CN113621665A - Lactobacillus plantarum acidic extracellular polysaccharide and application thereof - Google Patents
Lactobacillus plantarum acidic extracellular polysaccharide and application thereof Download PDFInfo
- Publication number
- CN113621665A CN113621665A CN202110938883.0A CN202110938883A CN113621665A CN 113621665 A CN113621665 A CN 113621665A CN 202110938883 A CN202110938883 A CN 202110938883A CN 113621665 A CN113621665 A CN 113621665A
- Authority
- CN
- China
- Prior art keywords
- lactobacillus plantarum
- polysaccharide
- dmdl9010
- acidic
- exopolysaccharide
- 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.)
- Granted
Links
- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 83
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 83
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 83
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 73
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 73
- 230000002378 acidificating effect Effects 0.000 title claims abstract description 59
- 150000004676 glycans Chemical class 0.000 title abstract description 11
- 238000000855 fermentation Methods 0.000 claims abstract description 30
- 230000004151 fermentation Effects 0.000 claims abstract description 30
- 239000002253 acid Substances 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 14
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims abstract description 9
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims abstract description 9
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims abstract description 9
- 150000002772 monosaccharides Chemical class 0.000 claims abstract description 9
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims abstract description 8
- 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 8
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims abstract description 8
- 235000013305 food Nutrition 0.000 claims abstract description 8
- 239000008103 glucose Substances 0.000 claims abstract description 8
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229930182830 galactose Natural products 0.000 claims abstract description 7
- 229930091371 Fructose Natural products 0.000 claims abstract description 6
- 239000005715 Fructose Substances 0.000 claims abstract description 6
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 6
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 claims abstract description 6
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims abstract description 6
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 claims abstract 2
- 150000004804 polysaccharides Chemical class 0.000 claims description 64
- 229920002444 Exopolysaccharide Polymers 0.000 claims description 50
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- 239000012071 phase Substances 0.000 claims description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- 239000002244 precipitate Substances 0.000 claims description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 229920001284 acidic polysaccharide Polymers 0.000 claims description 9
- 150000004805 acidic polysaccharides Chemical class 0.000 claims description 9
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 9
- 239000000935 antidepressant agent Substances 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 238000005342 ion exchange Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 claims description 7
- 230000003213 activating effect Effects 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 229920005654 Sephadex Polymers 0.000 claims description 5
- 239000012507 Sephadex™ Substances 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 5
- 239000003480 eluent Substances 0.000 claims description 5
- 235000019441 ethanol Nutrition 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 239000012074 organic phase Substances 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 241001052560 Thallis Species 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 241000186660 Lactobacillus Species 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 229940039696 lactobacillus Drugs 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 229940035901 lactobacillus sp Drugs 0.000 claims 1
- 241000699666 Mus <mouse, genus> Species 0.000 abstract description 24
- 230000000694 effects Effects 0.000 abstract description 21
- 238000002474 experimental method Methods 0.000 abstract description 14
- 238000002076 thermal analysis method Methods 0.000 abstract description 5
- 241000186610 Lactobacillus sp. Species 0.000 abstract description 3
- 230000001788 irregular Effects 0.000 abstract description 3
- 238000012545 processing Methods 0.000 abstract description 2
- TVZRAEYQIKYCPH-UHFFFAOYSA-N 3-(trimethylsilyl)propane-1-sulfonic acid Chemical compound C[Si](C)(C)CCCS(O)(=O)=O TVZRAEYQIKYCPH-UHFFFAOYSA-N 0.000 description 29
- 239000000243 solution Substances 0.000 description 28
- 241000699670 Mus sp. Species 0.000 description 20
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 230000011987 methylation Effects 0.000 description 6
- 238000007069 methylation reaction Methods 0.000 description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- 230000002093 peripheral effect Effects 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical compound [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- IAJILQKETJEXLJ-RSJOWCBRSA-N aldehydo-D-galacturonic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-RSJOWCBRSA-N 0.000 description 4
- 238000007664 blowing Methods 0.000 description 4
- 206010009887 colitis Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- GIYXAJPCNFJEHY-UHFFFAOYSA-N N-methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy]-1-propanamine hydrochloride (1:1) Chemical compound Cl.C=1C=CC=CC=1C(CCNC)OC1=CC=C(C(F)(F)F)C=C1 GIYXAJPCNFJEHY-UHFFFAOYSA-N 0.000 description 3
- 230000001430 anti-depressive effect Effects 0.000 description 3
- 229940005513 antidepressants Drugs 0.000 description 3
- 239000008346 aqueous phase Substances 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 229960000389 fluoxetine hydrochloride Drugs 0.000 description 3
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 238000012346 open field test Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000009777 vacuum freeze-drying Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- OVCJDQPBVXUBBF-UHFFFAOYSA-N 1,5-di-O-acetyl 2,3,4,6-tetra-O-methyl glucitol Natural products COCC(OC(C)=O)C(OC)C(OC)C(OC)COC(C)=O OVCJDQPBVXUBBF-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 206010009900 Colitis ulcerative Diseases 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 2
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 201000006704 Ulcerative Colitis Diseases 0.000 description 2
- JTIPYRUUQYKTIG-KBUPBQIOSA-N [(2r,3r,4r,5r)-2,5-diacetyloxy-3,4,6-trimethoxyhexyl] acetate Chemical compound COC[C@@H](OC(C)=O)[C@@H](OC)[C@H](OC)[C@H](OC(C)=O)COC(C)=O JTIPYRUUQYKTIG-KBUPBQIOSA-N 0.000 description 2
- OVCJDQPBVXUBBF-AAVRWANBSA-N [(2r,3r,4r,5r)-5-acetyloxy-2,3,4,6-tetramethoxyhexyl] acetate Chemical compound COC[C@@H](OC(C)=O)[C@@H](OC)[C@H](OC)[C@H](OC)COC(C)=O OVCJDQPBVXUBBF-AAVRWANBSA-N 0.000 description 2
- RUAAXNGDIMCTCD-GBJTYRQASA-N [(2s,3r,4r,5r)-5,6-diacetyloxy-2,3,4-trimethoxyhexyl] acetate Chemical compound CC(=O)OC[C@H](OC)[C@@H](OC)[C@H](OC)[C@@H](COC(C)=O)OC(C)=O RUAAXNGDIMCTCD-GBJTYRQASA-N 0.000 description 2
- RUAAXNGDIMCTCD-YJNKXOJESA-N [(2s,3r,4s,5r)-5,6-diacetyloxy-2,3,4-trimethoxyhexyl] acetate Chemical compound CC(=O)OC[C@H](OC)[C@@H](OC)[C@@H](OC)[C@@H](COC(C)=O)OC(C)=O RUAAXNGDIMCTCD-YJNKXOJESA-N 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 238000005349 anion exchange Methods 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 230000003542 behavioural effect Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 239000005018 casein Substances 0.000 description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
- 235000021240 caseins Nutrition 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000001307 helium Substances 0.000 description 2
- 229910052734 helium Inorganic materials 0.000 description 2
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 229940099596 manganese sulfate Drugs 0.000 description 2
- 239000011702 manganese sulphate Substances 0.000 description 2
- 235000007079 manganese sulphate Nutrition 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 2
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 2
- -1 polysaccharide monosaccharide Chemical class 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000012354 sodium borodeuteride Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000002411 thermogravimetry Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 2
- 239000001393 triammonium citrate Substances 0.000 description 2
- 235000011046 triammonium citrate Nutrition 0.000 description 2
- 230000002747 voluntary effect Effects 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 229940126673 western medicines Drugs 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 206010000117 Abnormal behaviour Diseases 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 206010002942 Apathy Diseases 0.000 description 1
- 238000011814 C57BL/6N mouse Methods 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000028399 Critical Illness Diseases 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 206010011971 Decreased interest Diseases 0.000 description 1
- 206010012741 Diarrhoea haemorrhagic Diseases 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 101000841301 Homo sapiens Utrophin Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 208000006083 Hypokinesia Diseases 0.000 description 1
- 208000036626 Mental retardation Diseases 0.000 description 1
- 229940123685 Monoamine oxidase inhibitor Drugs 0.000 description 1
- 208000019022 Mood disease Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229920002845 Poly(methacrylic acid) Polymers 0.000 description 1
- 206010057071 Rectal tenesmus Diseases 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 206010042458 Suicidal ideation Diseases 0.000 description 1
- OKJPEAGHQZHRQV-UHFFFAOYSA-N Triiodomethane Natural products IC(I)I OKJPEAGHQZHRQV-UHFFFAOYSA-N 0.000 description 1
- 102100029092 Utrophin Human genes 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical group OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 description 1
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical group OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 230000002567 autonomic effect Effects 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical group OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 1
- 208000027503 bloody stool Diseases 0.000 description 1
- 238000000861 blow drying Methods 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 208000035861 hematochezia Diseases 0.000 description 1
- 238000004192 high performance gel permeation chromatography Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000003483 hypokinetic effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000012844 infrared spectroscopy analysis Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000036649 mental concentration Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002899 monoamine oxidase inhibitor Substances 0.000 description 1
- 230000036651 mood Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 230000008450 motivation Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 230000009323 psychological health Effects 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 238000005173 quadrupole mass spectroscopy Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 230000008925 spontaneous activity Effects 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000012271 tenesmus Diseases 0.000 description 1
- 239000003029 tricyclic antidepressant agent Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/24—Antidepressants
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Microbiology (AREA)
- Animal Behavior & Ethology (AREA)
- Molecular Biology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nutrition Science (AREA)
- Pain & Pain Management (AREA)
- Tropical Medicine & Parasitology (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Virology (AREA)
- Psychiatry (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of food processing, and discloses a lactobacillus plantarum acidic extracellular polysaccharide and application thereof. The acidic extracellular polysaccharide is extracted from Lactobacillus plantarum (Lactobacillus sp.) DMDL9010 fermentation liquor for the first time, the acidic extracellular polysaccharide is novel in structure and uniform in components, the molecular weight is 61260Da, and the molar ratio of monosaccharide components is fucose: arabinose: galactose: glucose: mannose: fructose: galacturonic acid ═ 0.13: 0.69: 8.32: 27.57: 62.07:0.58:0.46. The purity of the lactobacillus plantarum DMDL9010 acid extracellular polysaccharide extracted by the method is as high as 99.41%. Scanning electron microscope and thermal analysis show that the polysaccharide is an irregular sheet structure, part of the polysaccharide is a rod-shaped structure with two expanded ends and a smooth surface, and the polysaccharide has certain thermal stability and potential food application value, and mouse animal experiments prove that the acidic extracellular polysaccharide has a good anti-depression effect.
Description
Technical Field
The invention belongs to the field of food processing, and particularly relates to lactobacillus plantarum acidic extracellular polysaccharide and application thereof.
Background
Depression is a psychiatric disorder characterized primarily by a marked and persistent decline in mood, a major mood disorder. Patients with depression also exhibit hypokinesia, lack of interest, and concomitant changes in cognition, physiology, and behavior, such as mental retardation, decreased mental concentration, decreased appetite, fatigue and pessimism, and even self-disability and suicidal tendencies in the critically ill. The incidence rate of depression rises with the age, and the incidence rate of 60-64-year-old women is close to 8 percent, which is a high risk group. Aging of society is also one of the risk factors for depression. Depression is taken as an epidemic disease in the 21 st century, and depression patients face a plurality of problems of lack of disease education, strong shame feeling, high misdiagnosis rate, high recurrence rate, strong side effect and the like. The life quality of the user is seriously influenced, the family happiness is good, and the social medical burden is increased.
Ulcerative Colitis (UC) has clinical manifestations mainly including mucopurulent bloody stool, diarrhea, and tenesmus, and has become a global disease with no significant difference in sex distribution and gradually younger onset age. Meanwhile, the psychological health of patients is influenced by inflammatory bowel diseases, statistics show that more than 25% of inflammatory bowel disease patients experience depression, and more than 30% of depression patients have gastrointestinal symptoms.
At present, western medicines for treating various depression mainly comprise tricyclic antidepressant drugs, monoamine oxidase inhibitors and 5-HT reuptake inhibitors, but the western medicines generally have the defects of drug resistance, large toxic and side effects and the like, so that the search for the antidepressant medicines with safety, high efficiency and small side effects is particularly important, and especially the natural antidepressant medicines becomes a research hotspot in the field.
Lactic Acid Bacteria (LAB) are widely present in the natural world, in various fermented foods, and in the intestinal tracts of higher animals, and their biological classifications belong to gram-positive bacteria and secrete Exopolysaccharides (EPS) during their growth and metabolism. The lactobacillus plantarum is one of common lactobacillus, and the lactobacillus plantarum extracellular polysaccharide belongs to prebiotics and has multiple functions of resisting oxidation free radicals, resisting tumors, regulating the immune system and the like. However, no depression research on extracellular polysaccharide of lactobacillus plantarum is reported at present.
Disclosure of Invention
Aiming at the application problem of microbial polysaccharides, the invention aims to provide a lactobacillus plantarum acidic extracellular polysaccharide and application thereof. The invention provides a preparation method and application of a natural antidepressant drug with safety, high efficiency and small side effect aiming at the defects of strong drug resistance, large toxic and side effect and the like of the existing antidepressant drugs.
The purpose of the invention is realized by the following technical scheme:
lactobacillus plantarum acid exopolysaccharide is prepared by activating Lactobacillus plantarum (Lactobacillus sp.) DMDL9010, inoculating to a fermentation culture medium, and fermenting to obtain a fermentation broth; and removing thallus, precipitating with ethanol, removing protein, and eluting with ion exchange column to obtain 0.1mol/L NaCl solution as eluent, to obtain extracellular acidic polysaccharide with molecular weight of 61260 Da.
Preferably, the monosaccharide composition of the acidic exopolysaccharide is fucose, arabinose, galactose, glucose, mannose, fructose and galacturonic acid, and the molar ratio is 0.13: 0.69: 8.32: 27.57: 62.07:0.58:0.46.
Preferably, the glycosidic bond of the acidic exopolysaccharide consists of t-Manp (1 →, t-Glcp (1 →, → 2) -Manp (1 →, → 6) -Galp (1 →, → 6) -Glcp (1 → and → 4) -Glcp (1 → composition, relative molar ratio 48.884: 4.667: 17.016: 7.650: 7.197: 14.565.
A method for preparing lactobacillus plantarum acidic exopolysaccharide, comprising the steps of:
(1) activating strains:activating lactobacillus plantarum DMDL9010 to obtain seed fermentation liquor, wherein the bacterium content of the seed fermentation liquor is 108～109CFU/mL；
(2) And (3) amplification culture: inoculating the seed fermentation liquor obtained in the step (1) into an amplification culture medium according to the volume ratio of (1-5): 100, and performing shake culture to obtain fermentation liquor;
(3) and (3) removing thalli: centrifuging the fermentation liquor obtained in the step (2), removing thallus precipitates, and reserving supernate;
(4) alcohol precipitation: adding absolute ethyl alcohol into the supernatant obtained in the step (3), standing, centrifuging to obtain a precipitate, collecting the precipitate, and dissolving the precipitate in water to obtain a crude polysaccharide solution;
(5) protein removal: adding Sevag reagent into the crude polysaccharide liquid obtained in the step (4), placing the crude polysaccharide liquid in a shaking table at room temperature, shaking and uniformly mixing the crude polysaccharide liquid and the shaking table to enable the protein to be fully adsorbed in an organic phase, centrifuging the organic phase, keeping a water phase, repeating the operation until the protein is completely removed, dialyzing the collected water phase, and freeze-drying the collected water phase to obtain crude extracellular polysaccharide;
(6) and (3) preparing the crude exopolysaccharide obtained in the step (5) into a solution of 10-30 mg/mL, eluting by using a DEAE-Cellulose 52 ion exchange column, wherein the eluent is 0.1mol/L NaCl solution, separating and purifying by using a Sephadex G-75 gel column, and then carrying out reduced pressure concentration and vacuum freeze drying to obtain the acidic exopolysaccharide freeze-dried powder.
Preferably, the culture conditions in step (2) are: the pH value is 5.8-6.2, the fermentation temperature is 32 +/-5 ℃, the inoculation amount is 7-11%, and the fermentation time is 28 +/-4 hours.
Preferably, the volume ratio of the supernatant to the absolute ethyl alcohol in the step (4) is 1: (4-6).
Preferably, the volume ratio of the crude polysaccharide liquid to the Sevag reagent in the step (5) is (4-5): 1.
Application of lactobacillus plantarum DMDL9010 acid exopolysaccharide in food.
Application of lactobacillus plantarum DMDL9010 acid exopolysaccharide in antidepressant drugs.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the acidic extracellular polysaccharide obtained by the invention is extracted from lactobacillus plantarum DMDL9010 fermentation liquor for the first time, has a new structure and uniform components, the molecular weight of the acidic extracellular polysaccharide is 61260Da, and the molar ratio of monosaccharide components is fucose (Fuc): arabinose (Ara): galactose (Gal): glucose (Glu): mannose (Man): fructose (Fru): galacturonic acid ═ 0.13: 0.69: 8.32: 27.57: 62.07:0.58:0.46.
(2) The purity of the lactobacillus plantarum DMDL9010 acid extracellular polysaccharide extracted by the method is as high as 99.41%.
(3) The structure of lactobacillus plantarum DMDL9010 acidic exopolysaccharide was analyzed by infrared, methylation and NMR and consisted of t-Manp (1 →, t-Glcp (1 →, → 2) -Manp (1 →, → 6) -Galp (1 →, → 6) -Glcp (1 → and → 4) -Glcp (1 → (relative molar ratio ═ 48.884: 4.667: 17.016: 7.650: 7.197: 14.565).
(4) Scanning electron microscopy and thermal analysis are adopted to find that the lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide shows an irregular sheet-shaped structure, a plurality of parts are rod-shaped structures with two expanded ends and smooth surfaces, and the lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide has certain thermal stability, has a decomposition amount of 68.14% at 188.8-800 ℃, and has potential practical application value in the food industry.
(5) Mouse and animal experiments prove that the lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide has a good anti-depression effect, and the effect of the low-concentration lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide group (80mg/kg, DSS +80EPS) is improved by 17.73 percent compared with that of an antidepressant fluoxetine hydrochloride.
DEAE-Cellulose 52 anion exchange column chromatography is based on the principle of ion exchange chromatography, and the matrix is composed of charged resin or Cellulose. The anion exchange matrix binds the negatively charged acidic polysaccharide, which is adsorbed onto the column and cannot be eluted by the deionized water. When the salt concentration in the eluent is increased, the acidic polysaccharide is eluted in sequence according to the different binding capacity of the negative charges in the acidic polysaccharide and the filler matrix.
And (3) thalli: lactobacillus plantarum (Lactobacillus sp.) DMDL9010 with the preservation number of CGMCC NO.5172, which is preserved in China general microbiological culture Collection center (CGMCC) at 8/19/2011, and the address is as follows: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences. The strain is disclosed in Chinese patent CN 102978134A.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention.
FIG. 1 DEAE-Cellulose 52 ion exchange column elution profile of Lactobacillus plantarum DMDL9010 acid exopolysaccharide.
FIG. 2 Sephadex G-75 gel column elution curve of Lactobacillus plantarum DMDL9010 acid exopolysaccharide.
FIG. 3 polysaccharide molecular weight standard curve.
FIG. 4 GPC high performance liquid chromatogram of Lactobacillus plantarum DMDL9010 acid exopolysaccharide.
FIG. 5 shows a high performance liquid chromatogram of the composition of acidic extracellular polysaccharide monosaccharide of Lactobacillus plantarum DMDL 9010.
FIG. 6 of Lactobacillus plantarum DMDL9010 acid exopolysaccharide1H NMR spectrum.
FIG. 7 of Lactobacillus plantarum DMDL9010 acid exopolysaccharide13C NMR spectrum.
FIG. 8 is an infrared spectrum of Lactobacillus plantarum DMDL9010 acid exopolysaccharide.
FIG. 9 scanning electron micrograph of Lactobacillus plantarum DMDL9010 acid exopolysaccharide (a: 500X, b: 2000X, c: 5000X, d: 10000X).
FIG. 10 thermal analysis graph of Lactobacillus plantarum DMDL9010 acid extracellular polysaccharide.
FIG. 11 is an open field experiment used for researching the influence of lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide on DSS-induced colitis mouse behavior disorder (A: the total movement distance of a mouse in the open field experiment; B: the movement average speed of the mouse in the open field experiment; C: the peripheral movement distance of the mouse in the open field experiment; D: the peripheral movement average speed of the mouse in the open field experiment). p<0.05,p**<0.01,p***<0.001 (compared to the DSS model group), p#<0.05,p##<0.01,p###<0.001 (compared to CK blank group)) Statistically significant differences were observed, with 95% confidence intervals.
FIG. 12 is a study of the effect of Lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide on DSS-induced colitis mouse behavioral disturbance by using a light and dark box experiment (A: total movement distance of mouse in light box; B: average movement speed of mouse in light box). p<0.05,p**<0.01,p***<0.001 (compared to the DSS model group), p#<0.05,p##<0.01,p###<0.001 (compared to the CK blank) was statistically significantly different with a 95% confidence interval.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto, and may be carried out with reference to conventional techniques for process parameters not particularly noted.
Example 1: lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide separation and molecular weight determination
The seed culture medium comprises the following components in parts by weight: 0.9 part of casein digest, 0.4 part of yeast extract, 1.8 parts of glucose, 0.15 part of triammonium citrate, 0.05 part of magnesium sulfate, 0.75 part of beef extract, 0.15 part of dipotassium hydrogen phosphate, 0.45 part of sodium acetate, 800.2 parts of tween, 0.02 part of manganese sulfate and the balance of water.
The fermentation medium formula comprises the following components in parts by weight: 0.9 part of casein digest, 0.4 part of yeast extract, 1.6 parts of glucose, 0.15 part of triammonium citrate, 0.055 part of magnesium sulfate, 0.8 part of beef extract, 0.15 part of dipotassium hydrogen phosphate, 0.45 part of sodium acetate, 800.2 parts of tween, 0.9 part of soybean protein peptide, 0.015 part of ascorbic acid, 0.25 part of manganese sulfate and the balance of water.
The Sevag reagent is obtained by mixing chloroform and n-butyl alcohol, and the volume ratio of the chloroform to the n-butyl alcohol is 5: 1.
(1) activating strains: activating lactobacillus plantarum DMDL9010 to obtain seed fermentation liquid with the bacterium content of 108CFU/mL；
(2) And (3) amplification culture: inoculating the seed fermentation liquor into a fermentation culture medium according to the volume ratio of 2:100, and culturing on a shaking table to obtain lactobacillus plantarum DMDL9010 fermentation liquor; the culture conditions were: the initial pH of the fermentation medium is 6.0, the fermentation temperature is 32 ℃, the inoculation amount is 9%, and the fermentation time is 28 h;
(3) and (3) removing thalli: centrifuging the fermentation liquor of Lactobacillus plantarum DMDL9010 (6000g,10min), removing thallus precipitate, and retaining supernatant;
(4) alcohol precipitation: adding anhydrous ethanol (supernatant: anhydrous ethanol 1: 4, v/v), standing, centrifuging to obtain precipitate, collecting precipitate, and dissolving in water to obtain crude polysaccharide solution;
(5) protein removal: adding a Sevag reagent (crude polysaccharide solution: Sevag reagent is 4: 1, v/v) into the crude polysaccharide solution obtained in the step (4), placing the crude polysaccharide solution in a shaking table at room temperature, shaking (220rpm/min,10min), uniformly mixing to enable the protein to be fully adsorbed in an organic phase, then centrifuging, retaining an aqueous phase, repeating the operation until the protein is completely removed, and dialyzing and freeze-drying the collected aqueous phase for later use.
(6) Separating and purifying a DEAE-Cellulose 52 ion exchange column and a Sephadex G-75 gel column: preparing the exopolysaccharide obtained in the step (5) into a10 mg/mL solution, loading 30mL of the solution into a DEAE-Cellulose 52 ion exchange column, sequentially carrying out gradient elution by using deionized water, 0.1mol/L NaCl solution, 0.3mol/L NaCl solution and 0.5mol/L NaCl solution at the flow rate of 1.0mL/min, collecting 10mL of the solution in each tube, collecting 20 tubes of each component, and tracking and detecting the content of the polysaccharide by adopting a phenol-sulfuric acid method, wherein four components including EPS-LP1, EPS-LP2, EPS-LP3, EPS-LP4 and the like are sequentially obtained as shown in figure 1. Collecting a component (EPS-LP2) eluted by 0.1mol/L NaCl solution, concentrating under reduced pressure, dialyzing for 1-3 d with deionized water, collecting dialysate, and carrying out vacuum freeze drying to obtain dry crude acid extracellular polysaccharide. Preparing the dried crude acid extracellular polysaccharide into a solution of 30mg/mL, loading 30mL of the solution to a Sephadex G-75 gel column, eluting with deionized water at a flow rate of 0.2mL/min, collecting 2mL of the solution in each tube, and detecting the polysaccharide content by tracking with a phenol-sulfuric acid method, as shown in FIG. 2. Collecting polysaccharide solutions in different tubes, and performing reduced pressure concentration and vacuum freeze drying to obtain the acid extracellular polysaccharide freeze-dried powder.
And (6) collecting the EPS-LP1 which is neutral exopolysaccharide through a DEAE-Cellulose 52 ion exchange column. EPS-LP3 and EPS-LP4 were darker in color, and it was speculated that EPS-LP3 and EPS-LP4 contained a large amount of pigment, while EPS-LP3 and EPS-LP4 contained less polysaccharide than EPS-LP1, and therefore no follow-up studies were performed.
(7) And (3) measuring the molecular weight: the uniformity and molecular weight of the Lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide (EPS-LP2) molecular weight are determined by high-phase liquid chromatography high-performance gel permeation chromatography (Waters1525 gel chromatograph). The chromatographic column is TSK G5000PWXL(6 μm, 7.8X 300mm) and TSK G3000PWXL(6 μm, 7.8X 300mm) was used in series with a differential Refractometer (RID) of Waters 2414, a column temperature of 35 ℃, a sample size of 10. mu.L, a mobile phase of 0.02mol/L of a dipotassium hydrogen phosphate buffer solution, and a flow rate of 0.6 mL/min. Respectively filtering the dextran standard substances with different molecular weights through 0.45 mu m filter membranes, and then loading the dextran standard substances on a machine, and recording the retention time. The retention time is plotted as the abscissa and the logarithm of the dextran molecular weight is plotted as the ordinate to form a standard curve. The acidic polysaccharide was tested for the time to peak in the same way, according to the standard curve: -0.2582x +10.14, R2The molecular mass of lactobacillus plantarum DMDL9010 acidic exopolysaccharide (EPS-LP2) was calculated at 0.9974(y is log (mol. weight), x is retention time (min), fig. 3).
The purity of the lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide separated and purified in the embodiment 1 of the invention is as high as 99.41%. As can be seen from fig. 4, the molecular weight of the extracellular acidic polysaccharide in lactobacillus plantarum DMDL9010 is 61260Da, which indicates that the extracellular acidic polysaccharide in lactobacillus plantarum DMDL9010 is homogeneous and can be further used for measuring the monosaccharide composition.
Example 2: lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide monosaccharide composition analysis
Preparing a standard substance: standards and reagents were prepared as shown in table 1.
TABLE 1 Standard and reagent information
Adding 8mL of sterile water into an EP tube, sequentially adding 100mg of each of fucose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid and glucuronic acid, dissolving, and diluting to 10mL to obtain a mother liquor of 10 mg/mL. The solution was diluted 100-fold to prepare a 100. mu.g/mL working solution, and the solution was diluted in the following gradient and placed in a 1.5mL EP tube. The gradient information (. mu.g/mL) for each monosaccharide mixture is shown in Table 2.
TABLE 2 monosaccharide Standard gradient concentration information (. mu.g/mL)
Sample pretreatment: the lactobacillus plantarum DMDL9010 acidic exopolysaccharide obtained in step (6) of example 1 was treated by the following specific steps: weighing polysaccharide samples 5mg each in a clean chromatographic bottle, adding TFA acid solution, heating at 121 ℃ for 2h, and blowing to dry by introducing nitrogen. Adding methanol, cleaning, blow-drying, and repeating for 2-3 times. Adding sterile water to dissolve, and transferring into a chromatographic bottle to be tested.
Extracting a liquid sample: taking a proper amount of supernatant, and blowing to dry by introducing nitrogen. The subsequent steps are consistent with solid sample extraction.
Analyzing and detecting: the chromatographic system used was a Thermo ICS5000+ ion chromatographic system (ICS5000+, (Thermo Fisher Scientific, USA) using DionexTMCarboPacTMPA10 (250X 4.0mm, 10 μm) liquid chromatography column, sample size 20 μ L. Mobile phase A (H)2O), mobile phase B (100mol/L NaOH), the column temperature is 30 ℃, and the monosaccharide components are analyzed and detected by an electrochemical detector. Specific gradients and data for the mobile phase are shown in the table below.
TABLE 3 gradient of mobile phase
TABLE 4 monosaccharide contents and molar ratios
As can be seen from fig. 5 and table 4, the lactobacillus plantarum DMDL9010 acidic exopolysaccharide has a composition molar ratio of fucose (Fuc): arabinose (Ara): galactose (Gal): glucose (Glu): mannose (Man): fructose (Fru): galacturonic acid ═ 0.13: 0.69: 8.32: 27.57: 62.07:0.58:0.46, wherein the uronic acid content is 0.46%, again demonstrating that the exopolysaccharide obtained by extraction is an acidic exopolysaccharide.
Example 3: lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide methylation and nuclear magnetic analysis
(1) Methylation and GC-MS analysis
Standards and reagents were prepared as shown in table 5.
TABLE 5 Standard and reagent information
Derivatization of polysaccharide samples: 10mg of the purified sample was weighed, dissolved in 1mL of deionized water, and reacted for 2 hours with 1mL of 100mg/mL carbodiimide. Then 1mL of 2mol/L imidazole is added, after the imidazole is divided into two parts on average, 1mL of 30mg/mL NaBH is added4And the same volume and concentration of NaBD4After 3 hours, 100. mu.L of glacial acetic acid was added to terminate the reaction. The samples were dialyzed for 48h, after which time the samples were freeze-dried and methylated. The lyophilized sample was dissolved in 500. mu.L of DMSO, incubated with 1mg of NaOH for 30min, and reacted with 50. mu.L of iodomethane solution for 1 h. 1mL of water and 2mL of methylene chloride were added, mixed and mixed, and the aqueous phase was centrifuged off. Washing with water for 3 times, sucking lower layer dichloromethane phase, evaporating to dryness, adding 100 μ L2 mol/L TFA, reacting at 121 deg.C for 90min, and evaporating to dryness at 30 deg.C; adding 50 mu L of 2mol/L ammonia water and 50 mu L of 1mol/L NaBD4Mixing and reacting for 2.5h at room temperature. Adding 20 mu L of acetic acid to terminate the reaction, blowing dry with nitrogen, washing twice with 250 mu L of methanol, blowing dry with nitrogen, adding 250 mu L of acetic anhydride, mixing uniformly by vortex, and reacting for 2.5h at 100 ℃. Adding 1mL of water, standing for 10min, adding 500 μ L of dichloromethane, vortex, mixing, centrifuging, and removing the water phase. After repeating the washing with water 3 times, the methylene chloride phase of the lower layer was taken off and preparedAnd (4) machine detection.
Gas chromatography-mass spectrometry analysis: an Agilent gas chromatography system (Agilent 7890A; Agilent Technologies, USA) is adopted, according to the properties of the compound, the sample injection amount is 1 mu L, the split ratio is 10:1, and the carrier gas is high-purity helium; keeping the initial temperature of the column incubator at 140 ℃ for 2.0min, and raising the temperature to 230 ℃ at the speed of 3 ℃/min and keeping the temperature for 3 min.
A quadrupole mass spectrometry detection system (Agilent 5977B; Agilent Technologies, USA) from Aiglent corporation, USA was used, equipped with an electron bombardment ion source (EI) and MassHunter workstation. The analytes are detected in a full SCAN (SCAN) mode using an electron impact ion source (EI) with a mass SCAN range (m/z) of 30-600.
Methylation analysis of lactobacillus plantarum DMDL9010 acidic exopolysaccharide is shown in table 6.
TABLE 6 methylation and GC-MS analysis of extracellular acidic polysaccharides
By comparison with the PMAA database, the 6 derivatives were 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl mannitol (1, 5-di-O-acetyl-2,3,4,6-tetra-O-methyl mannitol), 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol (1, 5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol), 1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl mannitol (1,2, 5-tri-O-acetyl-3,4,6-tri-O-methyl mannitol), 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl galactitol (1,5, 6-tri-O-acetyl-2,3,4-tri-O-methyl galactitol), 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl glucitol (1,5, 6-tri-O-acetyl-2,3,4-tri-O-methyl glucitol), 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol (1,4, 5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol).
As can be seen from Table 6, the linkage pattern of the glycosidic linkages of the acid exopolysaccharide of Lactobacillus plantarum DMDL9010 includes t-Manp, t-Glcp,2-Manp,6-Galp,6-Glcp,4-Glcp, and their relative molar ratio is 48.884: 4.667: 17.016: 7.650: 7.197: 14.565. which contain only the terminal units (t-Manp and t-Glcp) and no branch points. The branching Degree (DB) value of lactobacillus plantarum DMDL9010 acidic exopolysaccharide was calculated according to the equation DB ═ (NT + NB)/(NT + NB + NL) of 53.55%, where NT refers to the terminal residues t-Manp (1 → and t-Glcp (1 →, NB refers to branching residues (not contained), NL refers to the number of linear residues → 2) -Manp (1 →, → 6) -Galp (1 →, → 6) -Glcp (1 → and → 4) -Glcp (1 → a).
(2) Nuclear magnetic resonance analysis
The Lactobacillus plantarum DMDL9010 acidic exopolysaccharide prepared in the step (6) of example 1, 5mg, was respectively weighed and dissolved in 0.6mL of heavy water (D)2O), repeatedly freeze-drying and redissolving, adding 0.6mL of heavy water into a nuclear magnetic tube, and performing on a Bruker AV-600 nuclear magnetic resonance instrument1H NMR and13c NMR measurement.
Method for preparing lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide1The H NMR spectrum is shown in FIG. 6. Method for preparing lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide1The H NMR spectrum shows that 8 anomeric proton signals exist, and the chemical shifts are respectively 4.82, 4.83, 4.92, 4.96, 5.04, 5.21, 5.32 and 5.36ppm, which indicates that the acid extracellular polysaccharide chain of the lactobacillus plantarum DMDL9010 possibly comprises 8 sugar residues. As in fig. 713C NMR spectrum shows that the lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide has 8 anomeric carbon signals, which indicates that the lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide contains eight types of glycosidic bonds, and the chemical shifts are 107.91, 107.47, 103.00, 102.63, 102.16, 100.54, 99.34 and 98.19ppm respectively; the literature relating to binding methylation to hydrogen spectroscopy suggests that it contains, inter alia, α -D-mannose residues, 1, 6-linked α -D-glucose residues, 1, 4-linked β -D-galactose residues, 1,3, 6-linked α -D galactose residues, 1, 6-linked α -D galactose residues, and 1,3, 4-linked α -D galactose residues.
Example 4: infrared spectroscopic analysis of lactobacillus plantarum DMDL9010 acid extracellular polysaccharide
Respectively weighing 10mg of the lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide prepared in the step (6) in the example 1 by adopting a potassium bromide tabletting method, adding 100mg of KBr powder, pressing into uniform slices by using a tablet press, and performing 4000-500cm Fourier transform infrared spectroscopy by adopting a Bruker VERTEX 33 type Fourier transform infrared spectrometer-1Infrared spectrum scanning is carried out in the range, and a spectrogram is recorded. FIG. 8It can be seen that the length of the groove is 3417.82cm-1The broad stretching peak at (a) belongs to the hydroxyl stretching vibration. At 2928.91cm-1The peak at (A) is an aliphatic CH2The asymmetric C and H stretching vibration of the group indicates that organic matters such as sugar exist. Peak value of 1644.32cm-1Indicating the presence of mannose or galactose. 1374.28cm-1The vibration of (b) may be related to the symmetrical stretching of the carboxyl group. 1200-1000 cm-1The absorption peaks in the region may be caused by C-O-H and C-O-C stretching vibrations.
Example 5: lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide apparent morphology and thermal analysis
(1) Scanning electron microscope observation lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide apparent form
The scanning electron microscope is a commonly used method for observing the appearance and judging the types of the polysaccharides at present, has the advantages of simple operation, intuitive result and high resolution, and is widely applied to food science, chemistry, materials and biology. And (3) coating a small amount of the sufficiently dried lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide component obtained in the step (6) in the example 1 on conductive gel, spraying gold, and observing the surface morphology of the conductive gel by using a scanning electron microscope. As can be seen from FIG. 9, the Lactobacillus plantarum DMDL9010 acid exopolysaccharide sample has various structures, and some of the samples are observed to be irregular sheet-shaped structures with smooth surfaces, some of the structures are adhered to each other, and some of the structures are rod-shaped structures with two expanded ends and smooth surfaces as the magnification is increased.
(2) Thermal analysis
Thermogravimetric analysis (TGA) was used to start at 25 ℃ and heat up to 800 ℃ at a rate of 10 ℃/min, with the flow rate of helium set at 20 mL/min. As can be seen from FIG. 10, at 188.8 ℃, the weight loss of Lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide is 9.73%, mainly due to water evaporation, while at 188.8-800 ℃, the decomposition amount of Lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide is 68.14%, and the residual mass is 22.13%, which indicates that Lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide has layered thermal stability and has potential practical application value in the food industry.
Example 6: application of lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide to treatment of colitis depressive-like mice
C57BL/6N mice, male, 4-5 weeks old, were collected from Zhejiang Uvintland laboratory animal science and technology, Inc. All mice were acclimatized (23-25 ℃ and 12h light/dark cycle) with standard feed for 7 days. The mouse colitis model was activated with 4% (w/v) DSS. 40 mice were randomly divided into 5 groups (n-8/group): on day 1-7, the control group (CK) is perfused with stomach normal saline and stomach distilled water; (2) DSS group mice were given oral saline 4% DSS solution; (3) mice in the group of DSS + EPS (DSS +80EPS) were orally administered 80mg/kg lactobacillus plantarum DMDL9010 acidic exopolysaccharide, given 4% DSS solution; (4) mice in the group of DSS + EPS (DSS +160EPS) were orally administered 160mg/kg lactobacillus plantarum DMDL9010 acidic exopolysaccharide, given 4% DSS solution; (5) mice in the DSS + Flu group (DSS + Flu) were orally administered with 4.89mg/kg of fluoxetine hydrochloride (Flu) solution, and were given 4% DSS solution; and (5) treating the mice with distilled water on 8-10 days. All mice were weighed daily and observed. The Open Field Test (OFT) is performed in the morning on day 8, and the Light and Dark box Test (LDT) is performed in the morning on days 9-10. On day 10, all mice were euthanized.
The field test (OFT) is commonly used for the exploration of defined mice. A50 cm by 50cm movement monitoring box is evenly divided into 16 sections. The behavior of each mouse was monitored by a computer video tracking system (5 min). In the light and dark box test (LDT), each mouse was placed in the center of the apparatus (40 cm x 30 cm x 35 cm) for 10 minutes, containing two rooms of the same extent, one bright and one dark. Spontaneous movement of the mouse was monitored by a computer video tracking system.
In the open field experiment, the behavior such as the movement track, the distance, the average speed and the like of the mouse in the total open field, the central area and the peripheral area can be analyzed, so that the autonomous activity condition and the exploration desire of the mouse can be reflected. The remarkable features of depression patients are decreased interest and lack of motivation, which results in decreased voluntary activity and decreased search motivation, so open field experiments are often used in the assessment of behavioral disorders such as depression and anxiety. The more disorderly and disorderly the total movement distance of the mouse in the whole open field area, the faster the average speed, the more active the spontaneous activity of the mouse is; the distance and the average speed of the mouse in the peripheral area close to the wall in the open field can reflect the exploration desire of the mouse, the stronger the exploration desire is, the more frequently the mouse appears in the central area, and the total distance of the mouse appearing in the peripheral area is reduced.
As shown in fig. 11, the total course and average speed of the mice in the DSS model group moving throughout the open field were significantly reduced compared to the CK group, demonstrating that DSS significantly reduces the autonomic activity and search desire of the mice. Compared with the DSS group, the DSS + Flu group mice showed an increase in the overall course of movement and mean speed in the entire open field and central area, but the effect was not significant. The lactobacillus plantarum DMDL9010 acidic exopolysaccharide can effectively improve the autonomous activity and the exploration desire of a mouse, the treatment effect and the polysaccharide concentration have no obvious influence, but the treatment effect of the lactobacillus plantarum DMDL9010 acidic exopolysaccharide with low concentration (80mg/kg) is better than that of the lactobacillus plantarum DMDL9010 with high concentration (160mg/kg), and the effects of the lactobacillus plantarum DMDL9010 acidic exopolysaccharide and the polysaccharide with low concentration on the improvement of the total movement distance and the average speed of the whole open field and the central area are very obvious. As shown in Table 7, the autonomous activity (based on the total route) of the low-concentration Lactobacillus plantarum DMDL9010 acid exopolysaccharide group (80mg/kg, DSS +80EPS) mice can be restored to 52.35% of that of the CK group, and the effect of the low-concentration Lactobacillus plantarum DMDL9010 acid exopolysaccharide group is improved by 17.73% compared with that of the antidepressant drug fluoxetine hydrochloride.
TABLE 7 mean speed of movement and Total course of the open field laboratory mice
p*<0.05,p**<0.01,p***<0.001 (compared to the DSS model group), p#<0.05,p##<0.01,p###<0.001 (compared to the CK blank) was statistically significantly different with a 95% confidence interval.
In order to further explore the autonomous activities and the exploration desire of the mice, a bright-dark shuttle experiment is carried out. The results of the experiment after analyzing the movement of the mouse in the light and dark box are shown in fig. 12. The distance and average speed of the mice in the bright box were significantly reduced in the DSS group (p <0.001), which again demonstrated that DSS caused a reduction in the mice's voluntary activity and desire to explore, and lactobacillus plantarum DMDL9010 acidic exopolysaccharides (DSS +80EPS group and DSS +160EPS group) were able to significantly increase the distance and average speed of the mice in the bright box (p <0.05), second only to the CK group and superior to the effect of the antidepressant drug group (DSS + Flu).
By analyzing through an open field experiment and a bright-dark shuttle experiment, compared with the low-concentration lactobacillus plantarum DMDL9010 acidic exopolysaccharide (80mg/kg, DSS +80EPS), the action effect is more stable, and the anti-depression effect is obvious (p is less than 0.05).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (9)
1. The Lactobacillus plantarum acidic exopolysaccharide is characterized in that Lactobacillus plantarum (Lactobacillus sp) DMDL9010 is activated and inoculated to a fermentation medium for expanded fermentation culture to obtain fermentation liquor; and removing thallus, precipitating with ethanol, removing protein, and eluting with ion exchange column to obtain 0.1mol/L NaCl solution as eluent, to obtain extracellular acidic polysaccharide with molecular weight of 61260 Da.
2. The plant lactobacillus acidic exopolysaccharide according to claim 1, wherein the monosaccharide composition of the acidic exopolysaccharide is fucose, arabinose, galactose, glucose, mannose, fructose and galacturonic acid, in a molar ratio of 0.13: 0.69: 8.32: 27.57: 62.07:0.58:0.46.
3. The plant lactobacillus acidic exopolysaccharide according to claim 1, wherein the glycosidic bond of the acidic exopolysaccharide consists of t-Manp (1 →, t-Glcp (1 →, → 2) -Manp (1 →, → 6) -Galp (1 →, → 6) -Glcp (1 → and → 4) -Glcp (1 → relative molar ratio 48.884: 4.667: 17.016: 7.650: 7.197: 14.565.
4. A method for preparing the extracellular polysaccharide of lactobacillus plantarum of claims 1-3, comprising the steps of:
(1) activating strains: activating lactobacillus plantarum DMDL9010 to obtain seed fermentation liquor, wherein the bacterium content of the seed fermentation liquor is 108～109CFU/mL；
(2) And (3) amplification culture: inoculating the seed fermentation liquor obtained in the step (1) into an amplification culture medium according to the volume ratio of (1-5): 100, and performing shake culture to obtain fermentation liquor;
(3) and (3) removing thalli: centrifuging the fermentation liquor obtained in the step (2), removing thallus precipitates, and reserving supernate;
(4) alcohol precipitation: adding absolute ethyl alcohol into the supernatant obtained in the step (3), standing, centrifuging to obtain a precipitate, collecting the precipitate, and dissolving the precipitate in water to obtain a crude polysaccharide solution;
(5) protein removal: adding Sevag reagent into the crude polysaccharide liquid obtained in the step (4), placing the crude polysaccharide liquid in a shaking table at room temperature, shaking and uniformly mixing the crude polysaccharide liquid and the shaking table to enable the protein to be fully adsorbed in an organic phase, then centrifuging the organic phase, retaining the water phase, repeating the operation until the protein is completely removed, dialyzing the collected water phase, and freeze-drying the water phase to obtain crude extracellular polysaccharide;
(6) and (3) preparing the crude exopolysaccharide obtained in the step (5) into a solution of 10-30 mg/mL, eluting by using a DEAE-Cellulose 52 ion exchange column, wherein the eluent is 0.1mol/L NaCl solution, separating and purifying by using a Sephadex G-75 gel column, concentrating under reduced pressure, and freeze-drying under vacuum to obtain the acidic exopolysaccharide freeze-dried powder.
5. The method according to claim 4, wherein the culture conditions of step (2) are: the pH value is 5.8-6.2, the fermentation temperature is 32 +/-5 ℃, the inoculation amount is 7-11%, and the fermentation time is 28 +/-4 hours.
6. The method according to claim 4, wherein the volume ratio of the supernatant to the absolute ethyl alcohol in the step (4) is 1: (4-6).
7. The method as claimed in claim 4, wherein the volume ratio of the crude polysaccharide solution to the Sevag reagent in the step (5) is (4-5): 1.
8. The application of the lactobacillus plantarum DMDL9010 acid exopolysaccharide disclosed by claim 1-4 in food.
9. The Lactobacillus plantarum DMDL9010 acid exopolysaccharide disclosed in claim 1-4 for use in antidepressant drugs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110938883.0A CN113621665B (en) | 2021-08-16 | 2021-08-16 | Lactobacillus plantarum acidic extracellular polysaccharide and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110938883.0A CN113621665B (en) | 2021-08-16 | 2021-08-16 | Lactobacillus plantarum acidic extracellular polysaccharide and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113621665A true CN113621665A (en) | 2021-11-09 |
| CN113621665B CN113621665B (en) | 2023-06-20 |
Family
ID=78385811
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110938883.0A Active CN113621665B (en) | 2021-08-16 | 2021-08-16 | Lactobacillus plantarum acidic extracellular polysaccharide and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113621665B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114561436A (en) * | 2022-02-28 | 2022-05-31 | 西北农林科技大学 | Preparation method and application of lactobacillus plantarum exopolysaccharide |
| WO2023019987A1 (en) * | 2021-08-16 | 2023-02-23 | 华南理工大学 | Neutral exopolysaccharide of lactobacillus plantarum and use thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102702372A (en) * | 2010-10-28 | 2012-10-03 | 内蒙古蒙牛乳业（集团）股份有限公司 | Streptococcus thermophilus extracellular polysaccharide and preparation and detection method thereof |
| CN102978134A (en) * | 2012-11-21 | 2013-03-20 | 华南理工大学 | Lactobacillus and method for producing D-lactic acid by fermenting using lactobacillus |
| US20140322273A1 (en) * | 2011-12-06 | 2014-10-30 | Bright Dairy & Food Co., Ltd. | Strain of exopolysaccharide-secreting lactobacillus plantarum and application thereof |
| HUP1500332A2 (en) * | 2015-07-16 | 2017-01-30 | Feher Janos Dr | Compositions for maintaining and restoring microbiota-host symbiosis |
| WO2018225557A1 (en) * | 2017-06-09 | 2018-12-13 | 旭興産株式会社 | Extracellular polysaccharide of lactic acid bacteria and use thereof |
| CN112662717A (en) * | 2021-01-28 | 2021-04-16 | 华南理工大学 | Lactobacillus rhamnosus exopolysaccharide and preparation method and application thereof |
-
2021
- 2021-08-16 CN CN202110938883.0A patent/CN113621665B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102702372A (en) * | 2010-10-28 | 2012-10-03 | 内蒙古蒙牛乳业（集团）股份有限公司 | Streptococcus thermophilus extracellular polysaccharide and preparation and detection method thereof |
| US20140322273A1 (en) * | 2011-12-06 | 2014-10-30 | Bright Dairy & Food Co., Ltd. | Strain of exopolysaccharide-secreting lactobacillus plantarum and application thereof |
| CN102978134A (en) * | 2012-11-21 | 2013-03-20 | 华南理工大学 | Lactobacillus and method for producing D-lactic acid by fermenting using lactobacillus |
| HUP1500332A2 (en) * | 2015-07-16 | 2017-01-30 | Feher Janos Dr | Compositions for maintaining and restoring microbiota-host symbiosis |
| WO2018225557A1 (en) * | 2017-06-09 | 2018-12-13 | 旭興産株式会社 | Extracellular polysaccharide of lactic acid bacteria and use thereof |
| CN112662717A (en) * | 2021-01-28 | 2021-04-16 | 华南理工大学 | Lactobacillus rhamnosus exopolysaccharide and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 曹永强等: "植物乳杆菌胞外多糖的分离纯化及其乳化特性", 食品科学 * |
| 陈绮等: "植物乳杆菌XN1904E产胞外多糖发酵条件优化", 乳业科学与技术 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023019987A1 (en) * | 2021-08-16 | 2023-02-23 | 华南理工大学 | Neutral exopolysaccharide of lactobacillus plantarum and use thereof |
| CN114561436A (en) * | 2022-02-28 | 2022-05-31 | 西北农林科技大学 | Preparation method and application of lactobacillus plantarum exopolysaccharide |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113621665B (en) | 2023-06-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11111317B2 (en) | Cordyceps militaris medium polysaccharide, method for separating and purifying same, and use thereof | |
| CN111154676B (en) | Lactobacillus rhamnosus exopolysaccharide, preparation method thereof and bacteria used thereby | |
| CN109750070B (en) | Functional mulberry leaf oligosaccharide and preparation method and application thereof | |
| CN113621665B (en) | Lactobacillus plantarum acidic extracellular polysaccharide and application thereof | |
| CN113564069B (en) | Bifidobacterium longum, extracellular polysaccharide of bifidobacterium longum, and extraction method and application thereof | |
| CN110256593B (en) | Stropharia rugosoannulata polysaccharide and preparation method and application thereof | |
| CN112662717A (en) | Lactobacillus rhamnosus exopolysaccharide and preparation method and application thereof | |
| CN111363056B (en) | Rhodopseudomonas palustris exopolysaccharide and preparation method and application thereof | |
| CN113698503B (en) | Lactobacillus plantarum neutral exopolysaccharide and application thereof | |
| CN113831419B (en) | Armillariella tabescens mycelium polysaccharide and its preparation method and bacteriostatic use | |
| CN113265337B (en) | Marine aspergillus versicolor and isolated culture method and application thereof | |
| CN113896807A (en) | Fresh rehmannia root polysaccharide and preparation method and application thereof | |
| CN113861303B (en) | Extracellular polysaccharide separated from Lactobacillus delbrueckii and Streptococcus thermophilus fermented yogurt and application thereof | |
| AU2014378873A1 (en) | Extracellular polysaccharide with immunomodulatory effect and preparation method and use thereof | |
| CN113480672B (en) | Exopolysaccharide of bacillus and application thereof | |
| CN107937318B (en) | Bacillus subtilis MXT-1 for degrading wheat pentosan and application thereof | |
| CN117106666B (en) | Pediococcus pentosaceus LL-07, pediococcus pentosaceus LL-07 extracellular polysaccharide, and production method and application thereof | |
| CN113801798B (en) | Acidocella adephagia strain A50, extracellular polysaccharide produced by same and application thereof | |
| CN113151071B (en) | Bacillus belgii and application thereof | |
| CN114409824B (en) | Mucor exopolysaccharide and preparation method and application thereof | |
| CN116622001A (en) | Century sugarcane brown sugar polysaccharide, and preparation method, identification method and application thereof | |
| CN113372463B (en) | Method for extracting probiotic functional sugar from distilled rice wine distillation residual liquid | |
| CN117143260A (en) | Camphor tree seed kernel polysaccharide and preparation method and application thereof | |
| CN114561436A (en) | Preparation method and application of lactobacillus plantarum exopolysaccharide | |
| CN116555096A (en) | Paramycolatopsis Diels strain and application thereof |
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 |