CA2155134A1 - Branched polysaccharide, microorganism producing it and compositions containing them - Google Patents
Branched polysaccharide, microorganism producing it and compositions containing themInfo
- Publication number
- CA2155134A1 CA2155134A1 CA002155134A CA2155134A CA2155134A1 CA 2155134 A1 CA2155134 A1 CA 2155134A1 CA 002155134 A CA002155134 A CA 002155134A CA 2155134 A CA2155134 A CA 2155134A CA 2155134 A1 CA2155134 A1 CA 2155134A1
- Authority
- CA
- Canada
- Prior art keywords
- polysaccharide
- microorganism
- branched
- branched polysaccharide
- strain
- 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
- 150000004676 glycans Chemical class 0.000 title claims abstract description 73
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 73
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 73
- 244000005700 microbiome Species 0.000 title claims abstract description 28
- 239000000203 mixture Substances 0.000 title claims abstract description 24
- 235000013305 food Nutrition 0.000 claims abstract description 7
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 7
- GUBGYTABKSRVRQ-QKKXKWKRSA-N lactose group Chemical group OC1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@@H](O)[C@H](O2)CO)[C@H](O1)CO GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 30
- 240000002605 Lactobacillus helveticus Species 0.000 claims description 19
- 239000001257 hydrogen Substances 0.000 claims description 14
- 229910052739 hydrogen Inorganic materials 0.000 claims description 14
- 235000013336 milk Nutrition 0.000 claims description 13
- 210000004080 milk Anatomy 0.000 claims description 13
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Chemical group CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 claims description 10
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Chemical group CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 9
- 235000013967 Lactobacillus helveticus Nutrition 0.000 claims description 9
- 229930182830 galactose Natural products 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 9
- 229940054346 lactobacillus helveticus Drugs 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 9
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 7
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical group C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims description 6
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 claims description 5
- OVRNDRQMDRJTHS-KEWYIRBNSA-N N-acetyl-D-galactosamine Chemical group CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-KEWYIRBNSA-N 0.000 claims description 5
- 239000005862 Whey Substances 0.000 claims description 5
- 102000007544 Whey Proteins Human genes 0.000 claims description 5
- 108010046377 Whey Proteins Proteins 0.000 claims description 5
- 239000003826 tablet Substances 0.000 claims description 4
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims description 3
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 claims description 3
- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 claims description 3
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 claims description 3
- 239000002775 capsule Substances 0.000 claims description 3
- 235000007173 Abies balsamea Nutrition 0.000 claims description 2
- 239000004857 Balsam Substances 0.000 claims description 2
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- 229940112822 chewing gum Drugs 0.000 claims description 2
- 235000015218 chewing gum Nutrition 0.000 claims description 2
- 239000006071 cream Substances 0.000 claims description 2
- 239000002324 mouth wash Substances 0.000 claims description 2
- 239000002674 ointment Substances 0.000 claims description 2
- 239000006188 syrup Substances 0.000 claims description 2
- 235000020357 syrup Nutrition 0.000 claims description 2
- 239000000606 toothpaste Substances 0.000 claims description 2
- 229940034610 toothpaste Drugs 0.000 claims description 2
- LWGJTAZLEJHCPA-UHFFFAOYSA-N n-(2-chloroethyl)-n-nitrosomorpholine-4-carboxamide Chemical compound ClCCN(N=O)C(=O)N1CCOCC1 LWGJTAZLEJHCPA-UHFFFAOYSA-N 0.000 claims 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- 238000004458 analytical method Methods 0.000 description 17
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 16
- 241000194020 Streptococcus thermophilus Species 0.000 description 15
- 239000008267 milk Substances 0.000 description 12
- 229920001223 polyethylene glycol Polymers 0.000 description 12
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 10
- 238000000034 method Methods 0.000 description 10
- 102000004856 Lectins Human genes 0.000 description 9
- 108090001090 Lectins Proteins 0.000 description 9
- 238000005100 correlation spectroscopy Methods 0.000 description 9
- 239000002523 lectin Substances 0.000 description 9
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- 238000001228 spectrum Methods 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- 235000000346 sugar Nutrition 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 238000005481 NMR spectroscopy Methods 0.000 description 8
- 239000003480 eluent Substances 0.000 description 8
- 238000000855 fermentation Methods 0.000 description 8
- 230000004151 fermentation Effects 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- -1 methyl glycosides Chemical class 0.000 description 8
- 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 description 7
- 230000011987 methylation Effects 0.000 description 7
- 238000007069 methylation reaction Methods 0.000 description 7
- 150000002772 monosaccharides Chemical class 0.000 description 7
- 229930182470 glycoside Natural products 0.000 description 6
- 235000020183 skimmed milk Nutrition 0.000 description 6
- 229920002444 Exopolysaccharide Polymers 0.000 description 5
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical group [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 5
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 238000005903 acid hydrolysis reaction Methods 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 230000003595 spectral effect Effects 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 108010046569 Galectins Proteins 0.000 description 4
- 102000007563 Galectins Human genes 0.000 description 4
- 241000186660 Lactobacillus Species 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 230000027455 binding Effects 0.000 description 4
- 238000009739 binding Methods 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 125000002519 galactosyl group Chemical group C1([C@H](O)[C@@H](O)[C@@H](O)[C@H](O1)CO)* 0.000 description 4
- 239000013067 intermediate product Substances 0.000 description 4
- 230000007935 neutral effect Effects 0.000 description 4
- 229920001542 oligosaccharide Polymers 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 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 description 3
- 241000194017 Streptococcus Species 0.000 description 3
- 230000015271 coagulation Effects 0.000 description 3
- 238000005345 coagulation Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 210000002919 epithelial cell Anatomy 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 3
- FBPFZTCFMRRESA-UHFFFAOYSA-N hexane-1,2,3,4,5,6-hexol Chemical group OCC(O)C(O)C(O)C(O)CO FBPFZTCFMRRESA-UHFFFAOYSA-N 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 229940039696 lactobacillus Drugs 0.000 description 3
- 235000020191 long-life milk Nutrition 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 150000002482 oligosaccharides Chemical group 0.000 description 3
- 230000008569 process Effects 0.000 description 3
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- 239000000243 solution Substances 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- 235000013618 yogurt Nutrition 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 2
- WLAMNBDJUVNPJU-UHFFFAOYSA-N 2-methylbutyric acid Chemical compound CCC(C)C(O)=O WLAMNBDJUVNPJU-UHFFFAOYSA-N 0.000 description 2
- 241000186045 Actinomyces naeslundii Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- FKLJPTJMIBLJAV-UHFFFAOYSA-N Compound IV Chemical compound O1N=C(C)C=C1CCCCCCCOC1=CC=C(C=2OCCN=2)C=C1 FKLJPTJMIBLJAV-UHFFFAOYSA-N 0.000 description 2
- UDIPTWFVPPPURJ-UHFFFAOYSA-M Cyclamate Chemical compound [Na+].[O-]S(=O)(=O)NC1CCCCC1 UDIPTWFVPPPURJ-UHFFFAOYSA-M 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 241000194034 Lactococcus lactis subsp. cremoris Species 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
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- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 235000014962 Streptococcus cremoris Nutrition 0.000 description 2
- 235000020167 acidified milk Nutrition 0.000 description 2
- NIGUVXFURDGQKZ-UQTBNESHSA-N alpha-Neup5Ac-(2->3)-beta-D-Galp-(1->4)-[alpha-L-Fucp-(1->3)]-beta-D-GlcpNAc Chemical group O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@H]2[C@@H]([C@@H](O[C@]3(O[C@H]([C@H](NC(C)=O)[C@@H](O)C3)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](CO)O[C@@H](O)[C@@H]1NC(C)=O NIGUVXFURDGQKZ-UQTBNESHSA-N 0.000 description 2
- 125000004429 atom Chemical group 0.000 description 2
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- 238000001030 gas--liquid chromatography Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- AWUCVROLDVIAJX-UHFFFAOYSA-N glycerol 1-phosphate Chemical compound OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- 150000002339 glycosphingolipids Chemical class 0.000 description 1
- 229940037467 helicobacter pylori Drugs 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229940004208 lactobacillus bulgaricus Drugs 0.000 description 1
- 210000001821 langerhans cell Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000006140 methanolysis reaction Methods 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 229960002216 methylparaben Drugs 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 125000005629 sialic acid group Chemical group 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 229940100458 steareth-21 Drugs 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000007916 tablet composition Substances 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C21/00—Whey; Whey preparations
- A23C21/02—Whey; Whey preparations containing, or treated with, microorganisms or enzymes
- A23C21/026—Whey; Whey preparations containing, or treated with, microorganisms or enzymes containing, or treated only with, lactic acid producing bacteria, bifidobacteria or propionic acid bacteria
-
- 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
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/12—Antidiarrhoeals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/147—Helveticus
-
- 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
- C12R2001/225—Lactobacillus
Abstract
The present invention concerns a new branched natural soluble polysaccharide comprising a main chain having repeating side chains which are only made of lactose units, possibly substituted.
The present invention also concerns the microorganism by which this branched polysaccharide may be obtained and the food composition, the cosmetical composition and the pharmaceutical composition comprising said branched polysaccharide and/or microorganism.
The present invention also concerns the microorganism by which this branched polysaccharide may be obtained and the food composition, the cosmetical composition and the pharmaceutical composition comprising said branched polysaccharide and/or microorganism.
Description
, .
. ~ 1 OVM/WP-20 juillet 1995 P.NEST.05/EP
0 BRAN~n POLYSA~ RTn~, MI~ROORGANISM PRODUCING IT AND
~OMPOSITIONS ~ONTAINING T~.
Field of th~ invention.
The present invention concerns a new branched polysaccharide, a microorganism producing it, the food composition, the pharmaceutical composition and the cosmetical composition containing them.
Background of the ;nven~ion.
The biological communication (the possibility for a cell to recognize a molecule or another cell) is a central phenomenon in pathological as well as in normal state.
Among the various mechanisms of molecular recognition between cells, and/or between cells and molecules, the binding of specific glycosidic structures by specialized proteins (lectins) is today considered as a major molecular recognition system.
The lectins may be bound specifically and non-covalently to well-defined glycosidic sequences.
Some lectins are bound to oligosaccharides which contain elevated mannose amounts, to structures carrying sialic acids, to fucosylated glycosides, ... .
Other lectins can bind ~-galactosides and lactose.
tlS5134 .
Multigeneric coaggregations exist between oral bacterial cells (Actinomyces naeslundii or viscosus, Streptococcus mitis or sanguis, Fusobacterium nucleatum, Porphyromonas gingivalis, Bacteriodes intermedius, ...) which aggregate and form a network as the dental plaque.
Between these bacterial cells, ~he interaction is often obtained by a non-covalent bound between a ~-galactoside lectin on one cell and a glycosidic receptor on another cell (ref. 1).
Most of the infectious diseases are initiated by the adhesion of pathogeneous agents (Actinomyces naeslundii, Fusobacterium nuclea~um, Bacteriodes intermedius, Salmonella typhimurium, Vibrio Cholera, Campylobacter jejuni, Bacteriodes, Fusobacteria, Clostridia, Shigella, Yersinia, and Helicobacter pylori, ...) to the epithelial cells of the mucosa of its host, which allows then the colonisation of the animal tissues.
This adhesion is often obtained by a binding between a ~-galactoside lectin located at the surface of this pathogeneous agent and a glycosidic receptor located at the surface of the epithelial cell (ref. 2).
Various cells of the immune system (lymphocites T
and B, macrophages, neutrophils) are known either to be able to bind ~-galactoside lectins or to express at their surface such lectins of the galectin family.
In addition, some epithelial cells such as intestinal cells or keratinocytes produce these galectins which can also coat Langerhans cells, and immunoglobulins such as IgE can specifically bind to galectins (ref. 3, 4 and 5).
Stat~ o the art.
They have been many prior studies upon polysaccharides produced by micro-organisms and, in recent years, they have been several reports of studies on the structure of exocellular polysaccharides obtained by lactic acid bacteria and on their biological activities.
A polysaccharide consisting o~ galactose, glucose and N-acetylgalactosamine (2:1:1) is obtaihed by the strains of Streptococcus thermophilus CNCM I-733, CNCM I-734 and CNCM
I-735 (ref. 6 and 7);
a polysaccharide consisting of galactose only is 0 obtained by the strain Lactococcus cremoris H414 (ref. 8);
a polysaccharide consisting of galactose, glucose,rhamnose and phosphate (2:2:1:1) is obtained by the strain Lactococcus cremoris SBT 0495 (ref. 9);
a polysaccharide consisting of galactose, glucose 5 and rhamnose (5:1:1) is obtained by the strain Lactobacillus bulgaricus rr (ref. 10);
a polysaccharide consisting of glucose, rhamnose, 1-phosphoglycerol and a O-acetyl group (3:2:1:0.85) is obtained by the strain Lactobacillus sake 0-1 (ref. 11).
On the other hand, other polysaccharides obtained by a few strains of Lactobacillus helveticus were studied, but never their structural characterizations were performed.
For example, a polysaccharide of unknown structure consisting of glucose and galactose (2:1) used as anti-tumor agent is obtained by the strain Lactobacillus helveticus var. jugurti No 851 "FERM BP-66 (FERM-P No 5851)" (ref. 12 and 13).
Similarly, a polysaccharide of unknown structure consisting of galactose, glucose and N-acetylglucosamine (2.5-3.5:2.5-3.5:1) used in treating inflammation and to accelerate bone marrow growth is obtained by the strain Lactobacillus helveticus MIKI-010 (ref. 14).
Aims of the invention.
The present invention aims to provide a new branched polysacharide and/or the microorganism producing it, which can be used to inhibit the binding between ~-galactoside lectins and their receptor(s).
Another aim of the invention i~ to provide food compositions comprising said polysaccharide and/or microorganism, having improved organoleptic and texture properties.
A further aim of the invention is to provide a pharmaceutical composition and/or cosmetical composition, comprising said polysaccharide and/or microorganism.
A last aim of the invention is to provide a polysaccharide which can be used as an intermediate product for the production of polymerized derivatives of gangliotriose, Sd-a blood group, or sialyl- and sulfated-Lewis X.
Desc~iption of the invention.
The present invention concerns a new natural soluble branched polysaccharide comprising a main chain having repeating side chains which are only made of lactose units, possibly substituted.
According to a preferred embodiment of the present invention, the branched polysaccharide corresponds to the following formula :
- 3 Gal ~ 1 - 3 Glc ~ 1 - 3 Glc $ 1 - 5 Galf$ 1 Rl \ 3 Gal ~ 1 - 4 Glc ~ 1 R2 R3 n , where n ~ 1, Gal = galactose, Glc = glucose, R1 = Hydrogen or GalNAc~1 (N - acetylgalactosamine) R2 = Hydrogen, NeuNAc~2(N-acetylneur~m; n; c Acid) or HSO3 R3 = Hydrogen or Fuc~1 (fucose) When R1 = R2 = R3 =Hydrogen, the branched polysaccharide is characterized by the following physico-chemical properties:
- molecular weight higher than 2,000,000 , - soluble in water and solutions containing less than 20 ~ trichloroacetic acid , - insoluble in alcohol and in acetone , - neutral property , - the freeze-dried product is in the form of white powder , - component sugars and compositional ratio :
Glucose : Galactose (1 : 1.1).
When R1 is GalNAc~1 (N-acetyl galactosamine) and R2=R3=Hydrogen, the branched polysaccharide is a derivative of the gangliotriose determinant which is advantageously obtained from an intermediate product (the polysaccharide with R1=R2=R3=Hydrogen) by the methods described in the documents 15 and 16.
When R1is GalNAc~1 (N-acetyl galactosamine), R2 is NeuNAc~2 (N-acetyl neuraminic Acid) and R3 is Hydrogen, the branched polysaccharide is a derivative of the blood group Sd-a determinant, which is advantageously obtained from an intermediate product (the polysaccharide with R1=R2=R3=Hydrogen) by the methods described in the documents 17 and 18.
When Rl is Hydrogen R2 is NeuNAc~2 (N-acetylneuraminic Acid) or HSO3 and R' is Fuc~1 (fucose), the branched polysaccharide is a derivative of the pharmaceutical products sialyl- or sulfated- Lewis X described in the documents 19 and 20.
The bindings between Gal ~ 1 - 4 Glc and R1, R2 are advantageously obtained from an intermediate product (the polysaccharide with R1 = R2 = R3 = Hydrogen) by the method described in the document 17.
The present invention concerns also the microorganism producing the branched polysaccharide having R1 = R2 = R3 = Hydrogen.
Advantageously, said microorganism corresponds to a strain of Lactobacillus helveticus, preferably the strain o~ ~actobacillus helveticus CNCM I-1449.
A deposit of this microorganism has been made according to the Budapest Treaty on July 27, 1994, at the Collection Nationale de Culture de Microorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 PARIS CEDEX
15, FRANCE.
The present invention also concerns the food having improved organoleptic and texture properties comprising the polysaccharide and/or the microorganism according to the invention.
Preferably, said food composition is a set-style 215513~
.
acidified milk or a stirred acidified milk.
Another aspect of the present invention concerns the cosmetic composition comprising the polysaccharide and/or the microorganism according to the invention.
According to a preferred embodiment of the invention, said cosmetical composition is ~ cosmetic product intended for the buccal hygiene, choosen among the group consisting of a tooth paste, tooth gel, mouth rinse, chewing-gum and/or tablet.
ZlS5134 According to another preferred embodiment of the present invention, said cosmetical composition is a product intended for the skin hygiene, choosen among the group consisting of a cream, ointment or balsam.
Another aspect of the present invention concerns the pharmaceutical composition comprising the polysaccharide ~and/or the microorganism according to the present invention.
Advantageously, said composition is a antidiaorrheic product choosen amQng the group consisting of a capsule, syrup, powder and/or tablet.
A last aspect of the present invention concerns a diagnostic and/or analytic device comprising the branched polysaccharide according to the invention ~or the trapping of specific molecules and/or microorganisms.
Brief description of the drawings.
The figure 1 represents the chromatographic (FPLC) analysis of the polysaccharide according to the invention.
The figure 2 represents six major fractions separated by gel filtration from the polysaccharide hydrolysate.
The ~igure 3 represents the chromatographic (HPAE -PAD) analysis of the fraction II.
The figure 4 represents the chromatographic (HPAE
- PAD~ analysis o~ the fraction IV.
Description of a preferrçd embodime~ of the present invention.
The present invention concerns a new natural soluble branched polysaccharide with a main chain, having repeating side chains which are only lactose units; said lactose is possibly substituted.
The "branched polysaccharide" according to the invention is a saccharide having more than 10 repeating ~ g units, preferably more than 40 repeating units.
Said branched polysaccharide has preferably the structure of a "polymer'l consisting of the repetition of identical single "units" comprising one side chain 2 branched on a main chain as described below:
~ ~ n n>1 The production and the physico-chemical structure of a polysaccharide according to the invention will be described hereafter.
1. PR~PARATION AND PURIFICATION OF THF~ h~XOPOl,YSACCHARIDE
pRcDuc~n BY T,~CTO~ACIL~US HELV~TICUS CNCM I-1449.
1.1 Fermentation Co~dit ons.
Lactobacillus helveticus CNCM I-1449 was a ropy strain from the Nestlé strain collection. Among the 168 strains of L. helveticus of the Nestlé collection, only 2 strains produce exopolysaccharides.
The growth medium was 10~ reconstituted skim milk heat-treated (115C, 35 min) for sterilization prior to fermentation. A one-liter scale fermentor with a magnetic stirrer was used -for regulating the pH during the fermentation. The pH was maintained at 5.5 by using 2N sodium hydroxide. Stirring rate was maintained at 60 RPM. Incubation was made at 40C. The amount of starter culture inoculated to the medium was 1~. During the fermentation (time = 6h, 9h and 24h), several samples were taken and frozen for further analysis and polysaccharide extractior.
1.2. Ex~rac~ion of the Polysaccharide An equal volume of trichloroacetatic acid (40~ was added to the sample to remove proteins by precipitation, followed by centrifugation (17,000g, 20 minutes). To the supernatant fraction containing polysaccharides, the same volume o~ acetone was added. Precipitated polysaccharides were then separated by centrifugation (17,000g, 20 minutes).
The resulting precipitated fraction was dissolved in distilled water and pH was adjusted to 7.0 with sodium hydroxide solution. After dialysis against distilled water (overnight), insoluble substances were removed by ultracentrifugation (llO,OOOg, l hour).
The supernatant fraction containing polysaccharides was lyophilized and crude dehydrated polysaccharides were finally obtained. Total neutral sugar content was determined by the phenol-sulphuric acid method.
1.3. Size of the ~xopolysaccharide.
The filtration was conducted to confirm purity and estimate the molecular weight o~ polysaccharides using FPLC
system (Pharmacia). The column used was Superose 6 (l.0 cm x 30cm) (Fig. 1). 200 ~l samples containing 200-400 ~g dehydrated polysaccharides were applied on to the column, eluted with 50 mM phosphate buffer at pH 7.2 at the rate o~
0.5 ml/min. Fractions (l.0 ml) were collected in tubes.
Polysaccharide content in each tube was determined as total neutral sugar by the phenol-sulphuric acid method.
1.4. Monosaccharide Composition.
Monosaccharide composition o~ a freeze-dried polysaccharide was analyzed using gas-liquid chromatography technique (ref. 21).
The exopolysaccharide obtained ~rom the spent culture medium was examined after extraction ~rom three samples (time = 6h, 9h and 24h). The yield was found to increase as a function of the fermentation time, but the size as well as the monosaccharide composition of the polymer were found invariable:
Fermentation Crude Neutral Pure Monosaccharide Time Yield Sugars Yield Composition (hours) (mg/L) (~) (mg/L) Rha Gal Glc GalNAc 6 42 27.6 11.6 - 1.1 1 -9 142 70.4 100.0 - 1.1 24 272 83.3 226.6 - 1.1 The figure 1 shows the elution and the purity of the polysaccharide obtained at t = 24 h, by FPLC analysis (with a column of Superose 6). The polysaccharide was eluted at around the exclusion limit (approximately 2 X 106 M.W.).
1.5. Yields ~f Polysaccharide Ob~ained in Non-Regulated Fermenta~'ons.
The polysaccharide described above was also produced during fermentations in set-style conditions, by Lactobacillus helveticus CNCM I-1449 alone, or by this strain used together with a strain of Streptococcus thermophilus (for example S. thermophilus YS4).
For this purpose, the growth medium was 10 reconstituted MSK (skim milk powder: 100 g/l and yeast extract: 1 g/l) heat-treated (115C, 35 min) for sterilization prior to fermentation. The typical sample size was 250 ml, the incubation was made at 40C and the amount of starter culture inoculated to the medium was 1 ~. The yields of pure polysaccharide obtained in such conditions were the following:
Strains in theFermentation Time Pure Yield Starter Culture (hours) (mg/l) L . hel ve ti cus 8 8 5 L . hel ve ti cus 4 !23 CNCM I-1449 & S.
thermophi 1 us YS4 215513~
. ~ 1 OVM/WP-20 juillet 1995 P.NEST.05/EP
0 BRAN~n POLYSA~ RTn~, MI~ROORGANISM PRODUCING IT AND
~OMPOSITIONS ~ONTAINING T~.
Field of th~ invention.
The present invention concerns a new branched polysaccharide, a microorganism producing it, the food composition, the pharmaceutical composition and the cosmetical composition containing them.
Background of the ;nven~ion.
The biological communication (the possibility for a cell to recognize a molecule or another cell) is a central phenomenon in pathological as well as in normal state.
Among the various mechanisms of molecular recognition between cells, and/or between cells and molecules, the binding of specific glycosidic structures by specialized proteins (lectins) is today considered as a major molecular recognition system.
The lectins may be bound specifically and non-covalently to well-defined glycosidic sequences.
Some lectins are bound to oligosaccharides which contain elevated mannose amounts, to structures carrying sialic acids, to fucosylated glycosides, ... .
Other lectins can bind ~-galactosides and lactose.
tlS5134 .
Multigeneric coaggregations exist between oral bacterial cells (Actinomyces naeslundii or viscosus, Streptococcus mitis or sanguis, Fusobacterium nucleatum, Porphyromonas gingivalis, Bacteriodes intermedius, ...) which aggregate and form a network as the dental plaque.
Between these bacterial cells, ~he interaction is often obtained by a non-covalent bound between a ~-galactoside lectin on one cell and a glycosidic receptor on another cell (ref. 1).
Most of the infectious diseases are initiated by the adhesion of pathogeneous agents (Actinomyces naeslundii, Fusobacterium nuclea~um, Bacteriodes intermedius, Salmonella typhimurium, Vibrio Cholera, Campylobacter jejuni, Bacteriodes, Fusobacteria, Clostridia, Shigella, Yersinia, and Helicobacter pylori, ...) to the epithelial cells of the mucosa of its host, which allows then the colonisation of the animal tissues.
This adhesion is often obtained by a binding between a ~-galactoside lectin located at the surface of this pathogeneous agent and a glycosidic receptor located at the surface of the epithelial cell (ref. 2).
Various cells of the immune system (lymphocites T
and B, macrophages, neutrophils) are known either to be able to bind ~-galactoside lectins or to express at their surface such lectins of the galectin family.
In addition, some epithelial cells such as intestinal cells or keratinocytes produce these galectins which can also coat Langerhans cells, and immunoglobulins such as IgE can specifically bind to galectins (ref. 3, 4 and 5).
Stat~ o the art.
They have been many prior studies upon polysaccharides produced by micro-organisms and, in recent years, they have been several reports of studies on the structure of exocellular polysaccharides obtained by lactic acid bacteria and on their biological activities.
A polysaccharide consisting o~ galactose, glucose and N-acetylgalactosamine (2:1:1) is obtaihed by the strains of Streptococcus thermophilus CNCM I-733, CNCM I-734 and CNCM
I-735 (ref. 6 and 7);
a polysaccharide consisting of galactose only is 0 obtained by the strain Lactococcus cremoris H414 (ref. 8);
a polysaccharide consisting of galactose, glucose,rhamnose and phosphate (2:2:1:1) is obtained by the strain Lactococcus cremoris SBT 0495 (ref. 9);
a polysaccharide consisting of galactose, glucose 5 and rhamnose (5:1:1) is obtained by the strain Lactobacillus bulgaricus rr (ref. 10);
a polysaccharide consisting of glucose, rhamnose, 1-phosphoglycerol and a O-acetyl group (3:2:1:0.85) is obtained by the strain Lactobacillus sake 0-1 (ref. 11).
On the other hand, other polysaccharides obtained by a few strains of Lactobacillus helveticus were studied, but never their structural characterizations were performed.
For example, a polysaccharide of unknown structure consisting of glucose and galactose (2:1) used as anti-tumor agent is obtained by the strain Lactobacillus helveticus var. jugurti No 851 "FERM BP-66 (FERM-P No 5851)" (ref. 12 and 13).
Similarly, a polysaccharide of unknown structure consisting of galactose, glucose and N-acetylglucosamine (2.5-3.5:2.5-3.5:1) used in treating inflammation and to accelerate bone marrow growth is obtained by the strain Lactobacillus helveticus MIKI-010 (ref. 14).
Aims of the invention.
The present invention aims to provide a new branched polysacharide and/or the microorganism producing it, which can be used to inhibit the binding between ~-galactoside lectins and their receptor(s).
Another aim of the invention i~ to provide food compositions comprising said polysaccharide and/or microorganism, having improved organoleptic and texture properties.
A further aim of the invention is to provide a pharmaceutical composition and/or cosmetical composition, comprising said polysaccharide and/or microorganism.
A last aim of the invention is to provide a polysaccharide which can be used as an intermediate product for the production of polymerized derivatives of gangliotriose, Sd-a blood group, or sialyl- and sulfated-Lewis X.
Desc~iption of the invention.
The present invention concerns a new natural soluble branched polysaccharide comprising a main chain having repeating side chains which are only made of lactose units, possibly substituted.
According to a preferred embodiment of the present invention, the branched polysaccharide corresponds to the following formula :
- 3 Gal ~ 1 - 3 Glc ~ 1 - 3 Glc $ 1 - 5 Galf$ 1 Rl \ 3 Gal ~ 1 - 4 Glc ~ 1 R2 R3 n , where n ~ 1, Gal = galactose, Glc = glucose, R1 = Hydrogen or GalNAc~1 (N - acetylgalactosamine) R2 = Hydrogen, NeuNAc~2(N-acetylneur~m; n; c Acid) or HSO3 R3 = Hydrogen or Fuc~1 (fucose) When R1 = R2 = R3 =Hydrogen, the branched polysaccharide is characterized by the following physico-chemical properties:
- molecular weight higher than 2,000,000 , - soluble in water and solutions containing less than 20 ~ trichloroacetic acid , - insoluble in alcohol and in acetone , - neutral property , - the freeze-dried product is in the form of white powder , - component sugars and compositional ratio :
Glucose : Galactose (1 : 1.1).
When R1 is GalNAc~1 (N-acetyl galactosamine) and R2=R3=Hydrogen, the branched polysaccharide is a derivative of the gangliotriose determinant which is advantageously obtained from an intermediate product (the polysaccharide with R1=R2=R3=Hydrogen) by the methods described in the documents 15 and 16.
When R1is GalNAc~1 (N-acetyl galactosamine), R2 is NeuNAc~2 (N-acetyl neuraminic Acid) and R3 is Hydrogen, the branched polysaccharide is a derivative of the blood group Sd-a determinant, which is advantageously obtained from an intermediate product (the polysaccharide with R1=R2=R3=Hydrogen) by the methods described in the documents 17 and 18.
When Rl is Hydrogen R2 is NeuNAc~2 (N-acetylneuraminic Acid) or HSO3 and R' is Fuc~1 (fucose), the branched polysaccharide is a derivative of the pharmaceutical products sialyl- or sulfated- Lewis X described in the documents 19 and 20.
The bindings between Gal ~ 1 - 4 Glc and R1, R2 are advantageously obtained from an intermediate product (the polysaccharide with R1 = R2 = R3 = Hydrogen) by the method described in the document 17.
The present invention concerns also the microorganism producing the branched polysaccharide having R1 = R2 = R3 = Hydrogen.
Advantageously, said microorganism corresponds to a strain of Lactobacillus helveticus, preferably the strain o~ ~actobacillus helveticus CNCM I-1449.
A deposit of this microorganism has been made according to the Budapest Treaty on July 27, 1994, at the Collection Nationale de Culture de Microorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 PARIS CEDEX
15, FRANCE.
The present invention also concerns the food having improved organoleptic and texture properties comprising the polysaccharide and/or the microorganism according to the invention.
Preferably, said food composition is a set-style 215513~
.
acidified milk or a stirred acidified milk.
Another aspect of the present invention concerns the cosmetic composition comprising the polysaccharide and/or the microorganism according to the invention.
According to a preferred embodiment of the invention, said cosmetical composition is ~ cosmetic product intended for the buccal hygiene, choosen among the group consisting of a tooth paste, tooth gel, mouth rinse, chewing-gum and/or tablet.
ZlS5134 According to another preferred embodiment of the present invention, said cosmetical composition is a product intended for the skin hygiene, choosen among the group consisting of a cream, ointment or balsam.
Another aspect of the present invention concerns the pharmaceutical composition comprising the polysaccharide ~and/or the microorganism according to the present invention.
Advantageously, said composition is a antidiaorrheic product choosen amQng the group consisting of a capsule, syrup, powder and/or tablet.
A last aspect of the present invention concerns a diagnostic and/or analytic device comprising the branched polysaccharide according to the invention ~or the trapping of specific molecules and/or microorganisms.
Brief description of the drawings.
The figure 1 represents the chromatographic (FPLC) analysis of the polysaccharide according to the invention.
The figure 2 represents six major fractions separated by gel filtration from the polysaccharide hydrolysate.
The ~igure 3 represents the chromatographic (HPAE -PAD) analysis of the fraction II.
The figure 4 represents the chromatographic (HPAE
- PAD~ analysis o~ the fraction IV.
Description of a preferrçd embodime~ of the present invention.
The present invention concerns a new natural soluble branched polysaccharide with a main chain, having repeating side chains which are only lactose units; said lactose is possibly substituted.
The "branched polysaccharide" according to the invention is a saccharide having more than 10 repeating ~ g units, preferably more than 40 repeating units.
Said branched polysaccharide has preferably the structure of a "polymer'l consisting of the repetition of identical single "units" comprising one side chain 2 branched on a main chain as described below:
~ ~ n n>1 The production and the physico-chemical structure of a polysaccharide according to the invention will be described hereafter.
1. PR~PARATION AND PURIFICATION OF THF~ h~XOPOl,YSACCHARIDE
pRcDuc~n BY T,~CTO~ACIL~US HELV~TICUS CNCM I-1449.
1.1 Fermentation Co~dit ons.
Lactobacillus helveticus CNCM I-1449 was a ropy strain from the Nestlé strain collection. Among the 168 strains of L. helveticus of the Nestlé collection, only 2 strains produce exopolysaccharides.
The growth medium was 10~ reconstituted skim milk heat-treated (115C, 35 min) for sterilization prior to fermentation. A one-liter scale fermentor with a magnetic stirrer was used -for regulating the pH during the fermentation. The pH was maintained at 5.5 by using 2N sodium hydroxide. Stirring rate was maintained at 60 RPM. Incubation was made at 40C. The amount of starter culture inoculated to the medium was 1~. During the fermentation (time = 6h, 9h and 24h), several samples were taken and frozen for further analysis and polysaccharide extractior.
1.2. Ex~rac~ion of the Polysaccharide An equal volume of trichloroacetatic acid (40~ was added to the sample to remove proteins by precipitation, followed by centrifugation (17,000g, 20 minutes). To the supernatant fraction containing polysaccharides, the same volume o~ acetone was added. Precipitated polysaccharides were then separated by centrifugation (17,000g, 20 minutes).
The resulting precipitated fraction was dissolved in distilled water and pH was adjusted to 7.0 with sodium hydroxide solution. After dialysis against distilled water (overnight), insoluble substances were removed by ultracentrifugation (llO,OOOg, l hour).
The supernatant fraction containing polysaccharides was lyophilized and crude dehydrated polysaccharides were finally obtained. Total neutral sugar content was determined by the phenol-sulphuric acid method.
1.3. Size of the ~xopolysaccharide.
The filtration was conducted to confirm purity and estimate the molecular weight o~ polysaccharides using FPLC
system (Pharmacia). The column used was Superose 6 (l.0 cm x 30cm) (Fig. 1). 200 ~l samples containing 200-400 ~g dehydrated polysaccharides were applied on to the column, eluted with 50 mM phosphate buffer at pH 7.2 at the rate o~
0.5 ml/min. Fractions (l.0 ml) were collected in tubes.
Polysaccharide content in each tube was determined as total neutral sugar by the phenol-sulphuric acid method.
1.4. Monosaccharide Composition.
Monosaccharide composition o~ a freeze-dried polysaccharide was analyzed using gas-liquid chromatography technique (ref. 21).
The exopolysaccharide obtained ~rom the spent culture medium was examined after extraction ~rom three samples (time = 6h, 9h and 24h). The yield was found to increase as a function of the fermentation time, but the size as well as the monosaccharide composition of the polymer were found invariable:
Fermentation Crude Neutral Pure Monosaccharide Time Yield Sugars Yield Composition (hours) (mg/L) (~) (mg/L) Rha Gal Glc GalNAc 6 42 27.6 11.6 - 1.1 1 -9 142 70.4 100.0 - 1.1 24 272 83.3 226.6 - 1.1 The figure 1 shows the elution and the purity of the polysaccharide obtained at t = 24 h, by FPLC analysis (with a column of Superose 6). The polysaccharide was eluted at around the exclusion limit (approximately 2 X 106 M.W.).
1.5. Yields ~f Polysaccharide Ob~ained in Non-Regulated Fermenta~'ons.
The polysaccharide described above was also produced during fermentations in set-style conditions, by Lactobacillus helveticus CNCM I-1449 alone, or by this strain used together with a strain of Streptococcus thermophilus (for example S. thermophilus YS4).
For this purpose, the growth medium was 10 reconstituted MSK (skim milk powder: 100 g/l and yeast extract: 1 g/l) heat-treated (115C, 35 min) for sterilization prior to fermentation. The typical sample size was 250 ml, the incubation was made at 40C and the amount of starter culture inoculated to the medium was 1 ~. The yields of pure polysaccharide obtained in such conditions were the following:
Strains in theFermentation Time Pure Yield Starter Culture (hours) (mg/l) L . hel ve ti cus 8 8 5 L . hel ve ti cus 4 !23 CNCM I-1449 & S.
thermophi 1 us YS4 215513~
2. METHODS USED FOR TH~ STRUCTURAL CHARACTE~IZATION OF THE
F:~OPOI.YSACC~IARIDE PRODUCF~n BY LACTOBACILLI~S ~T,VETICUS CNCM
I-1449 .
2.1. Monosaccharide Analysis.
Polysaccharide (Q.1 mg) was methanolysed (methanolic 0.5 M HCl, 24h, 80C), N-reace~ylated (one night at room temperature). The trimethylsilylated methyl glycosides were analysed by gas phase chromatography (Varian 3400) using a BP1 fused-silica capillary column (25 m X 0.32 mm, SGE). The elution was performed by applying on the column a temperature gradient from 120C to 240C at 2C minl. The absolute configuration of the monosaccharides was determined by GLC of the trimethylsilylated (N-reacetylated) (-)-2-butyl glyclosides.
2.2 Methylation Analysis.
Samples (native polysaccharide and oligosaccharide-alditols) were permethylated, and methylated products were subjected either to methanolysis or acid hydrolysis (trifluoroacetic acid 4N, 4 h, 100C) followed by reduction with BD4Na.
The partially methylated and acetylated (pyridine.
anhydride acetic 1:1) methyl glycosides and alditols were identified by GLC (BP1 column) and GLC MS in e.i.mode on a Nermag R10-lOS mass spectrometer using an electron energy of 70 ev and an ionizing current of 0.2 mA.
2.3. Partial Acid Hydrolysis.
Polysaccharide (40mg) was hydrolysed in 4 ml of 0.5 M trifluoroacetic acid during lh30 at 100C. Complete hydrolysis and obtention of low mass oligosaccharides were monitored by thin ~ayer chromatography on Silica Gel 60 F254 aluminium sheets (Merck) using a butanol/water/acetic acid 2:1:1.5 solvant and detection with orcinol-sulfuric acid.
2.4. H.p.a.e-~.a.d. Chromatography.
Fractionation of HW40 peaks was performed on HPAE
- PAD Dionex LC system consisting of a Dionex Bio-LC
~uaternary gradient module, a model p.a.d. 2 detector and a Carbopac PA-1 pellicular anion exchange column (250 X 9 mm).
Two elution programs were used :
- progr~m 1 : 99 :1 eluent A (0.1 M NaOH) - eluent B (0.1 M
NaOH containing M CH3COONa) for 0.2 min then going to 65:35 eluent A (0.1 M NaOH containing M
CH3COONa) in 60 min at 3 ml min~1; 5 - proqr~m 2 : 98:2 eluent ~ - eluent B then going to 70:30 eluent A - eluent B in 60 min.
The eluted ~ractions were immediately neutralized with M acetic and liophilized. The fractions were successively desalted on a column (6 X 1 cm) of Dowex 50 X
8 (H') resin and on a column of Fractogel (55 X 2 cm) using deionised water as eluent.
~.5. 1H-Nuclear Maanetic Res~nn~nce SpectroscQpy.
For lH-NMR measurements, the deuterium-exchanged oligosaccharides were dissolved in 0.5 ml of 2H2O (99.96 ~
atom 2H, Aldrich). The 400 MHz 1H-NMR experiments were performed with a Bruker AM-400WB spectrometer, e~uipped with a 5 mm 1H-/l3C mixed probe head, operating in the pulsed Fourier transform mode and controlled by an Aspect 3000 computer (Centre Commun de Mesures, Université de Lille Flandres Artois).
215513~
All the spectra were obtained at a probe temperature of 353K. One dimensional spectra were obtained with a spectral width of 3000 Hz for a 16 K frequency-domain points and time-domain data points giving a final digital resolution of 0.365 Hz/point.
The 100 MHz l3C-NMR experiments ~ere obtained with the standard Brucker pulse program Powgate with lH composite pulse decoupling. The spectral width was 22.727 Hz for a 32 K frequency-domain data points and time-domain data giving a final digital resolution of 1.387 Hz/point; a ninety-degree pulse (6 ~s) and 1 s recycle delay were used. The chemical given relative to the signal of the methyl group of acetone (~ 2.225 for lH and ~ 31.55 for 13C) .
The 2D-homonuclear COSY 45, COSY with simple, double and triple relay transerts were performed by use of the standard Bruker pulse program library or the programs given by B. Perly (C.E.A., Saclay). For all RCT experiments, re~ocusing delays of 35 ms were chosen and the relaxation delay was 2 s. In all these experiments, the spectral width was 1840 Hz, the lH ninety-degree pulse was 10.6 ~s; 256 W X
2K data matrices were acquired, which were zero-filled prior to Fourier transform, to obtain a 1 K X 2 K spectral data matrix and a sine-bell squared function was used in both dimensions.
The 2D-l3C/lH COSY experiments were performed with simultaneous suppression of lH homonuclear couplings by use of the standard Bruker pulse program XHCORRD. Refocusing delays were adjusted to an average ljc~ coupling constant of 150 KHz. lH and 13C ninety-degree pulse width were 10.6 and 6 ~s. The relaxation delay was 0.8 s. 128 W X 4 K data matrix was acquired, which was zero-filled prior to Fourier transform, to obtain a 512 W X 4 K spectral data matrix. An exponential function (LB = 1 Hz) for l3C-subspectra and a sine-bell function for lH-spectra were applied to enhance the signal to noise ratio.
F:~OPOI.YSACC~IARIDE PRODUCF~n BY LACTOBACILLI~S ~T,VETICUS CNCM
I-1449 .
2.1. Monosaccharide Analysis.
Polysaccharide (Q.1 mg) was methanolysed (methanolic 0.5 M HCl, 24h, 80C), N-reace~ylated (one night at room temperature). The trimethylsilylated methyl glycosides were analysed by gas phase chromatography (Varian 3400) using a BP1 fused-silica capillary column (25 m X 0.32 mm, SGE). The elution was performed by applying on the column a temperature gradient from 120C to 240C at 2C minl. The absolute configuration of the monosaccharides was determined by GLC of the trimethylsilylated (N-reacetylated) (-)-2-butyl glyclosides.
2.2 Methylation Analysis.
Samples (native polysaccharide and oligosaccharide-alditols) were permethylated, and methylated products were subjected either to methanolysis or acid hydrolysis (trifluoroacetic acid 4N, 4 h, 100C) followed by reduction with BD4Na.
The partially methylated and acetylated (pyridine.
anhydride acetic 1:1) methyl glycosides and alditols were identified by GLC (BP1 column) and GLC MS in e.i.mode on a Nermag R10-lOS mass spectrometer using an electron energy of 70 ev and an ionizing current of 0.2 mA.
2.3. Partial Acid Hydrolysis.
Polysaccharide (40mg) was hydrolysed in 4 ml of 0.5 M trifluoroacetic acid during lh30 at 100C. Complete hydrolysis and obtention of low mass oligosaccharides were monitored by thin ~ayer chromatography on Silica Gel 60 F254 aluminium sheets (Merck) using a butanol/water/acetic acid 2:1:1.5 solvant and detection with orcinol-sulfuric acid.
2.4. H.p.a.e-~.a.d. Chromatography.
Fractionation of HW40 peaks was performed on HPAE
- PAD Dionex LC system consisting of a Dionex Bio-LC
~uaternary gradient module, a model p.a.d. 2 detector and a Carbopac PA-1 pellicular anion exchange column (250 X 9 mm).
Two elution programs were used :
- progr~m 1 : 99 :1 eluent A (0.1 M NaOH) - eluent B (0.1 M
NaOH containing M CH3COONa) for 0.2 min then going to 65:35 eluent A (0.1 M NaOH containing M
CH3COONa) in 60 min at 3 ml min~1; 5 - proqr~m 2 : 98:2 eluent ~ - eluent B then going to 70:30 eluent A - eluent B in 60 min.
The eluted ~ractions were immediately neutralized with M acetic and liophilized. The fractions were successively desalted on a column (6 X 1 cm) of Dowex 50 X
8 (H') resin and on a column of Fractogel (55 X 2 cm) using deionised water as eluent.
~.5. 1H-Nuclear Maanetic Res~nn~nce SpectroscQpy.
For lH-NMR measurements, the deuterium-exchanged oligosaccharides were dissolved in 0.5 ml of 2H2O (99.96 ~
atom 2H, Aldrich). The 400 MHz 1H-NMR experiments were performed with a Bruker AM-400WB spectrometer, e~uipped with a 5 mm 1H-/l3C mixed probe head, operating in the pulsed Fourier transform mode and controlled by an Aspect 3000 computer (Centre Commun de Mesures, Université de Lille Flandres Artois).
215513~
All the spectra were obtained at a probe temperature of 353K. One dimensional spectra were obtained with a spectral width of 3000 Hz for a 16 K frequency-domain points and time-domain data points giving a final digital resolution of 0.365 Hz/point.
The 100 MHz l3C-NMR experiments ~ere obtained with the standard Brucker pulse program Powgate with lH composite pulse decoupling. The spectral width was 22.727 Hz for a 32 K frequency-domain data points and time-domain data giving a final digital resolution of 1.387 Hz/point; a ninety-degree pulse (6 ~s) and 1 s recycle delay were used. The chemical given relative to the signal of the methyl group of acetone (~ 2.225 for lH and ~ 31.55 for 13C) .
The 2D-homonuclear COSY 45, COSY with simple, double and triple relay transerts were performed by use of the standard Bruker pulse program library or the programs given by B. Perly (C.E.A., Saclay). For all RCT experiments, re~ocusing delays of 35 ms were chosen and the relaxation delay was 2 s. In all these experiments, the spectral width was 1840 Hz, the lH ninety-degree pulse was 10.6 ~s; 256 W X
2K data matrices were acquired, which were zero-filled prior to Fourier transform, to obtain a 1 K X 2 K spectral data matrix and a sine-bell squared function was used in both dimensions.
The 2D-l3C/lH COSY experiments were performed with simultaneous suppression of lH homonuclear couplings by use of the standard Bruker pulse program XHCORRD. Refocusing delays were adjusted to an average ljc~ coupling constant of 150 KHz. lH and 13C ninety-degree pulse width were 10.6 and 6 ~s. The relaxation delay was 0.8 s. 128 W X 4 K data matrix was acquired, which was zero-filled prior to Fourier transform, to obtain a 512 W X 4 K spectral data matrix. An exponential function (LB = 1 Hz) for l3C-subspectra and a sine-bell function for lH-spectra were applied to enhance the signal to noise ratio.
3. STRUCTURE OF T~F~ EXOPOIIYSACCHARII~E PRoDucF~n BY
T,~CTO~ACITT~US ~TV~TICUS CNCM I-1~49.
3.1. Isolation and Composition Analysis of the Polysacchari~e.
GLC. analysis of the trimethylsilylated glycosides and (-)-2-butyl glyosides has confirmed the presence of D-galactose and D-glucose in a molar ratio 1:1.
Nl~ s~7ectros~0~v.
The 400 MHz 1H n.m.r. spectrum of the native polysaccharide recorded in D2O at 80C shows 4 signals at 5.201, 5.158, 4.568 and 4.350 ppm characteristic of anomeric protons in a ratio 1:2:2:1 suggesting a hexasaccharide repeating unit (table 1).
This is confirmed by the 100 MHz 13C spectrum (table 1) that exhibits six anomeric carbon signals (108.95, 103.74, 103.8, 102.52, 100.26 and 99.87 ppm).
According to the 1H spin system, on individual sugar units depicted on the two setaps relayed COSY sprectrum, the monosaccharides may be respectively identi~ied as ~-Galf (residue A), g-D-Glcp (residues B and E), ~-D-Glcp (residue C), ~-D-Galp (residue D) and g-D-Galp (residue F).
Examination o~ the relayed COSY spectra o~
oligosaccharide alditols IV B and II A obtained by partial acid hydrolysis (see below) leads to the obvious identi~ication of the g-D-Galp. As demonstrated by the COSY
spectrum of the polysaccharide, the H-2 and H-3 o~ the g-D-Galp F residue exhibit a strong coupling constant which does 215513~
not allow to analyse their multiple patterns.
Correlation peaks observed in the lH - 13C
heteronuclear COSY spectrum shows that one of the proton resonances (5.158 ppm) is connected to the carbon resonance deshielded at 108.95 ppm that proves a $-anomeric configuration ~or the Galf A residue.
Most of the proton resonances may be assigned in the homonuclear COSY spectrum except for the H-5 and H-6 spin systems of the $-D-Galp F residue. The carbon resonances may also be assigned by direct correlation to their attach protons.
The two remainding unassigned carbons at 73.52 and 61.12 ppm were deduced to correspond with the C-5 and C-6 atom resonances of $-D-Galp F residue.
In summary, all these assignments clearly furnish the substitution pattern of each sugar unit, according to their downfield shifted carbon resonances :
C-3 and C-5 for ~-D-Gal~ A, C-3 for ~-D-Glcp C, C-3 ~or ~-D-Galp D, C-3 for $-D-Glcp B and C-4 for $~D-Glcp E.
The sixth sugar unit $-D-Galp F, which does not possess any downfield shifted l3C resonance occurs consequently in non-reducing position.
3.2. Methylation Analysis.
The NMR results are supported and confirmed by GLC
MS analysis of the partially methylated alditol acetates and methyl glycosides (Table 2) obtained from the permethylated polysaccharides. Indeed, it demonstrates the presence of terminal galactosyl residue. 3-linked glucosyl residue, 4-.
linked glucosyl residue, 3-linked galactosyl residue and 3,5 linked galactosyl residue in a ratio 1:2:1:1:1 respectively.
3.3. Paxtial Acid Hydrolys-s.
In order to elucidate the position of the branched terminal galactosyl residue, oligosaccharldes were produced by partial acid hydrolysis of the native polysaccharide. Six major fractions were separated by gel filtration on Fractogel HW40 F from the hydrolysate (Fig.2).
Two of them (fractions II and IV) were subjected to HPAE - PAD chromatography (Fig. 3, 4 and table 3) and the structure of subfractions denoted II A, IV A and IV B was investigated both by NMR and methylation analysis.
Oligosaccharide-alditol IV A contains two Glc residues and one hexitol residue, as shown by the lH spin system depicted in the two-steps relayed COSY spectrum. The two Glc residues occur at the non-reducing terminal position, as confirmed by the methylation analysis which furnished 2, 3, 4, 6-tetra-O-methyl glucitol. The last derivative shows a pattern corresponding to a C-3 and C-5 substitution characteristic of a furanic sugar. From the previous n.m.r.
data, this hexitol originates from the ~-D-Galf A unit. These findings lead to the ~ollowing structure :
E A
$-D-Glcp-(1-3)-D-Gal5-ol B
i~-D-Glcp-l It must be noted that due to the symetry displayed by the lH spin system of galactitol, the lH NMR assignment of the compound was achieved after the elucidation of compound IV B structure.
Oligosaccharide-alditol IV B contains $-D-Galp and $-D-Glcp in the ration 1:1, as clearly shown by the pattern of the vicinal coupling constants. The methylation analysis (Table 4) indicated the presence of 2, 3, 4!~ 6-tetra-O-methyl galactose, 2, 3, 6-tri-O-methyl glucose and 1, 2, 4, 5, 6-penta-O-methyl galacticol. Therefore, oligosaccharide-alditol 2 possesses the following structure :
F E A
$-D-Galp-(1-4)-~-D-Glcp-(1-3)-Gal-ol The attachment of ~-D-Glcp at C-3 of Gal-ol is not susceptible to modify dramatically the chemical shift of the H-l and H-2 resonances of the hexitol, when compared with the NMR data from compound IV A. Therefore, the two signals at 3.77 and 4.056 ppm can be assigned to H-l and H-2 of Gal-ol, whereas the H-5 and H-6 atoms resonances are upfield shifted at 4.149 and 3.68 ppm, by comparison with commpound IV A
(Table 3).
In the NMR spectra of IV A and IV B, the H-5 resonance of Gal-ol is the most upfield shifted atom resonance of the molecule and this observation is in agreement with the assignments proposed by others.
Oligosaccharide-alditol II A (Table 2) contains 2 ~-D-Glcp, 1 $-D-Galp and 1 ~-D-Glcp residues, whereas the presence of C-3 and C-5 substituted Gal-ol can be easily deduced by the similarity in chemical shifts of H-l to H-6 of Gal-ol in compound 1. Since the two $-D-Glcp units and the non-reducing terminal $-D-Galp have already been located in oligosaccharide-alditols IV A and IV B, and according to the 215513~
.
fact that the ~-D-Glcp B residue is C-3 substituted, the oligosaccharide-alditol II A possesses the following structure :
C B A
~-D-Glcp-(1-3)-~-D-Glcp-(1-5)-Gal-ol F E :~3 ~-D-Galp-(1-4)-$-D-Glcp-l The sixth sugar unit, ~-D-Galp, was not observed among the products of the partial hydrolysis. Nevertheless, the fact that the ~-D-Glcp unit C is C-3 substituted and that the only ~-D-Galp present in the polysaccharide occupies the non-reducing position (from the NMR data) lead to conclude that the ~-D-Galp is attached to this ~-D-Glcp residue.
Therefore, the structural unit of the polysaccharide was defined as follows:
D C B A
-3)-~-D-Galp-(1-3)-~-D-Glcp-(1-3)-~-D-Glcp-(1-5)-~-D-Galf-(l-F E
~-D-Galp-(1-4)-~-D-Glcp 1 215~13~
Table 1 : NMR chemical shift of the polysaccharide from in D2O at 80C (internal standard : acetone) Residue .. .. . ............ ., ............ .
,~ssiynment $-Galp $-Glcp ~-Glcp ~-Galp ~-Glcp ~-Galp A B C D I E F
H (ppm) H-1 5.158 4.568 5.201 5.158 4.5684.350 H-2 4.240 3.289 3.542 3.888 3.2063.470 H-3 4.419 3.532 3.752 3.776 3.4883.470 H-4 4.125 3.533 3.512 3.999 3.5333.796 H-5 4.118 3.354 3.920 4.108 3.4553.582 H-6 3.723 3.84 3.61 3.62 3.903.67 H-6' 3.62 3.66 3.61 3.62 3.703.67 l3C (ppm) C-1 108.95 103.08 99.87 100.26 102.52 103.74 C-2 80.52 72.72 71.19 68.33 73.4771.19 C-3 85.08 85.15 82.90 77.31 74.9075.32 C-4 81.64 70.14 70.20 69.19 80.1968.69 C-5 78.84 76.43 72.48 71.27 73.5275.80 C-6 61.68 61.38 61.07 61.26 61.1260.99 215513~
Table 2 : Methylation analysis of the native polysaccharide ¦ derivative ¦ molar ratio partially partially methylated alditol- methylated acetates ~ethylglycosides 52, 3, 4, 6 Gal 2, 4, 6 Glc 22 19 2, 3, 6 Glc 9 13 2, 4, 6 Gal 7 16 2, 6 Gal 1 9 able 3 : NMR chemical shifts of the oligosaccharide-alditols obtained by acid partial hydrolysis of the polysaccharide from Lactobacillus helveticus in D20 at 353K.
Residue in IV A Residue in IV B Residue i~ II A
assignment Gal-ol ~-Glcp ~-Galp Gal-ol ~-Glcp ~-Glcp Gal-ol ~-Glcp ~-Glcp ~-Glcp ~-Galp A-ol E F A-ol B E A B C D E
H (ppm) H-1 3.77 4.6864.5573.77 4.539 4.4283.77 4.707 5.3404.590 4.460 H-1' 3.77 - - 3.77 - - 3.77 H-2 4.118 3.3283.3204.0563.421 3.5414.1203.423 3.5593.379 3.542 H-3 4.141 3.5003.4843.9153.661 3.6614.1483.647 3.7483.647 3.665 H-4 3.907 3.4243.4063.7973.674 3.9273.911,3.7483.4633.678 3.928 H-5 4.301 3.5433.4534.1493.62 3.7324.2993.47 4.0253.590 3.73 H-6 3.77 3.8993.8993.68 4.02 3.79 3.77 3.97 3.80 3.90 3.79 H-6' 3.77 3.7583.7583.68 3.80 3.79 3.77 3.85 3.80 3.75 3.79 e,~
Table 4 : Methylation analysis of oligosaccharide-alditol (partially methylated and acetylated methyl glycosides) IV A, IV B and II A obtained by partial acid hydrolysis of native polysaccharide.
¦ derivative ¦ molar ratio IV A IV B II A
1, 2, 4, 5, 6 Gal-ol - 1 -1, 2, 4, 6 Gal-ol 102, 3, 4, 6 Glc 1.2 - 1.0 2, 3, 4, 6 Gal (*) - 1.4 0.9 2, 4, 6 Glc - - 1.3 2, 3, 6 Glc - 1.0 1.2 (*) : Due to its high volatility the value is lower than expected.
The composition comprising the polysaccharide and/or microorganism according to the invention are described in the following non limiting examples.
Ex~mple 1: se~-s~yle a~ir~;fied milk.
Set-style acidifed milk comprising the L.
helveticus strain according to the invention and two S.
thermophilus strains, traditionnaly used for the production of a set-style yoghourt, was obtained by the following process.
To a whole milk comprising 3,7 ~ fats, 2,5 skimmed milk powder was added.
40 liters of this milk was pasteurized at 92C for six minutes, homogeneized at 75C and 150 bars (two levels) 215513~
and cooled at a temperature around 42C.
The ~reeze-dried S. thermophilus CNCM I-1422, S.
thermophilus CNCM I-1424 and L. helveticus CNCM I-1449 strains were reactived with several successive cultures in 5 a sterile MSK medium (skimmed milk powder reconstituted at 10~, comprising 0,1 ~ of a commercial yea$t extract).
A deposit of the microorganism has been made according to the Budapest Treaty on May 18, 1994, for the Streptococcus strains and on July 27, 1994, for the Lactobacillus strain at the Collection Nationale de Culture de Microorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 PARIS CEDEX 15, FRANCE.
The sterilized milk was inoculated with 1 ~ of the third culture of each S. thermophilus strain and with 2 ~ of the second culture of L. helveticus strain taken at the medium coagulation stage.
The milk was incubated at 42C and at a pH around 4.65, and then cooled at a temperature of 4C.
20 Ex~ple 2 : aci~;fied whe~ milk.
Whey milk comprising the L. helveticus strain acording to the invention and two S. thermophilus strains, traditionnaly used for the production of a yoghourt, was obtained by the following process.
A sweet lactoserum powder was reconstituted at 12,5~ in water.
40 liters of this whey was pasteurized at 92C for six minutes, homogeneized at 75C and 150 bars (two levels) and cooled at a temperature around 42C.
The freeze-dried S. thermophilus CNCM I-1422, S.
thermophilus CNCM I-1424 and L. helveticus CNCM I-1449 strains were reactived with several successive cultures in 215513~
a sterile MSK medium (skimmed milk powder reconstituted at 10~, comprising 0,1 ~ o~ a commercial yeast extract).
A deposit of the microorganism has been made according to the Budapest Treaty on May 18, 1994, for the Streptococcus strains and on July 27, 1994, for the Lactobacillus strain at the Collection Nationale de Culture de Microorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 PARIS CEDEX 15, FRANCE.
The sterilized milk was inoculated with 1 ~ of the third culture of each S. thermophilus strain and with 2 ~ of the second culture o~ L. helveticus strain taken at the medium coagulation stage.
The whey milk was incubated at 42C and at a pH
around 4. 65, and then cooled at a temperature of 4C.
Example 3 : stirred aci~ified milk.
A stirred acidifed milk comprisin~ the L.
helveticus CNCM I-1449 strain according to the invention and two commercialized S. thermophilus strains, traditionnaly used for the production of a stirred yoghourt, was obtained by the following process.
The milk was obtained from a whole milk comprising 3,7 ~ fats, by the addition of 2,5 ~ skimmed milk powder.
40 liters of this milk was pasteurized at 105C for two minutes, homogeneized at 75C and 300 bars (first level) and cooled at a temperature around 43C.
The lyophilized S. thermophilus CNCM I-1421, S.
thermophilus CNCM I-1423 and L. helveticus CNCM I-1449 strains were reactived with several successive cultures in a sterile MSK medium.
A deposit of the microorganism has been made according to the Budapest Treaty on May 18, 1994, for the 215513~
StreptococcUs strains and on July 27, 1994, for the LactobacillUs strain at the Collection Nationale de Culture de MicroorganiSmes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 PARIS CEDEX 15, FRANCE.
The sterilized milk was inoculated with 1 ~ of the third culture of each S. thermophilus stra~n and with 2 ~ o the third culture of L. helveticus strain taken at the medium coagulation stage.
The milk was incubated at 43C and at a pH around 4.65, and then cooled at a temperature of 4C during stirring.
The following table 5 represents the properties of the obtained products.
Table 5.
¦ ¦¦Example 1 ¦Example 3 Acidification 6 h 7 h 15 time at pH = 4.65 pH of the product 4.34 4.49 20 after 1 day at 4C
pH of the product 4.1 4.3 after 24 days at taste after 24 good taste, very good taste, 25 days slightly acid aromatic smooth texture smooth and onctuous texture F~xample 4 : cosrnetic composi tion for buccal hygiene .
CHEMICAL NA~E ¦ TRADE NAME ¦ 96 WEIGHT
PHASE A
PEG-40 Hydrogenated Cremophor RH 40 0.10 castor oil Flavour Strawberry E 2226 0.04 Flavour Raspberry 9/022436 0.10 PHASE B
10 Sodium Cyclamate Sodium Cyclamate 0.10 Exopolysaccharide -- 0.50-5.00 according to the present invention Demineralized water 94.66-99.16 Ex~mI~le 5 : co~netic composi tion for sk7 n hyaiene.
¦ % WEIGHT
OIL PHASE
BRIJ 721 (Steareth 21) 4.00 Cetyl alcohol 10.00 Mineral oil 5.00 Propyl parahydroxybenzoate 0.02 WATER PHASE
CARBOPOL 934 (Carbomer 934) 0.10 Sodium hydroxide (solution 0.10 at 10~) Methyl parahydroxybenzoate 0.18 Exopolysaccharide according 0.50-5.00 to the present invention ¦Demineralized water 75.60-80.10 TOTA~ 100 E~xample 6 : Pharmaceutical comF~osi tion for anti -diarrheoic usage .
A pharmaceutical composition was obtained as a capsule which was made with gelatine and! water, and which contained from 5 to 50 mg of the exopolysaccharide according to the present invention. Alternatively, powdered tablet formulations can be obtained directly from the acidified cultured milks described in the above examples 1, 2 and 3, by freeze-drying these fermented milks and whey.
References.
1. Kolenbrander, P.E., Ganeshkumar, N., Cassels, F.J. &
Hughes, C.V., Coaggregation: speciic adherence among human oral plaque bacteria. The FASEB Journal, 7 (1993) 406-413.
2. Karlsson, K.-A., ~n;m~l glycosphingolipids as membrane attachment sites for bacteria. Annual Review of Biochemistry, 58 (1989) 309-350.
3. Hughes, R.C., Mac-2: A versatile galactose-binding protein of mammalian tissues. Glycobiology, 4 (1994) 5-12.
4. Truong, M.-J., Gruart, V., Kusnierz, J.-P., Papin, J.-P., Loiseau, S., Capro~, A. & Capron, M., Human neutrophils express immunoglobulin E (IgE)-binding proteins (Mac-2/~BP) of the S-type lectin family: role in IgE-dependent activation. Journal of EXperimental Medicine, 177 (1993) 243-248.
T,~CTO~ACITT~US ~TV~TICUS CNCM I-1~49.
3.1. Isolation and Composition Analysis of the Polysacchari~e.
GLC. analysis of the trimethylsilylated glycosides and (-)-2-butyl glyosides has confirmed the presence of D-galactose and D-glucose in a molar ratio 1:1.
Nl~ s~7ectros~0~v.
The 400 MHz 1H n.m.r. spectrum of the native polysaccharide recorded in D2O at 80C shows 4 signals at 5.201, 5.158, 4.568 and 4.350 ppm characteristic of anomeric protons in a ratio 1:2:2:1 suggesting a hexasaccharide repeating unit (table 1).
This is confirmed by the 100 MHz 13C spectrum (table 1) that exhibits six anomeric carbon signals (108.95, 103.74, 103.8, 102.52, 100.26 and 99.87 ppm).
According to the 1H spin system, on individual sugar units depicted on the two setaps relayed COSY sprectrum, the monosaccharides may be respectively identi~ied as ~-Galf (residue A), g-D-Glcp (residues B and E), ~-D-Glcp (residue C), ~-D-Galp (residue D) and g-D-Galp (residue F).
Examination o~ the relayed COSY spectra o~
oligosaccharide alditols IV B and II A obtained by partial acid hydrolysis (see below) leads to the obvious identi~ication of the g-D-Galp. As demonstrated by the COSY
spectrum of the polysaccharide, the H-2 and H-3 o~ the g-D-Galp F residue exhibit a strong coupling constant which does 215513~
not allow to analyse their multiple patterns.
Correlation peaks observed in the lH - 13C
heteronuclear COSY spectrum shows that one of the proton resonances (5.158 ppm) is connected to the carbon resonance deshielded at 108.95 ppm that proves a $-anomeric configuration ~or the Galf A residue.
Most of the proton resonances may be assigned in the homonuclear COSY spectrum except for the H-5 and H-6 spin systems of the $-D-Galp F residue. The carbon resonances may also be assigned by direct correlation to their attach protons.
The two remainding unassigned carbons at 73.52 and 61.12 ppm were deduced to correspond with the C-5 and C-6 atom resonances of $-D-Galp F residue.
In summary, all these assignments clearly furnish the substitution pattern of each sugar unit, according to their downfield shifted carbon resonances :
C-3 and C-5 for ~-D-Gal~ A, C-3 for ~-D-Glcp C, C-3 ~or ~-D-Galp D, C-3 for $-D-Glcp B and C-4 for $~D-Glcp E.
The sixth sugar unit $-D-Galp F, which does not possess any downfield shifted l3C resonance occurs consequently in non-reducing position.
3.2. Methylation Analysis.
The NMR results are supported and confirmed by GLC
MS analysis of the partially methylated alditol acetates and methyl glycosides (Table 2) obtained from the permethylated polysaccharides. Indeed, it demonstrates the presence of terminal galactosyl residue. 3-linked glucosyl residue, 4-.
linked glucosyl residue, 3-linked galactosyl residue and 3,5 linked galactosyl residue in a ratio 1:2:1:1:1 respectively.
3.3. Paxtial Acid Hydrolys-s.
In order to elucidate the position of the branched terminal galactosyl residue, oligosaccharldes were produced by partial acid hydrolysis of the native polysaccharide. Six major fractions were separated by gel filtration on Fractogel HW40 F from the hydrolysate (Fig.2).
Two of them (fractions II and IV) were subjected to HPAE - PAD chromatography (Fig. 3, 4 and table 3) and the structure of subfractions denoted II A, IV A and IV B was investigated both by NMR and methylation analysis.
Oligosaccharide-alditol IV A contains two Glc residues and one hexitol residue, as shown by the lH spin system depicted in the two-steps relayed COSY spectrum. The two Glc residues occur at the non-reducing terminal position, as confirmed by the methylation analysis which furnished 2, 3, 4, 6-tetra-O-methyl glucitol. The last derivative shows a pattern corresponding to a C-3 and C-5 substitution characteristic of a furanic sugar. From the previous n.m.r.
data, this hexitol originates from the ~-D-Galf A unit. These findings lead to the ~ollowing structure :
E A
$-D-Glcp-(1-3)-D-Gal5-ol B
i~-D-Glcp-l It must be noted that due to the symetry displayed by the lH spin system of galactitol, the lH NMR assignment of the compound was achieved after the elucidation of compound IV B structure.
Oligosaccharide-alditol IV B contains $-D-Galp and $-D-Glcp in the ration 1:1, as clearly shown by the pattern of the vicinal coupling constants. The methylation analysis (Table 4) indicated the presence of 2, 3, 4!~ 6-tetra-O-methyl galactose, 2, 3, 6-tri-O-methyl glucose and 1, 2, 4, 5, 6-penta-O-methyl galacticol. Therefore, oligosaccharide-alditol 2 possesses the following structure :
F E A
$-D-Galp-(1-4)-~-D-Glcp-(1-3)-Gal-ol The attachment of ~-D-Glcp at C-3 of Gal-ol is not susceptible to modify dramatically the chemical shift of the H-l and H-2 resonances of the hexitol, when compared with the NMR data from compound IV A. Therefore, the two signals at 3.77 and 4.056 ppm can be assigned to H-l and H-2 of Gal-ol, whereas the H-5 and H-6 atoms resonances are upfield shifted at 4.149 and 3.68 ppm, by comparison with commpound IV A
(Table 3).
In the NMR spectra of IV A and IV B, the H-5 resonance of Gal-ol is the most upfield shifted atom resonance of the molecule and this observation is in agreement with the assignments proposed by others.
Oligosaccharide-alditol II A (Table 2) contains 2 ~-D-Glcp, 1 $-D-Galp and 1 ~-D-Glcp residues, whereas the presence of C-3 and C-5 substituted Gal-ol can be easily deduced by the similarity in chemical shifts of H-l to H-6 of Gal-ol in compound 1. Since the two $-D-Glcp units and the non-reducing terminal $-D-Galp have already been located in oligosaccharide-alditols IV A and IV B, and according to the 215513~
.
fact that the ~-D-Glcp B residue is C-3 substituted, the oligosaccharide-alditol II A possesses the following structure :
C B A
~-D-Glcp-(1-3)-~-D-Glcp-(1-5)-Gal-ol F E :~3 ~-D-Galp-(1-4)-$-D-Glcp-l The sixth sugar unit, ~-D-Galp, was not observed among the products of the partial hydrolysis. Nevertheless, the fact that the ~-D-Glcp unit C is C-3 substituted and that the only ~-D-Galp present in the polysaccharide occupies the non-reducing position (from the NMR data) lead to conclude that the ~-D-Galp is attached to this ~-D-Glcp residue.
Therefore, the structural unit of the polysaccharide was defined as follows:
D C B A
-3)-~-D-Galp-(1-3)-~-D-Glcp-(1-3)-~-D-Glcp-(1-5)-~-D-Galf-(l-F E
~-D-Galp-(1-4)-~-D-Glcp 1 215~13~
Table 1 : NMR chemical shift of the polysaccharide from in D2O at 80C (internal standard : acetone) Residue .. .. . ............ ., ............ .
,~ssiynment $-Galp $-Glcp ~-Glcp ~-Galp ~-Glcp ~-Galp A B C D I E F
H (ppm) H-1 5.158 4.568 5.201 5.158 4.5684.350 H-2 4.240 3.289 3.542 3.888 3.2063.470 H-3 4.419 3.532 3.752 3.776 3.4883.470 H-4 4.125 3.533 3.512 3.999 3.5333.796 H-5 4.118 3.354 3.920 4.108 3.4553.582 H-6 3.723 3.84 3.61 3.62 3.903.67 H-6' 3.62 3.66 3.61 3.62 3.703.67 l3C (ppm) C-1 108.95 103.08 99.87 100.26 102.52 103.74 C-2 80.52 72.72 71.19 68.33 73.4771.19 C-3 85.08 85.15 82.90 77.31 74.9075.32 C-4 81.64 70.14 70.20 69.19 80.1968.69 C-5 78.84 76.43 72.48 71.27 73.5275.80 C-6 61.68 61.38 61.07 61.26 61.1260.99 215513~
Table 2 : Methylation analysis of the native polysaccharide ¦ derivative ¦ molar ratio partially partially methylated alditol- methylated acetates ~ethylglycosides 52, 3, 4, 6 Gal 2, 4, 6 Glc 22 19 2, 3, 6 Glc 9 13 2, 4, 6 Gal 7 16 2, 6 Gal 1 9 able 3 : NMR chemical shifts of the oligosaccharide-alditols obtained by acid partial hydrolysis of the polysaccharide from Lactobacillus helveticus in D20 at 353K.
Residue in IV A Residue in IV B Residue i~ II A
assignment Gal-ol ~-Glcp ~-Galp Gal-ol ~-Glcp ~-Glcp Gal-ol ~-Glcp ~-Glcp ~-Glcp ~-Galp A-ol E F A-ol B E A B C D E
H (ppm) H-1 3.77 4.6864.5573.77 4.539 4.4283.77 4.707 5.3404.590 4.460 H-1' 3.77 - - 3.77 - - 3.77 H-2 4.118 3.3283.3204.0563.421 3.5414.1203.423 3.5593.379 3.542 H-3 4.141 3.5003.4843.9153.661 3.6614.1483.647 3.7483.647 3.665 H-4 3.907 3.4243.4063.7973.674 3.9273.911,3.7483.4633.678 3.928 H-5 4.301 3.5433.4534.1493.62 3.7324.2993.47 4.0253.590 3.73 H-6 3.77 3.8993.8993.68 4.02 3.79 3.77 3.97 3.80 3.90 3.79 H-6' 3.77 3.7583.7583.68 3.80 3.79 3.77 3.85 3.80 3.75 3.79 e,~
Table 4 : Methylation analysis of oligosaccharide-alditol (partially methylated and acetylated methyl glycosides) IV A, IV B and II A obtained by partial acid hydrolysis of native polysaccharide.
¦ derivative ¦ molar ratio IV A IV B II A
1, 2, 4, 5, 6 Gal-ol - 1 -1, 2, 4, 6 Gal-ol 102, 3, 4, 6 Glc 1.2 - 1.0 2, 3, 4, 6 Gal (*) - 1.4 0.9 2, 4, 6 Glc - - 1.3 2, 3, 6 Glc - 1.0 1.2 (*) : Due to its high volatility the value is lower than expected.
The composition comprising the polysaccharide and/or microorganism according to the invention are described in the following non limiting examples.
Ex~mple 1: se~-s~yle a~ir~;fied milk.
Set-style acidifed milk comprising the L.
helveticus strain according to the invention and two S.
thermophilus strains, traditionnaly used for the production of a set-style yoghourt, was obtained by the following process.
To a whole milk comprising 3,7 ~ fats, 2,5 skimmed milk powder was added.
40 liters of this milk was pasteurized at 92C for six minutes, homogeneized at 75C and 150 bars (two levels) 215513~
and cooled at a temperature around 42C.
The ~reeze-dried S. thermophilus CNCM I-1422, S.
thermophilus CNCM I-1424 and L. helveticus CNCM I-1449 strains were reactived with several successive cultures in 5 a sterile MSK medium (skimmed milk powder reconstituted at 10~, comprising 0,1 ~ of a commercial yea$t extract).
A deposit of the microorganism has been made according to the Budapest Treaty on May 18, 1994, for the Streptococcus strains and on July 27, 1994, for the Lactobacillus strain at the Collection Nationale de Culture de Microorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 PARIS CEDEX 15, FRANCE.
The sterilized milk was inoculated with 1 ~ of the third culture of each S. thermophilus strain and with 2 ~ of the second culture of L. helveticus strain taken at the medium coagulation stage.
The milk was incubated at 42C and at a pH around 4.65, and then cooled at a temperature of 4C.
20 Ex~ple 2 : aci~;fied whe~ milk.
Whey milk comprising the L. helveticus strain acording to the invention and two S. thermophilus strains, traditionnaly used for the production of a yoghourt, was obtained by the following process.
A sweet lactoserum powder was reconstituted at 12,5~ in water.
40 liters of this whey was pasteurized at 92C for six minutes, homogeneized at 75C and 150 bars (two levels) and cooled at a temperature around 42C.
The freeze-dried S. thermophilus CNCM I-1422, S.
thermophilus CNCM I-1424 and L. helveticus CNCM I-1449 strains were reactived with several successive cultures in 215513~
a sterile MSK medium (skimmed milk powder reconstituted at 10~, comprising 0,1 ~ o~ a commercial yeast extract).
A deposit of the microorganism has been made according to the Budapest Treaty on May 18, 1994, for the Streptococcus strains and on July 27, 1994, for the Lactobacillus strain at the Collection Nationale de Culture de Microorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 PARIS CEDEX 15, FRANCE.
The sterilized milk was inoculated with 1 ~ of the third culture of each S. thermophilus strain and with 2 ~ of the second culture o~ L. helveticus strain taken at the medium coagulation stage.
The whey milk was incubated at 42C and at a pH
around 4. 65, and then cooled at a temperature of 4C.
Example 3 : stirred aci~ified milk.
A stirred acidifed milk comprisin~ the L.
helveticus CNCM I-1449 strain according to the invention and two commercialized S. thermophilus strains, traditionnaly used for the production of a stirred yoghourt, was obtained by the following process.
The milk was obtained from a whole milk comprising 3,7 ~ fats, by the addition of 2,5 ~ skimmed milk powder.
40 liters of this milk was pasteurized at 105C for two minutes, homogeneized at 75C and 300 bars (first level) and cooled at a temperature around 43C.
The lyophilized S. thermophilus CNCM I-1421, S.
thermophilus CNCM I-1423 and L. helveticus CNCM I-1449 strains were reactived with several successive cultures in a sterile MSK medium.
A deposit of the microorganism has been made according to the Budapest Treaty on May 18, 1994, for the 215513~
StreptococcUs strains and on July 27, 1994, for the LactobacillUs strain at the Collection Nationale de Culture de MicroorganiSmes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 PARIS CEDEX 15, FRANCE.
The sterilized milk was inoculated with 1 ~ of the third culture of each S. thermophilus stra~n and with 2 ~ o the third culture of L. helveticus strain taken at the medium coagulation stage.
The milk was incubated at 43C and at a pH around 4.65, and then cooled at a temperature of 4C during stirring.
The following table 5 represents the properties of the obtained products.
Table 5.
¦ ¦¦Example 1 ¦Example 3 Acidification 6 h 7 h 15 time at pH = 4.65 pH of the product 4.34 4.49 20 after 1 day at 4C
pH of the product 4.1 4.3 after 24 days at taste after 24 good taste, very good taste, 25 days slightly acid aromatic smooth texture smooth and onctuous texture F~xample 4 : cosrnetic composi tion for buccal hygiene .
CHEMICAL NA~E ¦ TRADE NAME ¦ 96 WEIGHT
PHASE A
PEG-40 Hydrogenated Cremophor RH 40 0.10 castor oil Flavour Strawberry E 2226 0.04 Flavour Raspberry 9/022436 0.10 PHASE B
10 Sodium Cyclamate Sodium Cyclamate 0.10 Exopolysaccharide -- 0.50-5.00 according to the present invention Demineralized water 94.66-99.16 Ex~mI~le 5 : co~netic composi tion for sk7 n hyaiene.
¦ % WEIGHT
OIL PHASE
BRIJ 721 (Steareth 21) 4.00 Cetyl alcohol 10.00 Mineral oil 5.00 Propyl parahydroxybenzoate 0.02 WATER PHASE
CARBOPOL 934 (Carbomer 934) 0.10 Sodium hydroxide (solution 0.10 at 10~) Methyl parahydroxybenzoate 0.18 Exopolysaccharide according 0.50-5.00 to the present invention ¦Demineralized water 75.60-80.10 TOTA~ 100 E~xample 6 : Pharmaceutical comF~osi tion for anti -diarrheoic usage .
A pharmaceutical composition was obtained as a capsule which was made with gelatine and! water, and which contained from 5 to 50 mg of the exopolysaccharide according to the present invention. Alternatively, powdered tablet formulations can be obtained directly from the acidified cultured milks described in the above examples 1, 2 and 3, by freeze-drying these fermented milks and whey.
References.
1. Kolenbrander, P.E., Ganeshkumar, N., Cassels, F.J. &
Hughes, C.V., Coaggregation: speciic adherence among human oral plaque bacteria. The FASEB Journal, 7 (1993) 406-413.
2. Karlsson, K.-A., ~n;m~l glycosphingolipids as membrane attachment sites for bacteria. Annual Review of Biochemistry, 58 (1989) 309-350.
3. Hughes, R.C., Mac-2: A versatile galactose-binding protein of mammalian tissues. Glycobiology, 4 (1994) 5-12.
4. Truong, M.-J., Gruart, V., Kusnierz, J.-P., Papin, J.-P., Loiseau, S., Capro~, A. & Capron, M., Human neutrophils express immunoglobulin E (IgE)-binding proteins (Mac-2/~BP) of the S-type lectin family: role in IgE-dependent activation. Journal of EXperimental Medicine, 177 (1993) 243-248.
5. Wollenberg, A., de la Salle, H., Hanau, D., Liu, F.-T.
Bieber, T., Human keratinocytes release the endogenous $-galactoside-binding soluble lectin immunoglobulin E
(IgE-binding protein) which binds to Langerhans cells where it modulates their binding capacity for IgE
glycoforms. ~ournal of Experimental Medicine, 178 (1993) 777-785.
Bieber, T., Human keratinocytes release the endogenous $-galactoside-binding soluble lectin immunoglobulin E
(IgE-binding protein) which binds to Langerhans cells where it modulates their binding capacity for IgE
glycoforms. ~ournal of Experimental Medicine, 178 (1993) 777-785.
6. Doco, T., Fournet, B., Carcano, D., Ramos, P., Loones, A., Piot, J.-M. & Guillochon, D., Polysaccharide, application comme agent epaississant et comme agent anti-tumoral. Demande de brevet europeen, EUR 331 564, 06.09.1989.
7. Doco, T., Wieruszeski, J.-M., Fournet, B., Carcano, D., Ramos, P. & Loones, A., Structure of an exocellular polysaccharide produced by Streptococcus thermophilus.
215513~
Carbohydrate Research, 198 (1990) 313-321.
215513~
Carbohydrate Research, 198 (1990) 313-321.
8. Gruter, M., Leeflang, B.R., Kuiper, J., Kamerling, J.P.
~ Vliegenthart, J.F.G., Structure of the exopolysaccharide produced by Lactococcus lactis subspecies cremoris H414 grown in a defined medium or skimmed milk. Carbohydrate Research, 2!31 (1992) 273-291.
~ Vliegenthart, J.F.G., Structure of the exopolysaccharide produced by Lactococcus lactis subspecies cremoris H414 grown in a defined medium or skimmed milk. Carbohydrate Research, 2!31 (1992) 273-291.
9. Nakajima, H., Hirota, T, Toba, T., Itoh, T. & Adachi, S., Structure of the extracellular polysaccharide from slime-forming Lactococcus lactis subsp. cremoris SBT
0495. Carbohydrate Research, 224 (1992) 245-253.
0495. Carbohydrate Research, 224 (1992) 245-253.
10. Gruter, M., Leeflang, B.R., Kuiper, J., Kamerling, J.P.
& Vliegenthart, J.F.G., Structural characterization of the exopolysaccharide produced by Lactobacillus delbruckii subspecies bulgaricus rr grown in skimmed milk. Carbohydrate Research, 239 (1993) 209-226.
& Vliegenthart, J.F.G., Structural characterization of the exopolysaccharide produced by Lactobacillus delbruckii subspecies bulgaricus rr grown in skimmed milk. Carbohydrate Research, 239 (1993) 209-226.
11. Van den Berg, D.J.C., Ledeboer, A.M., Robijn, G.W. ~
Vreeker, R., Lactobacillus sake like strains, production and use of their exopolysaccharides. International Patent Application PCT No WO 94/12656, 9 June 1994.
Vreeker, R., Lactobacillus sake like strains, production and use of their exopolysaccharides. International Patent Application PCT No WO 94/12656, 9 June 1994.
12. Tsuchiya, F., Miyazawa, K., Kanbe, M., Oda, M. & Ebisawa, N., High molecular polysaccharide MPS-80. United States Patent No 4,396,763, August 2, 1983.
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Claims (10)
1. Branched natural soluble polysaccharide characterized in that it comprises a main chain having repeating side chains which are only made of lactose units, possibly substituted.
2. Branched polysaccharide according to claim 1, corresponding to the following formula :
where n > 1, Gal = galactose, Glc = glucose, R1 = Hydrogen or GalNAc.beta.1 (N-acetylgalactosamine) R2 = Hydrogen, NeuNAc.alpha.2(N-acetylneuraminic Acid) or HSO3 R3 = Hydrogen or Fuc.alpha.1 (fucose)
where n > 1, Gal = galactose, Glc = glucose, R1 = Hydrogen or GalNAc.beta.1 (N-acetylgalactosamine) R2 = Hydrogen, NeuNAc.alpha.2(N-acetylneuraminic Acid) or HSO3 R3 = Hydrogen or Fuc.alpha.1 (fucose)
3. Microorganism producing the branched polysaccharide according to claim 1 or 2.
4. Microorganism according to claim 3, characterized in that it is a strain of Lactobacillus helveticus, preferably the strain of Lactobacillus helveticus CNCM I-1449.
5. Food composition comprising the branched polysaccharide and/or the microorganism according to any of the preceding claims 1 to 4.
6. Food composition according to claim 5, characterized in that it is choosen among the group consisting of set-style or stirred-acidified milks or whey.
7. Cosmetical composition comprising the branched polysaccharide and/or the microorganism according to any of the preceding claims 1 to 4.
8. Cosmetical composition according to claim 7, characterized in that it is choosen among the group consisting of mouth rinse, tooth paste, tooth gel, chewing-gum, tablet, cream, ointment, balsam.
9. Pharmaceutical composition comprising the branched polysaccharide and/or the microorganism according to any of the preceding claims 1 to 4.
10. Pharmaceutical composition according to claim 9, characterized in that it is choosen among the group consisting of capsule, syrup, powder, tablet.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EPEP94870139.6 | 1994-08-19 | ||
| EP94870139A EP0699689B1 (en) | 1994-08-19 | 1994-08-19 | Branched polysaccharide, microorganism producing it and compositions containing them |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2155134A1 true CA2155134A1 (en) | 1996-02-20 |
Family
ID=8218667
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002155134A Abandoned CA2155134A1 (en) | 1994-08-19 | 1995-08-01 | Branched polysaccharide, microorganism producing it and compositions containing them |
Country Status (13)
| Country | Link |
|---|---|
| US (1) | US5955602A (en) |
| EP (1) | EP0699689B1 (en) |
| JP (1) | JPH08277303A (en) |
| AT (1) | ATE187177T1 (en) |
| AU (1) | AU700113B2 (en) |
| CA (1) | CA2155134A1 (en) |
| DE (1) | DE69421921T2 (en) |
| DK (1) | DK0699689T3 (en) |
| ES (1) | ES2141213T3 (en) |
| GR (1) | GR3032763T3 (en) |
| NO (1) | NO953264L (en) |
| NZ (1) | NZ272795A (en) |
| PT (1) | PT699689E (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009127057A1 (en) | 2008-04-15 | 2009-10-22 | Innovactiv Inc. | Cosmetic compositions comprising exopolysaccharides derived from microbial mats, and use thereof |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0889136A1 (en) * | 1997-07-05 | 1999-01-07 | Societe Des Produits Nestle S.A. | Exocellular polysaccharide produced by streptococcus thermophilus |
| US5972311A (en) * | 1998-01-26 | 1999-10-26 | Duke University | Method of preventing dental caries and other oral lesions |
| EP0957169A1 (en) * | 1998-04-22 | 1999-11-17 | Societe Des Produits Nestle S.A. | Identification of Lactobacillus helveticus LH59 genes involved in the biosynthesis of exopolysaccharides |
| WO1999062316A2 (en) * | 1998-04-22 | 1999-12-09 | Societe Des Produits Nestle S.A. | Identification of lactic acid bacteria genes involved in the biosynthesis of exopolysaccharides |
| JP4564169B2 (en) * | 1998-08-12 | 2010-10-20 | ソシエテ・デ・プロデュイ・ネスレ・エス・アー | Addition of exogenous lactic acid bacteria to oral microflora |
| EP1159951A1 (en) | 2000-06-02 | 2001-12-05 | Societe Des Produits Nestle S.A. | Use of exogenous lactic bacteria strain against Actinomyces naeslundii-related diseases |
| EP1142481B2 (en) * | 1999-01-11 | 2009-09-02 | Calpis Co., Ltd. | Process for producing fermented milk containing angiotensin converting enzyme inhibitory peptide and process for producing milk serum |
| AU2001239737B9 (en) * | 2000-02-02 | 2007-03-22 | The State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of Oregon State University | Biopolymer thickener |
| US6861077B1 (en) | 2000-03-17 | 2005-03-01 | L'oreal S.A. | Use of plant extracts in a cosmetic composition to protect keratinous fibers |
| CA2440656A1 (en) * | 2001-03-09 | 2002-09-19 | Apollo Biotechnology, Inc. | Conjugate probes and optical detection of analytes |
| ES2378854T3 (en) * | 2008-11-10 | 2012-04-18 | Nestec S.A. | Bacteria that produce sialic acid |
| CA2917268C (en) | 2013-07-05 | 2023-11-07 | Integra Medical Inc. | Oral compositions |
| CN104586642A (en) * | 2015-02-06 | 2015-05-06 | 武汉中科光谷绿色生物技术有限公司 | Applications of N-acetvlneuraminic acid monomer, N-acetvlneuraminic acid hydrate or N-acetvlneuraminic acid salt in personal care supplies |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4396763A (en) * | 1981-01-14 | 1983-08-02 | Meiji Milk Products Company Limited | High molecular polysaccharide MPS-80 |
| DE3750139T2 (en) * | 1986-04-16 | 1994-10-06 | Brigham & Womens Hospital | BACTERIAL ANTIGENS, ANTIBODIES, VACCINE AND THEIR PRODUCTION. |
| JP2518663B2 (en) * | 1987-12-24 | 1996-07-24 | 株式会社ヤクルト本社 | Method for producing processed milk containing galactooligosaccharide |
| JPH0539304A (en) * | 1991-08-05 | 1993-02-19 | Glyco Kyodo Nyugyo Kk | Soluble food fiber and its production |
| CH683223A5 (en) * | 1991-10-25 | 1994-02-15 | Nestle Sa | A process for preparing an acidified milk. |
-
1994
- 1994-08-19 PT PT94870139T patent/PT699689E/en unknown
- 1994-08-19 ES ES94870139T patent/ES2141213T3/en not_active Expired - Lifetime
- 1994-08-19 DE DE69421921T patent/DE69421921T2/en not_active Expired - Fee Related
- 1994-08-19 DK DK94870139T patent/DK0699689T3/en active
- 1994-08-19 EP EP94870139A patent/EP0699689B1/en not_active Expired - Lifetime
- 1994-08-19 AT AT94870139T patent/ATE187177T1/en not_active IP Right Cessation
-
1995
- 1995-07-17 AU AU25017/95A patent/AU700113B2/en not_active Ceased
- 1995-08-01 CA CA002155134A patent/CA2155134A1/en not_active Abandoned
- 1995-08-17 NZ NZ272795A patent/NZ272795A/en unknown
- 1995-08-18 US US08/516,783 patent/US5955602A/en not_active Expired - Fee Related
- 1995-08-18 NO NO953264A patent/NO953264L/en not_active Application Discontinuation
- 1995-08-21 JP JP7233177A patent/JPH08277303A/en active Pending
-
2000
- 2000-02-25 GR GR20000400465T patent/GR3032763T3/en not_active IP Right Cessation
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009127057A1 (en) | 2008-04-15 | 2009-10-22 | Innovactiv Inc. | Cosmetic compositions comprising exopolysaccharides derived from microbial mats, and use thereof |
| US11040058B2 (en) | 2008-04-15 | 2021-06-22 | Lucas Meyer Cosmetics Canada Inc. | Cosmetic compositions comprising exopolysaccharides derived from microbial mats, and methods of use thereof |
| US11752168B2 (en) | 2008-04-15 | 2023-09-12 | Lucas Meyer Cosmetics Canada Inc. | Methods of using cosmetic compositions comprising exopolysaccharides derived from microbial mats |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2141213T3 (en) | 2000-03-16 |
| ATE187177T1 (en) | 1999-12-15 |
| AU700113B2 (en) | 1998-12-24 |
| AU2501795A (en) | 1996-02-29 |
| DK0699689T3 (en) | 2000-05-08 |
| GR3032763T3 (en) | 2000-06-30 |
| JPH08277303A (en) | 1996-10-22 |
| NO953264D0 (en) | 1995-08-18 |
| NO953264L (en) | 1996-02-20 |
| US5955602A (en) | 1999-09-21 |
| DE69421921D1 (en) | 2000-01-05 |
| NZ272795A (en) | 1997-10-24 |
| DE69421921T2 (en) | 2000-05-11 |
| EP0699689B1 (en) | 1999-12-01 |
| PT699689E (en) | 2000-05-31 |
| EP0699689A1 (en) | 1996-03-06 |
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