菌落特征:厌氧培养36小时形成明显的菌落,直径在0.5-1mm之间,正面形态圆形,侧面形态呈突起状,边缘整齐,乳白色,半透明,表面湿润光滑,不产生色素,参见附图1;
(3)生长特性:在37℃恒温厌氧的条件下,在改良的mMRS培养基中培养约22小时达到对数末期。
(4)显著提高NAFLD小鼠肠道中Parabacteroides属的丰度,预防和减少肥胖、非酒精性脂肪性肝病、二型糖尿病以及癫痫等疾病发生;
(5)显著改善NAFLD小鼠的脂质代谢紊乱;
(6)显著改善NAFLD的胰岛素抵抗;
(7)能够回调血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)的升高;
(8)显著降低血清中低密度脂蛋白胆固醇的浓度,减少心血管疾病的风险;
(9)能够显著提高NAFLD小鼠肝脏中的超氧化物歧化酶(SOD)以及谷胱甘肽过氧化物酶(GSH-Px)的水平;
(10)能够显著改善NAFLD小鼠的肠道通透性增加;
(11)能够显著改善肝脏中IL-1β的水平;
(12)能够显著改善NAFLD小鼠肝脏组织损伤;
(13)能够显著改善脂肪肝细胞的Nrf2基因的表达。
(14)有良好的PFOA的吸附能力。
菌株的获取方法为:
(一)发酵用乳酸菌的分离筛选:
(1)取1g来自河南博爱县清化镇一个93岁女性的新鲜粪便。梯度稀释后涂布于mMRS固体培养基,置于厌氧环境下37℃培养72小时。
(2)观察记录菌落形态,挑取菌落划线纯化。
(3)在mMRS液体培养基中,37℃培养48小时,所得菌落进行革兰氏染色,记录菌落形态。
(4)弃除菌落中的革兰氏阴性菌菌株和革兰氏阳性球菌,挑选得到革兰氏阳性杆菌。
(5)过氧化氢酶分析后,弃除过氧化氢酶阳性菌株,保留过氧化氢酶阴性菌株。
(二)双歧杆菌的初步鉴定:果糖-6-磷酸盐磷酸酮酶测定法
(l)将步骤(一)所筛选得到的乳酸菌在液体mMRS培养液中培养24h,然后取lmL培养物8000rpm离心2min;
(2)用含0.05%(质量百分数)半胱氨酸的pH 6.5的0.05M KH2PO4溶液洗涤两次;
(3)重悬于200uL添加了0.25%(质量百分数)Triton X-100的上述磷酸盐缓冲液;
(4)添加50uL浓度为6mg/mL氟化钠和10mg/mL碘乙酸钠的混合液以及50uL浓度为80mg/mL的果糖-6-磷酸,37℃孵育1h;
(5)添加300uL浓度为0.139g/mL、pH 6.5的盐酸轻胺,并于室温放置10min;
(6)分别添加200uL15%(质量百分数)的三氯乙酸和4M HCI;
(7)添加200uL含有5%(质量百分数)三氯化铁的0.1M HCI,若体系迅速变为红色,即为F6PPK阳性,可初步断定其为双歧杆菌。
(三)发酵用乳酸菌的分子生物学鉴定:
(l)单菌基因组抽提:
A.将步骤(二)所筛选得到的乳酸菌培养过夜,取培养过夜的菌悬液lmL于1.5mL离心管,10000rpm离心2min,弃上清得菌体;
B.用lmL无菌水吹洗菌体后,10000rpm离心2min,弃上清得菌体;
C.加入200μLSDS裂解液,80℃水浴30min;
D.加入酚-氯仿溶液200μL于菌体裂解液中,其中酚-氯仿溶液的组成成分及体积比为Tris饱和酚:氯仿:异戊醇=25:24:1,颠倒混匀后,12000rpm离心5-10min,取上清200μL;
E.加入400μL冰乙醇或冰异丙醇于200uL上清中,-20℃静置1h,12000rpm离心5-10min,弃上清;
F.加入500μL70%(体积百分数)冰乙醇重悬沉淀,12000rpm离心1-3min,弃上清;
G.60℃烘箱烘干,或者自然晾干;
H.50μLddH2O重溶沉淀以备PCR;
(2)16S rDNA PCR
A.细菌16S rDNA 50μLPCR反应体系:
10×Taq buffer,5μL;dNTP,5μL;27F,0.5μL;1492R,0.5μL;Taq酶,0.5μL;模板,0.5μL;ddH2O,38μL。
B.PCR条件:
95℃ 5min;95℃10s;55℃ 30s;72℃ 30s;step2-4 30×;72℃ 5min;12℃ 2min;
(3)制备1%琼脂糖凝胶,之后将PCR产物与10000×loading buffer混合,上样量5μL,120V跑30min,然后进行凝胶成像;
(4)将16S rDNA的PCR产物进行测序分析,将得到的序列结果使用BLAST在GeneBank中进行搜索和相似性比对,选取测序结果鉴定为属于青春双歧杆菌的一个新菌株,-80℃保藏备用。
实施例1:青春双歧杆菌CCFM1062对C57BL/6J小鼠无毒副作用
将青春双歧杆菌CCFM1062菌体重悬于3%的蔗糖溶液中,制成浓度为3.0×109CFU/mL的菌悬液。取体重16-20g左右的健康雄性C57BL/6J小鼠8只,适应环境一周后,每日给予0.2mL青春双歧杆菌CCFM1062灌胃一次,观察一周,记录死亡和体重情况。
这些试验结果列于表1中。这些结果表明,每天喂食0.2mL 3.0×109CFU/mL的青春双歧杆菌CCFM1062未对小鼠造成明显影响,体重无显著变化,无死亡现象产生。小鼠外观无明显病理症状。
表1小鼠体重的变化及死亡情况
注:-:小鼠无死亡
实施例2:青春双歧杆菌CCFM1062对NAFLD小鼠肠道菌群的调节作用
取体重16-20g的健康雄性C57BL/6J小鼠48只,适应环境1周,随机分为6组:空白对照组(NC)、模型对照组(M)、罗格列酮对照组(RC)、辛伐他汀对照组(SC)、青春双歧杆菌CCFM1062干预组(CCFM1062)、青春双歧杆菌L10干预组(LC),每组含小鼠8只。实验动物分组及处理方法见表2:
表2实验动物分组
试验末期收集小鼠新鲜粪便冻存于-80℃,提取粪便中的宏基因组,并利用二代测序仪对肠道菌群结构进行分析。试验结束时,小鼠禁食不禁水12h,腹腔注射0.5mL/10g1%的戊巴比妥钠溶液麻醉后,心脏采血,辅以颈椎脱臼法处死。血液样本3000×g、4℃条件下离心15min,取上清,-80℃冻存用于测定相关血清指标。部分肝脏收集后迅速置于预冷的生理盐水中漂洗去血,放入4%中性多聚甲醛溶液中固定,剩余部分肝脏于液氮中速冻并转移至-80℃冻存,后续制成肝匀浆以用于测定相关指标,具体制备方法如下:称取一定量肝脏组织,按1:9比例加入生理盐水进行组织研磨,3000r离心10min,取上清冻存于-80℃备用。
菌群分析实验结果如图2所示,显著提高NAFLD小鼠肠道中Parabacteroides属的丰度,预防和减少肥胖、非酒精性脂肪性肝病、二型糖尿病以及癫痫等疾病发生。
与模型组相比,NAFLD小鼠经青春双岐杆菌CCFM1062制备的黄瓜梨汁乳酸菌饮料干预后,肠道中的Parabacteroides属丰度显著提高,而大量研究表明,Parabacteroides属与肥胖、非酒精性脂肪性肝病、二型糖尿病以及癫痫等疾病呈负相关,表明本发明制备的黄瓜梨汁乳酸菌饮料具有减少肥胖、非酒精性脂肪性肝病、二型糖尿病以及癫痫等疾病发生的功能。
实施例3:青春双岐杆菌CCFM1062降低NAFLD小鼠血清低密度脂蛋白胆固醇(LDL-C)的水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。按照试剂盒的检测方法测定低密度脂蛋白胆固醇(LDL-C)的含量。
实验结果如附图3所示。由实验结果可以看出,与正常对照组相比,模型组小鼠血清低密度脂蛋白胆固醇含量显著升高,灌胃青春双岐杆菌CCFM1062可降低血清低密度脂蛋白胆固醇的含量,且青春双岐杆菌CCFM1062对血清低密度脂蛋白胆固醇水平的回调能力明显优于青春双歧杆菌L10。
实施例4:青春双岐杆菌CCFM1062降低NAFLD小鼠血清谷丙转氨酶(ALT)水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。按照ALT试剂盒的检测方法测定血液中谷丙转氨酶(ALT)的含量。
实验结果如图4所示。模型组小鼠空腹ALT显著升高,青春双岐杆菌CCFM1062的干预明显降低了NAFLD小鼠的ALT水平,其降低小鼠空腹血糖水平的能力与辛伐他汀相似,而由青春双歧杆菌L10的摄入并未对ALT的升高有所逆转,值得注意的罗格列酮组的ALT水平显著高于模型组,提示长期服用罗格列酮会对肝脏造成损伤。
实施例5:青春双岐杆菌CCFM1062降低NAFLD小鼠血清谷草转氨酶(AST)的水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。按照试剂盒的检测方法测定血液中谷草转氨酶(AST)的含量。
实验结果如图5所示。由图5可以看出,模型组小鼠血清AST含量明显升高,灌胃青春双岐杆菌CCFM1062显著降低血清AST的含量,且其趋势和ALT趋势一致,提示青春双岐杆菌CCFM1062能够缓解肝脏损伤。
实施例6:青春双岐杆菌CCFM1062降低NAFLD小鼠的空腹血糖水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。
实验结果如图6所示。模型组小鼠空腹血糖显著升高,青春双岐杆菌CCFM1062的干预明显降低了NAFLD小鼠的空腹血糖水平,其空腹血糖控制能力显著强于青春双歧杆菌L10的干预,且其降低小鼠空腹血糖水平的能力与罗格列酮相似。
实施例7:青春双岐杆菌CCFM1062缓解NAFLD小鼠的胰岛素抵抗
C57BL/6J小鼠分组、造模及处理方法同实施例2。按照试剂盒的检测方法测定胰岛素(INS)的含量,并结合空腹血糖结果计算胰岛素抵抗指数。
实验结果如图7所示。与空白组相比,高脂高胆固醇饮食24周后,模型组小鼠胰岛素抵抗指数显著升高,青春双歧杆菌L10干预后NAFLD小鼠胰岛素抵抗指数有所降低,但其效果不如青春双岐杆菌CCFM1062,提示青春双岐杆菌CCFM1062能够提高NAFLD小鼠的胰岛素敏感性,可能对二型糖尿病有一定缓解效果。
实施例8:青春双岐杆菌CCFM1062显著降低NAFLD小鼠血清D-乳酸(D-LA)的水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。
实验结果如图8所示。模型组小鼠血清D-LA含量明显升高,灌胃青春双岐杆菌CCFM1062明显降低了模型小鼠的D-LA水平并接近于空白对照组。其降低小鼠血清D-LA的能力与辛伐他汀药物组相似,显著改善NAFLD小鼠肠道通透性的增加。青春双歧杆菌L10的干预没有明显的改善效果。
实施例9:青春双岐杆菌CCFM1062降低肝脏中总胆固醇(TC)的水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。按照试剂盒的检测方法测定总胆固醇(TC)的含量,以肝脏蛋白浓度进行校正。
实验结果如图9所示。与正常对照组相比,模型组小鼠肝脏TC显著升高,灌胃青春双岐杆菌CCFM1062降低NAFLD小鼠肝脏中TC的水平,且青春双岐杆菌CCFM1062对肝脏TC的调节能力与辛伐他汀相似。
实施例10:青春双岐杆菌CCFM1062降低肝脏中甘油三酯(TG)的水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。按照试剂盒的检测方法测定甘油三酯(TG)的含量,以肝脏蛋白浓度进行校正。
实验结果如图10所示。与正常对照组相比,模型组小鼠肝脏TG显著升高,灌胃青春双岐杆菌CCFM1062降低NAFLD小鼠肝脏中TG的水平,且青春双岐杆菌CCFM1062对肝脏TG的调节能力与辛伐他汀和罗格列酮相当。
实施例11:青春双岐杆菌CCFM1062提高了肝脏中超氧化物歧化酶(SOD)的水平,以肝脏蛋白浓度进行校正。
C57BL/6J小鼠分组、造模及处理方法同实施例2。按照超氧化物歧化酶(SOD)试剂盒的说明书测定肝脏中SOD的含量。
实验结果如图11所示。空白对照组的SOD水平显著高于模型组,灌胃青春双岐杆菌CCFM1062显著提高NAFLD小鼠肝脏中SOD的水平且高于辛伐他汀干预组,而青春双歧杆菌L10干预后并未呈现出相似的结果。
实施例12:青春双岐杆菌CCFM1062减轻NAFLD小鼠肝脏中的炎症水平
C57BL/6J小鼠分组、造模及处理方法同实施例2。按照白介素-1β(IL-1β)试剂盒的说明书测定肝脏中IL-1β的浓度,以肝脏蛋白浓度进行校正。
实验结果如图12所示。由实验结果可以看出,高脂高胆固醇饮食24周后,模型组的IL-1β水平显著升高,灌胃青春双岐杆菌CCFM1062显著降低NAFLD小鼠肝脏中IL-1β水平,且其炎症缓解效果强于辛伐他汀和罗格列酮,而青春双歧杆菌L10干预后NAFLD小鼠炎症缓解效果一般。
实施例13:青春双岐杆菌CCFM1062缓解NAFLD小鼠肝脏组织损伤
C57BL/6J小鼠分组、造模及处理方法同实施例2。取部分经4%中性多聚甲醛固定的肝脏制作石蜡切片,经HE染色后光镜下观察组织形态并拍照,进行病理学评价。具体步骤如下:
(1)固定:组织样品用生理盐水洗一下,立即投入4%中性多聚甲醛固定液中固定,一般固定时间在72h之内。
(2)洗涤:流水冲洗或浸泡数小时或过夜。
(3)脱水:样品依次经70%、80%、90%各级乙醇溶液脱水,各30min,再放入95%1次20min、100%2次每次10min。
(4)透明:1/2纯酒精+1/2二甲苯混合液10min、二甲苯Ⅰ10min、Ⅱ10min(至透明为止)。
(5)浸蜡:将样品放入石蜡(62℃)透蜡2h。
(6)包埋:以最大的面在底层,使切出的面组织面所占最大。
(7)切片:用手动切片机,将蜡块切成5μm厚度的片段。
(8)展片和粘片(捞片):打开水浴锅,使水温维持在42℃,使切片平整铺展在水面上。
(9)烤片:将载玻片连同载玻片架放入55℃的干燥箱,约2h至蜡熔化。
(10)水化:石蜡切片经二甲苯Ⅰ、Ⅱ脱蜡各10min,然后放入100%、95%、90%、80%、70%各级酒精溶液中各5min,再放入蒸馏水中3min。
(11)初染:切片放入苏木精中染色约20s。
(12)水洗:用自来水流水冲洗约15min。使切片颜色变蓝,但要注意流水不能过大,以防切片脱落。
(13)分化:将切片放入1%盐酸乙醇溶液中褪色,7s。见切片变红,颜色较浅即可。
(14)漂洗:切片再放入自来水流水中冲洗15-20min使其恢复蓝色。
(15)复染:浸入伊红染液,立即取出进行脱水。
(16)脱水:将切片依次过95%乙醇Ⅰ、95%乙醇Ⅱ、70%乙醇,再放入80%乙醇50s、无水乙醇2min。
(17)透明:切片放入1/2无水乙醇,1/2二甲苯中1min,二甲苯Ⅰ、Ⅱ中各2min。
(18)封片:切片经二甲苯透明后,使用中性树胶作为封藏剂,树胶可用二甲苯稀释至合适的稠度。
实验结果如图13所示。由实验结果可以看出,模型组小鼠肝细胞排列稀疏,肝脂滴数量多且大小不一,脂滴之间相互粘连,肝小叶有炎性细胞浸润,少量肝细胞发生气球样病变,而灌胃青春双岐杆菌CCFM1062可明显改善上述病变,且效果明显优于青春双歧杆菌L10干预组。值得注意的是,罗格列酮干预24周后显著加重肝脏损伤,而辛伐他汀并未显著改善肝脏损伤,提示长期服用药物对肝脏不利。
实施例14:青春双岐杆菌CCFM1062提高脂肪肝细胞中Nrf2的水平
将L02细胞在含有10%的FBS连续稳定传代三次后,接种于6孔板上,于37℃、5%CO2环境中培养24h,待细胞贴壁后,给予2mg/mL甘油三酯混合物孵育24h后再分别单独接入1mL的青春双岐杆菌CCFM1062和青春双歧杆菌L10(接入PBS作为空白对照)孵育24h。所有孵育均在37℃、5%CO2环境中进行。青春双岐杆菌CCFM1062刺激实验组、青春双歧杆菌L10刺激实验组和PBS对照组各三个孔,且重复三次。
弃去培养液,先用PBS缓冲液清洗每个孔,每次1mL,清洗3次后,加TRIZOL裂解,进行细胞RNA提取。将提取的RNA反转录为cDNA后进行qPCR测定青春双岐杆菌CCFM1062和青春双歧杆菌L10与脂肪肝细胞共孵育后Nrf2基因的表达水平。Nrf2引物信息如表3所示。
表3引物信息
实验结果如图14所示。由实验结果可以看出,青春双岐杆菌CCFM1062刺激显著提高了脂肪肝细胞的Nrf2基因的表达水平,青春双歧杆菌L10刺激组的Nrf2基因的表达水平也有升高但明显不及青春双岐杆菌CCFM1062刺激组,表明青春双岐杆菌CCFM1062可能具有一定抗氧化能力。
实施例15:青春双岐杆菌CCFM1062对PFOA具有良好的吸附能力
对青春双歧杆菌CCFM1062进行纯化和活化培养,按2%(v/v)接种量接种于MRS液体培养基中,37℃厌氧培养24h。然后在8000r/min离心5min收集菌体,取沉淀用生理盐水清理后继续在8000r/min离心5min,去沉淀得到活菌体细胞,即湿菌体。将湿菌体重悬于50mg/LPFOA溶液中,并使最终菌体浓度达到1g干菌体/L(将湿菌体重悬于不含PFOA的超纯水中作为空白对照)。使用0.1M的NaOH或HCl溶液将含菌液的PFOA溶液的pH迅速调整至3.0,添加少量的NaOH或HCl(少于0.5ml)其离子强度对PFOA吸附的影响可以忽略。随后将装有100ml样液的250ml锥形瓶置于37℃、150rpm厌氧摇床培养,6h后取样测定,2次平行试验取平均值。
PFOA吸附量的测定:吸附实验后,样液在8000r/min离心5min,并用0.22μm的水膜过滤,PFOA浓度用具有Waters SYNAPT MS系统的UPLC-MS测定,采用Acquity UPLC BEH c18柱(2.1×100mm,1.7μm,Waters Co.),柱温35℃,进样量1μL。用100%(v/v)的乙腈溶液(溶液A)和0.1%(v/v)甲酸水溶液(溶液B)作为洗脱液,进行梯度清洗,流速是0.3mL/min,梯度清洗条件如表4所示。
表4梯度洗脱条件
t/min | 0-0.5 | 0.5-5.0 | 5.0-7.0 | 7.0-7.5 |
溶剂A比例 | 70% | 70-100% | 100% | 100-70% |
质谱条件:电离源为ESI源;MRM检测;MS+检测;Capillary(毛细管);3.0kV;Conc(椎体):40.00V;Source Temperature(放射源温度):120℃;Desolvation(去溶剂化)温度:400℃;Conc Gas Flow:50L/h;Desolvation Gas Flow:700L/h.气体流速为0.1ml/min;质子比扫描范围:100-2000;扫面时间1s,间隔0.061s。结果用MassLynxV4.1(Waters公司)分析;根据吸附前后PFOA的浓度差异计算乳酸菌对PFOA的吸附量。测定结果如图15所示,青春双歧杆菌CCFM1062对50mg/L PFOA的吸附率为67.92%±4.61%,显著高于其他菌株。
实施例16:青春双歧杆菌CCFM1062可降低二型糖尿病小鼠(空腹)血糖水平
取体重16-20g的健康雄性C57BL/6J小鼠40只,适应环境1周,随机分为5组:空白对照组(NC)、模型对照组(M)、罗格列酮对照组(RH)、青春双歧杆菌CCFM1062干预组(CCFM1062)、青春双歧杆菌BA1对照组(BA1)每组含小鼠8只,灌胃菌悬液的剂量为3.0×109CFU/mL,重悬于3%的蔗糖溶液中。实验动物分组及处理方法见表5:
表5实验动物分组
第2-7周:正常组小鼠喂食普通饲料,其余小鼠喂食高脂饲料。
在第11周的第1d,所有小鼠禁食不禁水12h,正常组注射50mmol/L柠檬酸-柠檬酸钠缓冲液(pH 4.5),其余组注射按照100mg/(kg体重)的剂量注射50mmol/L STZ(冰上避光,现配现用),其中STZ的配制是用50mmol/L柠檬酸-柠檬酸钠缓冲液溶解。
试验末期收集小鼠新鲜粪便冻存于-80℃,提取粪便中的宏基因组,并利用二代测序仪对肠道菌群结构进行分析。试验结束时,小鼠禁食不禁水12h,腹腔注射0.5mL/10g1%的戊巴比妥钠溶液麻醉后,心脏采血,辅以颈椎脱臼法处死。血液样本3000×g、4℃条件下离心15min,取上清,-80℃冻存用于测定相关血清指标。部分肝脏收集后迅速置于预冷的生理盐水中漂洗去血,放入多聚甲醛中固定,剩余部分肝脏于液氮中速冻并转移至-80℃冻存,后续制成肝匀浆以用于测定相关指标,具体制备方法如下:称取一定量肝脏组织,按1:9比例加入生理盐水进行组织研磨,3000r离心10min,取上清冻存于-80℃备用。
实验结果如图16所示。模型组小鼠空腹血糖显著升高,灌胃青春双歧杆菌CCFM1062明显降低了模型小鼠的空腹血糖水平并接近于空白对照组。其降低小鼠空腹血糖水平的能力与罗格列酮药物组相似。
实施例17:青春双歧杆菌CCFM1062可缓解二型糖尿病小鼠胰腺和肝脏的组织损伤
C57BL/6J小鼠分组、造模及处理方法同实施例16。试验结束时,小鼠禁食不禁水12h,腹腔注射0.5mL/10g 1%的戊巴比妥钠溶液麻醉后,心脏采血,颈椎脱臼处死。取胰腺、肝脏等部分制作石蜡切片,经HE染色后光镜下观察组织形态并拍照,进行病理学评价。
实验结果如附图17和图18所示。由实验结果可以看出,模型组小鼠胰岛数量变少,出现萎缩现象,肝细胞出现小泡性脂变,有早期纤维化的形态学表现,而灌胃青春双歧杆菌CCFM1062可明显改善上述病变,且效果明显好于青春双歧杆菌BA1。
实施例18:
青春双歧杆菌CCFM1062菌剂的制备方法为:
①菌株培养基的制备:青春双歧杆菌的培养基为改良MRS培养基(mMRS),配方为胰蛋白胨10g、牛肉浸膏10g、酵母粉5g、葡萄糖20g、醋酸钠5g、柠檬酸氢二铵2g、磷酸氢二钾2g、七水硫酸镁0.5g、吐温-80 1mL、一水合硫酸锰0.25g、半胱氨酸盐酸盐0.5g,水1000mL;调整其pH为6.5±0.2。
②菌株保护剂的制备:保护剂配方为:120g/L脱脂奶粉、120g/L麦芽糊精、150g/L海藻糖、余量为水,冻干,得到冻干保护剂;
③青春双歧杆菌CCFM1062菌种按照以上所述培养基的质量计2%的接种量接种到在121℃条件下灭菌20min后的培养基中,在温度37℃厌氧条件下培养24h,用pH为6.8磷酸盐缓冲液清洗2~4次,用所述的保护剂重悬,使菌浓达到1010CFU/mL;接着,让该悬浮液在温度37℃厌氧条件下预培养60min,再在-15℃预冻12h,最后进行真空冷冻干燥得到所述的青春双歧杆菌CCFM1062菌剂。
实施例19:
核桃仁用0.5%的NaOH水溶液煮沸5min,将碱液去除,去除核桃仁表面的种皮,用清水洗涤去除核桃仁表面残留的碱液,按照料水比1:8打浆,西番莲果实洗净后打浆,核桃仁浆液与西番莲浆液以9:1的比例混合后过滤,均质,补加2%木糖醇,115℃,灭菌20min,将所述青春双歧杆菌CCFM1062菌剂以109CFU/m L接种到西番莲核桃仁的混合物中,37℃恒温培养箱中发酵12h。
应说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (10)
1.青春双歧杆菌CCFM1062(Bifidobacterium adoltescentis),于2019年06月28日保藏于广东省微生物菌种保藏中心,地址为广州市先烈中路100号大院59号楼5楼,广东省微生物研究所,保藏编号为GDMCC No:60707。
2.制备青春双歧杆菌CCFM1062菌剂的方法,其特征在于:包括,
菌株培养基的制备;
菌株保护剂的制备;
接种培养、冻干;
所述菌株培养基,为改良MRS培养基,其配方包括胰蛋白胨8~14 g、牛肉浸膏8~12 g、酵母粉4~7 g、葡萄糖15~25 g、醋酸钠4~6 g、柠檬酸氢二铵1.5~2.5 g、磷酸氢二钾1~3 g、七水硫酸镁0.4~0.6 g、吐温-80 0.8~1.2 mL、一水合硫酸锰0.2~0.3g、半胱氨酸盐酸盐0.4~0.6 g,水1000 mL;调整其pH为6.5±0.2;
其中,青春双歧杆菌CCFM1062(Bifidobacterium adoltescentis),于2019年06月28日保藏于广东省微生物菌种保藏中心,地址为广州市先烈中路100号大院59号楼5楼,广东省微生物研究所,保藏编号为GDMCC No:60707。
3.如权利要求2所述制备青春双歧杆菌CCFM1062菌剂的方法,其特征在于:所述菌株保护剂,其配方为100 g/ L~150g/ L脱脂奶粉、100 g/ L~150g/ L麦芽糊精、140 g/ L~160g/ L海藻糖、余量为水,冻干,得到冻干保护剂。
4.如权利要求3所述制备青春双歧杆菌CCFM1062菌剂的方法,其特征在于:所述菌株保护剂,其配方为120 g/ L脱脂奶粉、120 g/ L麦芽糊精、150 g/ L海藻糖、余量为水,冻干,得到冻干保护剂。
5.如权利要求2所述制备青春双歧杆菌CCFM1062菌剂的方法,其特征在于:所述接种培养、冻干,包括,青春双歧杆菌CCFM1062以5%的接种量接种到在119~123℃条件下灭菌15~25min后的所述菌株培养基中,在温度35~39℃厌氧条件下培养24~48 h,用pH为6.8~7.2磷酸盐缓冲液清洗2~4次,用所述菌株保护剂重悬,使菌浓达到1010 CFU/mL;接着,让该菌株重悬液在温度37℃厌氧条件下预培养50~70 min,再在-15~-20℃预冻8~14 h,之后真空冷冻干燥。
6.如权利要求5所述制备青春双歧杆菌CCFM1062菌剂的方法,其特征在于:青春双歧杆菌CCFM1062以5%的接种量接种到在121℃条件下灭菌20 min后的培养基中,在温度37℃厌氧条件下培养24 h,用pH为6.8磷酸盐缓冲液清洗2~4次,用所述的保护剂重悬,使菌浓达到1010 CFU/mL;接着,让该菌株重悬液在温度37℃厌氧条件下预培养60 min,再在-15℃预冻12 h,之后真空冷冻干燥。
7.一种发酵食品,其特征在于:使用权利要求2所述的青春双歧杆菌CCFM1062菌剂进行发酵,所述菌剂含有大于106 CFU/g的活性青春双歧杆菌CCFM1062。
8.如权利要求7所述的发酵食品,其特征在于:所述发酵食品包括乳制品、豆制品与果蔬制品。
9.如权利要求8所述的发酵食品,其特征在于:所述发酵食品,包括西番莲核桃发酵乳饮料。
10.如权利要求9所述的发酵食品,其特征在于:所述西番莲核桃发酵乳饮料,其制备方法为,核桃仁用0.5%的NaOH水溶液煮沸5min,将碱液去除,去除核桃仁表面的种皮,用清水洗涤去除核桃仁表面残留的碱液,按照料水比 1:8 打浆,西番莲果实洗净后打浆,核桃仁浆液与西番莲浆液以9:1的比例混合后过滤,均质,补加2%木糖醇,115℃,灭菌20min,将所述青春双歧杆菌CCFM1062菌剂以109CFU/m L接种到西番莲核桃仁的混合物中,37℃恒温培养箱中发酵12h。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910770725.1A CN110432332B (zh) | 2019-08-20 | 2019-08-20 | 青春双歧杆菌ccfm1062、其发酵食品及菌剂制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910770725.1A CN110432332B (zh) | 2019-08-20 | 2019-08-20 | 青春双歧杆菌ccfm1062、其发酵食品及菌剂制备方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110432332A CN110432332A (zh) | 2019-11-12 |
CN110432332B true CN110432332B (zh) | 2021-10-26 |
Family
ID=68436710
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910770725.1A Active CN110432332B (zh) | 2019-08-20 | 2019-08-20 | 青春双歧杆菌ccfm1062、其发酵食品及菌剂制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110432332B (zh) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109929883A (zh) * | 2019-04-02 | 2019-06-25 | 烟台华康荣赞生物科技有限公司 | 重组酵母、构建方法和其在制备酪醇及衍生物中的应用 |
CN111000247B (zh) * | 2019-12-31 | 2023-04-07 | 扬州江大食生一生食品有限公司 | 一种复合益生菌粉的制备方法 |
CN112715660A (zh) * | 2021-01-18 | 2021-04-30 | 润盈生物工程(上海)有限公司 | 一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合及应用 |
CN113005060B (zh) * | 2021-03-15 | 2022-11-01 | 江南大学 | 青春双歧杆菌ccfm1173在制备功能性菌剂、食品和或药物中的应用 |
CN113249264B (zh) * | 2021-06-03 | 2021-11-23 | 北京量化健康科技有限公司 | 一株青春双歧杆菌及其在代谢综合征中的应用 |
CN116875516B (zh) * | 2023-09-06 | 2023-12-08 | 上海上药信谊药厂有限公司 | 青春双歧杆菌ba-3及其在抗衰老、抗氧化和抗炎中的应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104046584A (zh) * | 2014-06-19 | 2014-09-17 | 北京工商大学 | 一种青春双歧杆菌细菌素及其生产方法与专用生产菌株 |
WO2016030504A1 (en) * | 2014-08-29 | 2016-03-03 | Chr. Hansen A/S | Probiotic bifidobacterium adolescentis strains |
-
2019
- 2019-08-20 CN CN201910770725.1A patent/CN110432332B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104046584A (zh) * | 2014-06-19 | 2014-09-17 | 北京工商大学 | 一种青春双歧杆菌细菌素及其生产方法与专用生产菌株 |
WO2016030504A1 (en) * | 2014-08-29 | 2016-03-03 | Chr. Hansen A/S | Probiotic bifidobacterium adolescentis strains |
CN107109348A (zh) * | 2014-08-29 | 2017-08-29 | 科.汉森有限公司 | 益生菌青春双歧杆菌菌株 |
Non-Patent Citations (1)
Title |
---|
青春双歧杆菌对2型糖尿病模型大鼠肠道菌群和脂质代谢的影响;刘伯阳等;《中国微生态学杂志》;20091031;第21卷(第10期);第877-879页 * |
Also Published As
Publication number | Publication date |
---|---|
CN110432332A (zh) | 2019-11-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110432332B (zh) | 青春双歧杆菌ccfm1062、其发酵食品及菌剂制备方法 | |
CN110368406B (zh) | 青春双歧杆菌ccfm1062在制备功能性菌剂、食品和/或药物中的应用 | |
CN111662850B (zh) | 一株能够缓解酒精性肠道损伤的副干酪乳杆菌及其应用 | |
CN110331119B (zh) | 两歧双歧杆菌ccfm1063及其应用 | |
CN110305820B (zh) | 鼠李糖乳杆菌ccfm1064及其应用 | |
CN108076643B (zh) | 用于治疗例如细菌性阴道病的鼠李糖乳杆菌细菌 | |
CN110638843A (zh) | 鼠李糖乳杆菌ccfm1060在制备功能性菌剂、食品和/或药物中的应用 | |
CN110354148B (zh) | 青春双歧杆菌ccfm1061在制备功能性菌剂、食品和或药物中的应用 | |
CN112574917B (zh) | 一株缓解高尿酸血症的鼠李糖乳杆菌ccfm1131 | |
CN110122877A (zh) | 鼠李糖乳杆菌及其用途 | |
CN110468070A (zh) | 鼠李糖乳杆菌ccfm1060、其发酵食品及菌剂制备方法 | |
CN112195139B (zh) | 植物乳杆菌菌株ldvs007及其应用 | |
CN110093287B (zh) | 假小链双歧杆菌ccfm1045、其组合物、发酵食品、用途、菌剂及其菌剂制备方法 | |
CN114107134B (zh) | 一株侧孢短芽孢杆菌及其应用 | |
CN109593678B (zh) | 长双歧杆菌yh295及其在制备降低腹型肥胖风险产品中的应用 | |
CN112760250B (zh) | 一株缓解结肠炎的瘤胃乳杆菌及应用 | |
CN113122478A (zh) | 一株能够缓解辣椒素致胃肠损伤的副干酪乳杆菌及其用途 | |
CN110331118B (zh) | 青春双歧杆菌ccfm1061、其发酵食品及菌剂制备方法 | |
CN111117918B (zh) | 缓解pfos毒害作用的多功能戊糖片球菌ccfm1107、其发酵食品及应用 | |
CN116064286A (zh) | 具有改善非酒精性肝病的瑞士乳杆菌zjuids11及其应用 | |
CN112195103B (zh) | 冻干保护剂、粪菌冷冻干燥产物及其制备方法 | |
CN116004472A (zh) | 一株缓解肥胖的丁酸梭菌及其应用 | |
CN116064313A (zh) | 植物乳杆菌ccfm1281在缓解运动性疲劳中的应用 | |
CN115418332A (zh) | 一种具有预防和改善化学性肝损伤的植物乳杆菌 | |
CN114921361A (zh) | 具有改善酒精性肝损伤的植物乳杆菌zy08及其应用 |
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 |