WO2019115770A1 - Separation of oligosaccharides - Google Patents

Separation of oligosaccharides Download PDF

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Publication number
WO2019115770A1
WO2019115770A1 PCT/EP2018/084966 EP2018084966W WO2019115770A1 WO 2019115770 A1 WO2019115770 A1 WO 2019115770A1 EP 2018084966 W EP2018084966 W EP 2018084966W WO 2019115770 A1 WO2019115770 A1 WO 2019115770A1
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Prior art keywords
oligosaccharide
less
compound
containing mixture
chromatographic support
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PCT/EP2018/084966
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French (fr)
Inventor
Ernst MEINJOHANNS
Kenneth Harlow
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Upfront Chromatography A/S
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Publication of WO2019115770A1 publication Critical patent/WO2019115770A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1807—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using counter-currents, e.g. fluidised beds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B01D15/3823—Affinity chromatography of other types, e.g. avidin, streptavidin, biotin

Definitions

  • the present invention relates to an improved process for isolating oligosaccharides from an oligosaccharide containing mixture.
  • the present invention relates to a method for isolating oligosaccharides from an oligosaccharide containing mixture using a chromatographic support providing an unspecific binding or a specific binding of oligosaccharides from the oligosaccharide containing mixture.
  • HMO Human milk oligosaccharides
  • HMO human milk oligosaccharides
  • HMO histoneum mellomycin
  • Several interesting bioactivities have been attributed to HMO. For example, they antagonize the binding of some strains of bacteria to epithelial cells, and a large epidemiologic study indicated that sugar epitopes resulting from an individual genotype appear to protect against diarrhoea in breast fed infants. Furthermore, those containing sialic acid may serve as immune modulators. It has also been suggested that HMO may serve as growth factors for colonic microbiota, as it has long been known that breast feeding results in increased levels of faecal bifidobacteria with respect to infant formula. Based on their structure and composition, HMO may affect the gut microbiota consortium either by selectively providing growth factors and energy substrates to some members, or alternatively via binding and eliminating others, or both.
  • HMO HMO
  • the activities modulated by HMO it is first necessary to isolate them in large enough quantities to serve as fermentation and or growth factor substrates in microbiological media, and to ensure that they are entirely free of milk mono and disaccharides.
  • isolating specific HMO's or some specific groups of HMOs' a more specific modelling of the HMO's activity may be investigated.
  • An interesting source of oligosaccharides may be milk obtained from a ruminant, such as a cow or a bovine or the oligosaccharides may be synthetized. The synthetized
  • oligosaccharides may be produced by chemical production, enzymatic production and/or by microbial fermentation.
  • oligosaccharides obtained from these other interesting sources are considered to be analogous to HMO, and it is suggested that the oligosaccharides, in particular the bovine milk oligosaccharides, has a similar protective role as suggested by human milk oligosaccharides.
  • an object of the present invention relates to an improved method for isolating oligosaccharides from an oligosaccharide containing mixture.
  • one aspect of the invention relates to a method for separating one or more oligosaccharide compound(s) from an oligosaccharide containing mixture, the method comprises the steps of:
  • chromatographic support Subjecting the chromatographic support to at least one elution buffer obtaining one or more oligosaccharide compound(s) from the chromatographic support; and wherein the chromatographic support comprises an adsorbent comprising one or more ligand capable of binding the one or more oligosaccharide compound(s) from the oligosaccharide containing mixture and wherein the one or more ligand comprise an a boronic acid compound, a serotonin compound, or a derivate thereof.
  • Another aspect of the present invention relates to an oligosaccharide compound comprising one or more moiety selected from the group consisting of a Hexose moiety (a Hex moiety); a HexNAc moiety; a fucose moiety (a Fuc moiety); and a NeuAc moiety.
  • Yet another aspect of the present invention relates to the use of the oligosaccharide according to the present invention as an ingredient, e.g. for infant formulas, a fortified or functional food or beverages, a health ingredients for human and animal or an over the counter food supplements.
  • HMO human milk oligosaccharides
  • the human milk oligosaccharides have been shown to play an important role in the early development of infants and young children, such as the maturation of the immune system.
  • the role is dependent on the structure and composition of the HMO many different kinds of HMOs are found in the human milk, where each individual oligosaccharide is based on a specific combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and/or N-acetylglucosamine with many and varied linkages between them, thus accounting for the enormous number of different oligosaccharides in human milk.
  • HMOs can be acidic (e.g. charged sialic acid containing oligosaccharide) or neutral (e.g. fucosylated oligosaccharide).
  • the one or more oligosaccharide compound obtained by the method according to the present invention has a significantly reduced content of lactose.
  • a preferred embodiment of the present invention a method for separating one or more oligosaccharide compound(s) from an oligosaccharide containing mixture, the method comprises the steps of: (i) Providing the oligosaccharide containing mixture;
  • the chromatographic support comprises an adsorbent comprising one or more ligand capable of binding the one or more oligosaccharide compound(s) from the oligosaccharide containing mixture and wherein the one or more ligand comprise an a boronic acid compound, a serotonin compound, or a derivate thereof.
  • oligosaccharide hence, oligosaccharides that are not bound as glycoproteins or glycolipids where the oligosaccharides may be attached on the surface of the respective molecules.
  • the concentration of free oligosaccharides may be increased by hydrolysing the covalent bond between the oligosaccharide and the protein, in a glycoprotein, or between the oligosaccharide and the lipid, in a glycolipid, liberating the oligosaccharide.
  • the hydrolysis may be performed by the action of an enzyme.
  • the one or more oligosaccharide compound(s) comprises 3-30 sugar moieties, such as 5-25 sugar moieties, e.g. 8-22 sugar moieties, such as 10-20 sugar moieties, e.g. 14-18 sugar moieties.
  • the oligosaccharide containing mixture may be a dairy source, , a fermentation broth, a plant material or a mixture obtained from a chemical reaction.
  • the dairy source may be selected from the group consisting of milk, whole milk, skimmed milk, milk concentrates, reconstituted milk powder, non-pasteurised milk, micro-filtrated milk, pH-adjusted milk, pre-treated dairy source, a whey material, and a fraction obtained from a whey material .
  • the fraction obtained from a whey material may comprise oligosaccharides in combination with one or more of protein, peptide, vitamins and/or minerals.
  • the fraction obtained from a whey material may preferably comprise oligosaccharides in combination with vitamins and/or minerals.
  • the oligosaccharide containing mixture may be a dairy source or synthetized oligosaccharides.
  • the synthetized oligosaccharides may be obtained from chemical production, enzymatic production and/or by microbial
  • the dairy source may be a milk source or a whey source.
  • the oligosaccharide containing mixture has not been subjected to
  • Oligosaccharides are found in the human and animal milk, and each individual
  • oligosaccharide may be based on a specific combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and/or N-acetylglucosamine coupled by different linkages.
  • oligosaccharide may be the preferred target available for interacting with the adsorbent and/or the ligand.
  • One challenge when being interested in separating oligosaccharides from oligosaccharide containing mixture may be the presence of enzymes capable of inactivating or degrading oligosaccharides.
  • the oligosaccharide containing mixture provided in step (i) may be subjected to a first pre-treatment.
  • the first pre treatment may involve a step of enzyme inactivation.
  • the enzyme inactivation involves inactivation of one or more of the enzymes selected from the group consisting of fucose-, sialic acid-, N-Acetylglucosamine-, lacto-N-biose-, glucose- and/or galactose-degrading enzymes in the oligosaccharide containing mixture.
  • the enzyme inactivation may involve addition of an enzyme degrading compound, a pre separation step removing the enzyme from the oligosaccharide containing mixture or a combination hereof.
  • the pre-separation step may preferably involve removing the enzyme from the oligosaccharide containing mixture. Such removal may involve a filtration process, a centrifugation process or a chromatographic process.
  • the pH of the oligosaccharide containing mixture provided in step (i) may be adjusted before contacting the oligosaccharide containing mixture with the chromatographic support in step (ii).
  • the oligosaccharide containing mixture provided in step (i) may be pH adjusted to a pH value below pH 6 before contacting the oligosaccharide containing mixture with the chromatographic support in step (ii), such as a pH value below pH 5, e.g. a pH value below pH 4.5, such as a pH value below pH 4, e.g. a pH value below pH 3.5, such as a pH value below pH 3.2, e.g. a pH value below pH 3.0.
  • a pH value below pH 6 such as a pH value below pH 6 before contacting the oligosaccharide containing mixture with the chromatographic support in step (ii), such as a pH value below pH 5, e.g. a pH value below pH 4.5, such as a pH value below pH 4, e.g. a pH value below pH 3.5, such as a pH value below pH 3.2, e.g. a pH value below pH 3.0.
  • the oligosaccharide containing mixture provided in step (i) may be pH adjusted to a pH value above pH 6 before contacting the oligosaccharide containing mixture with the chromatographic support in step (ii), such as a pH value above pH 7, e.g. a pH value above pH 7.5, such as a pH value above pH 8, e.g. a pH value above pH 8.3, such as a pH value above pH 8.5, e.g. a pH value above pH 8.75, such as a pH value above pH 9, e.g. a pH value in the range of pH 6-9.5, such as a pH value in the range of pH 7-9, e.g. a pH value in the range of pH 8-8.75, such as a pH value about pH 8.5.
  • a pH value above pH 7 e.g. a pH value above pH 7.5
  • a pH value above pH 8 e.g. a pH value above pH 8.3
  • a pH value above pH 8.5 e.g.
  • the adsorbent according to the present invention capable of binding the one or more oligosaccharide compound(s) from the oligosaccharide containing mixture may be essential for the effectivity of the invention.
  • the non-porous material may preferably be selected from a metal oxide; a silicate; a ceramic metal; alumina; or a magnetic material.
  • a metal oxide e.g., titanium oxide; silicates; ferric oxide, nickel oxide or cobalt oxide.
  • the magnetic material according to the present invention may be selected from ferric oxide, nickel oxide or cobalt oxide.
  • the non-porous material may be a high density non-porous material .
  • the high density non-porous material may have a density of at least 4.0 g/ml, such as at least 10 g/ml, e.g . at least 16 g/ml, such as at least 25 g/ml.
  • the non-porous core material has a density in the range of about 4.0-25 g/ml, such as about 4.0-20 g/ml, e.g . about 4.0-16 g/ml, such as 12-19 g/ml, e.g. 14-18 g/ml, such as about 6.0-15.0 g/ml, e.g . about 6.0-16 g/ml .
  • the high density non-porous material according to the present invention may be selected from tungsten carbide or steel .
  • the adsorbent may be a metal oxide capable of binding the one or more oligosaccharide compound(s) from the oligosaccharide containing mixture.
  • the adsorbent comprises a particle comprising a non-porous material surrounded by a porous polymeric material .
  • the porous polymeric material may be an organic porous polymeric material .
  • the porous polymeric material may be selected from agarose, alginate, chitosan, carrageenan, and/or pectin.
  • the one or more adsorbent may have a density of at least 1.3 g/ml, more preferably at least 1.5 g/ml, still more preferably at least 1.8 g/ml, even more preferably at least 2.0 g/ml, even more preferably at least 2.3 g/ml, even more preferably at least 2.5 g/ml, even more preferably at least 2.75 g/ml, even more preferably at least 3.0 g/ml, even more preferably at least 3.5 g/ml, even more preferably at least 4.0 g/ml, even more preferably at least 4.5 g/ml .
  • the density of the adsorbent according to the present invention relates to the density of an adsorbent in it's fully solvated (e.g. hydrated) state as opposed to the density of a dried adsorbent particle.
  • the desired density of the one or more adsorbent may be provided by inclusion of a certain proportion or a certain amount of the non-porous material in the porous polymeric material .
  • the adsorbent may comprise one or more ligands capable of binding the one or more oligosaccharide compound(s) from the oligosaccharide containing mixture.
  • the adsorbent comprises one or more ligands capable of binding the one or more oligosaccharide compound(s) from the oligosaccharide containing mixture.
  • the ligand may be an un-specifically binding ligand ; or the ligand may be a specifically binding ligand.
  • the one or more ligands may be un- specifically binding the oligosaccharide compound(s) from the oligosaccharide containing mixture.
  • un-specifically binding relates to the binding of the oligosaccharides present in the oligosaccharide containing mixture with limited or no (or substantially no) differentiation between the different oligosaccharide molecules present in the oligosaccharide containing mixture.
  • the one or more adsorbent capable of binding the one or more oligosaccharide compound(s) does not comprise an ion exchange ligand .
  • the one or more adsorbent capable of binding the one or more oligosaccharide compound(s) comprise a boronic acid compound or a derivate thereof.
  • the boronic acid compound or a derivative thereof comprises a boric acid and an organic substituent.
  • the organic substituent may preferably be an alkyl compound or an aryl compound, preferably, the organic substituent is an aryl compound .
  • the boronic acid compound or a derivative thereof may be a phenyl borate compound
  • the organic substituent constitutes the connection between the adsorbent and the boric acid.
  • the ligand may be a boronic acid compound or a derivative thereof.
  • the ligand may be specifically binding oligosaccharides.
  • the term "specifically binding” relates to a preference (a differentiation) in the binding a specific type of oligosaccharide by the specifically binding ligand.
  • the specifically binding ligand may be a serotonin compound or a derivative thereof.
  • the ligand concentration on the adsorbent may be in the range of 30-300 pmoles per ml sedimented adsorbent, e.g. in the range of 50-200 pmoles per ml sedimented adsorbent, such as 75-175 pmoles per ml sedimented adsorbent, e.g. 100-160 pmoles per ml sedimented adsorbent, such as 120-145 pmoles per sedimented adsorbent.
  • the adsorbent and/or the ligand according to the present invention may have a high specificity for one or more oligosaccharide compound(s) from the oligosaccharide containing mixture.
  • the term "high specificity for one or more oligosaccharide compound(s)" relates to more than 50% (w/w) of the molecules specifically bound to the adsorbent and/or the ligand may be one or more oligosaccharide compound(s), such as more than 55%, e.g. more than 60%, such as more than 65%, e.g. more than 70%, such as more than 75%, e.g. more than 80%, such as more than 90%, e.g. more than 95%.
  • the chromatographic support may be a membrane chromatography support, or a column chromatography support.
  • the column chromatography support includes a Packed Bed Chromatography, stirred tank adsorption, moving bed chromatography, simulated moving bed chromatography, Fluidized Bed Chromatography and/or Expanded Bed Chromatography.
  • the oligosaccharide containing mixture may be loaded on to the chromatographic support at a flow-rate in the range of 1-50 cm/min; preferably in the range of 5-30 cm/min; more in the range of 10-25 cm/min; even more preferably, in the range of 15-20 cm/min.
  • a loading buffer may be used when loading the oligosaccharide containing mixture on to the chromatographic support.
  • the loading buffer may comprise an organic compound.
  • the organic compound may be taurine.
  • the loading buffer may have a pH value in the range of pH 6-9.5, such as a pH value in the range of pH 7-9, e.g. a pH value in the range of pH 8-8.75, such as a pH value about pH 8.5.
  • the separation process according to the present invention may either be a batch separation process or a continuous separation process.
  • large-scale production may be conducted at a continuous process.
  • oligosaccharides When the oligosaccharides are retained by the chromatographic support an elution step at some point during the separation process may be necessary.
  • continuous separation By providing at least two chromatographic supports and placing them in parallel, such continuous separation may be provided where the flow of oligosaccharide containing mixture may be shifted from one chromatographic support, when this chromatographic material is loaded and ready for elution, to the other chromatographic support.
  • moving bed chromatography, simulated moving bed chromatography or the like may be used.
  • the oligosaccharide compound bound to the chromatographic support may be released from the chromatographic support by subjecting the chromatographic support to at least one elution buffer obtaining one or more oligosaccharide compound(s) from the chromatographic support, as described in step (v).
  • the elution in step (v) may be a sequential elution of one or more oligosaccharides providing one of more oligosaccharide compounds.
  • step (v) involves a change of the pH.
  • the change in pH for eluting the one or more oligosaccharides involves a change in pH to a pH above pH 3.0, such as a pH above pH 3.2, e.g. a pH above pH 3.5, such as a pH above pH 4.0, e.g. a pH above pH 4.5, such as a pH above pH 5.0, e.g. a pH above pH 6.0, such as a pH in the range of pH 3.0-9.0, e.g. a pH in the range of pH 3.2-8, such as a pH in the range of pH 3.5-7.5, e.g. a pH in the range of pH 4.0-7.2, such as a pH in the range of pH 4.5-7.0, e.g. a pH in the range of pH 5.0-6.5, such as about pH 6.0.
  • a pH above pH 3.0 such as a pH above pH 3.2, e.g. a pH above pH 3.5, such as a pH above pH 4.0, e.
  • an elution buffer may be used when elution the oligosaccharide retained on the chromatographic support.
  • the elution buffer may comprise an organic compound.
  • the organic compound may be taurine.
  • the elution buffer comprises a carbohydrate compound, such as sorbitol, lactose, and/or fucose.
  • the elution buffer may have a pH value in the range of pH 6-9.5, such as a pH value in the range of pH 7-9, e.g. a pH value in the range of pH 8-8.75, such as a pH value about pH 8.5.
  • a preferred embodiment of the present invention relates to an oligosaccharide compound comprising one or more moiety selected from the group consisting of a Hexose moiety (a Hex moiety); a HexNAc moiety; a fucose moiety (a Fuc moiety); and a NeuAc moiety.
  • the Hex moiety of the oligosaccharide compound may preferably be selected from a glucose residue, a galactose residue or a mannose residue.
  • the HexNAc moiety of the oligosaccharide compound may preferably be a N- acetylglucosamine residue or a N-acetylgalactosamine residue.
  • the fucose moiety of the oligosaccharide compound may preferably be a deoxyhexose residue.
  • the NeuAc moiety of the oligosaccharide compound may preferably be a N-acetyl neuraminic acid residue or a sialic acid residue.
  • the one or more oligosaccharide compound(s) comprises a fucose moiety (such as a deoxyhexose residue); and a NeuAc moiety (such as a N-acetyl neuraminic acid residue or a sialic acid residue).
  • a fucose moiety such as a deoxyhexose residue
  • a NeuAc moiety such as a N-acetyl neuraminic acid residue or a sialic acid residue.
  • the ligand is a specifically binding ligand, preferably serotonin or a derivative thereof, and the oligosaccharide compound retained is an oligosaccharide comprising a NeuAc moiety and/or a sialic acid residue in the terminal end of the oligosaccharide.
  • the one or more oligosaccharide compound(s) may comprise 3-30 sugar moieties, such as 5-25 sugar moieties, e.g. 8-22 sugar moieties, such as 10-20 sugar moieties, e.g. 14-18 sugar moieties.
  • the content of free lactose present in the oligosaccharide compound according to the present invention may be less than 5 mg/I, such as less than 4 mg/I, e.g. less than 3 mg/I, such as less than 2 mg/I, e.g. less than 1 mg/I, such as less than 0.5 mg/I, e.g. less than 0.1 mg/I.
  • the content of glucose present in the oligosaccharide compound according to the present invention may be less than 5 mg/I, such as less than 4 mg/I, e.g. less than 3 mg/I, such as less than 2 mg/I, e.g. less than 1 mg/I, such as less than 0.5 mg/I, e.g. less than 0.1 mg/I.
  • the content of galactose present in the oligosaccharide compound according to the present invention may be less than 5 mg/I, such as less than 4 mg/I, e.g. less than 3 mg/I, such as less than 2 mg/I, e.g. less than 1 mg/I, such as less than 0.5 mg/I, e.g. less than 0.1 mg/I.
  • the oligosaccharide compound according to the present invention may be used as an ingredient, e.g. for infant formulas, a fortified or functional food or beverages, a health ingredients for human and animal or an over the counter food supplements.
  • the oligosaccharide compound according to the present invention may be used as a prebiotic ingredient.
  • prebiotic means non-digestible carbohydrates that beneficially affect the host by selectively stimulating the growth and/or the activity of healthy bacteria such as bifidobacteria in the colon of humans.
  • the oligosaccharides of the nutritional composition of the present invention may act at different levels to support the natural defenses of the developing infant or young child : (i) boost commensals to obtain better colonization resistence, (ii) boost innate immunity to counteract pathogens and/or (iii) act directly on pathogens as decoy to deviate the pathogens from their natural target. It is also believed that the fucosylated oligosaccharide(s) and the particular N-acetylated oligosaccharide(s) act synergically for these purposes.
  • Example 1 coupling serotonin to a chromatographic support
  • Serotonin binds specifically to NeuAc and sialic acid residues in the terminal end of oligosaccharides.
  • serotonin is coupled to an agarose/tungsten carbide adsorbent matrix suitable for Expanded bed Adsorption and tested in a packed bed column of 1 ml .
  • the serotonin was coupled to the adsorbent by the following procedure: The following buffers were used :
  • Coupling buffer 0.2 M NaHCOs, 0.5 M NaCI, pH 8.3
  • Buffer A 0.5 M ethanolamine, 0.5 M NaCI, pH 8.3
  • Buffer B 0.1 M sodium acetate, 0.5 M NaCI, pH 4
  • Ligand coupling Isopropanol was washed out of the column comprising the adsorbent using the cleaning buffer, 1 mM HCI, ice-cold. 3 x 2 ml cleaning buffer was used at a flowrate of 1 ml/min. Immediately after cleaning 1 ml of ligand solution (63 mg of serotonin in 4 ml of coupling buffer) was injected onto the column and the run through was discarded . 3 ml coupling buffer left with recirculation by connecting a peristaltic pump over night at 4°C (cold room) was continuously added .
  • the column is left for 30 min at room temperature.
  • the flowrate during injection, recirculation, wash and deactivation was 0.5 ml/min .
  • the column was sealed with parafilm and stored until use.
  • Example 2 Capturing oligosaccharides having sialic acid in the terminal end of the oligo saccharide using the serotonin adsorbent provided in example 1.
  • oligosaccharides having terminal NeuAc and/or sialic acid residues and lactoferrin comprising oligosaccharides having terminal NeuAc and/or sialic acid residues were used as target molecule.
  • the oligosaccharide containing fetuin sample and lactoferrin sample were each dissolved in pure water and loaded on to each separate packed bed columns comprising the EBA adsorbent coupled with serotonin.
  • the flowrate during loading is 0.5 ml/min. After loading the columns were washed using 50 mM pure water. A flowrate of 0.5 ml/min and 6 column volumes were used during the washing.
  • the retained fetuin and the retained lactoferrin comprising oligosaccharides were eluted from the columns using a flowrate of 0.5 ml/min and a buffer comprising 100 mM sodium phosphate, pH 7.0. 7-18 column volumes of elution buffer were used in each column.
  • oligosaccharides having sialic acid in the terminal end of the oligosaccharide illustrated by both fetuin and lactoferrin comprising oligosaccharides, having NeuAc and/or sialic acid in the terminal end of the oligosaccharide, is retained by the adsorbent provided in Example 1, and the retained oligosaccharides were subsequently successfully eluted from the column.
  • Example 3 determination of unspecific protein binding of the serotonin adsorbent provided in example 1.
  • Example 2 The unspecific protein binding of the serotonin adsorbent produced above in Example 1 was demonstrated in the ability to bind fetuin and lactoferrin which has been stripped from NeuAc and sialic acid oligosaccharides.
  • the fetuin sample and lactoferrin sample were each dissolved in pure water and loaded on to each separate packed bed columns comprising the EBA adsorbent coupled with serotonin.
  • the flowrate during loading is 0.5 ml/min.

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Abstract

The present invention relates to a method for separating one or more oligosaccharide compound(s) from an oligosaccharide containing mixture, the method comprises the steps of (i) providing the oligosaccharide containing mixture; (ii) contacting the oligosaccharide containing mixture with a chromatographic support allowing one or more oligosaccharide compound(s) present in the oligosaccharide containing mixture to be retained by the chromatographic support; (iii) obtaining a unretained, flow through fraction from the chromatographic support comprising a protein, a cell, a cell debris, a nucleic acid and/or an enzyme; (iv) optionally washing the chromatographic support; (v) subjecting the chromatographic support to at least one elution buffer obtaining one or more oligosaccharide compound(s) from the chromatographic support; and wherein the chromatographic support comprises an adsorbent comprising one or more ligand capable of binding the one or more oligosaccharide compound(s) from the oligosaccharide containing mixture and wherein the one or more ligand comprise an a boronic acid compound, a serotonin compound, or a derivative thereof.

Description

SEPARATION OF OLIGOSACCHARIDES
Technical field of the invention
The present invention relates to an improved process for isolating oligosaccharides from an oligosaccharide containing mixture. In particular, the present invention relates to a method for isolating oligosaccharides from an oligosaccharide containing mixture using a chromatographic support providing an unspecific binding or a specific binding of oligosaccharides from the oligosaccharide containing mixture.
Background of the invention
Human milk oligosaccharides (HMO) constitute the third most abundant class of molecules in breast milk. Since infants lack the enzymes required for milk glycan digestion, this group of carbohydrate polymers passes undigested to the lower part of the intestinal tract, where they can be consumed by specific members of the infant gut microbiota, and is considered to provide the infant with nutritional and pharmacological activities.
Some analytical studies have indicated that there may be as much as 20 g/L of oligosaccharides in breast milk which makes it the third most concentrated component after lactose and fat. However, unlike lactose and fat, these molecules do not appear to provide energy to infants as they resist the action of human digestive enzymes.
Most human milk oligosaccharides (HMO) are elongation products of lactose, and are synthesized from glucose, galactose, glucosamine, fucose and sialic acid. In addition to being concentrated, HMO are also highly diverse and at least 200 individual structures have been detected using mass spectrometry. This diversity may come from a difference in the compositions of HMO from woman to woman as well as the stage of the lactation circle.
Several interesting bioactivities have been attributed to HMO. For example, they antagonize the binding of some strains of bacteria to epithelial cells, and a large epidemiologic study indicated that sugar epitopes resulting from an individual genotype appear to protect against diarrhoea in breast fed infants. Furthermore, those containing sialic acid may serve as immune modulators. It has also been suggested that HMO may serve as growth factors for colonic microbiota, as it has long been known that breast feeding results in increased levels of faecal bifidobacteria with respect to infant formula. Based on their structure and composition, HMO may affect the gut microbiota consortium either by selectively providing growth factors and energy substrates to some members, or alternatively via binding and eliminating others, or both.
Furthermore, in order to further investigate the activity of HMO and the activities modulated by HMO, it is first necessary to isolate them in large enough quantities to serve as fermentation and or growth factor substrates in microbiological media, and to ensure that they are entirely free of milk mono and disaccharides. Moreover, by isolating specific HMO's or some specific groups of HMOs' a more specific modelling of the HMO's activity may be investigated.
Previously attempts has been made to isolate HMO from other constituents in milk, although mostly for quantification as opposed to the production of substrates for biochemical assays.
An interesting source of oligosaccharides may be milk obtained from a ruminant, such as a cow or a bovine or the oligosaccharides may be synthetized. The synthetized
oligosaccharides may be produced by chemical production, enzymatic production and/or by microbial fermentation.
The oligosaccharides obtained from these other interesting sources are considered to be analogous to HMO, and it is suggested that the oligosaccharides, in particular the bovine milk oligosaccharides, has a similar protective role as suggested by human milk oligosaccharides.
The presently provided attempts to isolate oligosaccharides from milk has only been for analytical studies and there has been no focus on industrial process for isolating larger amounts of oligosaccharides for commercial use.
Hence, there is an interest in providing a method for isolating the oligosaccharides present in an oligosaccharide containing mixture in order to make the oligosaccharides available for analytical purposes and investigation as well as making the oligosaccharides available for commercial applications as an ingredient.
However, even it may be theoretically possible to isolate oligosaccharides from milk it has not yet been industrially and financially interesting. Hence, an improved method for isolating oligosaccharides from an oligosaccharide containing mixture would be advantageous, and in particular a faster, a more efficient and/or reliable method for isolating oligosaccharides from the oligosaccharide containing mixture would be advantageous.
Summary of the invention
Thus, an object of the present invention relates to an improved method for isolating oligosaccharides from an oligosaccharide containing mixture.
In particular, it is an object of the present invention to provide an improved method for isolating oligosaccharides from an oligosaccharide containing mixture, in particular a method which is faster, more efficient and/or reliable in the isolation of oligosaccharides from an oligosaccharide containing mixture and that solves the abovementioned problems of the prior art with effectivity and volume of the oligosaccharide product provided.
Thus, one aspect of the invention relates to a method for separating one or more oligosaccharide compound(s) from an oligosaccharide containing mixture, the method comprises the steps of:
(i) Providing the oligosaccharide containing mixture;
(ii) Contacting the oligosaccharide containing mixture with a chromatographic support allowing one or more oligosaccharide compound(s) present in the oligosaccharide containing mixture to be retained by the chromatographic support;
(iii) Obtaining a unretained, flow through fraction from the chromatographic support comprising a protein, a cell, a cell debris, a nucleic acid and/or an enzyme;
(iv) Optionally washing the chromatographic support;
(v) Subjecting the chromatographic support to at least one elution buffer obtaining one or more oligosaccharide compound(s) from the chromatographic support; and wherein the chromatographic support comprises an adsorbent comprising one or more ligand capable of binding the one or more oligosaccharide compound(s) from the oligosaccharide containing mixture and wherein the one or more ligand comprise an a boronic acid compound, a serotonin compound, or a derivate thereof.
Another aspect of the present invention relates to an oligosaccharide compound comprising one or more moiety selected from the group consisting of a Hexose moiety (a Hex moiety); a HexNAc moiety; a fucose moiety (a Fuc moiety); and a NeuAc moiety.
Yet another aspect of the present invention relates to the use of the oligosaccharide according to the present invention as an ingredient, e.g. for infant formulas, a fortified or functional food or beverages, a health ingredients for human and animal or an over the counter food supplements.
The present invention will now be described in more detail in the following.
Detailed description of the invention
As mentioned earlier, the human milk oligosaccharides (HMO's) have been shown to play an important role in the early development of infants and young children, such as the maturation of the immune system. As the role is dependent on the structure and composition of the HMO many different kinds of HMOs are found in the human milk, where each individual oligosaccharide is based on a specific combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and/or N-acetylglucosamine with many and varied linkages between them, thus accounting for the enormous number of different oligosaccharides in human milk.
Almost all human milk oligosaccharides have a lactose moiety at their reducing end while sialic acid and/or fucose (when present) occupy terminal positions at the non-reducing ends. The HMOs can be acidic (e.g. charged sialic acid containing oligosaccharide) or neutral (e.g. fucosylated oligosaccharide).
Thus, it is of interest to provide a method for isolating one or more oligosaccharides from milk. Preferably, the one or more oligosaccharide compound obtained by the method according to the present invention has a significantly reduced content of lactose. A preferred embodiment of the present invention a method for separating one or more oligosaccharide compound(s) from an oligosaccharide containing mixture, the method comprises the steps of: (i) Providing the oligosaccharide containing mixture;
(ii) Contacting the oligosaccharide containing mixture with a chromatographic support allowing one or more oligosaccharide compound(s) present in the oligosaccharide containing mixture to be retained by the chromatographic support;
(iii) Obtaining a unretained, flow through fraction from the chromatographic support comprising a protein, a cell, a cell debris, a nucleic acid and/or an enzyme;
(iv) Optionally washing the chromatographic support;
(v) Subjecting the chromatographic support to at least one elution buffer
obtaining one or more oligosaccharide compound(s) from the chromatographic support; and wherein the chromatographic support comprises an adsorbent comprising one or more ligand capable of binding the one or more oligosaccharide compound(s) from the oligosaccharide containing mixture and wherein the one or more ligand comprise an a boronic acid compound, a serotonin compound, or a derivate thereof.
In an embodiment of the present invention the oligosaccharide may be a free
oligosaccharide, hence, oligosaccharides that are not bound as glycoproteins or glycolipids where the oligosaccharides may be attached on the surface of the respective molecules.
In another embodiment of the present invention the concentration of free oligosaccharides may be increased by hydrolysing the covalent bond between the oligosaccharide and the protein, in a glycoprotein, or between the oligosaccharide and the lipid, in a glycolipid, liberating the oligosaccharide. The hydrolysis may be performed by the action of an enzyme.
In a further embodiment of the present invention the one or more oligosaccharide compound(s) comprises 3-30 sugar moieties, such as 5-25 sugar moieties, e.g. 8-22 sugar moieties, such as 10-20 sugar moieties, e.g. 14-18 sugar moieties. Preferably, the oligosaccharide containing mixture may be a dairy source, , a fermentation broth, a plant material or a mixture obtained from a chemical reaction. In an embodiment of the present invention the dairy source may be selected from the group consisting of milk, whole milk, skimmed milk, milk concentrates, reconstituted milk powder, non-pasteurised milk, micro-filtrated milk, pH-adjusted milk, pre-treated dairy source, a whey material, and a fraction obtained from a whey material . The fraction obtained from a whey material may comprise oligosaccharides in combination with one or more of protein, peptide, vitamins and/or minerals. The fraction obtained from a whey material may preferably comprise oligosaccharides in combination with vitamins and/or minerals. In an embodiment of the present invention the oligosaccharide containing mixture may be a dairy source or synthetized oligosaccharides. The synthetized oligosaccharides may be obtained from chemical production, enzymatic production and/or by microbial
fermentation. The dairy source may be a milk source or a whey source. Preferably, the oligosaccharide containing mixture has not been subjected to
pasteurisation.
Oligosaccharides are found in the human and animal milk, and each individual
oligosaccharide may be based on a specific combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and/or N-acetylglucosamine coupled by different linkages.
In a further embodiment of the present invention, the terminal moiety of the
oligosaccharide may be the preferred target available for interacting with the adsorbent and/or the ligand.
One challenge when being interested in separating oligosaccharides from oligosaccharide containing mixture may be the presence of enzymes capable of inactivating or degrading oligosaccharides.
Hence, in an embodiment according to the present invention the oligosaccharide containing mixture provided in step (i) may be subjected to a first pre-treatment. The first pre treatment may involve a step of enzyme inactivation. In an embodiment of the present invention the enzyme inactivation, involves inactivation of one or more of the enzymes selected from the group consisting of fucose-, sialic acid-, N-Acetylglucosamine-, lacto-N-biose-, glucose- and/or galactose-degrading enzymes in the oligosaccharide containing mixture.
The enzyme inactivation may involve addition of an enzyme degrading compound, a pre separation step removing the enzyme from the oligosaccharide containing mixture or a combination hereof. The pre-separation step may preferably involve removing the enzyme from the oligosaccharide containing mixture. Such removal may involve a filtration process, a centrifugation process or a chromatographic process.
In an embodiment of the present invention the pH of the oligosaccharide containing mixture provided in step (i) may be adjusted before contacting the oligosaccharide containing mixture with the chromatographic support in step (ii).
In a further embodiment of the present invention the oligosaccharide containing mixture provided in step (i) may be pH adjusted to a pH value below pH 6 before contacting the oligosaccharide containing mixture with the chromatographic support in step (ii), such as a pH value below pH 5, e.g. a pH value below pH 4.5, such as a pH value below pH 4, e.g. a pH value below pH 3.5, such as a pH value below pH 3.2, e.g. a pH value below pH 3.0.
In another embodiment of the present invention the oligosaccharide containing mixture provided in step (i) may be pH adjusted to a pH value above pH 6 before contacting the oligosaccharide containing mixture with the chromatographic support in step (ii), such as a pH value above pH 7, e.g. a pH value above pH 7.5, such as a pH value above pH 8, e.g. a pH value above pH 8.3, such as a pH value above pH 8.5, e.g. a pH value above pH 8.75, such as a pH value above pH 9, e.g. a pH value in the range of pH 6-9.5, such as a pH value in the range of pH 7-9, e.g. a pH value in the range of pH 8-8.75, such as a pH value about pH 8.5.
The adsorbent according to the present invention capable of binding the one or more oligosaccharide compound(s) from the oligosaccharide containing mixture may be essential for the effectivity of the invention.
The non-porous material may preferably be selected from a metal oxide; a silicate; a ceramic metal; alumina; or a magnetic material. However, further examples of non-porous materials, (non-porous core materials) are described in WO 2010/037736, which is hereby incorporated by reference. The metal oxide according to the present invention may be selected from titanium oxide; silicates; ferric oxide, nickel oxide or cobalt oxide.
The magnetic material according to the present invention may be selected from ferric oxide, nickel oxide or cobalt oxide.
In an embodiment of the present invention, the non-porous material may be a high density non-porous material .
In a further embodiment of the present invention the high density non-porous material may have a density of at least 4.0 g/ml, such as at least 10 g/ml, e.g . at least 16 g/ml, such as at least 25 g/ml. Typically, the non-porous core material has a density in the range of about 4.0-25 g/ml, such as about 4.0-20 g/ml, e.g . about 4.0-16 g/ml, such as 12-19 g/ml, e.g. 14-18 g/ml, such as about 6.0-15.0 g/ml, e.g . about 6.0-16 g/ml .
The high density non-porous material according to the present invention may be selected from tungsten carbide or steel .
In an embodiment of the present invention the adsorbent may be a metal oxide capable of binding the one or more oligosaccharide compound(s) from the oligosaccharide containing mixture.
In an embodiment of the present invention the adsorbent comprises a particle comprising a non-porous material surrounded by a porous polymeric material . Preferably, the porous polymeric material may be an organic porous polymeric material . The porous polymeric material may be selected from agarose, alginate, chitosan, carrageenan, and/or pectin.
In an embodiment of the present invention the one or more adsorbent may have a density of at least 1.3 g/ml, more preferably at least 1.5 g/ml, still more preferably at least 1.8 g/ml, even more preferably at least 2.0 g/ml, even more preferably at least 2.3 g/ml, even more preferably at least 2.5 g/ml, even more preferably at least 2.75 g/ml, even more preferably at least 3.0 g/ml, even more preferably at least 3.5 g/ml, even more preferably at least 4.0 g/ml, even more preferably at least 4.5 g/ml .
The density of the adsorbent according to the present invention relates to the density of an adsorbent in it's fully solvated (e.g. hydrated) state as opposed to the density of a dried adsorbent particle. The desired density of the one or more adsorbent may be provided by inclusion of a certain proportion or a certain amount of the non-porous material in the porous polymeric material .
In order to improve the capability of the adsorbent to bind the one or more oligosaccharide compound(s) from the oligosaccharide containing mixture the adsorbent may comprise one or more ligands capable of binding the one or more oligosaccharide compound(s) from the oligosaccharide containing mixture.
In an embodiment of the present invention the adsorbent comprises one or more ligands capable of binding the one or more oligosaccharide compound(s) from the oligosaccharide containing mixture.
In yet an embodiment of the present invention, the ligand may be an un-specifically binding ligand ; or the ligand may be a specifically binding ligand.
In a preferred embodiment of the present invention the one or more ligands may be un- specifically binding the oligosaccharide compound(s) from the oligosaccharide containing mixture.
In the context of the present invention the term "un-specifically binding" relates to the binding of the oligosaccharides present in the oligosaccharide containing mixture with limited or no (or substantially no) differentiation between the different oligosaccharide molecules present in the oligosaccharide containing mixture.
Preferably the one or more adsorbent capable of binding the one or more oligosaccharide compound(s) does not comprise an ion exchange ligand .
In a preferred embodiment of the present invention the one or more adsorbent capable of binding the one or more oligosaccharide compound(s) comprise a boronic acid compound or a derivate thereof. Preferably, the boronic acid compound or a derivative thereof comprises a boric acid and an organic substituent. The organic substituent may preferably be an alkyl compound or an aryl compound, preferably, the organic substituent is an aryl compound . Preferably, the boronic acid compound or a derivative thereof may be a phenyl borate compound
In an embodiment of the present invention the organic substituent constitutes the connection between the adsorbent and the boric acid. In yet an embodiment of the present invention the ligand may be a boronic acid compound or a derivative thereof.
In another preferred embodiment of the present invention the ligand may be specifically binding oligosaccharides.
In the present contest, the term "specifically binding" relates to a preference (a differentiation) in the binding a specific type of oligosaccharide by the specifically binding ligand.
In an embodiment of the present invention the specifically binding ligand may be a serotonin compound or a derivative thereof.
The ligand concentration on the adsorbent may be in the range of 30-300 pmoles per ml sedimented adsorbent, e.g. in the range of 50-200 pmoles per ml sedimented adsorbent, such as 75-175 pmoles per ml sedimented adsorbent, e.g. 100-160 pmoles per ml sedimented adsorbent, such as 120-145 pmoles per sedimented adsorbent.
The adsorbent and/or the ligand according to the present invention may have a high specificity for one or more oligosaccharide compound(s) from the oligosaccharide containing mixture.
In the present context, the term "high specificity for one or more oligosaccharide compound(s)" relates to more than 50% (w/w) of the molecules specifically bound to the adsorbent and/or the ligand may be one or more oligosaccharide compound(s), such as more than 55%, e.g. more than 60%, such as more than 65%, e.g. more than 70%, such as more than 75%, e.g. more than 80%, such as more than 90%, e.g. more than 95%.
In an embodiment of the present invention the chromatographic support may be a membrane chromatography support, or a column chromatography support. Preferably, the column chromatography support includes a Packed Bed Chromatography, stirred tank adsorption, moving bed chromatography, simulated moving bed chromatography, Fluidized Bed Chromatography and/or Expanded Bed Chromatography.
In order to quickly process the oligosaccharide containing mixture an increased loading speed may be preferred. Hence, in an embodiment of the present invention the oligosaccharide containing mixture may be loaded on to the chromatographic support at a flow-rate in the range of 1-50 cm/min; preferably in the range of 5-30 cm/min; more in the range of 10-25 cm/min; even more preferably, in the range of 15-20 cm/min. In an embodiment of the present invention a loading buffer may be used when loading the oligosaccharide containing mixture on to the chromatographic support. The loading buffer may comprise an organic compound. The organic compound may be taurine.
In another embodiment of the present invention the loading buffer may have a pH value in the range of pH 6-9.5, such as a pH value in the range of pH 7-9, e.g. a pH value in the range of pH 8-8.75, such as a pH value about pH 8.5. The separation process according to the present invention may either be a batch separation process or a continuous separation process.
In an embodiment of the present invention, large-scale production (industrial scale production) may be conducted at a continuous process. When the oligosaccharides are retained by the chromatographic support an elution step at some point during the separation process may be necessary. By providing at least two chromatographic supports and placing them in parallel, such continuous separation may be provided where the flow of oligosaccharide containing mixture may be shifted from one chromatographic support, when this chromatographic material is loaded and ready for elution, to the other chromatographic support. Alternatively, moving bed chromatography, simulated moving bed chromatography or the like may be used.
The oligosaccharide compound bound to the chromatographic support may be released from the chromatographic support by subjecting the chromatographic support to at least one elution buffer obtaining one or more oligosaccharide compound(s) from the chromatographic support, as described in step (v).
In an embodiment of the present invention the elution in step (v) may be a sequential elution of one or more oligosaccharides providing one of more oligosaccharide compounds.
In a further embodiment of the present invention the elution in step (v) involves a change of the pH.
In yet an embodiment of the present invention the change in pH for eluting the one or more oligosaccharides involves a change in pH to a pH above pH 3.0, such as a pH above pH 3.2, e.g. a pH above pH 3.5, such as a pH above pH 4.0, e.g. a pH above pH 4.5, such as a pH above pH 5.0, e.g. a pH above pH 6.0, such as a pH in the range of pH 3.0-9.0, e.g. a pH in the range of pH 3.2-8, such as a pH in the range of pH 3.5-7.5, e.g. a pH in the range of pH 4.0-7.2, such as a pH in the range of pH 4.5-7.0, e.g. a pH in the range of pH 5.0-6.5, such as about pH 6.0.
In an embodiment of the present invention an elution buffer may be used when elution the oligosaccharide retained on the chromatographic support. The elution buffer may comprise an organic compound. The organic compound may be taurine.
In a further embodiment of the present invention, the elution buffer comprises a carbohydrate compound, such as sorbitol, lactose, and/or fucose.
In another embodiment of the present invention the elution buffer may have a pH value in the range of pH 6-9.5, such as a pH value in the range of pH 7-9, e.g. a pH value in the range of pH 8-8.75, such as a pH value about pH 8.5.
A preferred embodiment of the present invention relates to an oligosaccharide compound comprising one or more moiety selected from the group consisting of a Hexose moiety (a Hex moiety); a HexNAc moiety; a fucose moiety (a Fuc moiety); and a NeuAc moiety.
The Hex moiety of the oligosaccharide compound may preferably be selected from a glucose residue, a galactose residue or a mannose residue.
The HexNAc moiety of the oligosaccharide compound may preferably be a N- acetylglucosamine residue or a N-acetylgalactosamine residue.
The fucose moiety of the oligosaccharide compound may preferably be a deoxyhexose residue.
The NeuAc moiety of the oligosaccharide compound may preferably be a N-acetyl neuraminic acid residue or a sialic acid residue.
In an embodiment of the present invention the one or more oligosaccharide compound(s) comprises a fucose moiety (such as a deoxyhexose residue); and a NeuAc moiety (such as a N-acetyl neuraminic acid residue or a sialic acid residue).
In yet an embodiment of the present invention the ligand is a specifically binding ligand, preferably serotonin or a derivative thereof, and the oligosaccharide compound retained is an oligosaccharide comprising a NeuAc moiety and/or a sialic acid residue in the terminal end of the oligosaccharide. In a further embodiment of the present invention the one or more oligosaccharide compound(s) may comprise 3-30 sugar moieties, such as 5-25 sugar moieties, e.g. 8-22 sugar moieties, such as 10-20 sugar moieties, e.g. 14-18 sugar moieties.
Preferably, the content of free lactose present in the oligosaccharide compound according to the present invention may be less than 5 mg/I, such as less than 4 mg/I, e.g. less than 3 mg/I, such as less than 2 mg/I, e.g. less than 1 mg/I, such as less than 0.5 mg/I, e.g. less than 0.1 mg/I.
Preferably, the content of glucose present in the oligosaccharide compound according to the present invention may be less than 5 mg/I, such as less than 4 mg/I, e.g. less than 3 mg/I, such as less than 2 mg/I, e.g. less than 1 mg/I, such as less than 0.5 mg/I, e.g. less than 0.1 mg/I.
Preferably, the content of galactose present in the oligosaccharide compound according to the present invention may be less than 5 mg/I, such as less than 4 mg/I, e.g. less than 3 mg/I, such as less than 2 mg/I, e.g. less than 1 mg/I, such as less than 0.5 mg/I, e.g. less than 0.1 mg/I.
The oligosaccharide compound according to the present invention may be used as an ingredient, e.g. for infant formulas, a fortified or functional food or beverages, a health ingredients for human and animal or an over the counter food supplements. The oligosaccharide compound according to the present invention may be used as a prebiotic ingredient.
In the context of the present invention, the term "prebiotic" means non-digestible carbohydrates that beneficially affect the host by selectively stimulating the growth and/or the activity of healthy bacteria such as bifidobacteria in the colon of humans.
Without being bound by theory, the oligosaccharides of the nutritional composition of the present invention may act at different levels to support the natural defenses of the developing infant or young child : (i) boost commensals to obtain better colonization resistence, (ii) boost innate immunity to counteract pathogens and/or (iii) act directly on pathogens as decoy to deviate the pathogens from their natural target. It is also believed that the fucosylated oligosaccharide(s) and the particular N-acetylated oligosaccharide(s) act synergically for these purposes. They could exert synergistic protection in that they target numerous diverse adhesins and quorum sensing mechanisms of pathogens, thereby reducing their virulence and competiveness, and they favour the establishment and competitiveness and metabolic activity of commensal microbiota leading to colonization resistance.
It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.
All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety. The invention will now be described in further details in the following non-limiting examples.
Examples
Example 1 - coupling serotonin to a chromatographic support
Serotonin binds specifically to NeuAc and sialic acid residues in the terminal end of oligosaccharides. In the Present example serotonin is coupled to an agarose/tungsten carbide adsorbent matrix suitable for Expanded bed Adsorption and tested in a packed bed column of 1 ml .
The serotonin was coupled to the adsorbent by the following procedure: The following buffers were used :
1. Coupling buffer: 0.2 M NaHCOs, 0.5 M NaCI, pH 8.3
2. Cleaning buffer: lmM HCI ice-cold
3. Buffer A: 0.5 M ethanolamine, 0.5 M NaCI, pH 8.3
4. Buffer B: 0.1 M sodium acetate, 0.5 M NaCI, pH 4
5. Storage buffer: 0.05 M Na2HP04, 0.1% NaN3, pH 7
Ligand coupling Isopropanol was washed out of the column comprising the adsorbent using the cleaning buffer, 1 mM HCI, ice-cold. 3 x 2 ml cleaning buffer was used at a flowrate of 1 ml/min. Immediately after cleaning 1 ml of ligand solution (63 mg of serotonin in 4 ml of coupling buffer) was injected onto the column and the run through was discarded . 3 ml coupling buffer left with recirculation by connecting a peristaltic pump over night at 4°C (cold room) was continuously added .
Any non-specifically bound ligands were washed out and any excess active groups on the adsorbent that had not been coupled to the ligand was deactivated, following the below procedure :
• 3 x 2 ml of Buffer A was injected.
• 3 x 2 ml of Buffer B was injected .
• 3 x 2 ml of Buffer A was injected .
The column is left for 30 min at room temperature.
• 3 x 2 ml of Buffer B was injected .
• 3 x 2 ml of Buffer A was injected.
• 3 x 2 ml of Buffer B was injected .
Finally, 2 ml of storage solution was injected to adjust the pH and to be stored .
The flowrate during injection, recirculation, wash and deactivation was 0.5 ml/min .
The column was sealed with parafilm and stored until use.
Example 2 - Capturing oligosaccharides having sialic acid in the terminal end of the oligo saccharide using the serotonin adsorbent provided in example 1.
The serotonin adsorbent produced above in Example 1 was demonstrated in the ability to bind and isolate NeuAc and sialic acid containing oligosaccharides. In order to determine the effectivity of the column in binding NeuAc and sialic acid, fetuin comprising
oligosaccharides having terminal NeuAc and/or sialic acid residues and lactoferrin comprising oligosaccharides having terminal NeuAc and/or sialic acid residues were used as target molecule.
The oligosaccharide containing fetuin sample and lactoferrin sample were each dissolved in pure water and loaded on to each separate packed bed columns comprising the EBA adsorbent coupled with serotonin. The flowrate during loading is 0.5 ml/min. After loading the columns were washed using 50 mM pure water. A flowrate of 0.5 ml/min and 6 column volumes were used during the washing. The retained fetuin and the retained lactoferrin comprising oligosaccharides were eluted from the columns using a flowrate of 0.5 ml/min and a buffer comprising 100 mM sodium phosphate, pH 7.0. 7-18 column volumes of elution buffer were used in each column.
The results show that oligosaccharides having sialic acid in the terminal end of the oligosaccharide, illustrated by both fetuin and lactoferrin comprising oligosaccharides, having NeuAc and/or sialic acid in the terminal end of the oligosaccharide, is retained by the adsorbent provided in Example 1, and the retained oligosaccharides were subsequently successfully eluted from the column.
Example 3 - determination of unspecific protein binding of the serotonin adsorbent provided in example 1.
The unspecific protein binding of the serotonin adsorbent produced above in Example 1 was demonstrated in the ability to bind fetuin and lactoferrin which has been stripped from NeuAc and sialic acid oligosaccharides.
The fetuin sample and lactoferrin sample were each dissolved in pure water and loaded on to each separate packed bed columns comprising the EBA adsorbent coupled with serotonin. The flowrate during loading is 0.5 ml/min.
After loading the columns were washed using 50 mM pure water. A flowrate of 0.5 ml/min and 6 column volumes were used during the washing. The retained fetuin and the retained lactoferrin substantially without any oligosaccharides were eluted from the columns using a flowrate of 0.5 ml/min and a buffer comprising 100 mM sodium phosphate, pH 7.0. 7-18 column volumes of elution buffer were used in each column. The results show that substantially no fetuin and substantially no lactoferrin were retained by the serotonin coupled adsorbent. A small signal of eluted proteins may be observed, which relates to in un-absolute oligosaccharide stripping of the fetuin and lactoferrin molecules and not to an unspecific protein binding by the serotonin coupled adsorbent. Hence, it concluded that serotonin may be successfully coupled to an expanded bed adsorbent and that the serotonin coupled adsorbent show to be a highly successful candidate for isolating oligosaccharides comprising NeuAc and/or sialic acid in the terminal end of the oligosaccharide (see example 2 above)
References
WO 2010/037736

Claims

Claims
1. A method for separating one or more oligosaccharide compound(s) from an oligosaccharide containing mixture, the method comprises the steps of:
(i) Providing the oligosaccharide containing mixture;
(ii) Contacting the oligosaccharide containing mixture with a chromatographic support allowing one or more oligosaccharide compound(s) present in the oligosaccharide containing mixture to be retained by the chromatographic support;
(iii) Obtaining a unretained, flow through fraction from the chromatographic support comprising a protein, a cell, a cell debris, a nucleic acid, minerals and/or an enzyme;
(iv) Optionally washing the chromatographic support;
(v) Subjecting the chromatographic support to at least one elution buffer obtaining one or more oligosaccharide compound(s) from the chromatographic support; and wherein the chromatographic support comprises an adsorbent comprising one or more ligand capable of binding the one or more oligosaccharide compound(s) from the oligosaccharide containing mixture and wherein the one or more ligand comprise a serotonin compound, or a derivative thereof, wherein the oligosaccharide containing mixture is a preliminary diary source or a whey material, and/or wherein the
chromatographic support is a column chromatography support, wherein the column chromatography support is an Expanded Bed Chromatography.
2. The method according to anyone of the preceding claims, wherein the adsorbent comprises a non-porous metal material.
3. The method according to claim 2, wherein the non-porous metal material is selected from a metal oxide; a silicate; a ceramic metal; alumina; a magnetic material; or high density non-porous metal material.
4. The method according to anyone of claims 2-3, wherein the high density non-porous metal material is surrounded by a porous polymeric material.
5. The method according to claim 4, wherein the porous polymeric material is an organic porous polymeric material, and wherein the porous polymeric material is selected from agarose, alginate, chitosan, carrageenan, and/or pectin.
6. The method according to anyone of the preceding claims, wherein the oligosaccharide containing mixture may be a preliminary diary source, a whey material, a fermentation broth, a plant material or a mixture obtained from a chemical reaction.
7. A oligosaccharide compound obtainable by a method according to anyone of claims 1-6.
8. The oligosaccharide compound according to claim 7, wherein the content of free lactose present in the oligosaccharide compound is less than 5 mg/I, such as less than 4 mg/I, e.g. less than 3 mg/I, such as less than 2 mg/I, e.g. less than 1 mg/I, such as less than 0.5 mg/I, e.g. less than 0.1 mg/I, or wherein the content of glucose present in the
oligosaccharide compound is less than 5 mg/I, such as less than 4 mg/I, e.g. less than 3 mg/I, such as less than 2 mg/I, e.g. less than 1 mg/I, such as less than 0.5 mg/I, e.g. less than 0.1 mg/I, or wherein the content of galactose present in the oligosaccharide compound is less than 5 mg/I, such as less than 4 mg/I, e.g. less than 3 mg/I, such as less than 2 mg/I, e.g. less than 1 mg/I, such as less than 0.5 mg/I, e.g. less than 0.1 mg/I.
9. Use of the oligosaccharide according to anyone of claims 7-8 as an ingredient, e.g. for infant formulas, a fortified or functional food or beverages, a health ingredient for human and animal or an over the counter food supplements.
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