CN113215030B - 约氏乳杆菌eu03及其用途 - Google Patents
约氏乳杆菌eu03及其用途 Download PDFInfo
- Publication number
- CN113215030B CN113215030B CN202110391018.9A CN202110391018A CN113215030B CN 113215030 B CN113215030 B CN 113215030B CN 202110391018 A CN202110391018 A CN 202110391018A CN 113215030 B CN113215030 B CN 113215030B
- Authority
- CN
- China
- Prior art keywords
- lactobacillus johnsonii
- myocardial
- rats
- group
- cardiac
- 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
- 241001468157 Lactobacillus johnsonii Species 0.000 title claims abstract description 45
- 230000002107 myocardial effect Effects 0.000 claims abstract description 25
- 230000004217 heart function Effects 0.000 claims abstract description 18
- 230000003680 myocardial damage Effects 0.000 claims abstract description 12
- 244000005700 microbiome Species 0.000 claims abstract description 4
- 239000003814 drug Substances 0.000 claims description 11
- 238000004321 preservation Methods 0.000 claims description 7
- 241000700159 Rattus Species 0.000 abstract description 47
- 208000010125 myocardial infarction Diseases 0.000 abstract description 45
- 230000003078 antioxidant effect Effects 0.000 abstract description 13
- 210000002216 heart Anatomy 0.000 abstract description 13
- 230000000405 serological effect Effects 0.000 abstract description 3
- 230000008439 repair process Effects 0.000 abstract description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 30
- 230000002861 ventricular Effects 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 230000000694 effects Effects 0.000 description 14
- 241000894006 Bacteria Species 0.000 description 13
- 238000005406 washing Methods 0.000 description 12
- 239000000523 sample Substances 0.000 description 11
- 235000019441 ethanol Nutrition 0.000 description 10
- 230000002829 reductive effect Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 102000008186 Collagen Human genes 0.000 description 9
- 108010035532 Collagen Proteins 0.000 description 9
- 230000000747 cardiac effect Effects 0.000 description 9
- 229920001436 collagen Polymers 0.000 description 9
- 229940079593 drug Drugs 0.000 description 9
- 230000006872 improvement Effects 0.000 description 9
- 238000010186 staining Methods 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 230000006378 damage Effects 0.000 description 8
- 239000000835 fiber Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 210000004165 myocardium Anatomy 0.000 description 8
- 241000186660 Lactobacillus Species 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000002791 soaking Methods 0.000 description 7
- 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 6
- 206010030113 Oedema Diseases 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 230000008595 infiltration Effects 0.000 description 6
- 238000001764 infiltration Methods 0.000 description 6
- 239000002207 metabolite Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000006041 probiotic Substances 0.000 description 6
- 235000018291 probiotics Nutrition 0.000 description 6
- 206010002091 Anaesthesia Diseases 0.000 description 5
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 5
- 102000002689 Toll-like receptor Human genes 0.000 description 5
- 108020000411 Toll-like receptor Proteins 0.000 description 5
- 230000037005 anaesthesia Effects 0.000 description 5
- 239000003963 antioxidant agent Substances 0.000 description 5
- 210000000038 chest Anatomy 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000002526 effect on cardiovascular system Effects 0.000 description 5
- 239000002158 endotoxin Substances 0.000 description 5
- 230000002757 inflammatory effect Effects 0.000 description 5
- 229940039696 lactobacillus Drugs 0.000 description 5
- 210000005240 left ventricle Anatomy 0.000 description 5
- 229920006008 lipopolysaccharide Polymers 0.000 description 5
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 5
- 230000036542 oxidative stress Effects 0.000 description 5
- 239000012188 paraffin wax Substances 0.000 description 5
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 5
- 238000004904 shortening Methods 0.000 description 5
- 239000008096 xylene Substances 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- 206010016654 Fibrosis Diseases 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 4
- 206010061216 Infarction Diseases 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 206010028851 Necrosis Diseases 0.000 description 4
- 208000027418 Wounds and injury Diseases 0.000 description 4
- 230000008602 contraction Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000004761 fibrosis Effects 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 description 4
- 230000007574 infarction Effects 0.000 description 4
- 230000000968 intestinal effect Effects 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 239000002068 microbial inoculum Substances 0.000 description 4
- 230000017074 necrotic cell death Effects 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 238000006722 reduction reaction Methods 0.000 description 4
- 238000007634 remodeling Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000002604 ultrasonography Methods 0.000 description 4
- 239000001993 wax Substances 0.000 description 4
- 206010067484 Adverse reaction Diseases 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 3
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 3
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 3
- 206010028594 Myocardial fibrosis Diseases 0.000 description 3
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 3
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000006838 adverse reaction Effects 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000017531 blood circulation Effects 0.000 description 3
- 210000004351 coronary vessel Anatomy 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 229960002725 isoflurane Drugs 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 208000037891 myocardial injury Diseases 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- -1 superoxide anions Chemical class 0.000 description 3
- 210000000115 thoracic cavity Anatomy 0.000 description 3
- YKPGZZXRWAOXRA-UHFFFAOYSA-N 1-[2-(4-iodophenyl)-3-nitrophenyl]-2H-tetrazol-1-ium chloride Chemical compound [Cl-].IC1=CC=C(C=C1)C1=C(C=CC=C1[N+](=O)[O-])[N+]=1NN=NC=1 YKPGZZXRWAOXRA-UHFFFAOYSA-N 0.000 description 2
- RVBUGGBMJDPOST-UHFFFAOYSA-N 2-thiobarbituric acid Chemical compound O=C1CC(=O)NC(=S)N1 RVBUGGBMJDPOST-UHFFFAOYSA-N 0.000 description 2
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 102000004420 Creatine Kinase Human genes 0.000 description 2
- 108010042126 Creatine kinase Proteins 0.000 description 2
- 101000990902 Homo sapiens Matrix metalloproteinase-9 Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 2
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 2
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 2
- 102000019197 Superoxide Dismutase Human genes 0.000 description 2
- 108010012715 Superoxide dismutase Proteins 0.000 description 2
- 239000002250 absorbent Substances 0.000 description 2
- 230000002745 absorbent Effects 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 230000001174 ascending effect Effects 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000005242 cardiac chamber Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 2
- 229960002327 chloral hydrate Drugs 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 230000003205 diastolic effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 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 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000021107 fermented food Nutrition 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000005003 heart tissue Anatomy 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 230000000004 hemodynamic effect Effects 0.000 description 2
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 description 2
- 210000004969 inflammatory cell Anatomy 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 230000007358 intestinal barrier function Effects 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 230000000302 ischemic effect Effects 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 210000000651 myofibroblast Anatomy 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 235000021391 short chain fatty acids Nutrition 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- UYPYRKYUKCHHIB-UHFFFAOYSA-N trimethylamine N-oxide Chemical compound C[N+](C)(C)[O-] UYPYRKYUKCHHIB-UHFFFAOYSA-N 0.000 description 2
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 1
- 238000011265 2D-echocardiography Methods 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 229940127291 Calcium channel antagonist Drugs 0.000 description 1
- 102000004082 Calreticulin Human genes 0.000 description 1
- 108090000549 Calreticulin Proteins 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 102000028526 Dihydrolipoamide Dehydrogenase Human genes 0.000 description 1
- 108010028127 Dihydrolipoamide Dehydrogenase Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 208000037487 Endotoxemia Diseases 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 208000012671 Gastrointestinal haemorrhages Diseases 0.000 description 1
- 206010058061 Gastrointestinal oedema Diseases 0.000 description 1
- 208000000435 Heart Rupture Diseases 0.000 description 1
- 208000035150 Hypercholesterolemia Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 206010022680 Intestinal ischaemia Diseases 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- 240000001046 Lactobacillus acidophilus Species 0.000 description 1
- 244000199866 Lactobacillus casei Species 0.000 description 1
- 241000218492 Lactobacillus crispatus Species 0.000 description 1
- 241000186673 Lactobacillus delbrueckii Species 0.000 description 1
- 241000186840 Lactobacillus fermentum Species 0.000 description 1
- 241000186606 Lactobacillus gasseri Species 0.000 description 1
- 241000186605 Lactobacillus paracasei Species 0.000 description 1
- 241000186604 Lactobacillus reuteri Species 0.000 description 1
- 241000186869 Lactobacillus salivarius Species 0.000 description 1
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000010750 Metalloproteins Human genes 0.000 description 1
- 108010063312 Metalloproteins Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 208000010428 Muscle Weakness Diseases 0.000 description 1
- 208000029549 Muscle injury Diseases 0.000 description 1
- 208000021642 Muscular disease Diseases 0.000 description 1
- 206010028372 Muscular weakness Diseases 0.000 description 1
- 208000000112 Myalgia Diseases 0.000 description 1
- 201000009623 Myopathy Diseases 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 201000001880 Sexual dysfunction Diseases 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 208000001871 Tachycardia Diseases 0.000 description 1
- 108010093894 Xanthine oxidase Proteins 0.000 description 1
- 102100033220 Xanthine oxidase Human genes 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000004716 alpha keto acids Chemical class 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 230000006538 anaerobic glycolysis Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 210000002376 aorta thoracic Anatomy 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000002876 beta blocker Substances 0.000 description 1
- 229940097320 beta blocking agent Drugs 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 230000007211 cardiovascular event Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000005254 chromizing Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000007691 collagen metabolic process Effects 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 230000002951 depilatory effect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 125000004925 dihydropyridyl group Chemical group N1(CC=CC=C1)* 0.000 description 1
- 230000000916 dilatatory effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000007323 disproportionation reaction Methods 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000005485 electric heating Methods 0.000 description 1
- 210000001174 endocardium Anatomy 0.000 description 1
- 230000003090 exacerbative effect Effects 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 235000021001 fermented dairy product Nutrition 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 235000010855 food raising agent Nutrition 0.000 description 1
- 239000008098 formaldehyde solution Substances 0.000 description 1
- 235000012055 fruits and vegetables Nutrition 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 208000030304 gastrointestinal bleeding Diseases 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 230000004110 gluconeogenesis Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000010247 heart contraction Effects 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 210000005007 innate immune system Anatomy 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000003601 intercostal effect Effects 0.000 description 1
- 210000005027 intestinal barrier Anatomy 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 229940035901 lactobacillus sp Drugs 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 229940118019 malondialdehyde Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000007102 metabolic function Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000004115 mitral valve Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000004118 muscle contraction Effects 0.000 description 1
- 208000013465 muscle pain Diseases 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 210000001087 myotubule Anatomy 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 1
- 235000015816 nutrient absorption Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 238000006395 oxidase reaction Methods 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 210000003540 papillary muscle Anatomy 0.000 description 1
- 210000002976 pectoralis muscle Anatomy 0.000 description 1
- 210000003516 pericardium Anatomy 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000036316 preload Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000001012 protector Effects 0.000 description 1
- 210000001147 pulmonary artery Anatomy 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
- 230000011506 response to oxidative stress Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 231100000872 sexual dysfunction Toxicity 0.000 description 1
- 150000004666 short chain fatty acids Chemical class 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 210000001562 sternum Anatomy 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 150000003536 tetrazoles Chemical class 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000000779 thoracic wall Anatomy 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- XOSXWYQMOYSSKB-LDKJGXKFSA-L water blue Chemical compound CC1=CC(/C(\C(C=C2)=CC=C2NC(C=C2)=CC=C2S([O-])(=O)=O)=C(\C=C2)/C=C/C\2=N\C(C=C2)=CC=C2S([O-])(=O)=O)=CC(S(O)(=O)=O)=C1N.[Na+].[Na+] XOSXWYQMOYSSKB-LDKJGXKFSA-L 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 229940075420 xanthine Drugs 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
- 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
- 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
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- 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
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/151—Johnsonii
Abstract
本发明属于微生物技术领域，公开了一种约氏乳杆菌EU03(Lactobacillus johnsonii EU03)及其用途。本发明所述约氏乳杆菌具有抗氧化活性，可改善心肌梗死(MI)大鼠血清酶学指标，修复心肌损伤；并可改善MI大鼠心功能指标；使受损心脏恢复正常心肌形态学作用。
Description
技术领域
本发明属于微生物技术领域，具体涉及一种约氏乳杆菌EU03(Lactobacillusjohnsonii EU03)及其用途，尤其是一种能够改善心肌损伤、心功能以及心肌形态学的约氏乳杆菌EU03及其用途。
背景技术
心血管疾病和相关疾病，如高胆固醇血症、高血压、肥胖、糖尿病，是全世界死亡的主要原因，并且造成了经济损失。心肌梗死(MI)作为引起心血管疾病死亡的重要原因，已受到广泛关注。目前，治疗MI的药物在开发过程中存在多种问题。临床上早期采用糖皮质激素类药物治疗MI，随后发现此类药物会增加MI患者心脏破裂的风险4]。非甾体类抗炎药也曾广泛应用于MI的治疗中。然而，在使用过程中发现此类药物也会增加不良心血管事件的发生，加剧胃肠道出血。冠状动脉扩张药物如硝酸酯类药物，在使用过程中会产生耐药性，此外，硝酸酯类药物的持续治疗还会对缺血后的心肌细胞活力产生负面影响，导致动物心肌梗塞模型中的梗塞面积增加。钙拮抗剂如二氢吡啶类在治疗时可引起反射性心动过速等不良反应。β受体阻滞剂被报道静脉应用存在一定风险且具有疲劳，头晕，抑郁和性功能障碍等不良反应。调节血脂他汀类药物近年也被证实可以降低心血管风险事件的发生，但有文献报道长期的他汀类药物疗法引起的唯一严重不良事件(即他汀类药物的不良反应)是肌病(定义为肌肉疼痛或无力并伴有血肌酸激酶血药浓度大幅增加)。综上，当前治疗MI药物都有一定程度的不良反应，因此，开发无副作用且改善MI的药物还需研究人员进一步探究。
补充益生菌可降低心血管风险且没有副作用。益生菌可能通过不同的机制发挥作用，包括建立适当的肠道平衡，改善营养吸收和免疫功能，以及抵御病原体。在肠道益生菌中，乳杆菌属(Lactobacillus sp.)是革兰氏阳性菌，被归类为乳酸菌。各种乳杆菌菌株通过影响脂质胆固醇代谢、免疫炎症反应、氧化应激反应以及涉及的肠道代谢物(包括氧化三甲胺TMAO、短链脂肪酸SCFAs、脂多糖LPS和胆汁酸BAs)在心血管相关疾病预防和治疗中得到广泛研究。乳杆菌产生的代谢物，尤其是抗菌物质，可以抑制病原体的生长，调节肠道菌群(GM)紊乱。多糖是一种类似益生元的代谢物，在心血管相关疾病的预防和治疗中也发挥着重要作用。它们是GM的保护者，是乳杆菌对心血管相关疾病的主要贡献者。
中国公布的细菌名录包括13种乳酸菌，它们被公认为安全成分，广泛用于食品生产。国家食品药品监督管理局以公告的形式对名单进行了补充(http://samr.cfda.gov.cn/WS01/CL1975/228275.html，[2010]65号)包括L.acidophilus，L.casei，L.crispatus，L.delbrueckii，L.fermentum，L.gasseri，L.helveticus，L.johnsonii，L.paracasei，L.plantarum，L.reuteri，L.rhamnosus和L.salivarius。鉴于研究报道对MI有防治作用的乳杆菌仅有L.rhamnosus，从肠道中筛选和鉴定功能性乳杆菌研究已成为热点领域。
因此本发明中的约氏乳杆菌具有对MI大鼠的心脏保护作用，改善MI大鼠心脏功能指标，具有开发成功能性食品的潜力，且对MI的临床治疗具有重要的指导意义。
发明内容
有鉴于此，本发明旨在克服上述现有技术中存在的缺陷，提供一种从肠道中分离的乳杆菌，该乳杆菌可改善心肌损伤、心功能以及心肌形态学。
为达到上述目的，本发明的技术方案是这样实现的：
本发明提供了一种约氏乳杆菌EU03(Lactobacillus johnsonii EU03)，于2020年10月9日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址为北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.20845，状态为存活。
在一个实施方案中，本案考察了约氏乳杆菌EU03对心肌梗死导致的心肌损伤的改善作用，结果显示，约氏乳杆菌EU03的摄入可使MI大鼠具有抗氧化活性，降低大鼠氧化应激，改善MI大鼠血清酶学指标，提高SOD含量，降低MDA和LDH含量，起到修复心肌损伤的作用。
在一个实施方案中，本案考察了约氏乳杆菌EU03对心肌梗死导致的心功能指标下降的改善作用，结果显示，约氏乳杆菌EU03的摄入可使心脏室壁变厚，并增强室壁运动和收缩能力，提高血流动力学指标。
在一个实施方案中，本案考察了约氏乳杆菌EU03对心肌梗死导致的心肌细胞缺血坏死、心脏形态和胶原纤维遭到破坏的改善作用，结果显示，约氏乳杆菌EU03的摄入可以降低MI大鼠的梗死面积，降低炎性浸润，改善了心肌间质水肿和纤维化程度。
因此本发明提供了约氏乳杆菌EU03在制备改善心肌损伤、心功能以及心肌形态学的产品中的应用；
其中，所述改善心肌损伤为提高MI大鼠的抗氧化活性和血清酶学指标，降低氧化应激，提高SOD含量，降低MDA和LDH含量；所述改善心功能为降低Toll样受体(TLR)的刺激，从而减少心脏重塑标志物MMP9的产生，改善心功能；所述改善心肌形态学为通过抑制TGF-β蛋白水平及其下游蛋白的表达水平，减少梗死心脏炎症期后伤口重塑中的胶原沉积，并抑制成肌纤维细胞的数量，改善心肌纤维化。
本发明所述产品包括但不限于药物。
进一步地，本发明提供了含有所述约氏乳杆菌EU03的菌剂。
优选的，所述菌剂中约氏乳杆菌EU03的活菌数大于108CFU/mL。
本发明所书菌剂可以按常规方法制备。
在一些实施方案中，所述菌剂的制备方法为，将约氏乳杆菌EU03按照体积分数2％的接种量接种至MRS液体培养基中，在37℃兼性厌氧条件下培养24h，取出离心，弃去上清，用灭菌水洗两次后，用灭菌水调整菌液浓度为108CFU/mL。
本发明所书制备方法中，所述离心为在4℃的条件下进行离心操作，离心机转速为10000r/min，离心的时间为10min。
本发明中，菌落的计数方法为：将冻存的约氏乳杆菌转移至灭菌的脱脂乳培养基中，42℃培养4h，反复活化3代，使菌种活力恢复，置于4℃保存备用。将活化的约氏乳杆菌分别转种至MRS液体培养基中，37℃静置培养16h，以此作为种子液。用灭菌水依次10倍稀释至106倍稀释液。取100～106倍稀释液50μL分别涂布于MRS琼脂平皿上，每个稀释度做3个平行，37℃恒温培养24h后，对菌落数在30～300个的平板计数，求平均值，以CFU/mL为单位，确定其中1μL乳杆菌培养液相当于105CFU活菌数。
在某些实施方案中，所述受试者是指哺乳动物，包括但不限于，人，啮齿类动物(小鼠，大鼠，豚鼠)，狗，马，牛，猫，猪，猴，黑猩猩。
在某些优选的实施方案中，所述受试者是人。
本发明还提供了一种发酵食品，以所述约氏乳杆菌EU03作为发酵剂发酵生产的食品。
所述发酵食品为发酵乳制品、发酵豆制品或发酵果蔬制品。
本发明还提供了一种药物制剂，包括有效剂量的约氏乳杆菌EU03和药学上可接受的辅料。
术语解释：
如本文中所使用的，“SOD”是指超氧化物歧化酶又称超氧歧化酶，是一类能催化超氧阴离子自由基(O2-)歧化为H2O2和O2的酶。“MDA”是指丙二醛，是膜脂过氧化最重要的产物之一，它的产生能加剧膜的损伤。“LDH”是指乳酸脱氢酶，为含锌离子的金属蛋白，分子量为135-140kD，是糖无氧酵解及糖异生的重要酶系之一，可催化丙酸与L-乳酸之间的还原与氧化反应，也可催化相关的α-酮酸。“CTn-T”是指心肌肌钙蛋T，是是心肌肌肉收缩的调节蛋白，CTn-T的大部分是以C-T-I的复合物形式存在于细丝上，6％—8％以游离的形式存在于心肌细胞浆中。“CK-MB”是指肌酸激酶同工酶，主要分布于心肌中，当心肌损伤后，CK-MB会释放到血液中。
“LVEF”是指左室射血分数，指每搏输出量占心室舒张末期容积量(即心脏前负荷)的百分比，射血分数与心肌的收缩能力有关，心肌收缩能力越强，则每搏输出量越多，射血分数也越大。“LVFS”是指左室短轴缩短率，是左心室收缩功能指标参数；若左室短轴缩短率＞25％为左心室收缩功能正常，若左室短轴缩短率＜25％为左心室收缩功能受损。“AVPeak Vel”指流出道血流峰值，“LVID；d”指舒张末期左室内径，“LVID；s”指收缩末期左室内径，以上超声参数均可作为评价心梗后大鼠心功能指标。
本发明的有益效果在于：
本发明所述约氏乳杆菌EU03可使MI大鼠具有抗氧化活性，降低大鼠氧化应激，改善MI大鼠血清酶学指标，提高SOD含量，降低MDA和LDH含量，起到修复心肌损伤的作用；且可使心脏室壁变厚，并增强室壁运动和收缩能力，提高血流动力学指标，改善心功能；并可降低MI大鼠的梗死面积，降低炎性浸润，改善了心肌间质水肿和纤维化程度，对心肌梗死导致的心肌细胞缺血坏死、心脏形态和胶原纤维遭到破坏的改善作用。本发明所述的约氏乳杆菌EU03可用于制备改善心肌损伤、心功能以及心肌形态学的药物，具有广泛的应用前景。
生物保藏说明：
EU03，分类命名：约氏乳杆菌EU03(Lactobacillus johnsonii EU03)，其保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为北京市朝阳区北辰西路1号院3号，有保藏号为CGMCC No.20845，保藏日期：2020年10月9日，状态为存活。
附图说明
构成本发明的一部分的附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1A-E为约氏乳杆菌EU03对MI大鼠SOD、MDA、LDH、CK-MB和CTn-T的影响；其中对照组为sham，模型组为model，给菌组为LJ,(vs sham#p<0.05,##p<0.01,###p<0.001；vsmodel*p<0.05,**p<0.01,**p<0.001)；
图2A为术后大鼠超声心动图(包括(1)切面图；(2)运动曲线图；(3)血流显像图；(4)多普勒频谱图；其中对照组为sham，模型组为model，给菌组为LJ)；
图2B-F为术后大鼠超声心功能参数变化(包括(B)EF％；(C)FS％；(D)AV；(E)LVID：d；(F)LVID：S；其中对照组为sham，模型组为model，给菌组为LJ,(vs sham#p<0.05,##p<0.01,###p<0.001；vs model*p<0.05,**p<0.01,**p<0.001))；
图3A-B为术后各组大鼠心肌左室部位细胞形态化(包括(A)HE染色图；(B)Masson染色图；其中对照组为sham，模型组为model，给菌组为LJ)。
具体实施方式
下面将结合实施例对本申请的实施方案进行详细描述，但是本领域技术人员将理解，下列实施例仅用于说明本申请，而不是对本申请的范围的限定。根据优选实施方案的下列详细描述，本申请的各种目的和有利方面对于本领域技术人员来说将变得显然。
为了进一步理解本发明，下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：约氏乳杆菌EU03具有抗氧化活性，改善MI后大鼠心肌损伤指标作用
如表1所示，实验共分三组，对照组(4周)、模型组(4周)和给菌组(4周)，分别以三组实验大鼠的血清作为检测样品。
模型组(4周)：大鼠适应性饲养一周，术前禁食12h，自由饮水。造模前对大鼠称重，并以现用现配5％的水合氯醛按照0.6mL/100g体重腹腔注射麻醉大鼠。麻醉后的大鼠胸部备皮，仰卧位固定，用酒精或碘伏进行胸前皮肤的消毒，沿左侧第四肋间(或心脏搏动最明显处)剪开，用止血钳逐层分离皮下组织，少量钝性分离胸肌。在心脏搏动最明显处用止血钳撑开胸廓，暴露心脏。剥开心包膜，轻压右胸，将心脏轻轻挤压出胸壁外，持无创缝合针丝线，于肺动脉圆锥与左心耳之间，在离左冠状动脉前降支(LAD)起始部2-3mm处进针。以5/0无损伤丝线结扎冠状动脉前降支，将心脏放回胸腔，抽空胸腔内空气，关闭胸腔，以2/0无损伤丝线分别缝合肌层及皮肤，假手术组仅在相应冠脉位置穿线，在MI后24小时，使用MS-250、16.0–21.0MHZ成像传感器评估左心室功能，该传感器连接到超声心动图系统，专门设计对小动物来说。麻醉下刮除大鼠胸部，进行二维超声心动图检查。通过室间隔和左室后壁的鉴别获得图像。超声心动图自动计算左室缩短率(FS，％)和射血分数(EF，％)左心室射血分数符合(30-45％)的大鼠予以保留，不符合的条件的大鼠视为不成模，剔除。模型组给予同体积的液体培基。每天定期喂养每只大鼠，连续喂养4周。
给菌组(4周)：造模后约氏乳杆菌灌胃组剂量为108CFU/mL，按每100g体重灌服1mL菌体计算，大鼠每天称重1次，根据体重变化及时调整给菌体积，连续喂养4周。
对照组(4周)：假手术组给予同体积的液体培基。每天定期喂养每只大鼠，连续喂养4周。饲料满足SPF级饲养和营养需要。
表1.约氏乳杆菌动物实验分组及剂量
指标检测原理：
SOD检测原理：通过黄嘌呤及黄嘌呤氧化酶反应系统产生超氧阴离子(O2-)，O2-可还原氮蓝四唑生成蓝色甲臜，后者在560nm处有吸收；SOD可清除O2-，从而抑制了甲臜的形成；反应液蓝色越深，说明SOD活性愈低，反之活性越高。
MDA检测原理：MDA与硫代巴比妥酸(thiobarbituric acid,TBA)缩合，生成红色产物，在532nm有最大吸收峰，进行比色后可估测样品中过氧化脂质的含量；同时测定600nm下的吸光度，利用532nm与600nm下的吸光度的差值计算MDA的含量。
LDH检测原理：在乳酸脱氢酶的作用下，NAD+被还原生成NADH，NADH和INT(2-p-iodophenyl-3-nitrophenyl tetrazolium chloride)被硫辛酰胺脱氢酶(diaphorase)催化反应生成NAD+和强生色物甲臜(formazan)，在490nm波长下产生吸收峰，从而可以通过比色来定量乳酸脱氢酶的活性。
CK-MB、CTn-T含量测定原理：采用ELISA法测定，CK-MB、CTn-T抗体包被于96孔微孔板中，制成固相载体，向微孔中分别加入标准品或样品，其中的CK-MB、CTn-T与连接于固相载体上的抗体结合，然后加入生物素化的CK-MB、CTn-T抗体，将未结合的生物素化抗体洗净后，加入HRP标记的亲和素，再次彻底洗涤后加入TMB底物显色。TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的CK-MB、CTn-T呈正相关。用酶标仪在450nm波长下测定吸光度(OD值)，计算样品浓度。
酶联免疫吸附测定(ELISA)法测定CK-MB、CTn-T的水平，具体操作如下溶液的制备：20X洗涤缓冲液用蒸馏水按1:20比例稀释。
(1)试剂盒室温平衡20min，从铝箔袋中取出所需板条。
(2)设置空白孔、标准品孔和样本孔，标准品孔中加不同浓度的标准样品50μL，样本孔先加入待测样品10μL，再加样品稀释液40μL。
(3)除空白孔外，向孔中加入辣根过氧化物酶(HRP)标记的检测抗体100μL，将反应孔用封板膜盖住封口，37℃恒温孵育60min。
(4)弃去液体，在吸水纸上拍干，每空加满洗涤液，静置1min，甩去洗涤液，吸水纸拍干，重复洗板5次。
(5)每孔加入底物A、B各50μL，37℃避光孵育15min。每孔加入终止液50μL，15min内，在450nm波长检测各孔OD值。
实验结果：
图1A显示，与对照组相比，模型组SOD水平较低，且与模型组相比，给菌组大鼠组织中的SOD水平显著升高(P<0.05)。图1B显示，MI术后给药28天时，与对照组相比，模型组大鼠组织MDA水平显著升高(P<0.001)。给菌后，MDA水平显著降低(P<0.01)。此外，与对照组相比，模型组LDH、CK-MB、CTn-T水平均较高，且给菌后有一定程度降低，说明给菌组有向假手术组回调的趋势(图1C-E)。
本领域，氧化程度越高心肌受损越严重，SOD较高，MDA较低，LDH较低，说明抗氧化活性高，氧化应激可以通过多种机制引起细胞凋亡或坏死，CK-MB和CTn-T是心肌损伤的标志物，越高表明受损越严重，可以推测氧化程度高，心肌损伤严重，从而使CK-MB和CTn-T升高。
综上，给菌组具有抗氧化活性，可改善MI大鼠血清酶学指标，修复心肌损伤。其抗氧化活性源于益生菌具有自己的抗氧化酶系统，可以产生各种抗氧化代谢物，增加抗氧化剂水平，抵抗ROS，包括过氧化物自由基，超氧阴离子和羟基自由基。同时益生菌还可以刺激宿主的抗氧化系统，刺激Nrf2-Keap1-ARE等抗氧化信号通路，同时也可调节肠道菌群，降低氧化应激。
实施例2：约氏乳杆菌EU03改善MI大鼠心功能作用
给药4周后进行大鼠心功能检测。大鼠经5％水合氯醛腹腔注射麻醉后脱毛膏脱毛。Vevo2100超高分辨率小动物超声实时影像仪(加拿大)开机后待用，将探头MS-250固定，升降台调节探头高度，旋转调节探头方向。超声采用异氟烷麻醉(完全麻醉：1％氧气+5％异氟烷于麻醉盒内麻醉；持续麻醉：1％氧气+2％异氟烷)。用胶带将大鼠腹部向上固定于操作台，设置温度为37℃；在大鼠胸部均匀涂抹一层超声耦合剂，将操作台向右下倾斜30-45°，并通过X、Y微调操作台位置。胸骨旁长轴，B-型超声长轴切面(二维成像)，可见左心室，主动脉弓和二尖瓣瓣叶，获得图像；M-型超声长轴切面，获得M-Mode图像，取样线位于最大腔径处(与乳头肌相切)。通过对大鼠收缩期和舒张期进行测量，获得相关心脏参数，以此评价约氏乳杆菌对MI心功能指标的改善作用。
实验结果：
MI大鼠超声M-Mode结果显示(图2A)，与对照组相比，模型组左心室室腔相较于Sham组呈现出左室容积扩张，室壁变薄，室壁运动减弱，收缩力降低。给菌组与模型组相比能明显改善左心室的损伤，提示约氏乳杆菌EU03在一定程度上可改善MI的损伤。统计出超声结果(图2B-F)来看，急性MI模型的射血分数(EF)、心室短轴缩短率(FS)、流出道血流峰值(AV Peak Vel)、舒张末期左室内径(LVID；d)、左室收缩末期内径(LVID；s)与对照组相比具有明显的显著性，给菌组与模型组相比能提高EF、FS、降低LVID；d、LVID；s，AV Peak Vel有上升趋势，但无显著性差异，提示约氏乳杆菌EU03可改善MI大鼠心功能指标。
研究表明心梗后的不良心输出量会导致肠缺血，水肿和炎症，通常称为肠屏障的“渗漏”，会引起肠道来源的菌群和有害代谢物移位，如革兰氏阴性菌细胞壁的脂多糖(LPS)，产生“代谢性内毒素血症”进而触发全身炎症。LPS可以和Toll样受体(TLR)结合，刺激先天免疫系统，引起代谢紊乱，而益生菌可以使TLR刺激下降，从而减少心脏重塑标志物MMP9的产生，改善心功能。除此之外，给菌后产生的有益代谢物可以恢复肠屏障功能，影响宿主心脏代谢功能。
实施例3：约氏乳杆菌EU03可使MI大鼠恢复正常心肌形态学作用
MI发生后，心脏功能发生障碍，导致心肌细胞缺血坏死，心脏形态和胶原纤维遭到破坏。受损的心脏为维持结构的完整性，病理性改善胶原代谢，形成胶原与心肌共存的病理学形态特征。本实验选择苏木精-伊红染色法(hematoxylin-eosin staining，HE)和Masson染色对心肌病变程度进行比较，从而观察约氏乳杆菌EU03对MI大鼠心肌形态学的影响。
取固定于10％甲醛溶液中至少48h的心脏组织，改刀切取厚约2-3mm的组织，按照以下顺序进行处理：75％乙醇1h→95％乙醇过夜→100％乙醇I1h→100％乙醇II 4h→二甲苯I15min→二甲苯II 45min→软蜡30min→硬蜡1h。随后进行包埋，将提前熔化的石蜡倒入包埋框，立即把浸入硬蜡的组织块同方向放入，待蜡块凝固后，固定于石蜡切片机上，切出5μm厚的石蜡组织切片，温水(50℃)中展片、分片、捞片，最后于电热干燥箱中(60℃)烤片4-6h。冷却后室温保存。
苏木精-伊红染色(HE染色)方法：
(1)脱蜡：将石蜡切片放于烤箱60℃10min；
(2)水化：将脱蜡后的切片取出，于二甲苯溶液Ⅰ、Ⅱ中各浸泡10min，再于无水乙醇Ⅰ、无水乙醇Ⅱ、95％、75％梯度乙醇中各浸泡5min，流水冲洗5min；
(3)伊红染胞质及脱水透明：l％伊红溶液染色1-2min，流水冲洗15-30sec；75％乙醇浸泡3min，95％乙醇浸泡3min，无水乙醇Ⅰ、无水乙醇Ⅱ各浸泡3-5min，上行梯度脱水；于二甲苯溶液Ⅰ、Ⅱ中各浸泡5min进行透明；
(4)封片：中性树胶滴加玻片组织周围，后用盖玻片覆盖封片；
(5)观察：光镜显微镜下观察并拍照、记录。
Masson染色：
胶原纤维被苏木素染成蓝紫色，观察蓝紫色视野相对比例大致判断心脏组织纤维化程度。具体步骤：
(1)石蜡切片脱蜡至水。
(2)铬化处理或去汞盐沉淀(甲醛固定的组织此步可略)。
(3)依次自来水和蒸馏水洗。
(4)用Regaud苏木精染液或Weigert苏木精液染核5-10min。
(5)充分水洗,如过染可盐酸酒精分化。
(6)蒸馏水洗。
(7)用Masson丽春红酸性复红液5-10min。
(8)以2％冰醋酸水溶液浸洗片刻。
(9)1％磷钼酸水溶液分化3-5min。
(10)不经水洗,直接用苯胺蓝或光绿液染5min。
(11)以0.2％冰醋酸水溶液浸洗片刻。
(12)95％酒精、无水酒精、二甲苯透明、中性树胶封固。
实验结果：
心肌组织经HE染色后，于显微镜下拍照，观察28天各组大鼠心肌组织的病理改变。如图3所示，对照组大鼠心肌纤维完整、紧密、整齐，无明显炎性细胞浸润和水肿，细胞核居中，胞质着色均匀，心外膜、心内膜无明显病理异常。模型组大鼠心肌坏死灶呈片状分布，病变累及心壁全层，肌纤维发生肿胀、断裂，心肌间质可见粘液变及炎细胞浸润伴有间质水肿，心外膜细胞死亡，呈现空泡样。给菌组病变面积明显缩小，降低了炎性浸润程度，改善了心肌间质水肿度程度。心肌细胞Masson染色结果显示假手术对照组心肌细胞排列整齐，模型组梗死灶周围均可见蓝染的胶原纤维，心肌纤维发生明显的断裂，排列紊乱。给菌组可明显减少胶原纤维产生，改善心肌纤维化。
研究结果显示，约氏乳杆菌EU03可以降低MI大鼠的梗死面积，降低炎性浸润，改善了心肌间质水肿和纤维化程度。研究表明，抑制TGF-β可以减少梗死心脏炎症期后伤口重塑中的胶原沉积并抑制成肌纤维细胞的数量，由此可推测给菌组可能通过降低TGF-β蛋白水平及其下游蛋白的表达水平，改善心肌纤维化，在一定阶段内对MI大鼠的心脏具有保护作用。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (1)
1.约氏乳杆菌EU03在制备改善心肌损伤、心功能以及心肌形态学的药物中的应用，所述约氏乳杆菌EU03(Lactobacillus johnsonii EU03)，保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.20845。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110391018.9A CN113215030B (zh) | 2021-04-12 | 2021-04-12 | 约氏乳杆菌eu03及其用途 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110391018.9A CN113215030B (zh) | 2021-04-12 | 2021-04-12 | 约氏乳杆菌eu03及其用途 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113215030A CN113215030A (zh) | 2021-08-06 |
| CN113215030B true CN113215030B (zh) | 2022-10-25 |
Family
ID=77087163
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110391018.9A Active CN113215030B (zh) | 2021-04-12 | 2021-04-12 | 约氏乳杆菌eu03及其用途 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113215030B (zh) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115500515A (zh) * | 2022-08-10 | 2022-12-23 | 华南理工大学 | 一种植物乳杆菌在调节肠道菌群中的应用 |
| CN116369427A (zh) * | 2022-12-30 | 2023-07-04 | 西南科技大学 | 一种约氏乳杆菌在制备抗氧化和提升免疫的制品中的应用 |
| CN116606776A (zh) * | 2023-06-20 | 2023-08-18 | 广东南芯医疗科技有限公司 | 约氏乳杆菌ls04在制备抗氧化和抗衰老产品中的应用 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2702977T3 (es) * | 2009-06-19 | 2019-03-06 | Dupont Nutrition Biosci Aps | Bifidobacterias para el tratamiento de diabetes y afecciones relacionadas |
| GB2599573A (en) * | 2019-05-31 | 2022-04-06 | Rhogen Biotech Llc | Compositions and methods for detoxifying bacterial endotoxins |
| EP4034253A4 (en) * | 2019-09-23 | 2024-02-07 | Univ Michigan Regents | COMPOSITIONS AND METHODS FOR INCREASING THE EFFECTIVENESS OF IMMUNOTHERAPIES AND VACCINES |
| CN112205561A (zh) * | 2020-09-26 | 2021-01-12 | 技源健康科技(江苏)有限公司 | 一种益生菌泡腾微片及其制备方法 |
-
2021
- 2021-04-12 CN CN202110391018.9A patent/CN113215030B/zh active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN113215030A (zh) | 2021-08-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113215030B (zh) | 约氏乳杆菌eu03及其用途 | |
| CN109666615B (zh) | 一种益生菌组合物及其应用 | |
| CN110272842A (zh) | 一株具有减肥降脂功能的植物乳杆菌lp104 | |
| JPS60501160A (ja) | 膣用カプセル | |
| WO2012002322A1 (ja) | 経口用皮膚性状改善剤 | |
| PL194697B1 (pl) | Zastosowanie szczepu Lactobacillus | |
| CN113308421B (zh) | 一种植物乳杆菌bufx及其在代谢综合征中的应用 | |
| CN110218681A (zh) | 一株发酵乳杆菌kp101及其应用 | |
| CN104430851A (zh) | 一种降胆固醇发酵乳及其制备方法 | |
| CN112553117B (zh) | 一株能够抑制皮肤角质层增厚的罗伊氏乳杆菌及其应用 | |
| AU2017337936A1 (en) | Novel Lactobacillus Sakei And Composition Comprising The Same | |
| CN113755409B (zh) | 一种缓解胰岛素抵抗的长双歧杆菌及其应用 | |
| CN110339216A (zh) | 可预防及治疗细菌性阴道炎的乳酸菌组合物 | |
| CN102526019A (zh) | 松萝酸及其衍生物在制备治疗马拉色菌引起皮肤疾病药物中的用途 | |
| CN113797232A (zh) | 具有缓解胰岛素抵抗功能的组合物及其应用 | |
| JP7383120B2 (ja) | 皮膚状態改善用組成物 | |
| CN113559045A (zh) | 燕麦麸皮发酵物，含其皮肤外用剂及其制备方法和应用 | |
| CN116019224B (zh) | 一种具有防腐和肠胃调节功能的后生元、其制备方法及应用 | |
| CN116747245A (zh) | 动物双歧杆菌乳亚种bx-245在抑菌和/或生产功能活性物质中的应用 | |
| CN115044507B (zh) | 一种治疗和/或预防糖脂代谢异常的微生物组合物及其应用 | |
| CN109475584A (zh) | 治疗由阴道加德纳氏菌导致的细菌性阴道感染和可能存在的并发真菌感染的乳酸菌组合物 | |
| CN106834186B (zh) | 一种植物乳杆菌微生态制剂及其制备方法和应用 | |
| CN115212237B (zh) | 普拉梭菌在制备治疗心肌梗死后的心室病理性重构和/或心力衰竭的药物中的应用 | |
| CN112402463B (zh) | 一种用于抑制阴道炎症的复合益生菌及其制品和应用 | |
| CN115838661A (zh) | 一种植物乳杆菌扁鹊君18、植物乳杆菌制剂及其应用 |
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 |