CN114717220A - Lactobacillus reuteri microcapsule and preparation method thereof - Google Patents
Lactobacillus reuteri microcapsule and preparation method thereof Download PDFInfo
- Publication number
- CN114717220A CN114717220A CN202110639675.0A CN202110639675A CN114717220A CN 114717220 A CN114717220 A CN 114717220A CN 202110639675 A CN202110639675 A CN 202110639675A CN 114717220 A CN114717220 A CN 114717220A
- Authority
- CN
- China
- Prior art keywords
- lactobacillus reuteri
- microcapsule
- vhprobi
- skin
- group
- 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
- 241000186604 Lactobacillus reuteri Species 0.000 title claims abstract description 107
- 229940001882 lactobacillus reuteri Drugs 0.000 title claims abstract description 107
- 239000003094 microcapsule Substances 0.000 title claims abstract description 55
- 238000002360 preparation method Methods 0.000 title claims abstract description 29
- 239000000463 material Substances 0.000 claims abstract description 25
- 229930006000 Sucrose Natural products 0.000 claims abstract description 18
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 18
- 239000005720 sucrose Substances 0.000 claims abstract description 18
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000004375 Dextrin Substances 0.000 claims abstract description 15
- 229920001353 Dextrin Polymers 0.000 claims abstract description 15
- 108010010803 Gelatin Proteins 0.000 claims abstract description 15
- 239000004368 Modified starch Substances 0.000 claims abstract description 15
- 229920000881 Modified starch Polymers 0.000 claims abstract description 15
- 235000019425 dextrin Nutrition 0.000 claims abstract description 15
- 239000008273 gelatin Substances 0.000 claims abstract description 15
- 229920000159 gelatin Polymers 0.000 claims abstract description 15
- 235000019322 gelatine Nutrition 0.000 claims abstract description 15
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 15
- 235000019426 modified starch Nutrition 0.000 claims abstract description 15
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 15
- 239000000661 sodium alginate Substances 0.000 claims abstract description 15
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 15
- 239000011162 core material Substances 0.000 claims abstract description 6
- 238000004321 preservation Methods 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 37
- 230000001580 bacterial effect Effects 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 17
- 239000011259 mixed solution Substances 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 10
- 238000004108 freeze drying Methods 0.000 claims description 10
- 238000010438 heat treatment Methods 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 9
- 239000010802 sludge Substances 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 4
- 230000000968 intestinal effect Effects 0.000 abstract description 16
- 235000011389 fruit/vegetable juice Nutrition 0.000 abstract description 15
- 210000004051 gastric juice Anatomy 0.000 abstract description 14
- 230000009759 skin aging Effects 0.000 abstract description 8
- 230000003712 anti-aging effect Effects 0.000 abstract description 6
- 230000008859 change Effects 0.000 abstract description 5
- 235000013305 food Nutrition 0.000 abstract description 4
- 235000013376 functional food Nutrition 0.000 abstract description 4
- 229960001231 choline Drugs 0.000 abstract description 3
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 abstract description 3
- 239000002537 cosmetic Substances 0.000 abstract description 3
- 210000004211 gastric acid Anatomy 0.000 abstract description 3
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 241000699670 Mus sp. Species 0.000 description 52
- 210000003491 skin Anatomy 0.000 description 46
- 241000894006 Bacteria Species 0.000 description 26
- 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 21
- 239000006041 probiotic Substances 0.000 description 21
- 235000018291 probiotics Nutrition 0.000 description 21
- 238000003304 gavage Methods 0.000 description 20
- 230000002829 reductive effect Effects 0.000 description 19
- 239000000243 solution Substances 0.000 description 19
- 210000004027 cell Anatomy 0.000 description 18
- 239000001963 growth medium Substances 0.000 description 18
- 239000007788 liquid Substances 0.000 description 18
- 239000000523 sample Substances 0.000 description 18
- 238000000855 fermentation Methods 0.000 description 17
- 230000004151 fermentation Effects 0.000 description 17
- 238000012360 testing method Methods 0.000 description 15
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 13
- 229960002591 hydroxyproline Drugs 0.000 description 13
- 230000000529 probiotic effect Effects 0.000 description 13
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 13
- 102000008186 Collagen Human genes 0.000 description 12
- 108010035532 Collagen Proteins 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 229920001436 collagen Polymers 0.000 description 12
- 230000000694 effects Effects 0.000 description 11
- 210000004209 hair Anatomy 0.000 description 11
- 235000012000 cholesterol Nutrition 0.000 description 10
- 239000000835 fiber Substances 0.000 description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 8
- 239000001888 Peptone Substances 0.000 description 8
- 108010080698 Peptones Proteins 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000011248 coating agent Substances 0.000 description 8
- 238000000576 coating method Methods 0.000 description 8
- 210000004207 dermis Anatomy 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 210000003780 hair follicle Anatomy 0.000 description 8
- 238000011081 inoculation Methods 0.000 description 8
- 235000019319 peptone Nutrition 0.000 description 8
- 238000012258 culturing Methods 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 238000005303 weighing Methods 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 6
- 230000032683 aging Effects 0.000 description 6
- 210000004969 inflammatory cell Anatomy 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 210000002435 tendon Anatomy 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 241000186660 Lactobacillus Species 0.000 description 5
- 239000008186 active pharmaceutical agent Substances 0.000 description 5
- 230000003115 biocidal effect Effects 0.000 description 5
- 210000002615 epidermis Anatomy 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 239000002932 luster Substances 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 108010019160 Pancreatin Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 230000033228 biological regulation Effects 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 230000035617 depilation Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 210000002950 fibroblast Anatomy 0.000 description 4
- 230000002496 gastric effect Effects 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 229940039696 lactobacillus Drugs 0.000 description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 4
- 235000019341 magnesium sulphate Nutrition 0.000 description 4
- 229940099596 manganese sulfate Drugs 0.000 description 4
- 235000007079 manganese sulphate Nutrition 0.000 description 4
- 239000011702 manganese sulphate Substances 0.000 description 4
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 229940055695 pancreatin Drugs 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000001953 sensory effect Effects 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- 244000068988 Glycine max Species 0.000 description 3
- 235000010469 Glycine max Nutrition 0.000 description 3
- 102000057297 Pepsin A Human genes 0.000 description 3
- 108090000284 Pepsin A Proteins 0.000 description 3
- 206010048669 Terminal state Diseases 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 210000000601 blood cell Anatomy 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 230000003859 lipid peroxidation Effects 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- 229940111202 pepsin Drugs 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000001732 sebaceous gland Anatomy 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 238000009423 ventilation Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 201000004384 Alopecia Diseases 0.000 description 2
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 2
- 108010006464 Hemolysin Proteins Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 241000186840 Lactobacillus fermentum Species 0.000 description 2
- 241000186869 Lactobacillus salivarius Species 0.000 description 2
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 2
- 230000002292 Radical scavenging effect Effects 0.000 description 2
- 241000196252 Ulva Species 0.000 description 2
- 231100000360 alopecia Toxicity 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000003833 bile salt Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 230000002500 effect on skin Effects 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 239000003228 hemolysin Substances 0.000 description 2
- 230000002949 hemolytic effect Effects 0.000 description 2
- 238000010562 histological examination Methods 0.000 description 2
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 229940012969 lactobacillus fermentum Drugs 0.000 description 2
- 229940118019 malondialdehyde Drugs 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 210000004877 mucosa Anatomy 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Inorganic materials [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 230000036559 skin health Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- OCZVHBZNPVABKX-UHFFFAOYSA-N 1,1-diphenyl-2-(2,4,6-trinitrophenyl)hydrazine;ethanol Chemical compound CCO.[O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1NN(C=1C=CC=CC=1)C1=CC=CC=C1 OCZVHBZNPVABKX-UHFFFAOYSA-N 0.000 description 1
- KEQXNNJHMWSZHK-UHFFFAOYSA-L 1,3,2,4$l^{2}-dioxathiaplumbetane 2,2-dioxide Chemical compound [Pb+2].[O-]S([O-])(=O)=O KEQXNNJHMWSZHK-UHFFFAOYSA-L 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- -1 DPPH free radical Chemical class 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000002815 broth microdilution Methods 0.000 description 1
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 229960003276 erythromycin Drugs 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 238000009650 gentamicin protection assay Methods 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000007407 health benefit Effects 0.000 description 1
- 230000008821 health effect Effects 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- OSWPSODKJQKKLC-UHFFFAOYSA-M sodium ethanol 2-hydroxybenzoate Chemical compound [Na+].CCO.OC1=CC=CC=C1C([O-])=O OSWPSODKJQKKLC-UHFFFAOYSA-M 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- 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
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/11—Encapsulated compositions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/60—Sugars; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/732—Starch; Amylose; Amylopectin; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/733—Alginic acid; Salts thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
-
- 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
- A23V2400/173—Reuteri
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Dispersion Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nutrition Science (AREA)
- Dermatology (AREA)
- Food Science & Technology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Polymers & Plastics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides a lactobacillus reuteri microcapsule and a preparation method thereof, the provided lactobacillus reuteri microcapsule comprises a core material and a wall material, wherein the core material is lactobacillus reuteri VHPribo E18 strain, and the preservation number is CCTCC NO: m2021153. The wall material comprises the components of sodium alginate, gelatin, dextrin, sucrose and modified starch. After the lactobacillus reuteri microcapsule provided by the invention is digested by artificial gastric juice and artificial intestinal juice, the viable count does not change obviously, and the lactobacillus reuteri microcapsule has strong gastric acid and choline resistance. The lactobacillus reuteri VHProbi E18 microcapsule provided by the invention has no toxic action on organisms, can be added into food to prepare functional food with an anti-aging effect, can also be added into cosmetics to delay skin aging, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of probiotic products, and particularly relates to a lactobacillus reuteri microcapsule and a preparation method thereof.
Background
With the gradual entering of China into the aging society, the proportion of the aged population in the population is getting larger, various health problems related to human aging such as skin relaxation, alopecia and the like gradually appear and disturb the daily life of people, and the quality of life is affected. In the aspects of improving skin health and preventing and treating alopecia, the existing medicines not only need to be taken for a long time, but also have the problem of serious side effect. The search for the development of anti-aging actives has become a research hotspot in dealing with the aging problem of the population.
Researches find that the probiotics play an important role in delaying human body aging. There is increasing evidence that the health effects of probiotics on humans are not limited to the gut, but also have a broader range of actions, such as endocrine balance regulation, immune balance regulation, nervous system regulation, respiratory system regulation, etc. Therefore, the method for delaying aging and improving the health condition of the skin by taking the probiotics becomes a new treatment idea.
Probiotics can only exert health benefits on a host when a sufficient amount of probiotics is applied, so that the amount of live bacteria is a precondition for the probiotic bacterial strains to exert the probiotic effect, and the guarantee of the activity of the probiotics has important significance. The activity of the probiotic strains is easily affected by factors such as air humidity, ambient temperature and oxygen to lose the activity. The microcapsule preparation prepared by reducing probiotics not only can improve the unit live bacterial quantity, but also can reduce the influence of environmental factors to the minimum extent, ensure the stability of the probiotics in shelf life, enable the probiotics to be more processing-resistant and lay the foundation for the development of downstream functional foods.
Disclosure of Invention
The invention aims to provide a lactobacillus reuteri microcapsule and a preparation method thereof. The lactobacillus reuteri is separated from fresh excrement of centenarians, can improve the collagen content of skin, improve skin relaxation and hair follicle health, and has remarkable anti-aging effect.
The lactobacillus reuteri microcapsule provided by the invention comprises a core material and a wall material, wherein the core material is lactobacillus reuteri VHPribo E18 strain; the Lactobacillus reuteri VHPribo E18 strain (Lactobacillus reuteri VHPribo E18) is preserved in China center for type culture Collection in 1 month and 25 days 2021, and the preservation number is CCTCC NO: m2021153.
The wall material comprises the components of sodium alginate, gelatin, dextrin, sucrose and modified starch;
wherein the mass percentage of the sodium alginate, the gelatin, the dextrin, the sucrose and the modified starch is 2-5%: 5% -10%: 5% -10%: 3% -6%: 8 to 12 percent.
Preferably, the mass percentages of the sodium alginate, the gelatin, the dextrin, the sucrose and the modified starch are 3%: 5%: 5%: 3%: 8 percent.
The microcapsule provided is prepared by adding water into sodium alginate, gelatin, dextrin, sucrose and modified starch, stirring to dissolve completely, mixing with Lactobacillus reuteri VHPribo E18 strain mud, and making into frozen mixed solution; then the frozen mixed solution is frozen and dried to obtain the product.
One specific freeze-drying procedure is as follows: precooling at-40 ℃ for 3h, heating to-30 ℃ at the speed of 1 ℃/min for primary drying, continuing for 800min, then heating to 25 ℃ at the speed of 1 ℃/min for secondary drying, continuing for 2h, keeping the temperature of a cold trap at-80 ℃ and the vacuum degree at 20Pa, and obtaining the lactobacillus reuteri VHProbi E18 microcapsule preparation.
The lactobacillus reuteri microcapsule provided by the invention has high unit viable bacteria amount, and the number of viable bacteria in the microcapsule counted by adopting a pouring method is 2.08 multiplied by 1010CFU/g, the survival rate of the lactobacillus reuteri is 95.5%. After the lactobacillus reuteri microcapsule provided by the invention is digested by artificial gastric juice and artificial intestinal juice, the viable bacteria amount does not change obviously, and the lactobacillus reuteri VHProbi E18 microcapsule prepared by the invention has strong gastric acid and choline resistance.
After the lactobacillus reuteri VHProbi E18 microcapsule provided by the invention is fed to the aged mice of 12 months old for 70 days, the glossiness of the fur of the aged mice of 12 months old is improved, and the depilation phenomenon is reduced. Meanwhile, the water content of the skin of the aged mouse is increased, the content of malondialdehyde is reduced, and the content of hydroxyproline in the skin and the tail tendon is increased. The epidermic structure integrity of the aged mice is improved, inflammatory cells are reduced, and fibroblasts are increased. The skin aging index of the aged mice is improved after the lactobacillus reuteri VHProbi E18 microcapsules are infused.
The lactobacillus reuteri VHProbi E18 microcapsule provided by the invention has no toxic action on organisms, can be added into food to prepare functional food with an anti-aging effect, can also be added into cosmetics to delay skin aging, and has wide application prospect.
Drawings
FIG. 1 is a diagram of carbon source metabolism of API 50CHL of E18 strain;
FIG. 2 is a Riboprinter fingerprint of E18 strain;
FIG. 3 is a RAPD fingerprint of E18 strain;
FIG. 4 is a rep-PCR fingerprint of E18 strain;
FIG. 5 is a graph showing sensory evaluation results of fur in mice of each group;
FIG. 6 is a graph showing the results of measurement of skin water content in mice of each group;
FIG. 7 is a graph showing the results of measuring the MDA content in the skin of mice of each group.
FIG. 8 is a graph showing the measurement results of hydroxyproline content in the skin of mice of each group.
FIG. 9 is a graph showing HE staining results of skin of mice of each group, wherein (A) 3-month-old control group; (B) a 12-month-old control group; (C) smearing group with 12 months old; (D) gavage group of 12 months old;
FIG. 10 is a graph showing the results of measurement of the number of hair follicles in the skin, the thickness of the epidermis, the thickness of the dermis and the content of collagen fibers in each group of mice, wherein (A) the control group was 3 months old; (B) a 12-month-old control group; (C) smearing group with 12 months old; (D) a gavage group of 12 months old and a control group of 3 months old, wherein p is less than 0.05; p <0.05 with 12-month-old control group.
Detailed Description
The applicant finds that the Lactobacillus reuteri (Lactobacillus reuteri) VHProbi E18 strain obtained by screening has anti-aging effect in previous research; and using the strain to prepare a related bacterial preparation.
The applicant reserves the lactobacillus reuteri VHProbi E18 in China center for type culture Collection of Wuhan university with the collection number of CCTCC NO: m2021153.
The screening method of the present invention is not limited to the examples, and any known method capable of achieving the screening purpose is possible, and the screening description of the examples is only illustrative of the present invention and is not limiting the scope of the present invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
The present invention will be described in detail with reference to specific examples.
Example 1 isolation screening of Lactobacillus reuteri VHProbi E18
1. Preliminary screening
MRS agar culture medium is prepared, pH is adjusted to 6.2-6.5, and autoclaving is carried out at 121 ℃ for 15 min.
Taking 1g of fresh excrement sample of the centenarian, diluting the sample by using sterile normal saline, putting the diluted sample into a sterile sample bag, and beating and uniformly mixing the sample by using a homogenizer; and (3) taking 100 mu L of the uniformly mixed solution, diluting in a gradient manner, coating the uniformly mixed solution on an MRS agar culture medium, performing anaerobic culture at 37 ℃ for 48h, and performing microscopic examination on a single colony grown on a plate. According to the microscopic examination result, the applicant screened 20 potential lactobacilli in total, which are named as E01, E02, … …, E18, E19 and E20.
2. Double sieve
Preparing 1L MRS liquid culture medium, autoclaving at 121 deg.C for 15min, cooling, adding 3.2g pig mucosa pepsin, shaking for dissolving, and placing in 37 deg.C water bath shaker in warm water bath for 1h to obtain acid-resistant culture medium.
The 20 screened lactobacillus strains E01, E02, … …, E18, E19 and E20 are respectively inoculated in the acid-resistant culture medium according to 6 percent of inoculum size, anaerobic static culture is carried out for 48 hours at the temperature of 37 ℃, and the bacterial count is carried out on the fermentation liquor.
The result shows that the E18 strain has the largest viable bacteria amount after being re-screened by the acid-resistant culture medium in the Log values of the viable bacteria amount in the 20 strains of lactobacillus fermentation liquor, and the Log value is as high as 8.01Log CFU/mL. Thus, the strain E18 has the highest acid resistance.
Example 2 Strain identification
1. Colony morphology identification
E18 strain is inoculated on MRS agar medium, after anaerobic culture at 37 ℃ for 24h, the single colony of E18 is seen to be milk white, the diameter of the colony is about 1.5-2mm, the surface is smooth, and the end of the Campylobacter under the microscope is round.
2. Physiological and biochemical characteristic identification
The inoculation solution in this example was prepared as follows: under the aseptic condition, taking a proper amount of fresh E18 bacterial liquid, centrifuging at 5000rpm/min for 5min, washing with PBS buffer for 2 times, and diluting by 50 times after thalli are re-weighted by the PBS buffer with the same volume to be used as inoculation liquid.
2.1 salinity tolerance test
Under sterile conditions, 190 μ L of BSM broth with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% was added to 96-well plates in 3 replicates per salt concentration, followed by 10 μ L of inoculum, non-inoculated wells as controls. 50 μ L of autoclaved paraffin oil was added to each well to prevent evaporation of water during the culture. Culturing at 37 deg.C, and observing whether the culture medium turns turbid. The results showed that the maximum salt concentration tolerated by the E18 strain was 5%.
2.2 Catalase assay
A drop of fresh bacterial liquid is dropped on a clean glass slide, then a drop of 3% hydrogen peroxide solution is dropped on the fresh bacterial liquid, and the E18 strain is observed not to generate bubbles and is a negative reaction.
2.3 carbon source metabolism test
The API 50CHL reagent strip is used for carrying out carbon source metabolism experiments on the E18 strain, and the experimental method and result interpretation are specifically referred to the API 50CHL kit instruction. The identification result of the E18 strain is as follows: when the% ID was 93 and the T value was 0.74, the API was lactobacillus fermentum, and the identification score was good (lactobacillus reuteri was not identified in the API-identifiable species so lactobacillus fermentum was identified). The results of carbon source metabolism of API 50CHL of strain E18 are shown in FIG. 1.
3 molecular biological identification
3.116 s rDNA Gene sequence analysis
3.1.1 extraction of genomic DNA
Reference was made to the Tiangen bacterium genomic DNA extraction kit (catalog No.: DP 302).
3.1.2, 16s rDNA Gene amplification
The primer sequence is as follows:
27F：AGAGTTTGATCCTGGCTCA；
1492R：GGTTACCTTGTTACGACTT。
the 16s rDNA sequence SEQ ID NO 1 of the E18 strain is obtained by sequencing, and the sequence is compared in NCBI database, thus preliminarily determining that the E18 strain is lactobacillus reuteri.
3.2 Riboprinter fingerprint
And (3) dipping the purified single colony from an agar culture medium plate by using a bacteria taking rod, putting the single colony into a sample tube with a buffer solution, stirring the single colony in the buffer solution by using a handheld stirrer to enable the single colony to be suspended in the buffer solution, putting a sample rack into a heater to inactivate, putting the sample rack into a Riboprinter system, and obtaining a bacteria identification result after DNA preparation, membrane conversion, imaging detection and data processing of the sample. The identification result shows that the E18 strain is Lactobacillus reuteri, and the Riboprinter fingerprint pattern result is shown in figure 2.
3.3 RAPD and rep-PCR fingerprinting
3.3.1 RAPD fingerprint identification
The primer sequence is as follows: GAGGGTGGCGGTTCT are provided.
The RAPD fingerprint of the E18 strain is shown in FIG. 3.
3.3.2 rep-PCR fingerprint
The primer sequences are as follows: CTACGGCAAGGCGACGCTGACG are provided.
The rep-PCR fingerprint of E18 strain is shown in FIG. 4.
The results of colony morphology and physiological and biochemical characteristics of the E18 strain were uploaded to the website http:// www.tgw1916.net/bacterial _ log _ desktop. htmL, and compared with the results published in De Clerck E, et al. And (3) determining the E18 strain as a new lactobacillus reuteri strain by integrating the identification result of molecular biology, and naming the new lactobacillus reuteri strain as lactobacillus reuteri VHProbi E18.
Example 3 tolerance test of Lactobacillus reuteri VHProbi E18 to artificial gastric and intestinal juices
1 preparation of artificial gastric juice
5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl were weighed, respectively, 1000mL of distilled water was added, pH3.0 was adjusted with dilute hydrochloric acid, and then sterilization was carried out at 115 ℃ for 20 min. Then 3.2g of pig mucosa pepsin is added before use, shaken up and dissolved, and placed in a water bath shaker at 37 ℃ for a water bath for 1h to simulate the temperature of a human body.
2 preparation of artificial intestinal juice
Separately weighing peptone 5g and yeast extract2.5g, glucose 1g, KH2PO46.8g and 3.0g of ox-gall salt, 77mL of 0.2mol/L NaOH solution is added, the volume is adjusted to 1000mL, the pH value is adjusted to 6.8 +/-0.1 by dilute hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized for 20min at 115 ℃. Then 1g pancreatin is added before use, shaken up and dissolved, and put into a water bath shaker at 37 ℃ for water bath for 1h to simulate the temperature of a human body.
3 test method
2mL of fresh bacterial liquid is taken, centrifuged at 5000rpm/min for 5min to collect thalli, the thalli are washed for 3 times by using normal saline, and then 2mL of normal saline is used for resuspension to serve as inoculation liquid. Taking 1mL of inoculation liquid, adding the inoculation liquid into 24mL of artificial intestinal juice, placing the artificial intestinal juice in a water bath shaker (200rpm/min) at 37 ℃ for 3h, sampling 1mL, and detecting the amount of live bacteria.
The viable bacteria counting method is used for measuring the bacterial quantity according to the national standard GB 4789.35-2016-food microorganism test lactobacillus test, and the viable bacteria quantity (Log CFU/mL) of the bacterial strain after being digested by artificial intestinal juice is shown in the table 1.
TABLE 1 viable cell count after digestion of artificial gastrointestinal fluids
As is clear from Table 1, the viable cell count of Lactobacillus reuteri VHProbi E18 screened by the present invention was increased after digestion with artificial gastric juice and artificial intestinal juice. Thereby showing that the strain can tolerate the artificial gastric juice and the artificial intestinal juice and can also carry out certain germination.
Example 4 hemolytic and antibiotic resistance experiments of Lactobacillus reuteri VHProbi E18
1. Hemolytic test
Weighing the components of TBS basic culture medium, dissolving, autoclaving at 121 deg.C for 15min, adding 5% sterile defibered sheep blood when the culture medium is cooled to 50 deg.C, mixing, and pouring into flat plate. And streaking the test strain on a prepared blood cell plate, culturing in an incubator at 37 ℃, and observing whether the test strain has hemolysis or not for 24-48 hours.
The results show that: lactobacillus reuteri VHProbi E18 could not grow and the blood cell plates were unchanged, indicating that Lactobacillus reuteri VHProbi E18 did not produce hemolysin and could not lyse blood cells.
2. Antibiotic resistance test
Specific results of determining the MIC value of minimum inhibitory concentration of antibiotic against Lactobacillus reuteri VHProbi E18 by broth dilution are shown in Table 2.
Table 2: antibiotic MIC value Table of Lactobacillus reuteri VHProbi E18
MIC unit μ g/mL
The results in table 2 show that lactobacillus reuteri VHProbi E18 provided by the present invention is sensitive to common antibiotics such as erythromycin and ampicillin, and has good biosafety.
Example 5 Lactobacillus reuteri VHProbi E18 assay for antioxidant function
1. Determination of DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) and Hydroxyl Radical (HRS) removing capability of strain
Determination of DPPH free radical scavenging ability of strain
Taking 1mL of PBS bacterial suspension of the strain to be detected, adding 1mL of 0.4mM of ready-prepared DPPH free radical solution, uniformly mixing, then placing at room temperature for shading reaction for 30min, then measuring the absorbance A sample of the sample at the wavelength of 517nm, and measuring for 3 times of parallelism. The control group samples were blank-zeroed with equal volumes of PBS solution and DPPH-ethanol mixture, and equal volumes of PBS suspension and ethanol mixture. The clearance was calculated according to the following formula: clearance%Sample (I)-ABlank space)/AControl]X 100%. The results are shown in Table 3.
TABLE 3 DPPH radical scavenging Table
3) Determination of HRS-removing ability of Strain
mu.L of 5mM sodium salicylate-ethanol solution, 100. mu.L of 5mM ferrous sulfate, 500. mu.L deionized water and 200. mu.L of lactic acid bacteria PBS suspension were mixed, 100. mu.L of hydrogen peroxide solution (3mM) was added, and absorbance of the sample was measured at a wavelength of 510nm after water bath at 37 ℃ for 15 min. The hydroxyl radical clearance was calculated according to the following formula.
Clearance%Sample(s)-AControlling)/(ABlank space-AControl of) X 100% where AControllingTo replace the sample with deionized water, ABlank spaceReplacement of sample and H for deionized Water2O2The results are shown in Table 4.
TABLE 4 HRS radical scavenging Table
2. Experimental identification of bacterial strain for resisting lipid peroxidation
Preparing a linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water.
0.5mL of PBS solution (pH 7.4) was added 1mL of an emulsion of linoleic acid, 1mL of LFeSO4(1%), adding 0.5mL of sample, carrying out water bath at 37 ℃ for 1.5h, adding 0.2mL of TCA (4%), 2mL of TBA (0.8%), carrying out water bath at 100 ℃ for 30min, rapidly cooling, centrifuging at 4000rpm/min for 15min, collecting supernatant, and measuring the absorbance at 532nm to obtain A; the control group uses 0.5mL of distilled water to replace the sample to obtain A0. Inhibition rate/% (A)0－A)/A0×100％
Note: a is the absorbance of the sample group; a. the0The results are shown in Table 5, which is the absorbance of the control group.
TABLE 5 inhibition of lipid peroxidation
Example 6 Lactobacillus reuteri VHProbi E18 in vitro cholesterol degradation experiment
1. Preparation of cholesterol micelle solution: accurately weighing 1g of cholesterol, dissolving in absolute ethyl alcohol, diluting to 100mL, and filtering and sterilizing with a 0.22-micron microporous filter membrane under aseptic condition.
2. 10.0g of peptone and 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 801.0 mL of Tween, 5.0g of sodium acetate, 0.1 g of magnesium sulfate, 0.05g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate, 1g of bile salt and 1000mL of distilled water are weighed, the pH value is adjusted to 7.3, sterilization is carried out at 115 ℃ for 30min, and then cholesterol solution is added to ensure that the final concentration of cholesterol is 0.1%.
Inoculating fresh bacterial liquid according to the inoculation amount of 0.1%, performing static culture at 37 ℃ for 48h, then taking 0.2mL of bacterial liquid, adding 1.8mL of absolute ethyl alcohol, uniformly mixing, standing for 10min, centrifuging for 5min at 3000 rpm, and taking supernatant for measuring the cholesterol content. Method for measuring cholesterol GB/T5009.128-2003 < determination of cholesterol in food >.
The results show that: the degradation rate of the lactobacillus reuteri VHProbi E18 on cholesterol provided by the invention reaches 16.24% (this is data without bile salt).
Example 7 hydrophobic cell surface testing of Lactobacillus reuteri VHProbi E18
1. Preparing bacterial liquid to be detected: and (3) selecting a purified lactobacillus reuteri VHProbi E18 colony, inoculating the colony in a newly prepared MRS liquid culture medium, and culturing at 37 ℃ for 24-48 h. Inoculating to MRS liquid culture medium according to the inoculation amount of 1% (V/V), continuously culturing at 37 deg.C for 24-48 hr, centrifuging at 6000 Xg for 10min, collecting thallus, washing with sterile normal saline for 2 times, and sterilizing with 0.1M KNO31mL of the solution was used as a suspension to be assayed.
2. Surface hydrophobicity measurement: sucking 50 μ L of the above bacterial suspension, adding 2450 μ L of 0.1M KNO3And record an OD600 of A01.5mL of the suspension was mixed with 500. mu.L of xylene and allowed to stand at room temperature for 10min (a two-phase system was formed). And (3) vortexing and oscillating the two-phase system for 2min, and then standing for 20min to form a water phase and an organic phase again. The absorbance A was measured at 600nm by carefully taking up the aqueous phase (not taking up the organic phase)1. Cell Hydrophobicity according to the formula (A)0-A1)/A1Calculated as% x, three experiments were measured and averaged.
The results show that: the cell surface hydrophobicity of the lactobacillus reuteri VHProbi E18 provided by the invention is 61.76%, and the standard deviation is 0.27%.
Example 8 cell adhesion test of Lactobacillus reuteri VHProbi E18
Recovering and subculturing Caco-2 cells, and expanding the number of the cultured cells to the required dosage. After adding pancreatin, the cells were returned to the incubator and were observed by eye to be in a single cell state as much as possible after the cells had completely fallen off. Cell counting was performed using a hemocytometer and the cell suspension was diluted with an appropriate amount of PBS. The cells were resuspended in MRS medium for use. And incubating the bacterial suspension to be detected and Caco-2 cells for 2h, and washing the non-adhered bacteria by PBS. Adding pancreatin for digestion, adding cell culture solution to stop digestion, collecting liquid, coating and counting. Capacity for attachment (CFU/cells) — total number of bacteria attached per culture well/total number of cells per culture well. The adhesion capability of lactobacillus reuteri VHProbi E18 was detected to be 4.7.
Example 9 use of Lactobacillus reuteri VHProbi E18 for improving the skin of aging mice
1.1 Experimental animals
C57 mice are of SPF grade, male, 6 mice at 3 months of age, 18 mice at 12 months of age and the weight of 19-25 g. Environmental conditions for experimental animal feeding management: the temperature of the room temperature is 20-26 ℃, the daily temperature difference is less than or equal to 4 ℃, the relative humidity is 40-70%, and the light and shade alternation time is 12/12 hours. Animals were housed in standard mouse cages, 6 animals per cage. Animal feed and drinking water: free ingestion and drinking. The feed is SPF-level rat growth breeding feed. The drinking water is urban tap water sterilized at high temperature.
1.2 Experimental methods
After the mice are adaptively fed for 7 days, the mice are randomly divided into a control group with the age of 3 months, a control group with the age of 12 months, a smearing group with the age of 12 months and an intragastric group with the age of 12 months, and each group contains 6 mice. The 12-month-old intragastric administration group is administered with probiotic bacteria liquid according to the intragastric administration of 0.2mL/10g, the 12-month-old smearing group is coated with probiotic bacteria liquid equal to the intragastric administration group, and the 3-month-old control group and the 12-month-old control group are administered with physiological saline equal to the intragastric administration and the probiotic bacteria. For a total of 70 days.
1.3 detection criteria
At the end, the mice were organoleptically scored for back coat, according to the scoring rules shown in table 6. Detecting the water content, SOD activity and MDA content change of the skin of the mouse, and detecting the hydroxyproline content in the skin and the tail tendon of the mouse.
TABLE 6 sensory evaluation of skin and hair on the mouse's back
| Rating of evaluation | Score of | Appearance of the |
| 0 | 1 | No luster, dry, no oil color, split, broken, white hair |
| + | 2 | Poor luster, no oily color and white hair |
| ++ | 3 | Glossy, soft, white and black hair-free |
| +++ | 4 | Good luster, smooth and bright hair, skin luster close-fitting, and bright black |
After the test is finished, the back skin tissue is taken, fixed by 4% paraformaldehyde, taken, dehydrated, embedded in paraffin, sliced, HE stained, and the number of hair follicles, the thickness of dermis and sebaceous gland cells are detected.
All experimental data are expressed as mean ± standard deviation, and data statistics and mapping were performed using Microsoft EXCEL, and the comparison between the two sets of data was determined as significant difference using t-test with P < 0.05.
1.4 results of the experiment
1.4.1 sensory evaluation of mouse Back fur
At the final stage, the hair of the 3-month-old mouse is dense and has regular luster; the hair of the control group mice of 12 months old is dry, has more depilation and has white hair; the hair of the 12-month-old smearing group mice is glossy and has no white hair; the hair of the mice in the gavage group aged 12 months is thick, glossy and black. The results of sensory evaluation of the back fur score of each group of mice are shown in fig. 5.
1.4.2 mouse skin moisture content, MDA and hydroxyproline content variation
Compared with the 12-month-old groups with the age of about 14-15 months at the end, the 3-month-old control group with the age of about 5-6 months at the end has obviously reduced skin moisture content (p is less than 0.05), and the reduction rate is about 15.95%; compared with the 12-month-old control group, the water content of the skin of the 12-month-old gavage group is increased, and the significant difference is realized (p is less than 0.05). The comparison of the skin moisture content of the mice in each group is shown in FIG. 6.
Compared with a 12-month-old gavage group, the skin MDA content of the 12-month-old gavage group is remarkably reduced and has a remarkable difference (p is less than 0.05), the skin MDA content of the 12-month-old smear group is remarkably reduced and also has a remarkable difference (p is less than 0.05), and the difference of the 12-month-old smear group and the skin MDA content of the 12-month-old gavage group in the terminal state is not remarkable (p is more than 0.05). A comparison of the MDA levels in the skin of the mice in each group is shown in FIG. 7.
Compared with a 12-month-old control group, the hydroxyproline content in the skin and the tail tendon of the gavage group and the daubing group at 12 months of age is increased and has significant difference (p is less than 0.05), and the hydroxyproline content of the skin at the terminal state of the gavage group at 12 months of age has no significant difference (p is more than 0.05) compared with the hydroxyproline content of the skin at the terminal state of the gavage group at 12 months of age. A comparison of hydroxyproline content in the skin and tendon of mice in each group is shown in FIG. 8.
1.4.3 histological examination of the dorsal skin of mice
As seen under an optical microscope, the epidermis structure of a control group mouse of 3 months old is complete, the cells are clearly layered, the control group mouse has obvious epidermal protrusion and dermal papilla, inflammatory cells are not infiltrated in a visual field, sebaceous gland hyperplasia can be seen, dermal collagen fibers can be seen in the visual field, and the control group mouse is uniformly arranged and distributed into a belt shape; the epidermis of a 12-month-old control group mouse is obviously thinned, the cuticle is visibly fallen off, the structural integrity is deficient, the cell number is reduced, the arrangement is irregular, the collagen fiber layer is obviously reduced, the fibers are broken, unevenly distributed and sparse, and the fibroblasts are reduced; epithelial cells of mice in the gavage group of 12 months old proliferate, the arrangement occasionally loosens and disorganizes, pilosebaceous glands occasionally proliferate, inflammatory cells occasionally appear, collagen fibers are loosely distributed, and the fracture is visible; epithelial cells of the 12-month-old smearing group are obviously proliferated, cuticle is separated, hair follicles and sebaceous glands are obviously proliferated, inflammatory cells are visible, skin is loose and visible, collagen fiber layers are loosely arranged and distributed, and the fracture phenomenon is obvious. Results of typical HE sections of mouse skin are shown in fig. 9.
At the end, compared with the control group with the age of 3 months, the skin hair follicle density of the mice in the control group with the age of 12 months is reduced, and the mice have significant difference (P < 0.05); compared with the mice of the 12-month-old control group, the density of hair follicles of the 12-month-old application group and the 12-month-old gavage group is increased, and the difference is significant (P < 0.05).
Compared with a control group with the age of 3 months, the thickness of the epidermis of the skin of the mice in the control group with the age of 12 months is reduced, but no significant difference exists; there was no difference in skin thickness among the groups of 12-month-old mice.
The skin dermis thickness of the mice of the 12-month-old control group is reduced compared with that of the control group of the 3-month-old group, and the mice have significant difference (P < 0.05); compared with the mice of the 12-month-old control group, the thickness of the dermis of the skin of the 12-month-old smear group and the dermis of the 12-month-old intragastric lavage group is increased, and the skin thickness is significantly different (P < 0.05).
Compared with a control group with the age of 3 months, the area of the collagen fibers of the skin of the mice in the control group with the age of 12 months is reduced, and the difference is significant (P < 0.05); compared with the mice of the 12-month-old control group, the skin collagen fiber areas of the 12-month-old smeared group and the 12-month-old gavage group are increased, and the differences are significant (P < 0.05). The results of comparison of skin aging degrees of mice of each group are shown in FIG. 10.
From the above results, it was found that the gloss of the fur was improved in both the gavage group and the application group at 12 months of age, and the depilation phenomenon was reduced without the occurrence of white hair, as compared with the control group at 12 months of age. The water content of the skin of mice in the gavage group with age of 12 months and the smearing group with age of 12 months is increased, and the MDA content is reduced. Hydroxyproline is used as amino acid with abundant and stable intradermal content, the content of hydroxyproline can directly reflect the content change of collagen in the dermis, and the hydroxyproline is one of indexes for detecting skin aging. The hydroxyproline content in the skin and the tail tendon of mice in the gavage group with the age of 12 months and the smearing group with the age of 12 months is increased, and the content of the collagen in the dermis can be increased by smearing and gavage the lactobacillus reuteri VHProbi E18. The histological examination of the skin of the mice shows that compared with the mice in a control group with the age of 12 months, the structural integrity of the epidermis of the mice in the gavage group with the age of 12 months and the mice in the smearing group with the age of 12 months is improved, inflammatory cells are reduced, fibroblasts are increased, and the effect of the gavage group is better than that of the smearing group. In addition, the number of hair follicles in the skin of mice in the gavage group of 12 months old and the application group of 12 months old was increased, the thickness of the dermis was increased, and the collagen fiber content was increased. The result shows that the lactobacillus reuteri VHProbi E18 can improve the skin indications of the aged mice to achieve the effect of delaying and repairing skin aging no matter the lactobacillus reuteri VHProbi E18 is used for gastric lavage or smearing.
In conclusion, the Lactobacillus reuteri VHProbi E18 provided by the invention has strong tolerance to simulated artificial intestinal gastric juice, which lays a foundation for probiotic strains to successfully colonize in the colon through the gastrointestinal tract to play a probiotic function. Antibiotic resistance tests prove that the Lactobacillus reuteri VHProbi E18 is sensitive to common antibiotics, does not produce hemolysin and has good biological safety. Meanwhile, lactobacillus reuteri VHProbi E18 can remove DPPH and HRS free radicals, inhibit lipid peroxidation, has certain antioxidant function activity, can degrade cholesterol, and has the probiotic characteristic of reducing serum cholesterol. Animal experiments prove that the lactobacillus reuteri VHProbi E18 has an anti-aging effect, can increase the content of skin collagen, improves the hair follicle health and improves the skin health state. The lactobacillus reuteri VHProbi E18 can be used for preparing functional food or cosmetics with the effect of delaying and repairing skin aging, and has a wide application prospect.
EXAMPLE 10 microcapsules and method of making the same
1. Preparation of Lactobacillus reuteri puree
Under the aseptic condition, inoculating the seed liquid of lactobacillus reuteri VHProbi E18 into a fermentation culture medium according to the volume ratio of 3%, wherein the fermentation culture medium comprises the following components in percentage by volume: 30g/L of sucrose, 25g/L of yeast powder, 15g/L of soybean peptone, 0.15g/L of magnesium sulfate and 0.08g/L of manganese sulfate; culturing at 37 deg.C for 24h, rotating speed of 100rpm, ventilation amount of 1L/min, and tank pressure of 0.05 MPa. And (3) obtaining fermentation liquor of the lactobacillus reuteri VHProbi E18 after fermentation is finished, centrifuging the fermentation liquor at the temperature of 4 ℃ for 10min at 4000g/min, and removing supernatant fluid to obtain lactobacillus reuteri VHProbi E18 bacterial sludge.
2. Preparation of probiotic microcapsules
Weighing five coating wall materials of sodium alginate, gelatin, dextrin, sucrose and modified starch according to a proportion, adding water, stirring until the wall materials are completely dissolved, and obtaining a wall material solution, wherein the wall material comprises the following components in percentage by mass: 3% of sodium alginate, 5% of gelatin, 5% of dextrin, 3% of sucrose and 8% of modified starch.
Adding the lactobacillus reuteri VHProbi E18 bacterial sludge prepared in the step 1 into the wall material solution, and uniformly stirring to obtain a frozen mixed solution; and (3) placing the frozen mixed solution into a freeze dryer for freeze drying, wherein the freeze drying procedure is as follows: precooling at-40 ℃ for 3h, heating to-30 ℃ at the speed of 1 ℃/min for primary drying for 800min, then heating to 25 ℃ at the speed of 1 ℃/min for secondary drying for 2h, keeping the temperature of a cold trap at-80 ℃ and the vacuum degree at 20Pa, and obtaining the lactobacillus reuteri VHProbi E18 microcapsule preparation.
EXAMPLE 11 preparation of Lactobacillus reuteri microcapsules
1. Preparation of Lactobacillus reuteri puree
Under the aseptic condition, inoculating the seed liquid of lactobacillus reuteri VHProbi E18 into a fermentation culture medium according to the volume ratio of 5%, wherein the fermentation culture medium comprises the following components in percentage by volume: 50g/L of sucrose, 30g/L of yeast powder, 25g/L of soybean peptone, 0.15g/L of magnesium sulfate and 0.10g/L of manganese sulfate; culturing at 37 deg.C for 36h, rotating speed of 150rpm, ventilation amount of 1.5L/min, and tank pressure of 0.08 MPa. And (3) obtaining fermentation liquor of the lactobacillus reuteri VHProbi E18 after fermentation is finished, centrifuging the fermentation liquor at the temperature of 4 ℃ at 6000g/min for 8min, and removing supernatant to obtain lactobacillus reuteri VHProbi E18 bacterial sludge.
2. Preparation of probiotic microcapsules
Weighing five coating wall materials of sodium alginate, gelatin, dextrin, sucrose and modified starch according to a proportion, adding water, stirring until the wall materials are completely dissolved, and obtaining a wall material solution, wherein the wall material comprises the following components in percentage by mass: 5% of sodium alginate, 10% of gelatin, 10% of dextrin, 6% of sucrose and 10% of modified starch.
Adding the lactobacillus reuteri VHProbi E18 bacterial sludge prepared in the step 1 into the wall material solution, and uniformly stirring to obtain a frozen mixed solution; placing the frozen mixed solution into a freeze dryer for freeze drying, wherein the freeze drying procedure is as follows: precooling at-60 ℃ for 2h, heating to-40 ℃ at the speed of 1 ℃/min for primary drying, continuing for 600min, then heating to 20 ℃ at the speed of 1 ℃/min for secondary drying, continuing for 3h, keeping the temperature of a cold trap at-80 ℃ and the vacuum degree at 20Pa, and obtaining the lactobacillus reuteri VHProbi E18 microcapsule preparation.
Example 12 Lactobacillus reuteri microcapsules and methods of making the same
1. Preparation of Lactobacillus reuteri puree
Under the aseptic condition, inoculating the seed liquid of lactobacillus reuteri VHProbi E18 into a fermentation culture medium according to the volume ratio of 8%, wherein the fermentation culture medium comprises the following components in percentage by volume: 50g/L of sucrose, 25g/L of yeast powder, 10g/L of soybean peptone, 0.05g/L of magnesium sulfate and 0.04g/L of manganese sulfate; culturing at 37 deg.C for 18h, rotating speed of 80rpm, ventilation amount of 2L/min, and tank pressure of 0.05 MPa. Obtaining fermentation liquor of the Lactobacillus reuteri VHProbi E18 after fermentation is finished, centrifuging the fermentation liquor at 4 ℃ for 5min at 8000g/min, and removing supernatant fluid to obtain the Lactobacillus reuteri VHProbi E18 bacterial sludge.
2. Preparation of probiotic microcapsules
Weighing five coating wall materials of sodium alginate, gelatin, dextrin, sucrose and modified starch according to a proportion, adding water, stirring until the wall materials are completely dissolved, and obtaining a wall material solution, wherein the wall material comprises the following components in percentage by mass: 2% of sodium alginate, 8% of gelatin, 10% of dextrin, 6% of sucrose and 12% of modified starch.
Adding the lactobacillus reuteri VHProbi E18 bacterial sludge prepared in the step 1 into the wall material solution, and uniformly stirring to obtain a frozen mixed solution; placing the frozen mixed solution into a freeze dryer for freeze drying, wherein the freeze drying procedure is as follows: precooling at-30 ℃ for 3h, heating to-20 ℃ at the speed of 1 ℃/min for primary drying, continuing for 1000min, then heating to 30 ℃ at the speed of 1 ℃/min for secondary drying, continuing for 1h, keeping the temperature of a cold trap at-80 ℃ and the vacuum degree at 20Pa, and obtaining the lactobacillus reuteri VHProbi E18 microcapsule preparation.
Example 13 Lactobacillus reuteri microcapsule survival assays
After 1g of each of the lactobacillus reuteri microcapsules prepared in examples 10 to 12 was diluted with physiological saline in a gradient manner, the number of viable bacteria in the microcapsules was counted by a pouring method, and the amount of viable bacteria per unit in the microcapsules and the survival rate of lactobacillus reuteri were calculated, and the results are shown in table 7.
TABLE 7 comparison of viable cell count per unit of Lactobacillus reuteri microcapsules and viability of the strains
| Lactobacillus salivarius preparation | Unit viable bacteria amount | Survival rate of Lactobacillus salivarius |
| Example 10 | 2.08×1010CFU/g | 95.5％ |
| Example 11 | 1.77×1010CFU/g | 89.1％ |
| Example 12 | 2.38×1010CFU/g | 94.2％ |
The results in table 7 show that the unit viable bacteria of the lactobacillus reuteri microcapsule provided by the present invention are high, and the survival rate of the lactobacillus reuteri VHProbi E18 after freeze drying can reach 95.5% after the lactobacillus reuteri VHProbi E18 is protected by the coating wall material, thereby demonstrating that the coating material and the freeze drying preparation process selected by the present invention have significant protective effects on lactobacillus reuteri VHProbi E18.
Example 14 detection of adverse resistance of Lactobacillus reuteri microcapsules
1. Stress resistance detection
1.1 preparation of Artificial gastric juice
5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl were weighed, respectively, 1000mL of distilled water was added, pH3.0 was adjusted with dilute hydrochloric acid, and then sterilization was carried out at 115 ℃ for 20 min. Then 3.2g of pig mucous membrane pepsin is added before use, shaken up and dissolved, and placed in a water bath shaker at 37 ℃ for water bath for 1 hour at medium temperature to simulate the temperature of a human body.
1.2 preparation of Artificial intestinal juice
Separately weighing peptone 5g, yeast extract 2.5g, glucose 1g, KH2PO46.8g and 3.0g of ox-gall salt, 77mL of 0.2mol/L NaOH solution is added, the volume is adjusted to 1000mL, the pH value is adjusted to 6.8 +/-0.1 by dilute hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized for 20min at 115 ℃. Then 1g pancreatin is added before use, shaken up and dissolved, and put into a water bath shaker at 37 ℃ for water bath for 1h to simulate the temperature of a human body.
1.3 test methods
2g of the Lactobacillus reuteri microcapsules prepared in examples 10 to 12 were separately taken and resuspended in 2mL of physiological saline to serve as a seed stock solution. Adding 1mL of inoculation liquid into 9mL of artificial gastric juice which is warmed in advance in a warm water bath for 1h, placing the artificial gastric juice in a water bath shaking table at 37 ℃ and oscillating the artificial gastric juice at the rotating speed of 200rpm/min for 2h, sampling 1mL at 0h and 2h respectively, and detecting the amount of viable bacteria. Then 1mL of the artificial gastric juice digested for 2h is taken and added into 24mL of the artificial intestinal juice, and the artificial intestinal juice is placed in a water bath shaker (200rpm/min) at 37 ℃ for 3h, and 1mL of the artificial gastric juice is sampled, and the amount of the live bacteria is detected. The viable bacteria counting method is used for measuring the bacterial load according to the national standard GB 4789.35-2016-food microorganism test lactobacillus test, and the viable bacteria load (LogCFU/g) of the microcapsule prepared in the embodiment 10-12 after the microcapsule is digested by the artificial gastric juice and the artificial intestinal juice is shown in the table 8.
TABLE 8 tolerance effect of Lactobacillus reuteri microcapsules on artificial gastric and intestinal juices
The results in table 8 show that after the lactobacillus reuteri microcapsule provided by the present invention is digested by artificial gastric juice and artificial intestinal juice, the viable count does not change significantly, so that the lactobacillus reuteri VHProbi E18 microcapsule prepared by the present invention has strong gastric acid and choline resistance.
After the lactobacillus reuteri VHProbi E18 microcapsule provided by the invention is fed to the aged mice of 12 months old for 70 days, the glossiness of the fur of the aged mice of 12 months old is improved, and the depilation phenomenon is reduced. Meanwhile, the water content of the skin of the aged mice is increased, the malondialdehyde content is reduced, and the hydroxyproline content in the skin and the tail tendon is increased. The epidermic structure integrity of the aged mice is improved, inflammatory cells are reduced, and fibroblasts are increased. The skin aging index of the aged mice is improved after the mice are infused with the lactobacillus reuteri VHProbi E18 microcapsules.
The preparation process of the lactobacillus reuteri microcapsule provided by the invention is simple, the coating material is cheap, the industrialization cost is low, the stability of the lactobacillus reuteri VHProbi E18 in the storage, processing and transportation process can be effectively improved, the actual using effect of the lactobacillus reuteri VHProbi E18 is powerfully improved, and the lactobacillus reuteri microcapsule can be widely applied to the fields of food, health care products and the like.
Sequence listing
<110> Islands Ulva Biometrics group, Inc. of Islands Ulva Biometrics group
<120> Lactobacillus reuteri microcapsule and preparation method thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1428
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aaaggttagg ccaccgactt tgggcgttac aaactcccat ggtgtgacgg gcggtgtgta 60
caaggcccgg gaacgtattc accgcggcat gctgatccgc gattactagc gattccgact 120
tcgtgtaggc gagttgcagc ctacagtccg aactgagaac ggctttaaga gattagctta 180
ctctcgcgag tttgcgactc gttgtaccgt ccattgtagc acgtgtgtag cccaggtcat 240
aaggggcatg atgatctgac gtcgtcccca ccttcctccg gtttgtcacc ggcagtctca 300
ctagagtgcc caactcaatg ctggcaacta gtaacaaggg ttgcgctcgt tgcgggactt 360
aacccaacat ctcacgacac gagctgacga cgaccatgca ccacctgtca ttgcgtcccc 420
gaagggaacg ccttatctct aaggttagcg caagatgtca agacctggta aggttcttcg 480
cgtagcttcg aattaaacca catgctccac cgcttgtgcg ggcccccgtc aattcctttg 540
agtttcaacc ttgcggtcgt actccccagg cggagtgctt aatgcgttag ctccggcact 600
gaagggcgga aaccctccaa cacctagcac tcatcgttta cggcatggac taccagggta 660
tctaatcctg ttcgctaccc atgctttcga gcctcagcgt cagttgcaga ccagacagcc 720
gccttcgcca ctggtgttct tccatatatc tacgcattcc accgctacac atggagttcc 780
actgtcctct tctgcactca agtcgcccgg tttccgatgc acttcttcgg ttaagccgaa 840
ggctttcaca tcagacctaa gcaaccgcct gcgctcgctt tacgcccaat aaatccggat 900
aacgcttgcc acctacgtat taccgcggct gctggcacgt agttagccgt gactttctgg 960
ttggataccg tcactgcgtg aacagttact ctcacgcacg ttcttctcca acaacagagc 1020
tttacgagcc gaaacccttc ttcactcacg cggtgttgct ccatcaggct tgcgcccatt 1080
gtggaagatt ccctactgct gcctcccgta ggagtatgga ccgtgtctca gttccattgt 1140
ggccgatcag tctctcaact cggctatgca tcatcgcctt ggtaagccgt taccttacca 1200
actagctaat gcaccgcagg tccatcccag agtgatagcc aaagccatct ttcaaacaaa 1260
agccatgcgg cttttgttgt tatgcggtat tagcatctgt ttccaaatgt tatcccccgc 1320
tccggggcag gttacctacg tgttactcac ccgtccgcca ctcactggtg atccatcgtc 1380
aatcaggtgc aagcaccatc aatcagttgg gccagtgcgt acgactgc 1428
Claims (6)
1. The lactobacillus reuteri microcapsule is characterized by comprising a core material and a wall material, wherein the core material is a material with a preservation number of CCTCC NO: m2021153 of Lactobacillus reuteri.
2. The lactobacillus reuteri microcapsule according to claim 1, wherein the wall material comprises the components of sodium alginate, gelatin, dextrin, sucrose and modified starch.
3. The lactobacillus reuteri microcapsule according to claim 2, wherein the mass percentage of the sodium alginate, the gelatin, the dextrin, the sucrose and the modified starch is 2-5%: 5% -10%: 5% -10%: 3% -6%: 8 to 12 percent.
4. The lactobacillus reuteri microcapsule according to claim 2, wherein the mass percentage of the sodium alginate, the gelatin, the dextrin, the sucrose and the modified starch is 3%: 5%: 5%: 3%: 8 percent.
5. The Lactobacillus reuteri microcapsule according to any of claims 1 to 4, wherein the microcapsule is prepared by adding water to sodium alginate, gelatin, dextrin, sucrose, and modified starch, stirring until completely dissolved, mixing with the bacterial sludge of Lactobacillus reuteri, and making into frozen mixed solution; then the frozen mixed solution is frozen and dried to obtain the product.
6. Lactobacillus reuteri microcapsules according to claim 5, wherein the freeze-drying procedure is as follows: pre-cooling at-40 deg.C for 3h, heating to-30 deg.C at 1 deg.C/min for primary drying for 800min, heating to 25 deg.C at 1 deg.C/min for secondary drying for 2h, cooling at-80 deg.C, and vacuum degree of 20Pa to obtain microcapsule preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110639675.0A CN114717220B (en) | 2021-06-09 | 2021-06-09 | Lactobacillus reuteri microcapsule and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110639675.0A CN114717220B (en) | 2021-06-09 | 2021-06-09 | Lactobacillus reuteri microcapsule and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114717220A true CN114717220A (en) | 2022-07-08 |
| CN114717220B CN114717220B (en) | 2023-06-27 |
Family
ID=82234892
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110639675.0A Active CN114717220B (en) | 2021-06-09 | 2021-06-09 | Lactobacillus reuteri microcapsule and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114717220B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115584333A (en) * | 2022-10-19 | 2023-01-10 | 廖梅香 | Lactobacillus reuteri and application thereof |
| WO2023240936A1 (en) * | 2022-11-30 | 2023-12-21 | 广西爱生生命科技有限公司 | Lactobacillus reuteri for prolonging lifespan, resisting aging and reducing fat, and product thereof and use thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101249109A (en) * | 2008-03-14 | 2008-08-27 | 华侨大学 | Lactobacillus reuteri hollow micro-capsule and its preparing method |
| CN105219683A (en) * | 2015-11-04 | 2016-01-06 | 广东省农业科学院动物科学研究所 | One strain has L. reuteri strain and the application thereof of prebiotic characteristics |
| US20180148801A1 (en) * | 2016-11-29 | 2018-05-31 | Wedea Inc. | Composition for Preventing and Treating Degenerative Brain Disease Using Novel Lactic Acid Bacteria |
| CN111534446A (en) * | 2019-12-11 | 2020-08-14 | 中国农业科学院特产研究所 | Lactobacillus reuteri and application thereof |
-
2021
- 2021-06-09 CN CN202110639675.0A patent/CN114717220B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101249109A (en) * | 2008-03-14 | 2008-08-27 | 华侨大学 | Lactobacillus reuteri hollow micro-capsule and its preparing method |
| CN105219683A (en) * | 2015-11-04 | 2016-01-06 | 广东省农业科学院动物科学研究所 | One strain has L. reuteri strain and the application thereof of prebiotic characteristics |
| US20180148801A1 (en) * | 2016-11-29 | 2018-05-31 | Wedea Inc. | Composition for Preventing and Treating Degenerative Brain Disease Using Novel Lactic Acid Bacteria |
| CN111534446A (en) * | 2019-12-11 | 2020-08-14 | 中国农业科学院特产研究所 | Lactobacillus reuteri and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115584333A (en) * | 2022-10-19 | 2023-01-10 | 廖梅香 | Lactobacillus reuteri and application thereof |
| WO2023240936A1 (en) * | 2022-11-30 | 2023-12-21 | 广西爱生生命科技有限公司 | Lactobacillus reuteri for prolonging lifespan, resisting aging and reducing fat, and product thereof and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114717220B (en) | 2023-06-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114717128B (en) | Lactobacillus reuteri with effects of improving aging skin and enhancing hair health and application thereof | |
| CN110835616B (en) | Active substance of lactobacillus paracasei GKS6, composition containing same and application of active substance in promoting longevity | |
| CN114717220B (en) | Lactobacillus reuteri microcapsule and preparation method thereof | |
| CN114081901B (en) | Probiotic composition, preparation method and application thereof | |
| CN114672443B (en) | Lactobacillus plantarum with functions of preventing or improving facial redness and type I rose acnes | |
| CN114806977B (en) | Lactobacillus salivarius and application thereof in preparation of anti-dermatitis products | |
| CN110833565B (en) | Active substance of lactobacillus plantarum GKM3, composition containing same and application of active substance in promoting longevity | |
| CN109481476A (en) | Application of the lactobacillus fermenti CQPC04 in the food or drug that preparation improves ulcerative colitis | |
| CN112708590A (en) | Composition for promoting growth of Achiminotropic bacteria and application thereof | |
| CN116515666A (en) | Lactobacillus helveticus with acne treatment effect and application thereof | |
| CN114703108A (en) | Lactobacillus mucilaginosus strain and application thereof in improvement of facial redness and type I rosacea | |
| CN110835615B (en) | Active substance of bifidobacterium lactis GKK2, composition containing same and application of active substance and composition in promoting longevity | |
| CN114717132A (en) | Bifidobacterium lactis capable of preventing and relieving constipation symptoms and application thereof | |
| CN114717219B (en) | Lactobacillus reuteri preparation with antioxidant and cholesterol reducing functions | |
| CN115786182B (en) | Bifidobacterium animalis and application thereof | |
| CN114711429B (en) | Lactobacillus reuteri with bone health enhancing effect and application thereof | |
| CN114657106B (en) | Lactobacillus plantarum and application thereof in preventing and treating acne | |
| CN115093985A (en) | Bifidobacterium lactis and fermentation method and application thereof | |
| CN114717134A (en) | Bifidobacterium lactis and application thereof in preventing and treating acne | |
| CN109749955A (en) | Lactobacillus brevis ZJ401 and its application | |
| WO2023093566A1 (en) | Lactobacillus helveticus having effect of treating acne and use thereof | |
| CN114657107B (en) | Lactobacillus plantarum with acne treatment effect and application thereof | |
| CN109161501A (en) | A kind of feeding bacillus licheniformis and its application | |
| CN114703079B (en) | Lactobacillus paracasei and application thereof in relieving skin injury | |
| CN117603824B (en) | Candida alcoholism and application thereof in aquaculture |
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 |