US20220160610A1 - Exopolysaccharide-protein complex, a method of preparing said complex and uses thereof - Google Patents
Exopolysaccharide-protein complex, a method of preparing said complex and uses thereof Download PDFInfo
- Publication number
- US20220160610A1 US20220160610A1 US17/570,048 US202217570048A US2022160610A1 US 20220160610 A1 US20220160610 A1 US 20220160610A1 US 202217570048 A US202217570048 A US 202217570048A US 2022160610 A1 US2022160610 A1 US 2022160610A1
- Authority
- US
- United States
- Prior art keywords
- exopolysaccharide
- kda
- protein complex
- epcx
- cells
- 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.)
- Abandoned
Links
- 238000000034 method Methods 0.000 title abstract description 20
- 229920002444 Exopolysaccharide Polymers 0.000 claims abstract description 55
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 33
- 241000894006 Bacteria Species 0.000 claims abstract description 29
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 28
- 239000012528 membrane Substances 0.000 claims abstract description 15
- 239000000203 mixture Substances 0.000 claims description 32
- 241000206596 Halomonas Species 0.000 claims description 18
- 239000002537 cosmetic Substances 0.000 claims description 17
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims 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 description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 235000000346 sugar Nutrition 0.000 claims description 6
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 claims description 5
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 5
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 claims description 5
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 5
- 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 5
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 claims description 5
- 229930182830 galactose Natural products 0.000 claims description 5
- 229960002442 glucosamine Drugs 0.000 claims description 5
- 239000002417 nutraceutical Substances 0.000 claims description 5
- 235000021436 nutraceutical agent Nutrition 0.000 claims description 5
- 102000015696 Interleukins Human genes 0.000 claims description 4
- 108010063738 Interleukins Proteins 0.000 claims description 4
- 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
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 2
- 150000007513 acids Chemical class 0.000 claims description 2
- 230000008512 biological response Effects 0.000 claims description 2
- 239000003102 growth factor Substances 0.000 claims description 2
- 229920000140 heteropolymer Polymers 0.000 claims description 2
- 239000003607 modifier Substances 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 description 50
- 150000004676 glycans Chemical class 0.000 description 22
- 229920001282 polysaccharide Polymers 0.000 description 22
- 239000005017 polysaccharide Substances 0.000 description 22
- 230000003110 anti-inflammatory effect Effects 0.000 description 17
- 239000002158 endotoxin Substances 0.000 description 17
- 229920006008 lipopolysaccharide Polymers 0.000 description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 102000004127 Cytokines Human genes 0.000 description 13
- 108090000695 Cytokines Proteins 0.000 description 13
- 206010028980 Neoplasm Diseases 0.000 description 13
- 230000002519 immonomodulatory effect Effects 0.000 description 13
- 239000000306 component Substances 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 238000004519 manufacturing process Methods 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 244000005700 microbiome Species 0.000 description 10
- 235000002639 sodium chloride Nutrition 0.000 description 10
- 201000011510 cancer Diseases 0.000 description 9
- 230000014509 gene expression Effects 0.000 description 9
- 230000012010 growth Effects 0.000 description 9
- 241000206595 Halomonas elongata Species 0.000 description 8
- 102000003814 Interleukin-10 Human genes 0.000 description 8
- 108090000174 Interleukin-10 Proteins 0.000 description 8
- 108090001007 Interleukin-8 Proteins 0.000 description 8
- 102000004890 Interleukin-8 Human genes 0.000 description 8
- 108020004999 messenger RNA Proteins 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 7
- 102000004889 Interleukin-6 Human genes 0.000 description 7
- 108090001005 Interleukin-6 Proteins 0.000 description 7
- 241000589516 Pseudomonas Species 0.000 description 7
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 7
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 7
- 238000000855 fermentation Methods 0.000 description 7
- 230000004151 fermentation Effects 0.000 description 7
- 230000034190 positive regulation of NF-kappaB transcription factor activity Effects 0.000 description 7
- 230000000770 proinflammatory effect Effects 0.000 description 7
- 238000003753 real-time PCR Methods 0.000 description 7
- 108010057466 NF-kappa B Proteins 0.000 description 6
- 102000003945 NF-kappa B Human genes 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 238000002955 isolation Methods 0.000 description 6
- 229920002477 rna polymer Polymers 0.000 description 6
- 241000590031 Alteromonas Species 0.000 description 5
- 241000193830 Bacillus <bacterium> Species 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 241000193004 Halobacillus Species 0.000 description 5
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 5
- 241001135624 Marinomonas Species 0.000 description 5
- 241000519590 Pseudoalteromonas Species 0.000 description 5
- 241000499366 Salinivibrio Species 0.000 description 5
- 241000607598 Vibrio Species 0.000 description 5
- 230000000259 anti-tumor effect Effects 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 5
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 210000002510 keratinocyte Anatomy 0.000 description 5
- 230000000813 microbial effect Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 4
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 4
- 238000000134 MTT assay Methods 0.000 description 4
- 231100000002 MTT assay Toxicity 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000001093 anti-cancer Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 229960002949 fluorouracil Drugs 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 230000002757 inflammatory effect Effects 0.000 description 4
- 239000008101 lactose Substances 0.000 description 4
- 201000005249 lung adenocarcinoma Diseases 0.000 description 4
- 210000002540 macrophage Anatomy 0.000 description 4
- 201000001441 melanoma Diseases 0.000 description 4
- 230000036961 partial effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000000638 stimulation Effects 0.000 description 4
- 230000035882 stress Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 102000019034 Chemokines Human genes 0.000 description 3
- 108010012236 Chemokines Proteins 0.000 description 3
- 108020004635 Complementary DNA Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 238000010804 cDNA synthesis Methods 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 239000002299 complementary DNA Substances 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
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 238000003468 luciferase reporter gene assay Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 210000004882 non-tumor cell Anatomy 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 241000186660 Lactobacillus Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 108010013639 Peptidoglycan Proteins 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 102000002689 Toll-like receptor Human genes 0.000 description 2
- 108020000411 Toll-like receptor Proteins 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000001994 activation Methods 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000001028 anti-proliverative effect Effects 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 206010003246 arthritis Diseases 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 229920001222 biopolymer Polymers 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 201000008274 breast adenocarcinoma Diseases 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000009295 crossflow filtration Methods 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 210000005175 epidermal keratinocyte Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 229920000912 exopolymer Polymers 0.000 description 2
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 2
- 230000009036 growth inhibition Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000003308 immunostimulating effect Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000028709 inflammatory response Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 229940039696 lactobacillus Drugs 0.000 description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000008177 pharmaceutical agent Substances 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 101150090724 3 gene Proteins 0.000 description 1
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- 101150033839 4 gene Proteins 0.000 description 1
- 208000002874 Acne Vulgaris Diseases 0.000 description 1
- 206010048998 Acute phase reaction Diseases 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 102100032252 Antizyme inhibitor 2 Human genes 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102100035882 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 206010007882 Cellulitis Diseases 0.000 description 1
- 208000015943 Coeliac disease Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 102000013382 Gelatinases Human genes 0.000 description 1
- 108010026132 Gelatinases Proteins 0.000 description 1
- 108010015899 Glycopeptides Proteins 0.000 description 1
- 102000002068 Glycopeptides Human genes 0.000 description 1
- 108010008488 Glycylglycine Proteins 0.000 description 1
- 241001420004 Halomonas anticariensis Species 0.000 description 1
- 241000251511 Holothuroidea Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000798222 Homo sapiens Antizyme inhibitor 2 Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 102000013462 Interleukin-12 Human genes 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 208000005615 Interstitial Cystitis Diseases 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 240000001046 Lactobacillus acidophilus Species 0.000 description 1
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 description 1
- 240000001929 Lactobacillus brevis Species 0.000 description 1
- 235000013957 Lactobacillus brevis Nutrition 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 108010048581 Lysine decarboxylase Proteins 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910017621 MgSO4-7H2O Inorganic materials 0.000 description 1
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 108090000913 Nitrate Reductases Proteins 0.000 description 1
- 102000052812 Ornithine decarboxylases Human genes 0.000 description 1
- 108700005126 Ornithine decarboxylases Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 241000944748 Quesada Species 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000009651 Voges-Proskauer test Methods 0.000 description 1
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 1
- 230000035508 accumulation Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000004658 acute-phase response Effects 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 150000001298 alcohols Chemical class 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
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003444 anaesthetic effect Effects 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000000767 anti-ulcer Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 206010003230 arteritis Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 230000002113 chemopreventative effect Effects 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000002301 combined effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 239000000490 cosmetic additive Substances 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 231100000050 cytotoxic potential Toxicity 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000011026 diafiltration Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- QELUYTUMUWHWMC-UHFFFAOYSA-N edaravone Chemical compound O=C1CC(C)=NN1C1=CC=CC=C1 QELUYTUMUWHWMC-UHFFFAOYSA-N 0.000 description 1
- 230000000459 effect on growth Effects 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 208000018685 gastrointestinal system disease Diseases 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 229940043257 glycylglycine Drugs 0.000 description 1
- 239000007999 glycylglycine buffer Substances 0.000 description 1
- 230000007407 health benefit Effects 0.000 description 1
- 229920006158 high molecular weight polymer Polymers 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000005918 in vitro anti-tumor Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 229940076144 interleukin-10 Drugs 0.000 description 1
- 229940117681 interleukin-12 Drugs 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229940039695 lactobacillus acidophilus Drugs 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- AIHDCSAXVMAMJH-GFBKWZILSA-N levan Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@@H]1[C@@H](O)[C@H](O)[C@](CO)(CO[C@@H]2[C@H]([C@H](O)[C@@](O)(CO)O2)O)O1 AIHDCSAXVMAMJH-GFBKWZILSA-N 0.000 description 1
- 230000006372 lipid accumulation Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000004130 lipolysis Effects 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002705 metabolomic analysis Methods 0.000 description 1
- 230000001431 metabolomic effect Effects 0.000 description 1
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 238000013048 microbiological method Methods 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 239000006837 my medium Substances 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 235000013557 nattō Nutrition 0.000 description 1
- 229920001206 natural gum Polymers 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 231100000065 noncytotoxic Toxicity 0.000 description 1
- 230000002020 noncytotoxic effect Effects 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 102000007863 pattern recognition receptors Human genes 0.000 description 1
- 108010089193 pattern recognition receptors Proteins 0.000 description 1
- 239000000816 peptidomimetic Substances 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000019612 pigmentation Effects 0.000 description 1
- 244000000003 plant pathogen Species 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 230000003938 response to stress Effects 0.000 description 1
- 239000012465 retentate Substances 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 230000037393 skin firmness Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000005758 transcription activity Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000037331 wrinkle reduction Effects 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative 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
-
- 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
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- 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
-
- 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
- A61Q19/06—Preparations for care of the skin for countering cellulitis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- 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
-
- 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
- C12P21/00—Preparation of peptides or proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Definitions
- the present invention relates to the field of biotechnology.
- the present invention relates to an exopolysaccharide-protein complex obtained from bacteria.
- the present invention further relates to a method of preparing said complex, a composition comprising thereof and uses thereof.
- EPS Microbial exopolysaccharides
- EPS production is among the biochemical strategies used by microorganisms present in 25 hypersaline environments, in order to survive in high saline conditions.
- the genus Halomonas has received increasing interest as several species are able to produce significant quantities of EPS with high surface activity and/or have rheological properties and applications in food, cosmetic and pharmaceutical sectors (Poli, Anzelmo, & Nicolaus, 2010).
- Microbial EPS are biopolymers with a high molecular weight having an extreme diversity in terms of chemical structure and composition.
- Polysaccharides are the most abundant component of the EPS but previous electron microscopy studies (Nevot, Deroncele, Lopez-Iglesias, et al., 2006; Nevot, Deroncele, Messner, Guinea, & Mercade, 2006), heavily emphasized that other macromolecules such as proteins can also be present.
- EPS also offer some important biomedical properties, including antitumor activities (Bazani Cabral de Melo et al., 2015; Ye et al., 2016), antimutagenicity (Miranda et al., 2008), anti-ulcer (Rasulov et al., 1993), anti-inflammatory properties and immune-modulating activities (Ciszek-Lenda, Nowak, Srottek, Gamian, & Marcinkiewicz, 2011).
- EPS Anti-inflammatory and immune-modulating activities of EPS are drawing much attention and in lactic acid bacteria were related to the physicochemical properties and structural characteristics of their EPS (Gorska et al., 2014; Shao et al., 2014; Yasuda, Serata. & Sako, 2009).
- An important mechanism involved in the immunostimulatory activity of polysaccharides is their ability to enhance macrophage function (Beutler, 2004). Polysaccharides were reported to be the active immunomodulators that potentiate both innate and adaptive immunity.
- NF- ⁇ B a transcription factor that promote the expression of variety of molecules involved in immune, inflammatory and acute phase responses, including NO and tissue necrosis factor alpha (Li et al., 2015), plays an important role in this activation process.
- chemopreventive activity of polysaccharides is based on their tumor anti-initiating activity through their modulation of carcinogen metabolism, in addition to the tumor anti-promoting activity through their anti-inflammatory activity (Gamal-Eldeen, Ahmed, & Abo-Zeid, 2009; Raafat, Gamal-Eldeen, El-Hussieny, Ahmed, & Eissa, 2014).
- U.S. Pat. No. 7,348,420 B2 describes the recombinant expression of cell wall, cell surface, and secreted proteins of Lactobacillus acidophilus potentially useful for the treatment or prevention of cancer, particularly colon cancer.
- this document does not disclose the combination of extracellular polysaccharides and selected associated-proteins and its use.
- U.S. Pat. No. 8,129,518 B2 provided synthetic polysaccharide antigens with anti-inflammatory or inflammatory immunomodulatory properties.
- this document does not disclose the combination of extracellular polysaccharides and selected associated-proteins and its use.
- the synthetic polysaccharide is composed mainly of lipopolysaccharide, and bacterial cell wall glycopeptides, also known as murein or peptidoglycan, from both Gram negative and Gram positive bacteria.
- U.S. Pat. No. 8,088,605 B2 discloses a delivery system for active molecule comprising exopolysaccharide micelles produced by a Lactobacillus strain. These includes active molecules such as DNA, RNA, protein, peptide, peptidomimetic, virus, bacteria, nutraceutical product and pharmaceutical agent with analgesic. anesthetic, antibiotic, anticancer, anti-inflammatory, and antiviral properties. However, these pharmaceutical agents are not produced by the bacterium.
- WO 2009/127057 A1 discloses a skin care composition comprising one exopolysaccharide derived from a microbial mat. This document discusses the use of different compositions for cosmetic or therapeutic approaches and provides examples for evaluating the effects of the EPSs on the synthesis of hyaluronic acid, lipid synthesis, among others, related with cosmeceuticals applications. However, this document does not disclose the combination of extracellular polysaccharides and selected associated-proteins and its use as anti-inflammatory or immunomodulatory agents.
- CN 104694594 discloses a preparation method of a sea cucumber epiphytic Bacillus subtilis exopolysaccharide that can be used in the anti-tumor medicines, cosmetic additives and other fields.
- the principal application exposed in this document is related with the application of this EPS in bacterial and plant pathogen growth inhibition, a peroxide scavenger, anti-tumor and inhibition of microapplication.
- U.S. Pat. No. 9,095,733 discloses methods of using polysaccharides for applications in topical personal care products, cosmetics, and for wrinkle reduction compositions.
- this document provides compositions of microalgal cells with high value cosmeceutical ingredients such as carotenoids, polyunsaturated fatty acids, moisturizing polysaccharides, superoxide dismutase, and other components.
- this document does not disclose the combination of extracellular polysaccharides and selected associated-proteins and its use as anti-inflammatory or immunomodulatory agents.
- WO 2013/082915 A1 discloses a strain of exopolysaccharide-secreting Lactobacillus brevis , and its applications in medicaments, healthcare products and food products for immunity enhancement.
- the exopolysaccharide extract is obtained after precipitating protein via trichloroacetic acid method to remove the protein.
- WO 2016/067218 discloses EPSs of Halomonas eurhalina containing rhamnose, galactose, glucose, D-glucosamine and glucouronic acid with low molecular weight proteins (10 kDa), being higher molecular weight proteins removed from the polymer.
- OMVs outer membrane vesicles
- the protein composition of these exopolimeric matrix has been studied to a considerably lesser degree than its polysaccharide composition, although protein content in some biopolymers may exceed their polysaccharide content.
- the production of these proteins inside of OMVs is one of the general envelope stress responses.
- Various stress factors such as temperature, nutrient depletion and exposure to harmful chemical agents may induce accumulation and aggregation of misfolded proteins in the periplasm.
- Packaging of these stress-products into OMV and their releases represents an efficient mechanism of alleviating stress.
- the present invention intends to preserve the entire composition of the exopolimeric material, i.e., the capsular polysaccharide with the OMVs, in order to use the proteins contained thereof. Accordingly, the present invention makes use of low-speed centrifugations, and differential filtration steps to remove residual bacteria and preserve the proteins inside the polymer, i.e as an exopolysaccharidic complex, decorated with membrane vesicles, in which proteins are concentrated in order to reduce adverse conditions generated by certain factors, such as ultraviolet radiation, oxidative stress, hypersalinity, among others.
- an exopolysaccharide-protein complex secreted by bacteria exhibits unique immunomodulating properties, is non-cytotoxic and non-proliferating to normal cell lines.
- the exopolysaccharide-protein complex ameliorates pro-inflammatory chemokines expression and induces the production of other anti-inflammatory cytokines of cells in culture. It has the activity of inhibiting growth of tumor cell lines.
- the complex can be used for treatment or prevention of diseases in which inflammation and immunomodulation are critical or as adjuvant in medical treatment, such as those related to an imbalance of the production of anti-inflammatory or proinflammatory cytokines, and also for preparing anti-tumor drugs.
- FIG. 1 shows sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel of EPCx. Analysis from SDS-PAGE gel indicates that the two principal bands have molecular weights of ⁇ 33 kDa and ⁇ 55 kDa, respectively.
- FIG. 2 shows the effect of EPCx on NF ⁇ B activation in LPS-stimulated keratinocytes.
- FIG. 3 shows the effect of EPCx on NF ⁇ B activation in LPS-stimulated THP-1 cells.
- FIG. 4 shows the growth inhibition of EPCx at different concentrations against cancer cell lines in vitro.
- FIG. 5 shows the partial 16S rRNA gene sequence of the isolate according to example 1.
- a first object of the invention relates to an exopolysaccharide-protein complex obtained from a bacterium comprising: (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins which are derived from outer membrane vesicles and have a molecular weight between 30 and 250 kDa.
- a second object of the invention relates to a method of preparing an exopolysaccharide-protein complex according to the first object.
- a third object of the invention relates to a composition (pharmaceutical, nutraceutical or cosmeceutical) comprising an exopolysaccharide-protein complex according to the first object.
- a fourth object of the invention relates to the use of the composition according to the third object of the invention and the exopolysaccharide-protein complex according to the first object of the invention.
- isolated should be considered to mean material removed from its original environment in which it naturally occurs, for example, a bacterial strain from hypersaline environment.
- EPS or “exopolysaccharide” or “EPSx” should be understood to mean high molecular weight polymers that are composed of sugar residues and expressed by bacteria.
- activation of NF- ⁇ B means the process by which stimulation of NF- ⁇ B mediated by Toll-like receptors activates NF- ⁇ B, subsequently facilitating increased transcription of mRNA coding for intracellular production of particular chemokines and cytokines and subsequent translation of the transcribed mRNA, resulting in increased amounts of particular cytokines and chemokines that are both present intracellularly and released by the eukaryotic cell into the intercellular environment.
- interleukin means any of a group of cytokines (secreted signaling molecules) that were first seen to be expressed by white blood cells. Interleukins are commonly designated using an abbreviation: e.g. IL-6, IL-8, etc.
- immunomodulatory refers to its ability to modulate the response of cells of the human immune system.
- anti-inflammatory refers to the ability to induce the production of interleukin-10, a potent anti-inflammatory cytokine and to block the production of interleukin 12, a cytokine pro-inflammatory nature.
- cancer and “tumor” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
- anticancer activity refers to preferential cytotoxic effect against tumor cells without any significant adverse effects to normal cells under the same conditions of exposure.
- the term “nutraceutical” refers to any compounds or chemicals that can provide dietary or health benefits when consumed by humans or animals.
- composition refers to a composition that is employed as both a cosmetic composition and as a pharmaceutical composition.
- adjuvant refers to an embodiment of the invention provided to a subject in conjunction with a medical treatment plan.
- immunomodulation refers to the ability to modify the immune responses in a subject in a way that may have healthful benefits, such as to produce an anti-inflammatory or an immunostimulatory effect.
- RT-PCR means reverse transcription polymerase chain reaction (RT-PCR), a laboratory technique for amplifying a defined piece of a ribonucleic acid (RNA) molecule.
- RNA ribonucleic acid
- RT-PCR reverse transcription polymerase chain reaction
- the RNA strand is first reverse transcribed into its DNA complement or complementary DNA, followed by amplification of the resulting DNA using polymerase chain reaction. This can either be a 1 or 2 step process.
- exopolysaccharide-protein complex should be understood as an entity in which the proteins are not found as mere impurities. In contrast, they contribute to the biological effect/activity of the complex which is different from that obtained for the exopolysaccharide alone.
- the present invention relates to an exopolysaccharide-protein complex (also called herein EPCx) obtained from a bacterium, preferably wherein said bacterium is from a genus selected from Halomonas, Pseudoalteromonas, Vibrio, Salinivibrio, Marinomonas, Alteromonas, Pseudomonas, Halobacillus , and Bacillus , more preferably from genus Halomonas , still more preferably from Halomonas elongata sp., comprising: (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins which are derived from outer membrane vesicles and have a molecular weight between 30 and 250 kDa.
- EPCx exopolysaccharide-protein complex
- exopolysaccharide-protein complex are not limited to (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins which are derived from outer membrane vesicles and have a molecular weight between 30 and 250 kDa.
- said exopolysaccharide-protein complex obtained from a bacterium consists of: (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins which are derived from outer membrane vesicles and have a molecular weight between 30 and 250 kDa.
- the term “consists of” should be understood as the components of the exopolysaccharide-protein complex are exclusively (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins which are derived from outer membrane vesicles and have a molecular weight between 30 and 250 kDa.
- the exopolysaccharide-protein complex of the present invention relates to an exopolysaccharide-protein complex comprising: (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins which are derived from outer membrane vesicles and have a molecular weight between 30 and 250 kDa, wherein the crude exopolysaccharide is a heteropolymer comprising or consisting of:
- the exopolysaccharide-protein complex of the invention comprises or consists of:
- the components (a), (b), (c) and (d) must amount 100 wt % if components (a), (b), (c) and (d) are the only components in the exopolysaccharide-protein complex or the components (a), (b), (c) and (d) plus any further component(s) must amount 100 wt % if components (a), (b), (c) and (d) are not the only components in the exopolysaccharide-protein complex.
- the crude exopolysaccharide further comprises sulfate, preferably at a concentration from 2 to 10 wt %.
- the exopolysaccharide-associated proteins which are derived from outer membrane vesicles and have a molecular weight between 30 and 250 kDa include two bands of molecular weight as obtained by SDS-PAGE.
- the EPCx has a protein profile comprising at least, according to the SDS-PAGE technique, 12 detectable bands, including two principal bands, corresponding, respectively, to molecular weights (approximate molecular weights given in relation to molecular standards, notably provided by Bio-Rad Laboratories) ranging between:
- the present invention relates to a method of preparing an exopolysaccharide-protein complex according to the first aspect of the invention, including each of the embodiments comprised in said first aspect and combinations thereof.
- the method of preparing an exopolysaccharide-protein complex comprises the steps of:
- the genus is selected from Halomonas, Pseudoalteromonas, Vibrio, Salinivibrio, Marinomonas, Alteromonas, Pseudomonas, Halobacillus , and Bacillus.
- said bacteria genus is Halomonas and in a more preferred embodiment, Halomonas elongata sp. is used as a bacterium belonging to Halomonas genus.
- the medium suitable to cultivate the above mentioned bacteria includes a synthetic medium comprising a carbon source selected from the group consisting of: lactose, maltose, glucose, galactose, sucrose, glycerol and mixtures thereof.
- a carbon source selected from the group consisting of: lactose, maltose, glucose, galactose, sucrose, glycerol and mixtures thereof.
- the carbon source is selected from the group consisting of: glucose, lactose, sucrose, and mixtures thereof.
- the carbon source is lactose.
- the other fermentation medium components are: potassium phosphate dibasic (K 2 HPO 4 ), 0.5-1.0 wt %; potassium phosphate monobasic (KH 2 PO 4 ), 0.1-0.5 wt %; sodium chloride (NaCl), 5.0-10.0 wt %; magnesium sulfate heptahydrate (MgSO 4 -7H 2 O). 0.01-0.05 wt %; ammonium sulfate (NH 4 ) 2 SO 4 , 0.05 wt %-0.5 wt % and peptone, 0.02-0.1 wt %.
- K 2 HPO 4 potassium phosphate dibasic
- KH 2 PO 4 potassium phosphate monobasic
- MgSO 4 -7H 2 O magnesium sulfate heptahydrate
- the suitable medium for culturing is a synthetic medium with a pH value ranging from 6 to 8. In a more preferred embodiment, the suitable medium has a pH value of 7.
- the culture step is conducted in fermenters operating at a temperature ranging from 25° C. to 37° C. In a more preferred embodiment, the temperature is 32° C.
- the present invention provides a fermentation process comprising a fermentation step allowing to grow a strain of the corresponding bacteria, preferably wherein said bacteria is from a genus selected from Halomonas, Pseudoalteromonas, Vibrio, Salinivibrio, Marinomonas, Alteromonas, Pseudomonas, Halobacillus , and Bacillus , more preferably from genus Halomonas , still more preferably from Halomonas elongata sp., in a suitable medium in a fermenter under conditions of agitation sufficient to maintain a homogenous culture and limited aeration such that dissolved oxygen pressure (pO2) within the culture is around 20 to 40% for most of the fermentation step.
- pO2 within the culture is 30% in the fermentation step.
- the inventive process of preparing an exopolysaccharide-protein complex comprises the step of isolating the exopolysaccharide-protein complex from the secreted fraction of the culture.
- Said isolation can be carried by removing other molecules present in the culture media by alcohol precipitation.
- alcohols which can be used include ethanol, isopropanol, and methanol.
- the isolation and purification of the EPCx after alcohol precipitation can be conducted by tangential flow filtration methods using ultrafiltration membranes.
- said membranes have a MWCO of 30 kDa and the retentate recovered after the ultrafiltration comprises the exopolysaccharide and the EPS-associate proteins which can be recovered.
- the tangential flow filtration can act to both diafilter and concentrate the EPCx.
- the present invention relates to a composition (pharmaceutical, nutraceutical or cosmeceutical) comprising an exopolysaccharide-protein complex according to the first object of the invention, including each of the embodiments comprised in said first aspect and combinations thereof.
- said composition further comprises a biological response modifier selected from the group consisting of lymphokine, interleukin, growth factor and NFkB factor.
- the present invention relates to a composition comprising an exopolysaccharide-protein complex according to the first object of the invention, including each of the embodiments comprised in said first aspect and combinations thereof, for use in stimulating an immune response in a subject for preventing or treating a disease selected from cancer or a disease associated to undesirable inflammatory activity.
- the present invention also relates to a method for preventing or treating a disease selected from cancer or a disease associated to undesirable inflammatory activity, the method comprising administering to a subject in need thereof a therapeutically effective amount of an exopolysaccharide-protein complex according to the first object of the invention, including each of the embodiments comprised in said first aspect and combinations thereof, or a composition comprising thereof.
- said disease associated to undesirable inflammatory activity is selected from allergy, Alzheimer's disease, arthritis, autoimmune deficiency syndrome, celiac disease, diabetes mellitus, gastrointestinal disorder, inflammatory bowel disease, interstitial cystitis, skin disorders, acne, arteritis, arthritis, cancer, cellulitis, dermatitis and cardiovascular diseases.
- said cancer is liver cancer or a hepatocellular carcinoma.
- said exopolysaccharide-protein complex according to the first object of the invention is used as an anticancer agent or adjuvant agent for cancer therapies.
- the present invention provides an in vitro toxicity test to evaluate the cytotoxic potential of EPCx on non-tumor cell lines.
- the cytotoxicity evaluation of each concentration of EPCx was performed by using the MTT Cell Proliferation Assay in order to determine the concentrations which are not harmful to Human Epidermal Keratinocytes (HEK) and human monocyte-like cells (THP-1).
- HEK Human Epidermal Keratinocytes
- THP-1 human monocyte-like cells
- the EPCx show in vitro anti-proliferative activity against a panel of one or more cancer cell lines, including human lung adenocarcinoma cell line (H1975), Human melanoma cell line (A375), and human hepatocellular carcinoma cell line (HepG2).
- human lung adenocarcinoma cell line H1975)
- Human melanoma cell line A375
- human hepatocellular carcinoma cell line HepG2
- the anticancer properties of EPCx was ascertained by MTT assay and showed that EPCx significantly inhibit the growth of human lung adenocarcinoma cell line (H1975), Human melanoma cell line (A375), and human hepatocellular carcinoma cell line (HepG2).
- EPCx The immunomodulatory and anti-proliferative properties of EPCx were ascertained by the analysis of the induced activation of human primary keratinocytes (HEK) and human monocyte-like cells (THP-1) stressed with LPS (lipopolysaccharide), analyzing the expression of mRNA for the cytokines IL-6, IL-8, IL-10 and TNF- ⁇ , by real-time PCR and the NF- ⁇ B activation using a luciferase reporter gene assay.
- HEK human primary keratinocytes
- TNF- ⁇ human monocyte-like cells
- the immunomodulatory and anti-cancer properties of the EPCx are showed preferably at EPCx concentrations between 0.01 mg/ml to 1.0 mg/ml.
- an EPCx obtained from a bacterium preferably wherein said bacterium is from a genus selected from Halomonas, Pseudoalteromonas, Vibrio, Salinivibrio, Marinomonas, Alteromonas, Pseudomonas, Halobacillus , and Bacillus , more preferably from genus Halomonas , still more preferably from Halomonas elongate sp., in accordance with the invention, can be used as anti-inflammatory and/or inmunomodulator agent, and is in particular able to inhibit a pro-inflammatory stimulation of normal cells in culture.
- EPCx exhibits tumor anti-promoting properties in cancer cell lines culture.
- a microorganism included in the present invention (deposited with number DSM 32408 at the depositary institution Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH by INKEMIA IUCT (Institut Univ. de Ciència i Tecnologia) Group) is isolated from a rock salt from solar salternsin Cardona (Spain) by serial dilution and plating on MH agar medium (Ventosa, Garcia, Kamekura, Onishi, & Ruizberraquero, 1989). The plates were incubated at 32° C. for 3 to 5 days, and bacterial colonies were isolated in pure form and maintained on slopes of the same medium.
- the bacterium isolates was grown in MY medium (Moraine & Rogovin, 1966) supplemented with 5-10% NaCl for 3-5 days at 32° C. under continuous shaking (120 rpm).
- the EPS from the growing culture was isolated using the method as described by Quesada et al. (Quesada, Bejar, & Calvo, 1993) and used for quantification and chemical analysis.
- the selected microorganism was analyzed by physiological and biochemical methods following standard microbiological methods (Table 1). As a result, based on physiological and biochemical analysis thereof, the microorganism of the present invention was confirmed to have similarity to Halomonas genus.
- the present inventors analyzed base sequence of ribosome small subunit gene. Particularly, genomic DNA was separated by using MasterPureTM DNA purification from Epicentre @ Biotechnologies Germany according to the manufacturer's instruction. PCR mediated amplification of 16S rDNA and purification of the PCR product was carried out as described previously (Rainey, WardRainey, Kroppenstedt, & Stackebrandt, 1996). As a result of database analysis on the decided base sequence using NCBI (National Center for Biological Information) BLAST, the microorganism was confirmed to have similarity to Halomonas genera. However, it had a little difference from the typical Halomonas elongata , so that the microorganism of the present invention was finally named as Halomonas elongata.
- NCBI National Center for Biological Information
- the partial 16S rRNA gene sequence is shown in FIG. 5 .
- Example 2 Preparation and Isolation of the Exopolysaccharide-Protein Complex (EPCx) Excreted by Halomonas Elongata According to Example 1
- the strain of the species Halomonas elongate (deposited with number DSM 32408 at the depositary institution Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH by INKEMIA IUCT (Institut Univ. de Ciència i Tecnologia) Group) was cultured in a fermenter, at 32° C. and at a pH of 7.5, whose broth contained (g L ⁇ 1 ): 100 NaCl; 50 Lactose; 7 K 2 HPO4; 2 KH 2 PO4; 0.1 MgSO4.7H 2 O; 1 (NH 4 )SO 4 and 0.5 Peptone.
- An inoculum was prepared with 10% (v/v) of a pre-culture and the duration of the fermentation was extended to 72 hours.
- the reactors are operated in batch mode, and dissolved oxygen was controlled by the agitation (300 to 900 rpm) at pO 2 ⁇ 30%.
- the bacteria were separated from the broth by centrifugation at 12,000 g for 45 min.
- the resulting clear solution was subjected to ultrafiltration and dialysis using an installation for ultrafiltration (Sartocon® Slice Cassette, Sartorius Stedim), membrane exclusion limit 30 KDa. If necessary, the final solution may be lyophilized and purified obtaining an exopolysaccharide-protein complex with a yield of 60-80%.
- the content of neutral and acid monosaccharides of the exopolysaccharide obtained according to Example 2 was determined by a method described by Honda et al. (Honda et al., 1989) and Yang et al. (X. B. Yang, Zhao, Wang, Wang, & Mei, 2005; X. B. Yang, Zhao, Zhou, et al., 2005). Briefly, the purified polysaccharide sample (1 mg) was hydrolyzed with 1 ml of 2 M trifluoroacetic acid at 120° C. for 2 h, derivatized with 1-phenyl-3-methyl-5-pyrazolone, and subsequently analyzed by high-performance liquid chromatography with detection by absorbance monitoring at 245 nm.
- the percent relationship of sugars obtained was 30-60% of glucose, 30-50% galactose, 5-10% or glucuronic acid, 1-10% of rhamnose and 1-10% of glucosamine, being the amounts consistent with a total of 100%.
- SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Laemmli, 1970) was carried out with a 4% stacking and a 9% separating gel.
- the EPCx sample was dissolved at 10 mg/mL in distilled water and added at 1:3 volume ratio into a buffer solution of 0.5% SDS with 1%-mercaptoethanol, and then heated to boiling for 5 min.
- the gels were stained with Coomassie Brilliant Blue R-250 to visualize proteins. 12 detectable bands, including three principal bands of 38 kDa, 46 kDa and 54 kDa were observed (see FIG. 1 ).
- the cytotoxicity evaluation of exopolysaccharide-protein complex was performed by using the MTT assay in order to determine the concentrations which are not harmful to Human Epidermal Keratinocytes (HEK) and human monocyte-like cells (THP-1). Proliferation of cells lines was measured based on the mitochondria-dependent reduction of yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide), to generate reducing equivalents such as NADH and NADPH. Briefly, 5 ⁇ 10 3 cells in 100 ⁇ L per well were plated in 96-well tissue culture plates for 24 h.
- EPS EPS to modulate the inflammatory response was evaluated in vitro in human primary keratinocytes (HEK) and human monocyte-like cells (THP-1) stressed with LPS (lipopolysaccharide).
- the cells were pretreated with different concentrations of exopolysaccharide-protein complex (1, 0.1 and 0.01 mg/ml) for 24 h and with 10 ⁇ g/ml of LPS for 24 h.
- Controls of HEK and THP-1 cells treated only with the different concentrations of exopolysaccharide-protein complex were also prepared.
- mRNA for the cytokines IL-6, IL-8, IL-10 and TNF- ⁇ was analyzed by real-time PCR and the NF- ⁇ B activation was measured using a luciferase reporter gene assay.
- CT threshold cycles
- EPCx exopolysaccharide-protein complex
- NF- ⁇ B activation was measured using a luciferase reporter gene assay.
- THP-1 macrophages (1 ⁇ 10 5 cells well ⁇ 1 ) were transfected with the pNF- ⁇ Bluciferase reporter gene construct (Stratagene) using Lipofectamine LTX plus (Invitrogen).
- EPS 1, 0.1 and 0.01 mg/ml
- lysis buffer 200 ⁇ L ⁇ well ⁇ 1
- lysis buffer 25 mM glycylglycine, 15 mM MgSO 4 , 4 mM EGTA, 1 mM dithiothreitol and 1% Triton X-100.
- Lysed cells were centrifuged (5 min, 9000 g) and stored at ⁇ 80° C. until assay.
- Luciferase activity was measured using luciferin (1 mM in glycylglycine buffer. 300 ⁇ L ⁇ sample ⁇ 1 ) in a luminometer at 562 nm.
- EPCx dose-dependently downregulated the NF- ⁇ B transcription activity in keratinocyte cells ( FIG. 2 ), whereas in EPCx-treated human monocyte-like cells, the NF-kB levels were comparable to those obtained in the THP-1 cells treated with LPS ( FIG. 3 ).
- the MTT assay was used for measuring the proliferation of the tumor cells. Briefly, human hepatocellular carcinoma cell line (HepG2), human breast adenocarcinoma cell line (MCF7), human lung adenocarcinoma cell line (H1975) and Human melanoma cell line (A375) were seeded at a density of 4 ⁇ 10 4 cells/mL in a volume of 0.1 mL in 96-well plates, respectively. After 24 h, different concentrations of EPCx (1.0, 0.1 and 0.01 mg/ml) were dissolved in the medium was added to each well and incubated for 48 h at 37° C. in a CO 2 incubator. 5-Fu (5-fluorouracil) was used as the positive control.
- EPCx 1.0, 0.1 and 0.01 mg/ml
- EPCx markedly inhibited proliferation of human hepatocellular carcinoma cell line (HepG2) in a dose-dependent manner ( FIG. 4 ), with little effect on growth of human breast adenocarcinoma cell line (MCF7), human lung adenocarcinoma cell line (H1975) and Human melanoma cell line (A375).
- MCF7 human breast adenocarcinoma cell line
- H1975 human lung adenocarcinoma cell line
- A375 Human melanoma cell line
- the potency of EPCx (at 1.0 mg/mL) to HepG2 cells was found to be similar to 5-fluorouracil (5-FU, IC50 was 10.0 ⁇ mol/L).
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Birds (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Dermatology (AREA)
- Molecular Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Mycology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
- The present invention relates to the field of biotechnology. In particular, the present invention relates to an exopolysaccharide-protein complex obtained from bacteria. The present invention further relates to a method of preparing said complex, a composition comprising thereof and uses thereof.
- Microbial exopolysaccharides (EPS) are synthesized by a wide variety of bacteria in natural ecological environments, mainly involved in the prevention of desiccation, protection against toxic and/or environmental stresses and adherence to surfaces (De Vuyst, De Vin, Vaningelgem, & Degeest, 2001)(Nwodo, Green, & Okoh, 2012).
- The number of EPSs produced by microbial fermentation has gradually increased with many applications in the food and pharmaceutical industries, among others, as their physiological activities differ from those of natural gums and synthetic polymers (Freitas, Alves, & Reis, 2011).
- EPS production is among the biochemical strategies used by microorganisms present in 25 hypersaline environments, in order to survive in high saline conditions. Among them, the genus Halomonas has received increasing interest as several species are able to produce significant quantities of EPS with high surface activity and/or have rheological properties and applications in food, cosmetic and pharmaceutical sectors (Poli, Anzelmo, & Nicolaus, 2010).
- Microbial EPS are biopolymers with a high molecular weight having an extreme diversity in terms of chemical structure and composition. Polysaccharides are the most abundant component of the EPS but previous electron microscopy studies (Nevot, Deroncele, Lopez-Iglesias, et al., 2006; Nevot, Deroncele, Messner, Guinea, & Mercade, 2006), heavily emphasized that other macromolecules such as proteins can also be present.
- Along with the general properties such as bio-compatibility, bio-degradability, renewability, flexibility, and eco-friendliness, EPS also offer some important biomedical properties, including antitumor activities (Bazani Cabral de Melo et al., 2015; Ye et al., 2016), antimutagenicity (Miranda et al., 2008), anti-ulcer (Rasulov et al., 1993), anti-inflammatory properties and immune-modulating activities (Ciszek-Lenda, Nowak, Srottek, Gamian, & Marcinkiewicz, 2011).
- Anti-inflammatory and immune-modulating activities of EPS are drawing much attention and in lactic acid bacteria were related to the physicochemical properties and structural characteristics of their EPS (Gorska et al., 2014; Shao et al., 2014; Yasuda, Serata. & Sako, 2009). An important mechanism involved in the immunostimulatory activity of polysaccharides is their ability to enhance macrophage function (Beutler, 2004). Polysaccharides were reported to be the active immunomodulators that potentiate both innate and adaptive immunity. They can bind to pattern recognition receptors on the surface of macrophages, such as toll-like receptors, neutrophils, monocytes, NK cells and dendritic cells, and then trigger several signaling pathways to activate macrophages (Kim, Hong, Kim, & Han, 2011). NF-κB, a transcription factor that promote the expression of variety of molecules involved in immune, inflammatory and acute phase responses, including NO and tissue necrosis factor alpha (Li et al., 2015), plays an important role in this activation process.
- Recently, a number of studies on the action mechanisms of polysaccharides have demonstrated that polysaccharides could also inhibit the tumor growth in vivo for their immunomodulatory activities (Sun, Li, Qi, Gao, & Lin, 2014; J. Yang, Li, Xue, Wang, & Liu, 2014; Zheng, Wang, & Li, 2015). They exert anti-tumor activity by boosting host's natural immune defense. Other work has suggested that chemopreventive activity of polysaccharides is based on their tumor anti-initiating activity through their modulation of carcinogen metabolism, in addition to the tumor anti-promoting activity through their anti-inflammatory activity (Gamal-Eldeen, Ahmed, & Abo-Zeid, 2009; Raafat, Gamal-Eldeen, El-Hussieny, Ahmed, & Eissa, 2014).
- Document WO2015063240A1 describes the cosmetic and/or dermopharmaceutical use of an EPS produced by Halomonas anticariensis, specifically, for the treatment and/or care of the skin, and in particular, its use for inflammation, lipolysis, lipid accumulation and skin firmness. However, this document does not disclose the combination of extracellular polysaccharides and selected associated-proteins and its use.
- Document WO 2015/117985 relates to a strain of Pseudomonas, a cold-adapted bacteria, useful for cosmetic compositions. However, this document does not encompass the use of the crude exopolymeric material, including the associated protein. The composition discloses the use of a partial or completely hydrolized EPS derived from Pseudomonas for several purposes, including dermoprotection.
- Document WO 2010/023178 A1 describes the use of bacterial polysaccharide derived from species of the genera Bifidobacterium. Streptococcus and Lactobacillus, some of which are considered as probiotic bacteria for treating inflammatory diseases, specifically, colitis or Crohn's disease. The application evaluated the anti-inflammatory potential of the purified EPS in a murine dendritic cell assay and/or an assay involving a human intestinal epithelial cell line. However, this document does not disclose the combination of extracellular polysaccharides and selected associated-proteins and its use.
- U.S. Pat. No. 7,348,420 B2 describes the recombinant expression of cell wall, cell surface, and secreted proteins of Lactobacillus acidophilus potentially useful for the treatment or prevention of cancer, particularly colon cancer. However, this document does not disclose the combination of extracellular polysaccharides and selected associated-proteins and its use.
- U.S. Pat. No. 8,129,518 B2 provided synthetic polysaccharide antigens with anti-inflammatory or inflammatory immunomodulatory properties. However, this document does not disclose the combination of extracellular polysaccharides and selected associated-proteins and its use. In this case the synthetic polysaccharide is composed mainly of lipopolysaccharide, and bacterial cell wall glycopeptides, also known as murein or peptidoglycan, from both Gram negative and Gram positive bacteria.
- U.S. Pat. No. 8,088,605 B2 discloses a delivery system for active molecule comprising exopolysaccharide micelles produced by a Lactobacillus strain. These includes active molecules such as DNA, RNA, protein, peptide, peptidomimetic, virus, bacteria, nutraceutical product and pharmaceutical agent with analgesic. anesthetic, antibiotic, anticancer, anti-inflammatory, and antiviral properties. However, these pharmaceutical agents are not produced by the bacterium.
- WO 2009/127057 A1 discloses a skin care composition comprising one exopolysaccharide derived from a microbial mat. This document discusses the use of different compositions for cosmetic or therapeutic approaches and provides examples for evaluating the effects of the EPSs on the synthesis of hyaluronic acid, lipid synthesis, among others, related with cosmeceuticals applications. However, this document does not disclose the combination of extracellular polysaccharides and selected associated-proteins and its use as anti-inflammatory or immunomodulatory agents.
- CN 104694594 discloses a preparation method of a sea cucumber epiphytic Bacillus subtilis exopolysaccharide that can be used in the anti-tumor medicines, cosmetic additives and other fields. The principal application exposed in this document is related with the application of this EPS in bacterial and plant pathogen growth inhibition, a peroxide scavenger, anti-tumor and inhibition of microapplication.
- U.S. Pat. No. 9,095,733 discloses methods of using polysaccharides for applications in topical personal care products, cosmetics, and for wrinkle reduction compositions. Particularly, this document provides compositions of microalgal cells with high value cosmeceutical ingredients such as carotenoids, polyunsaturated fatty acids, moisturizing polysaccharides, superoxide dismutase, and other components. However, this document does not disclose the combination of extracellular polysaccharides and selected associated-proteins and its use as anti-inflammatory or immunomodulatory agents.
- WO 2013/082915 A1 discloses a strain of exopolysaccharide-secreting Lactobacillus brevis, and its applications in medicaments, healthcare products and food products for immunity enhancement. The exopolysaccharide extract is obtained after precipitating protein via trichloroacetic acid method to remove the protein.
- Filomena Freitas et al. “Advances in bacterial exopolysaccharides: from production to biotechnological applications”, Trends In Biotechnology, vol. 29, no. 8, 2011, discloses EPSs composed of carbohydrates like glucose, galactose, mannose, rhamnose and fucose which in the purification process may contain impurities of proteins.
- Anthony Courtois et al. “Exopolysaccharides isolated from hydrothermal vent bacteria can modulate the complement system”, Plos One, vol. 9, No. 4, 2014, disclose EPSs from Atleromonas infernus wherein the proteins are also considered as impurities since no biological function/activity is determined.
- WO 2016/067218 discloses EPSs of Halomonas eurhalina containing rhamnose, galactose, glucose, D-glucosamine and glucouronic acid with low molecular weight proteins (10 kDa), being higher molecular weight proteins removed from the polymer.
- It is well known that the protein content detected in the EPS are released to the media through membrane vesicles. Gram-negative bacteria constitutively secrete native outer membrane vesicles (OMVs) into the extracellular milieu, and recent progress in this area has revealed that OMVs are essential for bacterial survival.
- The protein composition of these exopolimeric matrix has been studied to a considerably lesser degree than its polysaccharide composition, although protein content in some biopolymers may exceed their polysaccharide content. The production of these proteins inside of OMVs is one of the general envelope stress responses. Various stress factors such as temperature, nutrient depletion and exposure to harmful chemical agents may induce accumulation and aggregation of misfolded proteins in the periplasm. Packaging of these stress-products into OMV and their releases represents an efficient mechanism of alleviating stress.
- The present invention intends to preserve the entire composition of the exopolimeric material, i.e., the capsular polysaccharide with the OMVs, in order to use the proteins contained thereof. Accordingly, the present invention makes use of low-speed centrifugations, and differential filtration steps to remove residual bacteria and preserve the proteins inside the polymer, i.e as an exopolysaccharidic complex, decorated with membrane vesicles, in which proteins are concentrated in order to reduce adverse conditions generated by certain factors, such as ultraviolet radiation, oxidative stress, hypersalinity, among others.
- Thus, the above prior art known processes relating to the production of EPS from microbial sources do not disclose the combined effect of polysaccharide fraction with selected extracellular EPS-associated proteins which are derived from outer membrane vesicles and have a molecular weight between 30 and 250 kDa.
- The present inventors have surprisingly found that an exopolysaccharide-protein complex secreted by bacteria exhibits unique immunomodulating properties, is non-cytotoxic and non-proliferating to normal cell lines. The exopolysaccharide-protein complex ameliorates pro-inflammatory chemokines expression and induces the production of other anti-inflammatory cytokines of cells in culture. It has the activity of inhibiting growth of tumor cell lines. These properties have been found to be enhanced when the crude exopolymer are associated with the proteins derived from outer membrane vesicles.
- Accordingly, the complex can be used for treatment or prevention of diseases in which inflammation and immunomodulation are critical or as adjuvant in medical treatment, such as those related to an imbalance of the production of anti-inflammatory or proinflammatory cytokines, and also for preparing anti-tumor drugs.
-
FIG. 1 shows sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel of EPCx. Analysis from SDS-PAGE gel indicates that the two principal bands have molecular weights of ˜33 kDa and ˜55 kDa, respectively. -
FIG. 2 shows the effect of EPCx on NFκB activation in LPS-stimulated keratinocytes. -
FIG. 3 shows the effect of EPCx on NFκB activation in LPS-stimulated THP-1 cells. -
FIG. 4 shows the growth inhibition of EPCx at different concentrations against cancer cell lines in vitro. -
FIG. 5 shows the partial 16S rRNA gene sequence of the isolate according to example 1. - A first object of the invention relates to an exopolysaccharide-protein complex obtained from a bacterium comprising: (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins which are derived from outer membrane vesicles and have a molecular weight between 30 and 250 kDa.
- A second object of the invention relates to a method of preparing an exopolysaccharide-protein complex according to the first object.
- A third object of the invention relates to a composition (pharmaceutical, nutraceutical or cosmeceutical) comprising an exopolysaccharide-protein complex according to the first object.
- A fourth object of the invention relates to the use of the composition according to the third object of the invention and the exopolysaccharide-protein complex according to the first object of the invention.
- In order to facilitate the comprehension of this invention, the meanings of some terms and expressions as used in the context of the invention are included.
- As used herein, the term “isolated” should be considered to mean material removed from its original environment in which it naturally occurs, for example, a bacterial strain from hypersaline environment.
- As used herein, the term “EPS” or “exopolysaccharide” or “EPSx” should be understood to mean high molecular weight polymers that are composed of sugar residues and expressed by bacteria.
- As used herein, “activation of NF-κB (nuclear factor-kappa B)” means the process by which stimulation of NF-κB mediated by Toll-like receptors activates NF-κB, subsequently facilitating increased transcription of mRNA coding for intracellular production of particular chemokines and cytokines and subsequent translation of the transcribed mRNA, resulting in increased amounts of particular cytokines and chemokines that are both present intracellularly and released by the eukaryotic cell into the intercellular environment.
- As used herein, “interleukin” means any of a group of cytokines (secreted signaling molecules) that were first seen to be expressed by white blood cells. Interleukins are commonly designated using an abbreviation: e.g. IL-6, IL-8, etc.
- As used herein, the term “immunomodulatory” refers to its ability to modulate the response of cells of the human immune system.
- As used herein, “anti-inflammatory” refers to the ability to induce the production of interleukin-10, a potent anti-inflammatory cytokine and to block the production of interleukin 12, a cytokine pro-inflammatory nature.
- As used herein, “cancer” and “tumor” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
- As used herein, the term “anticancer activity” as used herein refers to preferential cytotoxic effect against tumor cells without any significant adverse effects to normal cells under the same conditions of exposure.
- As used herein, the term “nutraceutical” refers to any compounds or chemicals that can provide dietary or health benefits when consumed by humans or animals.
- As used herein, the term “cosmeceutical composition” refers to a composition that is employed as both a cosmetic composition and as a pharmaceutical composition.
- As used herein, the term “adjuvant” refers to an embodiment of the invention provided to a subject in conjunction with a medical treatment plan.
- As used herein. “immunomodulation”, “immunomodulatory”, and similar terms refer to the ability to modify the immune responses in a subject in a way that may have healthful benefits, such as to produce an anti-inflammatory or an immunostimulatory effect.
- As used herein, “RT-PCR” means reverse transcription polymerase chain reaction (RT-PCR), a laboratory technique for amplifying a defined piece of a ribonucleic acid (RNA) molecule. The RNA strand is first reverse transcribed into its DNA complement or complementary DNA, followed by amplification of the resulting DNA using polymerase chain reaction. This can either be a 1 or 2 step process.
- As used herein, the term “exopolysaccharide-protein complex” should be understood as an entity in which the proteins are not found as mere impurities. In contrast, they contribute to the biological effect/activity of the complex which is different from that obtained for the exopolysaccharide alone.
- As used herein “preventing or treating” does not exclude that both actions “preventing” and “treating” can be carried out on the same subject.
- In a first aspect the present invention relates to an exopolysaccharide-protein complex (also called herein EPCx) obtained from a bacterium, preferably wherein said bacterium is from a genus selected from Halomonas, Pseudoalteromonas, Vibrio, Salinivibrio, Marinomonas, Alteromonas, Pseudomonas, Halobacillus, and Bacillus, more preferably from genus Halomonas, still more preferably from Halomonas elongata sp., comprising: (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins which are derived from outer membrane vesicles and have a molecular weight between 30 and 250 kDa. In this context, the term “comprising” should be understood as the components of the exopolysaccharide-protein complex are not limited to (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins which are derived from outer membrane vesicles and have a molecular weight between 30 and 250 kDa.
- In another embodiment, said exopolysaccharide-protein complex obtained from a bacterium consists of: (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins which are derived from outer membrane vesicles and have a molecular weight between 30 and 250 kDa. In this context, the term “consists of” should be understood as the components of the exopolysaccharide-protein complex are exclusively (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins which are derived from outer membrane vesicles and have a molecular weight between 30 and 250 kDa.
- In a preferred embodiment, the exopolysaccharide-protein complex of the present invention relates to an exopolysaccharide-protein complex comprising: (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins which are derived from outer membrane vesicles and have a molecular weight between 30 and 250 kDa, wherein the crude exopolysaccharide is a heteropolymer comprising or consisting of:
-
- glucose units;
- galactose units;
- uronic acid units; and
- other sugar units selected from rhamnose, glucosamine and a mixture of rhamnose and glucosamine.
- In a further preferred embodiment, the exopolysaccharide-protein complex of the invention comprises or consists of:
- (a) 30 to 60 wt % glucose;
(b) 30 to 50 wt % galactose;
(c) 5 to 10 wt % uronic acids; and
(d) 1 to 10 wt % of other sugar units,
providing that the sum of the components of the crude exopolysaccharide is 100 wt %, i.e. the components (a), (b), (c) and (d) must amount 100 wt % if components (a), (b), (c) and (d) are the only components in the exopolysaccharide-protein complex or the components (a), (b), (c) and (d) plus any further component(s) must amount 100 wt % if components (a), (b), (c) and (d) are not the only components in the exopolysaccharide-protein complex. - In a further preferred embodiment, in the exopolysaccharide-protein complex, the crude exopolysaccharide further comprises sulfate, preferably at a concentration from 2 to 10 wt %.
- In a further preferred embodiment, in the exopolysaccharide-protein complex, the exopolysaccharide-associated proteins which are derived from outer membrane vesicles and have a molecular weight between 30 and 250 kDa include two bands of molecular weight as obtained by SDS-PAGE. In particular, the EPCx has a protein profile comprising at least, according to the SDS-PAGE technique, 12 detectable bands, including two principal bands, corresponding, respectively, to molecular weights (approximate molecular weights given in relation to molecular standards, notably provided by Bio-Rad Laboratories) ranging between:
-
- band 1: 30 kDa and 40 kDa, in particular 33 kDa:
- band 2: 51 kDa and 60 kDa, in particular 55 kDa.
- In a second aspect, the present invention relates to a method of preparing an exopolysaccharide-protein complex according to the first aspect of the invention, including each of the embodiments comprised in said first aspect and combinations thereof.
- The method of preparing an exopolysaccharide-protein complex comprises the steps of:
-
- culturing one or more bacteria from genus including, but not limited thereto, Halomonas. Pseudoalteromonas, Vibrio, Salinivibrio, Marinomonas, Alteromonas, Pseudomonas. Halobacillus, Bacillus or any other bacterium isolated from a hypersaline environment, the term “hypersaline” referred to a kind of extreme environments that have salt concentrations much greater than that of seawater, often close to or exceeding salt saturation;
- isolating the exopolysaccharide-protein complex from the secreted fraction of the culture.
- In a preferred embodiment, the genus is selected from Halomonas, Pseudoalteromonas, Vibrio, Salinivibrio, Marinomonas, Alteromonas, Pseudomonas, Halobacillus, and Bacillus. In a more preferred embodiment, said bacteria genus is Halomonas and in a more preferred embodiment, Halomonas elongata sp. is used as a bacterium belonging to Halomonas genus.
- The medium suitable to cultivate the above mentioned bacteria includes a synthetic medium comprising a carbon source selected from the group consisting of: lactose, maltose, glucose, galactose, sucrose, glycerol and mixtures thereof. Preferably, the carbon source is selected from the group consisting of: glucose, lactose, sucrose, and mixtures thereof. In a more preferred embodiment, the carbon source is lactose.
- In a particular embodiment, the other fermentation medium components are: potassium phosphate dibasic (K2HPO4), 0.5-1.0 wt %; potassium phosphate monobasic (KH2PO4), 0.1-0.5 wt %; sodium chloride (NaCl), 5.0-10.0 wt %; magnesium sulfate heptahydrate (MgSO4-7H2O). 0.01-0.05 wt %; ammonium sulfate (NH4)2SO4, 0.05 wt %-0.5 wt % and peptone, 0.02-0.1 wt %.
- In a preferred embodiment, before culturing bacteria, the suitable medium for culturing is a synthetic medium with a pH value ranging from 6 to 8. In a more preferred embodiment, the suitable medium has a pH value of 7.
- In another preferred embodiment, the culture step is conducted in fermenters operating at a temperature ranging from 25° C. to 37° C. In a more preferred embodiment, the temperature is 32° C.
- In a particular embodiment, the present invention provides a fermentation process comprising a fermentation step allowing to grow a strain of the corresponding bacteria, preferably wherein said bacteria is from a genus selected from Halomonas, Pseudoalteromonas, Vibrio, Salinivibrio, Marinomonas, Alteromonas, Pseudomonas, Halobacillus, and Bacillus, more preferably from genus Halomonas, still more preferably from Halomonas elongata sp., in a suitable medium in a fermenter under conditions of agitation sufficient to maintain a homogenous culture and limited aeration such that dissolved oxygen pressure (pO2) within the culture is around 20 to 40% for most of the fermentation step. Preferably, pO2 within the culture is 30% in the fermentation step.
- As mentioned above, the inventive process of preparing an exopolysaccharide-protein complex comprises the step of isolating the exopolysaccharide-protein complex from the secreted fraction of the culture. Said isolation can be carried by removing other molecules present in the culture media by alcohol precipitation. Non-limiting examples of alcohols which can be used include ethanol, isopropanol, and methanol. In particular, the isolation and purification of the EPCx after alcohol precipitation can be conducted by tangential flow filtration methods using ultrafiltration membranes. Preferably, said membranes have a MWCO of 30 kDa and the retentate recovered after the ultrafiltration comprises the exopolysaccharide and the EPS-associate proteins which can be recovered. The tangential flow filtration can act to both diafilter and concentrate the EPCx.
- In a third aspect, the present invention relates to a composition (pharmaceutical, nutraceutical or cosmeceutical) comprising an exopolysaccharide-protein complex according to the first object of the invention, including each of the embodiments comprised in said first aspect and combinations thereof.
- In a preferred embodiment, said composition further comprises a biological response modifier selected from the group consisting of lymphokine, interleukin, growth factor and NFkB factor.
- In a fourth aspect, the present invention relates to a composition comprising an exopolysaccharide-protein complex according to the first object of the invention, including each of the embodiments comprised in said first aspect and combinations thereof, for use in stimulating an immune response in a subject for preventing or treating a disease selected from cancer or a disease associated to undesirable inflammatory activity.
- Accordingly, the present invention also relates to a method for preventing or treating a disease selected from cancer or a disease associated to undesirable inflammatory activity, the method comprising administering to a subject in need thereof a therapeutically effective amount of an exopolysaccharide-protein complex according to the first object of the invention, including each of the embodiments comprised in said first aspect and combinations thereof, or a composition comprising thereof.
- In a preferred embodiment, said disease associated to undesirable inflammatory activity is selected from allergy, Alzheimer's disease, arthritis, autoimmune deficiency syndrome, celiac disease, diabetes mellitus, gastrointestinal disorder, inflammatory bowel disease, interstitial cystitis, skin disorders, acne, arteritis, arthritis, cancer, cellulitis, dermatitis and cardiovascular diseases.
- In another preferred embodiment, said cancer is liver cancer or a hepatocellular carcinoma.
- In another preferred embodiment, said exopolysaccharide-protein complex according to the first object of the invention, including each of the embodiments comprised in said first aspect and combinations thereof, is used as an anticancer agent or adjuvant agent for cancer therapies.
- In another embodiment, the present invention provides an in vitro toxicity test to evaluate the cytotoxic potential of EPCx on non-tumor cell lines. The cytotoxicity evaluation of each concentration of EPCx was performed by using the MTT Cell Proliferation Assay in order to determine the concentrations which are not harmful to Human Epidermal Keratinocytes (HEK) and human monocyte-like cells (THP-1).
- As shown in the example section, the EPCx show in vitro anti-proliferative activity against a panel of one or more cancer cell lines, including human lung adenocarcinoma cell line (H1975), Human melanoma cell line (A375), and human hepatocellular carcinoma cell line (HepG2). The anticancer properties of EPCx was ascertained by MTT assay and showed that EPCx significantly inhibit the growth of human lung adenocarcinoma cell line (H1975), Human melanoma cell line (A375), and human hepatocellular carcinoma cell line (HepG2).
- The immunomodulatory and anti-proliferative properties of EPCx were ascertained by the analysis of the induced activation of human primary keratinocytes (HEK) and human monocyte-like cells (THP-1) stressed with LPS (lipopolysaccharide), analyzing the expression of mRNA for the cytokines IL-6, IL-8, IL-10 and TNF-α, by real-time PCR and the NF-κB activation using a luciferase reporter gene assay.
- The immunomodulatory and anti-cancer properties of the EPCx, are showed preferably at EPCx concentrations between 0.01 mg/ml to 1.0 mg/ml.
- Accordingly, the inventors have surprisingly and unexpectedly found that an EPCx obtained from a bacterium, preferably wherein said bacterium is from a genus selected from Halomonas, Pseudoalteromonas, Vibrio, Salinivibrio, Marinomonas, Alteromonas, Pseudomonas, Halobacillus, and Bacillus, more preferably from genus Halomonas, still more preferably from Halomonas elongate sp., in accordance with the invention, can be used as anti-inflammatory and/or inmunomodulator agent, and is in particular able to inhibit a pro-inflammatory stimulation of normal cells in culture. In addition EPCx exhibits tumor anti-promoting properties in cancer cell lines culture.
- The present invention will be more clearly understood with the help of the following examples, without being the present invention limited thereto and included only for illustrative purposes only, showing isolation and characterization of bacteria, the preparation and characterization of EPCx and assays for biological activities in accordance with the invention.
- A microorganism included in the present invention (deposited with number DSM 32408 at the depositary institution Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH by INKEMIA IUCT (Institut Univ. de Ciència i Tecnologia) Group) is isolated from a rock salt from solar salternsin Cardona (Spain) by serial dilution and plating on MH agar medium (Ventosa, Garcia, Kamekura, Onishi, & Ruizberraquero, 1989). The plates were incubated at 32° C. for 3 to 5 days, and bacterial colonies were isolated in pure form and maintained on slopes of the same medium.
- To evaluate the EPS production capability, the bacterium isolates was grown in MY medium (Moraine & Rogovin, 1966) supplemented with 5-10% NaCl for 3-5 days at 32° C. under continuous shaking (120 rpm). The EPS from the growing culture was isolated using the method as described by Quesada et al. (Quesada, Bejar, & Calvo, 1993) and used for quantification and chemical analysis.
- The selected microorganism was analyzed by physiological and biochemical methods following standard microbiological methods (Table 1). As a result, based on physiological and biochemical analysis thereof, the microorganism of the present invention was confirmed to have similarity to Halomonas genus.
-
TABLE 1 Morphological and physiological characteristics of the isolated microorganism. Character Morphological characters Colony morphology Cream, circular Gram nature − Cell shape Rod Cell arrangement Single Motility + Pigmentation − Physiological characters − pH range for growth 5-11 pH optimum for growth 8 Temp. range for growth (° C.) 22-40 Temp. optimum for growth (° C. 32 NaCl range for growth (%) 1-20 NaCl optimum for growth (%) 5 Growth on King's B medium + Growth on McConky agar − Biochemical characters Voges-Proskauer test − Citrate utilization + Methyl red test − Production of Gelatinase − Urease − Catalase + Nitrate reductase + H2S − Lysine decarboxylase − Arginine decarboxylase − Ornithine decarboxylase − Indole − Phenylalanine deaminase + Tryptophan deaminase − Tentative identity Halomonas - In order to identify more precisely, the present inventors analyzed base sequence of ribosome small subunit gene. Particularly, genomic DNA was separated by using MasterPure™ DNA purification from Epicentre @ Biotechnologies Germany according to the manufacturer's instruction. PCR mediated amplification of 16S rDNA and purification of the PCR product was carried out as described previously (Rainey, WardRainey, Kroppenstedt, & Stackebrandt, 1996). As a result of database analysis on the decided base sequence using NCBI (National Center for Biological Information) BLAST, the microorganism was confirmed to have similarity to Halomonas genera. However, it had a little difference from the typical Halomonas elongata, so that the microorganism of the present invention was finally named as Halomonas elongata.
- Partial sequencing of the 16S rRNA gene (844 bp) indicated that the strain belongs to the species Halomonas elongate. The partial 16S rRNA gene sequence is shown in
FIG. 5 . - a) Method of Culturing of Strain the Species Halomonas elongata
- The strain of the species Halomonas elongate (deposited with number DSM 32408 at the depositary institution Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH by INKEMIA IUCT (Institut Univ. de Ciència i Tecnologia) Group) was cultured in a fermenter, at 32° C. and at a pH of 7.5, whose broth contained (g L−1): 100 NaCl; 50 Lactose; 7 K2HPO4; 2 KH2PO4; 0.1 MgSO4.7H2O; 1 (NH4)SO4 and 0.5 Peptone. An inoculum was prepared with 10% (v/v) of a pre-culture and the duration of the fermentation was extended to 72 hours. The reactors are operated in batch mode, and dissolved oxygen was controlled by the agitation (300 to 900 rpm) at pO2≈30%.
- The bacteria were separated from the broth by centrifugation at 12,000 g for 45 min. The resulting clear solution was subjected to ultrafiltration and dialysis using an installation for ultrafiltration (Sartocon® Slice Cassette, Sartorius Stedim), membrane exclusion limit 30 KDa. If necessary, the final solution may be lyophilized and purified obtaining an exopolysaccharide-protein complex with a yield of 60-80%.
- The content of neutral and acid monosaccharides of the exopolysaccharide obtained according to Example 2 was determined by a method described by Honda et al. (Honda et al., 1989) and Yang et al. (X. B. Yang, Zhao, Wang, Wang, & Mei, 2005; X. B. Yang, Zhao, Zhou, et al., 2005). Briefly, the purified polysaccharide sample (1 mg) was hydrolyzed with 1 ml of 2 M trifluoroacetic acid at 120° C. for 2 h, derivatized with 1-phenyl-3-methyl-5-pyrazolone, and subsequently analyzed by high-performance liquid chromatography with detection by absorbance monitoring at 245 nm.
- The percent relationship of sugars obtained was 30-60% of glucose, 30-50% galactose, 5-10% or glucuronic acid, 1-10% of rhamnose and 1-10% of glucosamine, being the amounts consistent with a total of 100%.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970) was carried out with a 4% stacking and a 9% separating gel. The EPCx sample was dissolved at 10 mg/mL in distilled water and added at 1:3 volume ratio into a buffer solution of 0.5% SDS with 1%-mercaptoethanol, and then heated to boiling for 5 min. The gels were stained with Coomassie Brilliant Blue R-250 to visualize proteins. 12 detectable bands, including three principal bands of 38 kDa, 46 kDa and 54 kDa were observed (see
FIG. 1 ). - The cytotoxicity evaluation of exopolysaccharide-protein complex was performed by using the MTT assay in order to determine the concentrations which are not harmful to Human Epidermal Keratinocytes (HEK) and human monocyte-like cells (THP-1). Proliferation of cells lines was measured based on the mitochondria-dependent reduction of yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide), to generate reducing equivalents such as NADH and NADPH. Briefly, 5×103 cells in 100 μL per well were plated in 96-well tissue culture plates for 24 h. Cells were incubated for 72 h in the presence of different concentrations of EPS (1, 0.1 and 0.01 mg/ml). After that, 20 μL of MTT solution (5 mg/mL in PBS) were added into each well and followed by further incubation for 4-5 h. The resulting intracellular purple formazan can be solubilised and quantified by spectrophotometric means. The results are given in Table 2 below.
-
TABLE 2 Effect of exopolysaccharide-protein complex non-tumor cell lines. Cell viability % of MTT Cell line Concentration (mg/ml) conversion HEK 1 101 0.1 98 0.01 102 THP-1 1 102 0.1 97 0.01 104 - No viability alteration and no significant changes in cells proliferation were observed at the exopolysaccharide-protein complex concentrations tested in this assay on normal cell lines (table 2).
- The capacity of EPS to modulate the inflammatory response was evaluated in vitro in human primary keratinocytes (HEK) and human monocyte-like cells (THP-1) stressed with LPS (lipopolysaccharide). The cells were pretreated with different concentrations of exopolysaccharide-protein complex (1, 0.1 and 0.01 mg/ml) for 24 h and with 10 μg/ml of LPS for 24 h. Controls of HEK and THP-1 cells treated only with the different concentrations of exopolysaccharide-protein complex (1.0, 0.1 and 0.01 mg/ml) were also prepared.
- Expression of mRNA for the cytokines IL-6, IL-8, IL-10 and TNF-α was analyzed by real-time PCR and the NF-κB activation was measured using a luciferase reporter gene assay.
- Real-time PCR: Total RNA was extracted with the RNA Isolation Kit (ThermoFischer Scientific) according to the manufacturer's recommendations. 1 ng of RNA was reverse transcribed into complementary DNA (cDNA) using Superscript One-Step RT-PCR kit with platinum Taq according to the instructions (Invitrogen). Quantification rests on the measure of threshold cycles (CT), which are measured at the beginning of the exponential phase of the reaction and on the normalization of the internal standard curve obtained with the reference gene.
- When HEK and THP-1 cells were pretreated with exopolysaccharide-protein complex (EPCx) before stimulation with LPS, we did not observe the upregulation of pro-inflammatory cytokines (IL-6, IL-8, and TNF-α), as occurred in the LPS-treated cells. However, cells pretreated with EPCx before stimulation with LPS shows a downregulation of anti-inflammatory cytokine, IL-10 (tables 3 and 4).
-
TABLE 3 Gene expression (mRNA, real-time PCR) of pro-inflammatory cytokines (IL-6, IL-8, and TNF-α) and anti-inflammatory cytokine (IL-10) in keratinocytes stresses with LPS and treated with different concentrations of EPCx (1.0, 0.1 and 0.01 mg/ml). Gene expression (mRNA, real-time PCR) IL-6 IL-8 TNF-α IL-10 Control 3 2 4 1 LPS 62 59 67 7 EPCx 0.01 12 17 21 11 EPCx 0.1 17 14 27 18 EPCx 1.0 15 18 31 22 -
TABLE 4 Gene expression (mRNA, real-time PCR) of pro-inflammatory cytokines (IL-6, IL-8, and TNF-α) and anti- inflammatory cytokine ( IL-10) in human monocyte-like cells stresses with LPS and treated with different concentrations of EPCx (1, 0.1 and 0.01 mg/ml). Gene expression (mRNA, real-time PCR) 1L-6 IL-8 TNF-α IL-10 Control 1.5 3 2 1 LPS 73 82 67 5 EPCx 0.01 21 27 32 12 EPCx 0.1 27 30 44 12 EPCx 1.0 15 18 31 15 - Measurement of NF-κB activation: NF-κB activation was measured using a luciferase reporter gene assay. For this assay THP-1 macrophages (1×105 cells well−1) were transfected with the pNF-κBluciferase reporter gene construct (Stratagene) using Lipofectamine LTX plus (Invitrogen). Sixteen hours after transfection, different concentrations of EPS (1, 0.1 and 0.01 mg/ml) were added and the incubation was continued for a further 8 h. The cells were then washed with NaC/P1 and lysed using lysis buffer (200 μL·well−1) (25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1 mM dithiothreitol and 1% Triton X-100). Lysed cells were centrifuged (5 min, 9000 g) and stored at −80° C. until assay. Luciferase activity was measured using luciferin (1 mM in glycylglycine buffer. 300 μL·sample−1) in a luminometer at 562 nm.
- EPCx dose-dependently downregulated the NF-κB transcription activity in keratinocyte cells (
FIG. 2 ), whereas in EPCx-treated human monocyte-like cells, the NF-kB levels were comparable to those obtained in the THP-1 cells treated with LPS (FIG. 3 ). - The MTT assay was used for measuring the proliferation of the tumor cells. Briefly, human hepatocellular carcinoma cell line (HepG2), human breast adenocarcinoma cell line (MCF7), human lung adenocarcinoma cell line (H1975) and Human melanoma cell line (A375) were seeded at a density of 4×104 cells/mL in a volume of 0.1 mL in 96-well plates, respectively. After 24 h, different concentrations of EPCx (1.0, 0.1 and 0.01 mg/ml) were dissolved in the medium was added to each well and incubated for 48 h at 37° C. in a CO2 incubator. 5-Fu (5-fluorouracil) was used as the positive control. After the incubation, 20 μL of MTT solution (5 mg/mL) were added into each well and followed by further incubation for 4-5 h. The culture media were then removed and 100 μL of DMSO was added to each well for 1 h. Absorbance at 570 nm was detected by microplate ELISA reader. The inhibition ratio of the tumor cells proliferation was determined, the results are shown in
FIG. 4 . - MTT assay showed that EPCx markedly inhibited proliferation of human hepatocellular carcinoma cell line (HepG2) in a dose-dependent manner (
FIG. 4 ), with little effect on growth of human breast adenocarcinoma cell line (MCF7), human lung adenocarcinoma cell line (H1975) and Human melanoma cell line (A375). The potency of EPCx (at 1.0 mg/mL) to HepG2 cells was found to be similar to 5-fluorouracil (5-FU, IC50 was 10.0 μmol/L). - The foregoing description of preferred embodiments and examples is intended only to acquaint others skilled in the art with Applicants' invention, its principles, and its practical application so that others skilled in the art may adapt and apply the invention in its numerous forms, as they may be best suited to the requirements of a particular use. This detailed description and its specific examples, while indicating preferred embodiments of this invention, are intended for purposes of illustration only. This invention, therefore, is not limited to the preferred embodiments described in this specification, and may be variously modified.
-
- Bazani Cabral de Melo, F. C., Borsato, D., de Macedo Junior, F. C., Mantovani, M. S., Luiz, R. C., & Pedrine Colabone Celligoi, M. A. (2015). Study of levan productivity from Bacillus subtilis Natto by surface response methodology and its antitumor activity against HepG2 cells using metabolomic approach. Pakistan Journal of Pharmaceutical Sciences, 28(6), 1917-1926.
- Beutler, B. (2004). Innate immunity: an overview. Molecular Immunology, 40(12), 845-859. Ciszek-Lenda, M., Nowak, B., Srottek, M., Gamian, A., & Marcinkiewicz, J. (2011). Immunoregulatory potential of exopolysaccharide from Lactobacillus rhamnosus KL37. Effects on the production of inflammatory mediators by mouse macrophages. International Journal of Experimental Pathology, 92(6), 382-391.
- De Vuyst, L., De Vin, F., Vaningelgem, F., & Degeest, B. (2001). Recent developments in the biosynthesis and applications of heteropolysaccharides from lactic acid bacteria. International Dairy Journal, 11(9), 687-707.
- Freitas, F., Alves, V. D., & Reis, M. A. M. (2011). Advances in bacterial exopolysaccharides: from production to biotechnological applications. Trends in Biotechnology, 29(8), 388-398.
- Gamal-Eldeen, A. M., Ahmed, E. F., & Abo-Zeid, M. A. (2009). In vitro cancer chemopreventive properties of polysaccharide extract from the brown alga, Sargassum latifolium. Food and Chemical Toxicology, 47(6), 1378-1384.
- García Sanz, M. N., Ferrer Montiel Antonio, V., Soley Astals, A., & AlmiÑAna DomÉNech, N. (2015). WO Patent No. WO 2015/063240 A1.
- Gorska, S., Schwarzer, M., Jachymek, W., Srutkova, D., Brzozowska, E., Kozakova, H., et al. (2014). Distinct Immunomodulation of Bone Marrow-Derived Dendritic Cell Responses to Lactobacillus plantarum WCFS1 by Two Different Polysaccharides Isolated from Lactobacillus rhamnosus LOCK 0900. Applied and Environmental Microbiology, 80(20), 6506-8516.
- Honda, S., Akao, E., Suzuki, S., Okuda, M., Kakehi, K., & Nakamura. J. (1989). HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF REDUCING CARBOHYDRATES AS STRONGLY ULTRAVIOLET-ABSORBING AND ELECTROCHEMICALLY SENSITIVE 1-PHENYL-3-METHYL-5-PYRAZOLONE DERIVATIVES. Analytical Biochemistry, 180(2), 351-357.
- Kim, H. S., Hong, J. T., Kim, Y., & Han, S.-B. (2011). Stimulatory Effect of beta-glucans on Immune Cells. Immune network, 11(4), 191-195.
- Laemmli, U. K. (1970). CLEAVAGE OF STRUCTURAL PROTEINS DURING ASSEMBLY OF HEAD OF BACTERIOPHAGE-T4. Nature, 227(5259), 680-&.
- Li, J., Qian, W., Xu, Y., Chen, G., Wang, G., Nie, S., et al. (2015). Activation of RAW 264.7 cells by a polysaccharide isolated from Antarctic bacterium Pseudoaltermonas sp S-5. Carbohydrate Polymers, 130, 97-103.
- Loing, E., Briatte, S., Vayssier, C., Beaulieu, M., & Dionne, P. (2009). WO Patent No. WO 2009/127057 A1.
- MercadÉ Gil Ma, E., CarriÓN Fonseca, O., & Montes LÓPez Ma, J. (2015). WO Patent No. WO 2015/117985 A1.
- Miranda. C. C. B. O, Dekker, R. F. H., Serpeloni, J. A., Fonseca, E. A. L., Couls, L. M. S., & Barbosa, A. M. (2008). Anticlastogenic activity exhibited by botryosphaeran, a new exopolysaccharide produced by Botryosphaeria rhodina MAMB-05. International Journal of Biological Macromolecules, 42(2), 172-177.
- Moraine, R. A., & Rogovin, P. (1966). KINETICS OF POLYSACCHARIDE B-1459 FERMENTATION. Biotechnology and Bioengineering, 8(4), 511-&.
- Nevot, M., Deroncele, V., Lopez-Iglesias, C., Bozal, N., Guinea, J., & Mercade, E. (2006). Ultrastructural analysis of the extracellular matter secreted by the psychrotolerant bacterium Pseudoalteromonas antarctica NF3. Microbial Ecology, 51(4), 501-507.
- Nevot, M., Deroncele, V., Messner, P., Guinea, J., & Mercade, E. (2006). Characterization of outer membrane vesicles released by the psychrotolerant bacterium Pseudoalteromonas antarctica NF3. Environmental Microbiology, 8(9), 1523-1533.
- Nwodo, U. U., Green, E., & Okoh, A. I. (2012). Bacterial Exopolysaccharides: Functionality and Prospects. International Journal of Molecular Sciences, 13(11), 14002-14015.
- Poll, A., Anzelmo, G., & Nicolaus, B. (2010). Bacterial Exopolysaccharides from Extreme Marine Habitats: Production, Characterization and Biological Activities. Marine Drugs, 8(6), 1779-1802.
- Quesada, E., Bejar, V., & Calvo, C. (1993). EXOPOLYSACCHARIDE PRODUCTION BY VOLCANIELLA EURIHALINA. Experientia, 49(12), 1037-1041.
- Raafat, E. M., Gamal-Eldeen, A. M., El-Hussieny, E. A., Ahmed, E. F., & Eissa, A. A. (2014). Polysaccharide extracts of the brown alga Sargassum asperifolium possess in vitro cancer chemopreventive properties. Natural Product Research, 28(24), 2304-2311.
- Rainey, F. A., WardRainey. N., Kroppenstedt, R. M., & Stackebrandt, E. (1996). The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: Proposal of Nocardiopsaceae fam nov. International Journal of Systematic Bacteriology, 46(4), 1088-1092.
- Rasulov, M. M., Kuznetsov, I. G., Slutskii. L. I., Velikaia, M. V., Zabozlaev, A. G., & Voronkov, M. G. (1993). The ulcerostatic effect of the exopolysaccharide from Bacillus mucilaginosus and its possible mechanisms. Biulleten' eksperimental'noi biologii i meditsiny, 116(11), 504-505.
- Shao, L., Wu. Z., Zhang, H., Chen, W., Ai, L., & Guo, B. (2014). Partial characterization and immunostimulatory activity of exopolysaccharides from Lactobacillus rhamnosus KF5. Carbohydrate Polymers, 107, 51-56.
- Sun, Z., Li, M. J., Qi, Q. S., Gao, C. J., & Lin, C. S. K. (2014). Mixed Food Waste as Renewable Feedstock in Succinic Acid Fermentation. Applied Biochemistry and Biotechnology, 174(5), 1822-1833.
- Ventosa, A., Garcia, M. T., Kamekura. M., Onishi, H., & Ruizberraquero, F. (1989). BACILLUS-HALOPHILUS SP-NOV, A MODERATELY HALOPHILIC BACILLUS SPECIES. Systematic and Applied Microbiology, 12(2), 162-166.
- Yang, J., U, X., Xue, Y., Wang, N., & Liu, W. (2014). Anti-hepatoma activity and mechanism of corn silk polysaccharides in H22 tumor-bearing mice. International Journal of Biological Macromolecules. 64, 276-280.
- Yang, X. B., Zhao, Y., Wang, Q. W., Wang, H. F., & Mei, Q. B. (2005). Analysis of the monosaccharide components in Angelica polysaccharides by high performance liquid chromatography. Analytical Sciences, 21(10), 1177-1180.
- Yang, X. B., Zhao, Y., Zhou, S. Y., Liu, L., Wang, H. F., & Mei, Q. B. (2005). Analysis of monosaccharide composition in Angelica polysaccharides by precolumn derivatization high performance liquid chromatography. Chinese Journal of Analytical Chemistry, 33(9), 1287-1290.
- Yasuda, E., Serata, M., & Sako, T. (2009). Suppressive Effect on Activation of Macrophages by Lactobacillus casei Strain Shirota Genes Determining the Synthesis of Cell Wall-Associated Polysaccharides (vol 74, pg 4746, 2008). Applied and Environmental Microbiology, 75(4), 1221-1221.
- Ye, S., Zhang, J., Liu, Z., Zhang, Y., Li, J., & Li, Y. O. (2016). Biosynthesis of selenium rich exopolysaccharide (Se-EPS) by Pseudomonas PT-8 and characterization of its antioxidant activities. Carbohydrate Polymers, 142, 230-239.
- Zhang, F., Liu, Y. E., Wang, W., Zhao, Y. U., & Li, Z. (2015). CN Patent No. CN 104694594 A.
- Zheng, Y., Wang, W.-d., & Li, Y. (2015). Antitumor and immunomodulatory activity of polysaccharide isolated from Trametes orientalis. Carbohydrate Polymers, 131, 248-254.
Claims (7)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/570,048 US20220160610A1 (en) | 2016-09-19 | 2022-01-06 | Exopolysaccharide-protein complex, a method of preparing said complex and uses thereof |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP16382436.0 | 2016-09-19 | ||
EP16382436.0A EP3295926A1 (en) | 2016-09-19 | 2016-09-19 | Exopolysaccharide-protein complex, a method of preparing said complex and uses thereof |
USPCT/US2017/073606 | 2017-09-19 | ||
US201916334346A | 2019-03-18 | 2019-03-18 | |
US17/570,048 US20220160610A1 (en) | 2016-09-19 | 2022-01-06 | Exopolysaccharide-protein complex, a method of preparing said complex and uses thereof |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
USPCT/US2017/073606 Continuation | 2016-09-19 | 2017-09-19 | |
US16/334,346 Continuation US11253457B2 (en) | 2016-09-19 | 2017-09-19 | Exopolysaccharide-protein complex, a method of preparing said complex and uses thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
US20220160610A1 true US20220160610A1 (en) | 2022-05-26 |
Family
ID=81657866
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/570,048 Abandoned US20220160610A1 (en) | 2016-09-19 | 2022-01-06 | Exopolysaccharide-protein complex, a method of preparing said complex and uses thereof |
Country Status (1)
Country | Link |
---|---|
US (1) | US20220160610A1 (en) |
-
2022
- 2022-01-06 US US17/570,048 patent/US20220160610A1/en not_active Abandoned
Non-Patent Citations (1)
Title |
---|
Llamas, I. et al "The potential biotechnological applications of the exopolysaccharide ..." Molecules, vol 17, pp 7103-7120. (Year: 2012) * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Zhang et al. | Production and characterization of exopolysaccharides from Chlorella zofingiensis and Chlorella vulgaris with anti-colorectal cancer activity | |
Zhang et al. | Characterization of exopolysaccharides produced by microalgae with antitumor activity on human colon cancer cells | |
AU2011230685B2 (en) | Anti-allergic agent | |
Shao et al. | Partial characterization and immunostimulatory activity of exopolysaccharides from Lactobacillus rhamnosus KF5 | |
Lam et al. | Non-digestible long chain beta-glucans as novel prebiotics | |
Bajpai et al. | Exopolysaccharide and lactic acid bacteria: Perception, functionality and prospects | |
Zhang et al. | Antioxidant capacity and prebiotic effects of Gracilaria neoagaro oligosaccharides prepared by agarase hydrolysis | |
Kook et al. | Immunomodulatory effects of exopolysaccharides produced by Bacillus licheniformis and Leuconostoc mesenteroides isolated from Korean kimchi | |
CN107137628B (en) | Hyaluronidase inhibitor derived from symbiotic fermentation product and application thereof | |
Jiang et al. | Optimization production of exopolysaccharide from Leuconostoc lactis L2 and its partial characterization | |
Priyanka et al. | Sulfated exopolysaccharide produced by Labrenzia sp. PRIM-30, characterization and prospective applications | |
Shankar et al. | Biomedical and therapeutic potential of exopolysaccharides by Lactobacillus paracasei isolated from sauerkraut: Screening and characterization | |
Zhang et al. | Characterization and antioxidant activity of released exopolysaccharide from potential probiotic Leuconostoc mesenteroides LM187 | |
Cimini et al. | Optimization of growth of Levilactobacillus brevis SP 48 and in vitro evaluation of the effect of viable cells and high molecular weight potential postbiotics on Helicobacter pylori | |
US11253457B2 (en) | Exopolysaccharide-protein complex, a method of preparing said complex and uses thereof | |
Bawadekji et al. | A review of the bioactive compound and medicinal value of Cordyceps militaris | |
US20220160610A1 (en) | Exopolysaccharide-protein complex, a method of preparing said complex and uses thereof | |
Talbi et al. | Bacterial exopolysaccharides: from production to functional features | |
Gao et al. | Structural characterization and in vitro evaluation of the prebiotic potential of an exopolysaccharide produced by Bacillus thuringiensis during fermentation | |
Yehia et al. | Isolation and characterization of anti-proliferative and anti-oxidative mannan from Saccharomyces cerevisiae | |
JP2015117234A (en) | Collagen production promoter | |
JP2012031089A (en) | Immunostimulator | |
JP3802011B2 (en) | Whitening agent | |
Abdel-Aziz et al. | Production and assessment of antioxidant activity of exopolysaccharide from marine Streptomyces globisporus bu2018 | |
KR20130059641A (en) | Fermentation method for improvement of bioactivity of fucoidan as cosmetic material |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
AS | Assignment |
Owner name: INSTITUT UNIV. DE CIENCIA I TECNOLOGIA, S.A., SPAIN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:MONTILLA AREVALO, RAFAEL;CASTELLS BOLIART, JOSEP;GARCIA DE LA MARINA, ADRIAN;AND OTHERS;SIGNING DATES FROM 20190415 TO 20190423;REEL/FRAME:059137/0521 |
|
AS | Assignment |
Owner name: LABORATORIO TECNICO DE SEGURIDAD Y ESTANDARIZACION, S.L.U., SPAIN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:RCD CONCURSAL, S.L.P AS TRUSTEE INSOLVENCY ADMINISTRATOR OF INSTITUT UNIVERSITARI DE CIENCIA I TECNOLOGIA;REEL/FRAME:059167/0044 Effective date: 20220114 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |