NZ625977B2 - Methods for providing sialylated oligosaccharides - Google Patents

Methods for providing sialylated oligosaccharides Download PDF

Info

Publication number
NZ625977B2
NZ625977B2 NZ625977A NZ62597712A NZ625977B2 NZ 625977 B2 NZ625977 B2 NZ 625977B2 NZ 625977 A NZ625977 A NZ 625977A NZ 62597712 A NZ62597712 A NZ 62597712A NZ 625977 B2 NZ625977 B2 NZ 625977B2
Authority
NZ
New Zealand
Prior art keywords
gal
neu5ac
gos
glc
sialic acid
Prior art date
Application number
NZ625977A
Other versions
NZ625977A (en
Inventor
Johannes Adrianus Hage
Johannis Paulus Kamerling
Erik Sallomons
Peter Sanders
Vuure Catherina Anna Van
Maarten Hotse Wilbrink
Original Assignee
Darling Ingredients Nederland Bv
Friesland Brands Bv
Rijksuniversiteit Groningen
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from NL2007931A external-priority patent/NL2007931C2/en
Application filed by Darling Ingredients Nederland Bv, Friesland Brands Bv, Rijksuniversiteit Groningen filed Critical Darling Ingredients Nederland Bv
Publication of NZ625977A publication Critical patent/NZ625977A/en
Publication of NZ625977B2 publication Critical patent/NZ625977B2/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/163—Sugars; Polysaccharides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
    • 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
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
    • 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
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • 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
    • 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
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • 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
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • 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
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01018—Exo-alpha-sialidase (3.2.1.18), i.e. trans-sialidase

Abstract

The disclosure relates to a method for providing a composition comprising sialic acid containing oligosaccharides, comprising the steps of: a) providing a source of non-digestible galactooligosaccharides (GOS) containing at least two terminally bonded beta-linked galactose residues; b) providing a sialic acid donor having (alpha2-3)-sialylated O-glycans; c) contacting said GOS with said sialic acid donor in the presence of an enzyme having trans-sialidase activity in an enzyme reaction mixture; and d) obtaining from said enzyme reaction mixture a fraction comprising at least 5 percent by weight of disialylated galactooligosaccharides (di-Sia-GOS) based on the dry matter. ialic acid donor having (alpha2-3)-sialylated O-glycans; c) contacting said GOS with said sialic acid donor in the presence of an enzyme having trans-sialidase activity in an enzyme reaction mixture; and d) obtaining from said enzyme reaction mixture a fraction comprising at least 5 percent by weight of disialylated galactooligosaccharides (di-Sia-GOS) based on the dry matter.

Description

WO 85384 METHODS FOR PROVIDING SIALYLATED OLIGOSACCHARIDES The invention relates to a method for providing analogs of human milk oligosaccharides (HMO), in particular oligosaccharides containing terminal sialic acid nafter: sialic acid containing oligosaccharides), especially sialylated galactooligosaccharides (Sia-GOS). In on, the invention relates to the obtainable sialylated oligosaccharides and the use thereof: in especially infant foods and animal feed.
Human milk contains a large quantity and diversity (>100 structures) of oligosaccharides with different physiological functions, including as prebiotic components and antiadhesive components for enic microorganisms. Especially sialic-acid-containing oligosaccharides (SOS) were shown to inhibit adhesion ofpathogenic microorganisms, including E. coli and Salmonella spp. in the small intestine. SOS are abundantly present in human milk (0.6-8.3 g/l) and can reach even higher trations in milk of the earliest stage of lactation (colostrum). In cow milk, however, SOS are present only in very small amounts. Therefore, a great discrepancy between human milk and infant a based on cow milk exists concerning the abundance of SOS.
In the sialylated oligosaccharides of human milk, N-acetyl— neuraminic acid c) is attached to a penultimate ose residue via 052-3 or «2-6 linkages or to an internal ylglucosamine residue via an a2- 6 linkage, formed by the action of sialyltransferases. Several pathogenic microorganisms, such as Vibrio cholerae, Escherichia coli, bacter pylori: and influenza viruses A and B, recognize a sialic-acid-containing carbohydrate receptor structure on target cells; sialylated components in human milk may therefore function as soluble receptor analogs to inhibit the attachment of these pathogens to cell surface receptors. It has been proposed that sialic acid is used for the synthesis of infant brain gangliosides and sialylglycoproteins.
The fact that human milk is a rich source of sialic acid for infants suggests that the rapid formation of brain gangliosides during first month post partum depends on metabolic utilization of milk sialyl-oligosaccharides and sialylglycoconjugates.
Since, as a rule, the aim is to make infant and baby foods resemble human milk as much as possible, several investigators recognized the need (especially with infant milk formulations) to enrich such foods with sialic-acid- containing oligosaccharides.
The focus has been predominantly on sialyllactoses since these are present at ably higher trations in human milk ed to other mammalian s. Sialyllactose is known to have anti-adhesive properties for specific pathogenic bacteria. For example, sialyllactose acts to inhibit cholera toxin in vitro (Idota et a1., Biosci. Biotech. Biochem. 59: 9, 1995) and Helicobacter pylori (Simon et al., Infection and Immunity 65: 750-757, 1997).
In light of its anti-adhesive properties, sialyllactose has been used to treat or prevent a number of medical conditions. For example, US 5,260,280 ses a composition containing -acid-containing oligosaccharides that neutralizes the s of bacterial enterotoxin. US 5,514,660, US 5,753,630 and US 5, 888,079 disclose methods for treating or preventing an ulcer in the stomach or duodenum or inhibiting Helicobacter pylori infection, respectively, by administering an effective amount of a sialic-acid-containing oligosaccharide. US 965 relates to compositions for inhibiting binding of the bacterium Helicobacter pylori to stomach or duodenal cells by administering an effective amount of certain oligosaccharides. US 5,834, 423 describes sialic acid derivatives that promote the proliferation of bifidobacteria and the use of effective amounts of certain sialylated oligosaccharides as an arrheal agent. The sialylated oligosaccharides comprise 3’-sialyllactose and 6’—sialyllactose (sialic acid = Neu5Ac). W02001060846 discloses a nutritional composition comprising the tic nces oligofructose and lactose that act synergistically to stimulate the growth of the ial bifidobacteria. WOO 1/60846 discloses nutritional compositions, such as an infant formula, comprising oligofructose and sialyllactose. EP 1549151 describes that the combination of ructose, sialyllactose and probiotic ia eradicates intestinal infection with pathogenic bacteria, particularly enteropathogenic E. coli, and may therefore be used for the prophylaxis of diarrhea due to enteropathogenic E. coli. USZOO70104843 relates to a milk- derived sialyllactose concentrate for the use in foods especially intended for infants, children or elderly persons as well as foods for medical or dietetic purposes and other food applications. It also discloses a process for producing the lactose concentrate comprising ultrafiltration of a milk product containing naturally occurring sialyllactose followed by diafiltration of the ultrafiltration retentate. W02009/1 13861 in the name of the applicant relates to a process for ing sialic-acid-containing oligosaccharides and in particular sialyllactose from a milk stream and especially from a whey stream.
The process yields a product having a high content of sialyllactose and a low content of phosphorus compounds. This t is highly suitable for supplementing infant foods. relates to a process of synthesis of sialyl-oligosaccharides and in particular of 6’-sialyllactose and its salts comprising a step of coupling by Koenigs-Knorr reaction under conditions that allow its use on an industrial scale.
Thurl et al. (British J. Nutr. 104: 1261-1271, 2010) reported a detailed investigation of the milk oligosaccharide composition of a large number of human milk samples obtained during the first 3 months of lactation. The sialylated accharides analyzed were lyllactose (3’- SL): 6’-sialyllactose (6’-SL), sialyl-lacto—N-tetraoses a-c (LSTa-c) and disialyllacto-N-tetraose (DSLNT). It was found that, s sialyllactose, DSLNT was the most prominent ated oligosaccharide. DSLNT exhibited 80 a maximum time curve 15 days postpartum. Asakuma et al. (Biosci. 2012/050857 Biotechnol. Biochem, 71: 1447-1451, 2007) investigated changes in concentration in sialyl-oligosaccharides of human colostrum during the first three days of lactation. Of the colostrum —oligosaccharides determined, LSTc was present at the highest concentration, followed by DSLNT, 3’-SL and 6’-SL.
Hence, a goal of the present invention is to provide a process for the manufacture of (artificial) disialylated oligosaccharides which can be used as analogs of disialylated HMO’S. Preferably, the process can be easily scaled up to generate disialylated oligosaccharides at an rial scale.
Advantageously, said process uses relatively cheap by-products from other kinds of industry, like the meat or dairy industry.
It was surprisingly found that the above goals can be met by decorating galacto-oligosaccharides (GOS) with sialic acids. GOS are oligosaccharides containing multiple galactose (Gal) units, ranging from DP 2~9 and are synthesized from lactose by the enzyme [i-galactosidase. GOS are produced on an industrial scale and have applications in various oducts, such as infant formula and dairy based drinks. Since GOS are not degraded in the human small intestine, they reach the colon largely intact where they serve as prebiotic stimulating growth of beneficial bacteria, like lactobacilli and bacteria. The Gal/Gal e type in GOS are , (Bl-3), ([314) and/or (Ell-6), depending on the source of the used B~galactosidase In the case of B- galactosidase from Bacillus circulans; yielding Vivinal GOS landCampina Domo), the major l linkage type is (Bl-4) and to a lesser extent (Bl-3) and (131-6), present in reducing linear and branched oligosaccharides (Table 1). The glucose moiety of GOS may also be connected via an (GI-1B) linkage with a Gal moiety that can be further extended with additional Gal residues en et al., Carbohydr. Res. 314: 101-114, 1998).
The inventors recognized that both the reducing branched oligosaccharides 80 and the non-reducing linear oligosaccharides result in GOS molecules that posses two terminal Gal units and hence two non-reducing ends, each of which can be modified With a sialic acid residue using a sialic acid donor and trans— sialidase (TS) enzyme activity.
Table 1. Elucidated GOS structures ed with Bacillus circulans [3- galactosidase (Yanahira et a1., Biosc. Biotech. Biochem. 59: 1021-1026, 1995; Fransen et a1., Carbohydr. Res. 314: 101-114, 1998; Fransen, PhD thesis Utrecht sity, 1999; Couher et 31., J. Agr. Food Chem. 57: 8488-8495, 2009 Disaccharides Gal(B1-2)Glc, Gal(Bl-8)Glc, Ga1(Bl-4)G1c, Ga1(Bl-6)G1c, -3)Ga1, Ga1(B1-4)Gal, G1c(oc1-1B)Gal, G1c(B1-1B)Ga1, Ga1(fi 1-4)Fru Trisaccharides Gal(l31 -4)Ga1(fi 1-2)G1c, Gal(|31-4)Ga1(}3 1-3)Glc, Ga1(B 1 -4) Galfli 1 -4) G10, Ga1(B 1—4) Ga1([5 1-6)Glc, Ga1(Bl-6)Ga1(f31 -4)G1c, Galfli 1~6)Ga1(B 1-6)G1c, -2)G1c(a1- 1B)Ga1, —4)Glc(0(1- 1B)Ga1, (331105 1 -4)Ga1([31 ~4)Fru Ga1(B1-2)[Ga1(B1-6)]G1c, Galfli 1-8) [Gal(B 1-6)]G1c, Gal(B1~4)[Gal(B1-6)]G1c, —2)[Ga1(B1-4)]Glc Tetrasaccharides Ga1(B1-4)Ga1([31-4)Ga1(Bl-4)G1c, Ga1(B 1-6) Gal(B 1 -6)Ga1(B1-4)Glc, Gala?) 1(131-4)G1c(ocl- 13)Ga1, Ga1(131 -4) G1c(o:1- 1[3)Ga1(4— 1 [3) Gal Pentasaccharides Ga1(B 1 -4)Ga1(B 1-4)Gal([31-4)Gal(B1-4)G1c, Gal([31 -6) Ga1(B 1-6) Ga1(B 1 -8)Gal(B 1—4) G10, Ga1([3 1-4) Ga1(B 1 -4)Ga1([31 -4)G1c(oc1- 1 B) Gal, -4)Gal(B1-4)G1c(oc1- 1 4— 13)Gal, Gal([31-4)Glc(oc1 — 1B)Gal(4- 1 B)Gal(4- 1 B)Gal Hexasaccharides Gal(B 1 -4)Ga1([31 -4) Gal(B 1 -4) Gal(B 1 —4) Gal(B 1-4)Glc, Gal(B 1-4)Gal(B 1 -4) Gal(B 1-4)Gal(fi c(oc1- 1B)Gal, Gal(B1-4)Gal(B 1-4)Gal(B 1 ~4)Glc(oc1- 1B)Gal(4— 1B) Gal, Gala} 1 -4)Gal([31 -4) Glc(oc 1- 1 B)Gal(4- 1B)Ga1(4— lfi) Gal, Ga1(B1-4)Glc(cx1- 1B)Gal(4- 1 B)Gal(4- 1 B)Gal(4- 1B)Gal Accordingly, in one ment the invention s to a method for providing a composition comprising sialic-acid-containing oligosaccharides, comprising the steps of : a) providing a source of non-digestible galactooligosaccharides containing at least two ally bound B-linked galactose residues; b) providing a sialic acid donor; c) contacting said galactooligosaccharide with said sialic acid donor in the presence of an enzyme having trans-sialidase activity in an enzyme reaction mixture; and d) obtaining from said enzyme reaction e a fraction comprising at least 5 percent by weight of disialylated galactooligosaccharides (di—Sia-GOS) based on the dry matter.
In one embodiment, the source of non-digestible galactooligosaccharide is obtained by tic treatment of e with B— galactosidase (EC 8.2123). B-Galactosidase from different sources such as fungi: yeast and/or bacteria, may be used yielding a mixture of oligomers with varied chain lengths and different glycosidic linkage ratios. For example) the commercial galactooligosaccharide preparation Vivinal GOS (FrieslandCampina Domo), comprising 57% G08, 21% lactose, and 22% WO 85384 glucose and galactose can be used. The GOS part contains in the DP>2 fractions reasonable amounts of reducing branched and non-reducing galacto- oligosaccharides having two al galactose units. In one embodiment, a mixture of lactose and econ-trehalose [Glc(oc1-1oc)Glc] is treated with B- galactosidase.
The GOS starting material may be pretreated to enrich for those species having two terminally bound B-linked galactose residues. For example, the DPl and/or DP2 species may be removed prior to trans-sialidase treatment. In another embodiment, a GOS starting composition having a low lactose content (eg. Vivinal—GOS 90) is suitably used. Individual GOS structures can be separated and isolated from a mixture of GOS species by methods known in the art. For example, tration can be used as described in Goulas et al.
(J. Membr. Sc. 209: 321-835, 2002). In a preferred embodiment, a GOS mixture is fractionated using cation exchange chromatography, preferably wherein the counter ion of the cation exchange resin is potassium. W02008/041848 discloses s for isolating various DP fractions from a mixture of GOS species.
Trans-sialidases (Figure l) are enzymes with the unique ability to ently er sialic acids (Neu5Ac or Neu5Gc) from various donors to an acceptor substrate containing al B-linked Gal residues. The best studied TS is that from the human parasite Trypanosoma cruzi (TcTS), while also TS s are known from closely d Trypanosoma species, such as Trypanosoma brucei and Trypanosoma congolense (Schauer and Kamerling, ChemBioChem 12: 2246-2264, 2011).
TcTS was previously shown to be active s donor substrates that posses (0L2-3)-linked sialic acids only (both Neu5Ac and Neu5Gc) (Scudder et al., J. Biol. Chem. 268: 9886-9891, 1998; Agusti et al., ydr. Res. 842: 2465-2469, 2007). The enzyme has a retaining mechanism, meaning that the 80 products formed also have an (052-8) bound sialic acid (Scudder et al, 1993). In the absence of a suitable acceptor, TcTS acts as a hydrolase (sialidase), releasing free sialic acid from its ates. Because of its sialic acid transferring capabilities and broad substrate range (both donor and acceptor), the TcTS enzyme is widely used in glycobiology to synthesize sialylated oligosaccharides (e.g. Neubacher et al, Org. Biomol. Chem. 8: 1551-1556, 2005). Other enzymes capable of transferring sialic acid to acceptor molecules are sialyltransferases. These enzymes, however, require activated sialic acid (CMP-Neu5Ac). Since nucleotide sugars are expensive, transferases are less le for (large-scale) applications in glycobiology.
The enzymatic sialylation With trans-sialidases of acceptor molecules is known in the art. See USZOO7/OOO4656, disclosing a novel enzyme isolated from Tryparwsoma congolese and the application thereof to produce sialylated products for use in vaccines, medicaments, foodstuffs or food additives. Although GOS are included in the list of possible sialic acid acceptors. the generation and isolation of a fraction enriched in ylated products is not disclosed or ted. In fact, the present ation that trans-sialylation of GOS results not only in monosialylated GOS (mono-Sia- GOS) but also in disialylated species (di—Sia-GOS) has heretofore never been reported.
The degree of rization (DP) of the di-Sia-GOS species obtainable according to the invention Will of course depend on the DP or range of DPs of the GOS acceptor molecules used. As said, typical GOS preparations contain oligosaccharides ranging between DP 2 and 8. For Vivinal GOS, the mixture can roughly be described as Gal(B1-X)Gal(fi 1-x)... .Gal(Bl-X)Glc (major amount; reducing linear and branched (DP>2) GOS components) with X = 4 (mainly), 8, and 6; and Gal(B1-4)Gal(B1-4)... 1-4)Glc(oc1—1[3)Gal(4- 113)... .Gal(4-1[3)Gal (minor amount; non-reducing GOS components) (Table 1).
In one ment, a method of the invention ses isolation of 80 a on comprising at least 5 percent by weight, preferably at least 7, 10, 12, WO 85384 18, 20, 22, 25, 27 or 30% by weight of di-Sia-GOS based on the dry matter, wherein said -GOS have a degree of polymerization (DP) within the range ofDP5 to DP11, preferably DP6 to DPS.
Exemplary disialylated GOS structures that can be formed based on Table 1, include the following: Pentasaccharide from GOS DP3 Neu5Ac(0L2-8)Gal(B 1-4)G1c(0¢1 -1fi)Gal(8-20L)Neu5Ac Neu5Ac(oc2-8)Gal(B 1-2) [Neu5Ac(a2—8) Gal(B 1-6)] Glc Neu5Ac(oc2-3)Gal([3 1-3) [Neu5Ac(oc2-3)Gal(B 1-6)]Glc, Neu5Ac(a2-8)Ga1(B1-4) [Neu5Ac(a2-8)Gal(B1-6)]Glc Neu5Ac(oc2-3)Ga1(B1-2) [Neu5Ac(0L2-3)Gal([31-4)]Glc Hexasaccharides from GOS DP4 Neu5Ac(oc2-3)Gal(l3 1-4) Gal([3 1-4)Glc(oc1 - l B)Gal(3-20L)Neu5Ac Neu5Ac(a2-3)Gal([3 1-4) Glc(oc1 - 1B)Gal(4— 1 [3) Gal(3-20L)Neu5Ac accharides from GOS DP5 (0cZ-3)Gal([3 1-4)Gal(13 l-4)Gal(B1-4)Glc((x1- 113)Ga1(3-20c)Neu5Ac Neu5Ac(oc2-8)Gal(l31-4)Gal(B1-4)Glc(oc1- 1 B)Gal(4- 113)Gal(8~20t)Neu5Ac Neu5Ac(oc2-3)Gal(B 1-4)Glc(0c1 l(4- 1B)Gal(4—1B)Gal(3—20c)Neu5Ac Octasaccharides from GOS DP6‘ Neu5Ac(oc2-3)Gal(B 1~4)Gal([3 1-4)Ga1(B 1-4)Gal(B 1-4) 1- 1B)Gal(3-2oc)Neu5Ac Neu5Ac(oc2-8)Gal(B1-4)Gal(B1-4)Gal(B1-4)Glc(0c1 - 1 B) Gal(4—1B)Gal(8—20t)Neu5Ac Neu5Ac(oc2-8)Gal(B 1-4)Gal(13 1-4)Glc(oc1- 1 B) Gal(4- 15)Gal(4—16)Ga1(8-2(X)Neu5Ac (0L2-3)Ga1([3 1-4) Glc(oc1- 1B)Gal(4- 1B)Gal(4— 1B)Gal(4—1B)Ga1(3-20()Neu5Ac In View of the enzyme specificity of the trans-sialidase used, the term “sialic acid donor” refers to any compound having one or more (a2-3)-sialylated glycans (glycoproteins, glycopeptides: glycolipids) or synthetic sialic acid glycosides (eg. 2’-(4-methylumbelliferyl)-oc-N-acetylneuraminic acid (4MU- 80 ) (Schauer and Kamerling, ChemBioChem 12: 2246-2264, 2011). These include (d2-8)-linked N-acetylneuraminic acid (Sia = ) donors and (0:2- 8)-linked N—acetylneuraminic acid/N-glycolylneuraminic acid (Sia = /Neu5Gc) donors. For human ations, preferably, the sialic acid donor contains (d2-3)-linked Neu5Ac, but for feed applications, the sialic acid donors may contain (0L2-8)-linked Neu5Ac/Neu5Gc. Combinations of different donors may also be used.
In one ment step b), the sialic acid donor is a naturally ing compound, preferably selected from sialic acids bound to oligosaccharides, polysaccharides, polysialic acids, glycoproteins. For example, the sialic acid donor is selected from whole animal blood plasma or bloodplasma-derived glycoproteins, being typical ts of the slaughterhouse, and milk glycoproteins from the dairy industry, or glycolipids.
Milk glycoproteins occur in a large variety in cow milk. The N- and O-linked carbohydrate chains are frequently terminated with members of the sialic acid (Sia) family. For example, the sialic acid donor is selected from the group ting of glycosylated whey proteins and caseins, and fragments of the same.
A highly advantageous sialic acid donor for use in the present invention is glycomacropeptide (GMP) from K-casein, which is produced as a by-product in the dairy industry. GMP is decorated with O-glycans, containing both Neu5Ac(oc2-8)Gal([31- and (oc2-6)GalNAc(oc1- units (van Halbeek et al., Biochim. Biophys. Acta 628: 295-800, 1980). With GMP as (0L2-3)-linked N- acetylneuraminic acid (Sia = Neu5Ac) donor in a method of the invention, the resulting di-Sia-GOS will find its way in among others infant nutrition and functional food.
In another preferred embodiment, the sialic acid donor is selected from the group consisting of glycosylated animal mucus proteins, and fragments of the same. Animal mucins are glycoproteins with a vely high carbohydrate content. The O-linked carbohydrate chains are frequently terminated with members of the sialic acid (Sia) family, of which N- acetylneuraminic acid c) and N-glycolylneuraminic acid (Neu5Gc) are the most important ones. Different mucins can have different sialylation patterns. Of particular st are pig and cow small inal mucin glycoproteins, typical by-products of the slaughterhouse, as sialic-acid- containing material. For instance, according to the literature, the sialylation n of pig small intestinal mucin glycoprotein (PSMG) comprises Neu5Ac(oc2-8)Gal({3 1-, Neu5Gc(oc2-8)Gal(B1-, (oc2«6)GalNAc(ocl-, and Neu5Gc(on2-6)GalNAc(ocl- units (Hansson et al., Carbohydr. Res. 221: 179-189, 1991). With PSMG as (d2-8)—linked N—acetylneuraminic acid/N- glycolylneuraminic acid (Sia = Neu5Ac/Neu5Gc) donor, the resulting di-Sia— GOS will find its way among others in the animal feed industry.
For example, the sialic acid donor is obtained from mucin in a production process whereby pig intestines are cleaned for the production of casings (removal of unborn manure; pressing mucin, pressing the caul, further processing of casing), followed by concentration and/or further purification/enzymatic degradation of sialic-acid—containing mucin biopolymers.
Thus, in one embodiment the ion involves enzymatic trans-glycosylation using a sialic acid donor obtained from relatively cheap by-products from the slaughterhouse and the dairy industry to e functional food.
In one embodiment step c) of a method disclosed herein uses an enzyme having trans-sialidase activity which is encoded by a gene product from microorganisms of the Trypanosoma genus, preferably Trypanosoma cruzi or Trypanosoma congolense. ably, said enzyme is inantly produced in a host cell e.g. a bacterial host cell like E. coli.
The tion conditions can vary depending on the enzyme source.
In one embodiment using TcTS, the pH of the enzyme reaction mixture ranges between 4 and 6, preferably between 4.8 and 5.8. Preferred buffers e Na- citrate, Pipes and TrisHCl, and mixtures thereof. In one embodiment, the enzyme incubation is performed in a mixture of Na-citrate, Pipes and Tris-HG] 2012/050857 (25 mM each) pH 5.5. It is also possible to carry out the enzyme on in an aqueous solution. For example, incubation of TcTS With GMP and GOS in water gave the same high conversion as observed for Na-citrate at pH 5.0. The enzyme may be stabilized by protein components, for example bovine serum albumin, but in the case of using rotein donors this is not necessary.
As the sialic acid transfer from donor to acceptor will not be complete, a mixture of GOS, ated GOS, sialic acid donor and asialo-donor can in theory be formed. For example, in case Vivinal GOS is applied as source of GOS, the product mixture will contain GOS, sialylated-GOS, lactose, galactose, glucose, and sialyllactose (lactose is also an acceptor for sialic acid). In case of GMP, Sia stands for Neu5Ac, in case of PSMG for Neu5Ac/Neu5Gc. This mixture may be ted via centrifugal ion (spin filters) into a high- molecular glycoprotein/enzyme fraction and a low-molecular carbohydrate fraction prior to the step of ed a fraction enriched in disialylated GOS. In principle, the Neu5Ac/Neu5Gc mixture can be ted in its Neu5Ac and Neu5Gc components Via lectin or antibody chromatography.
Step (1) of a method of the invention comprises isolating from said enzyme reaction mixture a fraction comprising at least 5 percent by weight of disialylated galactooligosaccharides based on the dry . Since disialylated oligosaccharides have two negative s per molecule at a physiological pH, they are suitably isolated using a separation technique based on a difference in charge between components to be ted, such as preferably anion-exchange chromatography. Exemplary anion-exchange resins include Resource Q. For example, after 21 h of incubation of GOS DP5 with TcTS and 4MU-Neu5Ac as artificial donor, the mixture can be separated in neutral GOS DP5, a mono-Sia-GOS DP5 (monosialylation of the reducing linear and branched as well as the non-reducing GOS components of DP5), a free Sia, and a di-Sia-GOS DP5 fraction (disialylation of the reducing branched and ducing GOS components of DP5), as checked by MALDI-TOF—MS and NMR spectroscopy after desalting.
Other le separation techniques are based on a difference in size between components to be separated, preferably size-exclusion chromatography.
Depending on the separation procedure employed and the intended application, the di-Sia—GOS enriched on may be subjected to a further up treatment, e.g. a desalting procedure in case anion-exchange chromatography has been used.
A further embodiment relates to a composition sing at least 5 percent by weight of disialylated galactooligosaccharides, obtainable by a method according to the invention. The composition preferably comprises at least 7, 10, 12, 18, 20 22, 25, 27 wt%, more preferably at least 30wt% of disialylated galactooligosaccharides. In one embodiment, the composition comprises less than 80wt%, preferably less than 60wt% of mono—sialylated oligosaccharides.
The weight ratio of di—sialylated to mono-sialylated oligosaccharides is for example between 1:20 and 100:1, more preferably n 1:10 and 9:1. In another preferred embodiment, the composition is essentially devoid of mono sialylated oligosaccharides. The composition ably does not contain (active) enzyme.
Also provided is the use of a composition enriched in disialylated galactooligosaccharides as active ingredient, for example as nutritional, pharmaceutical or nutraceutical additive.
In one ment, the ion provides a nutritional product comprising a composition enriched in disialylated galactooligosaccharides able as described herein. Besides the di-Sia-GOS species the food product may comprise other oligosaccharides, in particular prebiotic oligosaccharides and oligosaccharide mixtures. The addition of probiotics is also envisaged.
The nutritional product can be for human or animal purposes. For human applications, the ce of high concentrations of Neu5Gc should be prevented since NeuEGc does not or hardly occur in humans. Hence, for use in human products it is preferred to use a (0L2~3)-linked N-acetylneuraminic acid (Sia = ) donor as sialic acid donor, for example GMP. In case of using a sialic acid donor with (0L2-8)-linked N-acetylneuraminic acid/N- glycolylneuraminic acid (Sia = Neu5Ac/Neu5Gc), for example PSMG, the Neu5Gc-containing GOS species should be removed e.g. by lectin chromatography. In a specific aspect, the nutritional product is an infant formula. For example, a fraction enriched in di-Sia-GOS wherein Sia is Neu5Ac is added as supplement to a conventional infant formula in order to more closely resemble the composition of human milk, thereby enhancing the beneficial properties of the a. With the di—Sia-GOS concentrate of the present invention, infant as can be enriched with disialylated oligosaccharides in concentrations matching human milk, i.e. the concentration of disialylated oligosaccharides can be increased to 200-500 mg/l matching concentrations of human milk of various lactation stages. Of course, the concentrate is also suitably used to bring the total concentration of oligosaccharide bound sialic acid to 100-1500 mg/l. However, the scope of the present invention is not limited to this range of ment due to the great variations in human milk ition and also due to the fact that other (food) applications may require other oligosaccharide bound (di)sialic acid concentrations. In the present invention sialylated oligosaccharide trations have been measured using high mance liquid chromatography (HPLC) equipped with a UV detection system and a Resource Q column, however and state of the art technique with acceptable accuracy may be employed, The di-Sia-GOS fraction of the invention can be used as such, or it can be further treated by for example reverse osmosis, crystallisation, affinity chromatography or a ation there of to remove water and/or salts, or it can be dried alone or together with one or more carriers. Any carrier can be used, such as oil, fat, whey, demineralised whey, whey n concentrate, Whey n isolate, other whey fractions, Whey or milk permeate or concentrate, skimmed milk, whole milk, semi-skimmed milk, milk fractions, maltodextrins, sucrose, lactose, native and atinised starches, glucose syrups, casein and casein fractions.
The di-Sia-GOS fraction of the invention, including a dried concentrate thereof, can be used in any nutritional compositions, such as products for infant ion, protein bars, sports nutrition, drinks, health supplements, food for medical purposes and clinical nutrition. Infant nutrition can be, but is not restricted to, infant formulas, follow-on formulas, infant cereal products or growing-up milk, i.e. modified milk or milk powder suitable for children of 1-3 years.
Such formula is particularly suitable for administration to pre-term infants since the di-Sia-GOS enriched fraction may help to combat infections by harmful micro-organisms, including protozoa such as Entamoeba histolytica the parasite that causes Amebiasis. In one embodiment, the di-Sia-GOS fraction is used to protect the body from infection by inhibiting the adhesion of a microbial pathogen to human intestinal lial cells. For example, it is used to block binding of E. histolytica to host cells through interaction with the te’s Gal/GalNAc - lectin, a major nt protein that mediates adhesion and cytotoxicity. Hence, also encompassed is a method for providing an infant formula, sing isolating a fraction comprising at least 50 percent by weight of disialylated galactooligosaccharides based on the dry matter and ating said fraction into an infant formula together with a protein source, a fat source, a ydrate source and other tional 3O ingredients such as vitamins and minerals. Therapeutically or prophylactically effective s will depend on various factors eg. age and body weight of the t to be treated, the disease to be prevented or treated, the type of dosage form and the like.
The ion also relates to the medical use of a composition comprising a di-Sia-GOS fraction as disclosed herein. For instance, it is suitably used in a method of treating or preventing necrotizing colitis (NEC) in a subject, preferably a human subject, more preferably a preterm infant. NEC is a serious bacterial infection in the intestine, primarily of sick or premature newborn infants. It can cause the death (necrosis) of intestinal tissue and progress to blood poisoning (septicemia). It has a high mortality rate, especially among very low birth weight babies. Some 20 to 40 t of these infants die. NEC develops in approximately 10% of newborns weighing less than 800 g (under 2 lbs). Necrotizing colitis almost always occurs in the first month of life. Infants who require tube feedings may have an increased risk for the disorder. Hence, the risk for necrotizing enterocolitis may be diminished by using an enteral nutrition comprising a di-Sia-GOS— containing formula as provided herein.
For animal ations, the presence of high concentrations of Neu5Gc does not form a problem. It can even be an advantage, as it has been 2O reported that especially Neu5Gc-containing compounds are effective in the battle against specific infections leading to diarrhoea in piglets and calves.
Hence, for use in animal feed ts it is red to use a donor comprising (a2-8)-linked N-acetylneuraminic acid and N—glycolylneuraminic acids (Sia = Neu5Ac/Neu5Gc), such as PSMG. Provided herein is an animal feed product comprising a composition enriched in di-Sia-GOS wherein Sia is Neu5Ac and/or Neu5Gc, preferably Neu5Ac and Neu5Gc. The feed product is preferably formulated for piglets or calves and may comprise one or more r beneficial ingredients to enhance animal performance, meat quality and animal health.
LEGEND TO THE FIGURES Figure 1. Reversible glycosylation of (a2-8)-linked N-acetylneuraminic acid between Neu5Ac(oc2-8)Gal—OR1 and Neu5Ac(a2—8)Gal-OR2, catalyzed by trypanosomal trans-sialidases.
Figure 2. Separation of Vivinal-GOS on Bio-Gel P2 into 8 fractions of different DP. Demi water was used as eluent at a flow rate of 1 ml/min at 40°C.
Figure 3. HPAEC-PAD profiles of a TcTS tion of GOS DP5 with 4MU- Neu5Ac at t(0)(d0tted line) and after 21 h of incubation (solid line), showing the formation of monosialylated GOS DP5 (15-18 min) and disialylated GOS DP5 (20-28 min).
Figure 4‘ Separation of sialylated GOS DP5 on Resource Q, using a NaCl nt and monitored at 214 nm.
Figure 5. 500 MHZ 1H NMR spectrum of Vivinal-GOS DP5.
Figure 6. 500 MHZ 1H NMR spectrum of mono-Sia-GOS DP5.
Figure 7. 500 MHz 1H NMR spectrum of di-Sia—GOS DP5. 2012/050857 EXPERIMENTAL SECTION Example 1: logous expression and purification of Trypanosoma cruzi sialidase (TcTS) A DNA clone of TcTS containing a N-terminal 6x His-tag was obtained from Professor A.C.C. Frasch (Buenos Aires, Argentina). To express the protein, the growth and induction conditions as described in the literature (Paris et al., Glycobiology 11: 805-311, 2001) were slightly modified. Construct pTrcTSGll/2 was transformed into E. coli TOPlO or 8L2 103133) and cultures were inoculated in terrific broth medium, supplemented with llin (100 ug/ml) and 0.1 mM isopropyl fi-D—l-thiogalactopyranoside (IPTG) as inducer. The medium was inoculated ly from fresh colonies from Luria i agar plates, then incubated for 24 h at 80°C (ODeoo of 0.6) with shaking (200 rpm).
After ting the cells, the pellets were resuspended in lysis buffer rial protein extraction reagent / B-PER), and allowed to lyse at room temperature according to the manufacturer’s protocol. Cell debris was removed by ultracentrifugation for 80 min at 40,000 g, and the supernatant was subjected to gravity flow NiZt-nitrilotriacetic acid (NTA) column affinity chromatography. The recombinant TcTS was eluted from the column using 100 mM imidazole, yielding an enzyme preparation that was not completely pure (SDS-PAGE), But, as it is known that the enzyme becomes instable in purified form (Turnbull et al., Tetrahedron 58: 8207-3216, 2002), r purification steps were omitted. e 2: Activity tests and kinetic studies of TcTS preparations Activity assays with partially purified TcTS (2 mU/ml) were conducted with 1 mM 2’-(4-methylumbelliferyl)-0c-N-acetylneuraminic acid (4MU-Neu5Ac) commercial synthetic sialic acid model donor and lactose as acceptor, with subsequent formation of the product Neu5Ac(oc2-3)1actose (3’—sialyllactose, 3’SL). In the presence of a 10-fold excess of lactose, nearly 80% of the Neu5Ac unit of 4MU—Neu5Ac was transferred to lactose.
In kinetic experiments with partially purified TcTS, 4MU-Neu5Ac or industrially available K~casein-derived acropeptide (GMP) as donors and lactose as acceptor, the formation of B’SL was quantitatively ined after 15 and 30 min of incubation, making use of high-pH anion-exchange tography With pulsed amperometric detection -PAD). For both donors, the optimal temperature for the er reaction, deduced from a series of atures ranging from l5-50°C, With ents of 5°C, turned out to be 25°C, which is similar to the optimal temperature reported in literature (Scudder et al., J. Biol. Chem. 268: 891, 1993).
With respect to the thermal stability of TcTS, estimated in the range 15- 50°C with 5°C intervals (80 min treatments; remaining activity determined at 25°C), the enzyme retained its full activity until 25°C and then steadily decreased. After incubation at 50°C only 6% of the initial activity remained.
When using 4MU—Neu5Ac as donor, the on of protein (BSA) has a stabilizing effect on the TcTS activity; when using GMP, the on of BSA is not necessary.
The optimal pH for the transfer reaction, as deduced from incubations of TcTS, 4MU-Neu5Ac, and lactose (25°C) in 50 mM sodium citrate buffers (pH 4.5, 5.0, 5.5), Pipes buffers (pH 6.0, 6.5, 7.0, 7.5), and Tris-HG] buffers (pH 8.0, 8.5, 9.0) in the presence of BSA, turned out to be pH 5.0. A determination independent on the buffer type, as deduced from incubations using mixed buffers, containing 25 mM sodium citrate, Pipes, and Tris—HCl, ranging from pH 4.5 to 9.0, led to an optimal pH of 5.5.
The affinity of TcTS for the sialic acid donor substrates 4MU-Neu5Ac and GMP was tested by performing incubations (4.5 mU/ml) With an appropriate range of these substrates and a fixed concentration of lactose as 80 acceptor. For 4MU~Neu5Ac the incubation mixtures contained 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 and 10.0 mM 4MU-Neu5Ac and 1 mM lactose; the Km was found to be 1.65 mM and the Vmax 1.5 U/mg. For GMP the incubation mixtures contained 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 1.0, 2.5, and 5.0 mM GMP and mM lactose; the Km was found to be 0.18 mM and the Vmax 3.0 U/mg.
Example 3: Analysis of the sialic acid specifications of the donor GMP Mild acid hydrolysis of GMP (MW = 10 kDa), using 0.1 M HCl (1 h; 80 °C), ed by quantitative analysis of the released sialic acids by HPAEC-PAD, yielded a sialic acid content of 4.4%, built up from 99% Neu5Ac and 1% Neu5Gc.
From the literature it is known that the O-glycans of GMP contain an array of oligosaccharides with both (a2-8)- and/or (0L2-6)-linked sialic acid (van Halbeek et al., Biochim. Biophys. Acta 623: 295-300, 1980). Of these two linkage ons, only the (0L2-3)-linked sialic acid acts as an ive donor for TcTS (Schauer and Kamerling, ChemBioChem 12: 2246-2264, 2011). The amount of (a2-8)-linked , as determined by incubation with the (oc2—3)« specific sialidase from Salmonella typhimurium, followed by HPAEC-PAD analysis, turned out to be 72%.
TcTS (8 mU/ml) incubations with GMP (sialic acid content: 4.4%; 1 mM total sialic acid, (a2-8)- and (d2-6)—linked) and 10 mM e resulted in the transfer of around 60% of the total Neu5Ac into S’SL -PAD) (t = 0 h: 0% 3’SL, 0% free Neu5Ac; t = 1 h: 42% S'SL, 0% free Neu5Ac; t = 2 h: 53% 8’SL, 0% free Neu5Ac; t = 4 h: 58% B’SL, 0% free Neu5Ac; t = 24 h: 51% B’SL, % free Neu5Ac). Taking into t the linkage specificity, the actual conversion ency of (a2—8)-linked Neu5Ac into 8’SL is 81% at t = 4 h.
Example 4: Evaluation of sialylated products obtained with acceptor Vivinal-GOS and 4MU-Neu5Ac as donor Commercially available Vivinal-GOS, being a mixture of 57% galacto- 80 oligosaccharides (GOS) with different degrees of polymerization (DP), is 57% G08, 21% lactose, and 22% galactose + glucose, was separated into the DP fractions 2-8 on Bio-Gel P2 (Figure 2). The fractions were d by MALDI- TOF-MS analysis, trating separate GOS DP pools, with low ination of the adjacent DP’s.
Recombinant TcTS incubations (2 mU/ml) were conducted with GOS DP2 to GOS DP6 as acceptor substrate (3 umol) and u5Ac as donor substrate (6 umol). Besides reducing linear oligosaccharides: parts of DP3 to DP6 are reducing branched and non-reducing linear oligosaccharides, containing two terminally bound B-linked ose (Gal) residues (Table 1). As demonstrated by HPAEC-PAD, in all cases product formation could be observed, accompanied with a decrease of the G08 substrates The sialylated GOS products of the higher DPs consist, besides starting material, of two charged pools, a major alylated pool and a minor disialylated pool. As a typical example, in Figure 3, the results for GOS DP5 are depicted. The chromatogram shows a major charged pool with retention times between 15 and 20 min, and a minor charged pool with retention times between 20 and 22 min. Further structural analysis of these pools demonstrated the occurrence of monosialylated GOS DPS (mono—Sia-GOS DP5) and disialylated GOS DP5 (di— Sia-GOS DP5) (Sia = Neu5Ac).
Example 5: TcTS-catalyzed incubation of Vivinal—GOS DP5 and 4MU- Neu 5Ac, analyzed by 1H NMR oscopy For 1H NMR analysis, TcTS (2 mU/ml) was incubated with Vivinal-GOS DP5 (1 umol) and 4MU—Neu5Ac (2 umol) for 16 h at 25°C and pH 5. The monocharged and dicharged fractions were collected via anion-exchange tography (Figure 4), aliquots of which were analyzed by PAD for ation.
Figures 5, 6, and 7 present the 1H NMR spectra of GOS DPS, mono-Sia-GOS 80 DP5, and di—Sia-GOS DP5 (Sia = Neu5Ac), respectively. In earlier studies (Fransen et al., ydr. Res. 814: 101-114, 1998) it has been shown that the major components of GOS DP5 comprise the following structures (the 4- substituted Gal residues dominate over the 6-substituted ones): Gal(B1-4)Ga1(B1-4)Gal(fi1—4)Gal(B1-4)Glc Ga1(B1-6)Gal([31-6)Gal(B1-6)Gal(B 1-4)Glc Gal([31-4)Gal([31-4)Ga1([31~4)Glc(ocl- 1 fi)Ga1 Gal(B 1~4)Gal(B 1-4)Glc(al- l B)Gal(4— l [3)Gal Gal(fil—4) Glc(oc1~1B)Gal(4-113)Gal(4—1B)Gal However, as shown in example 10, also reducing branched structures do occur (see examples 8 and 9 for the occurrence of reduced ed structures in GOS DPS and DP4).
The spectra of the sialylated GOS DP5 probes reflect the presence of (C(23)- linked NeuBAc only, Based on the surface ratios of the Gal H-3 and H-4 and the Neu5Ac H-3e and H—Bax signals, the assignment for the mono—Sia-GOS DP5 and di-Sia-GOS DP5 pools could be made. Based on the UV responses on Resource Q, the molar ratio of mono-Sia—GOS DP5 and di-Sia-GOS DP5 is about 8.5: 1.
Example 6: Analysis of the sialic acid specifications of the donor PSMG Small ines were retrieved from casing material, d by means of pressing and washing in water of 44-48°C.
The mucus is subsequently d out the small intestines in 2 or 8 steps, ing on equipment installed, at increasing pressures. The final step of the cleaning process is to remove the mucus membrane.
The mucus resulting from these pressing steps is collected and an aqueous 80 solution of sodium meta bisulphite is added.
Mild acid hydrolysis of industrially ble pig small intestinal mucin glycoprotein (PSMG) material (MW = 1.7 X 103 kDa), using 0.1 M HCl (1 h; 80 °C), followed by quantitative analysis of the released sialic acids by HPAEC- PAD; yielded a sialic acid content of 1.4%, built up from 72% Neu5Ac and 28% Neu5Gc.
From the literature it is known that the O-glycans of PSMG contain an array of oligosaccharides with both (a2-3)— and/or (a2-6)-linked sialic acid (Hansson et al., Carbohydr. Res. 221: 9, 1991). Of these two linkage ons, only the (0:2-3)-1inked sialic acid acts as an effective donor for TcTS. TcTS (2-8 mU/ml) incubations with PSMG (0.5 mM total Neu5Ac, (0:243)- and (062-6)- linked) and 10 mM lactose resulted after 24 h in the transfer of up to 10% of the total Neu5Ac into 3’Neu5Ac-lactose (HPAEC-PAD). Similar levels of Neu5Gc were transferred to lactose, ng 3’Neu5Gc-lactose. When using partially purified PSMG, obtained via Sepharose CL-4B chromatography and concentrated on a spin—filter with a cut-off value of 50 kDa, or ethanol- precipitated PSMG, the er of total Neu5Ac to lactose could be sed to about 20% and of total Neu5Gc to about 15%. Taking into account the linkage specificity, the actual conversion efficiency of (a2-3)-linked Neu5Ac into 3’SL (NeuSAc) is 45%.
Example 7: Production of Sia-GOS (Sia = Neu5Ac) at the gram scale using GMP as donor, Vivinal—GOS as or, and TcTS as a biocatalyst E. coli TOP 10 cells harboring pTrcTS611/2 were inoculated in terrific broth medium (50 ml), containing 0.1 mM IPTG and 100 ug/ml ampicillin, and cultured for 20 h at 30°C on a rotary shaker The cells were harvested by centrifugation and ally lysed using bacterial protein extraction reagent 80 (B-PER, Thermo scientific). The TcTS protein was purified using His-tagged immobilized metal affinity chromatography (eluent, 100 mM imidazole). The purified protein fractions obtained from 10 ent 50-ml cultures were pooled and, to remove imidazole, subsequently washed and concentrated using an Amicon 50 kDa spinfilter. The washed protein was eventually buffered in 50 mM Tris-H01 (pH 8) containing 15% glycerol.
To ine the optimal sialic acid transfer in time, in test experiments TcTS was added (final cone of 2 mU/ml) to various concentrations of GMP-bound Neu5Ac (donor; a commercial GMP product (sialic acid content: 4.0%; containing 10% (w/w) NaCl) and lactose (acceptor). An excess of donor substrate led to high conversion of the acceptor. lncubations for more than 48 h under the ions tested led to hydrolysis (a side reaction of the TcTS enzyme) of the product. Typically, a mM ratio of Neu5Ac : lactose = 5:1 yielded 82% B’SL (Neu5Ac) after 24 h, 100% after 48 h, and 76% after 70 h; and a mM ratio of Neu5Ac : lactose = 5:2 gave 56% 8’SL c) after 24 h, 75% after 48 h, and 61% after 70 h.
An BOO-ml reaction mixture, ning 5 mM (d2—3)-linked Neu5Ac (~ 46 g/l (EMF), 2 mM GOS (Vivinal—GOS) and 2 mU/ml TcTS in 50 mM Na-citrate (pH ), was incubated for 48 at 25°C. Before use, crude GMP, containing relatively high amounts of NaCl, was desalted via a hersulfone membrane filter with a 8 kDa f value. After a heat inactivation step (20 min at 60°C), partially desialylated and starting GMP were removed via a filtration step (3 kDa ne filter). Qualitative analysis of the total permeate before and after mild acid hydrolysis se of Neu5Ac), using the AOAC method (employing B-galactosidase from Aspergillus myzae) to quantitatively measure GOS contents, showed that 48% non-sialylated GOS was present and 52% sialylated GOS. The total permeate was lyophilized and desalted by gel- filtration on Bio-Gel P-EZ using demiwater as eluent. As checked by HPAEC- 80 PAD, the first eluting ons contained the mixture of mono-Sia—GOS and di- S (Sia : Neu5Ac), essentially free of NaCl, followed by free Neu5Ac and decreasing DPS of non-sialylated GOS‘ MALDl-TOF-MS analysis showed ated GOS products obtained from GOS up to GOS DP7.
Example 8: TcTS-catalyzed incubation of Vivinal GOS DPS and GMP A TcTS (2.5 mU/ml) catalyzed incubation of GOS DP3 (3 mM) with GMP (sialic acid content: 4.0%; 6 mM (a2-3)~linked NeuSAc) was carried out for 23 h at °C and pH 5. After work-up, the oligosaccharide product was subjected to anion-exchange chromatography on Resource Q, with detection at 214 nm, yielding a mono-Sia-GOS DPS and a di—Sia-GOS DP8 fraction in a molar ratio of 88: 12. Verifications were carried out by MALDI-TOF-MS and 1H NMR spectroscopy. Further analysis of the di—Sia-GOS DPS on with a focus on “branched ng" and "non-branched non—reducing” forms, making use of a NaBH4 reduction step followed by HPAEC-PAD and MALDI-TOF-MS, showed that the major part of di—Sia-GOS DPS consists of branched structures.
Example 9: TcTS—catalyzed incubation of Vivinal GOS DP4 and GMP A TcTS (2.5 mU/ml) catalyzed incubation of GOS DP4 (3 leI) with GMP (sialic acid content: 4.0%; 6 mM (a2-8)-linked Neu5Ac) was carried out for 23 h at 2O 25°C and pH 5. After work-up, the oligosaccharide product was subjected to anion-exchange chromatography on Resource Q, with detection at 214 nm, yielding a mono-Sia-GOS DP4 and a di—Sia-GOS DP4 fraction in a molar ratio of 91:9. Verifications were carried out by MALDI-TOF-MS and 1H NMR oscopy. Further analysis of the di—Sia-GOS DP4 fraction with a focus on “branched reducing” and ranched non-reducing” forms, making use of a NaBH4 reduction step followed by PAD and MALDI-TOF-MS, showed that the major part of -GOS DP4 ts of branched structures.
Example 10: TcTS—catalyzed tion of Vivinal GOS DPS and GMP A TcTS (2.5 mU/ml) catalyzed incubation of GOS DP5 (8 mM) with GMP (sialic acid content: 4.0%; 6 mM (a2-3)-linked Neu5Ac) was carried out for 28 h at °C and pH 5. After work-up, the oligosaccharide product was subjected to anion-exchange chromatography on Resource Q, with detection at 214 nm, yielding a mono-Sia-GOS DP5 and a —GOS DP5 fraction in a molar ratio of 88: 12. Verifications were carried out by MALDl-TOF-MS and 1H NMR oscopy. Further analysis of the di-Sia-GOS DP5 fraction with a focus on “branched reducing” and “non-branched non-reducing” forms, making use of a NaBH4 reduction step followed by HPAEC-PAD and MALDI-TOF—MS, showed that the major part of di—Sia-GOS DP5 consists of branched structures. e 11: TcTS-catalyzed incubation of Vivinal GOS DP6-8 and Individual incubations of Vivinal GOS DP6, DP7 and DP8 with TcTS and GMP, according to the ol described in examples 8-10 d a ia- GOS DPS and a di-Sia-GOS DP6 fraction in a molar ratio of 87:18, a mono—Sia- GOS DP7 and a di-Sia-GOS DP7 fraction in a molar ratio of 84:16, and a mono-Sia-GOS DPS and a di-Sia-GOS DP8 fraction in a molar ratio of 81: 19.
Example 12: TcTS~catalyzed incubation of Vivinal GOS DP3 and DP4 and PSMG A TcTS (3 mU/ml) catalyzed tion of GOS DPS (8 mM) or GOS DP4 (8 mM) with ethanol-precipitated PSMG (2 mM) was carried out for 28 h at 25°C and pH 5. After work-up, the oligosaccharide products were subjected to MALDI-TOF-MS analysis showing in each case Neu5Ac and Neu5Gc sialylation.
Example 13: Nutritional Formulas for infants Table 2 shows the composition (per 100 ml) of three exemplary nutritional formulas according to the ion, eg. infant formulas for the age group between 0-6 months, for supporting or enhancing the infant’s immune .
Table 2: composition of the formulas (per 100 ml) Ba sic Formula a B: Formula C: a D: A: (11' sialo di Sialo disialo disialo ingredien 1: ingredient + ingredient + ingredient + sialyllactose sialyllacmse + sialyllactose + GOS GOS + fucos Hactose Fat (g) 3.5 3.5 3.5 35 8.5 Disialo 0.25 0.13 0.1 0.1 infldient (Q Sialyflactose 0.18 0.1 0. 1 Vivinal GOS 0.5 0.4 Fucosyllactose 0. 1 Example 14: Animal feed formula The animal feed is composed of a basic feed, supplemented with amongst others a disialo ingredient.
Basic feed Barley 27.45 % Wane i 20.00 % Wheat 24.84 % Soya meal, high protein 4.82 % Sunflower seed, 2.00 % Whexpowder 7.44 "/0 Molasses 1.00 % Feed fats 1.04 % Ea oil 0.50 % Premix 0.50 0/0 m carbonate 1.08 % Mono calcium hos hate Feed for Young piglets Hi Potato flotein Whey Meier 4. 37 i % Soycomill P 0.50 | % I Disialo ingredient [8.00 I % Feed for young piglets Low level of sialic acid in inredient Basic feed Feed for young piglets Lower level of sialic acid in in redient Potatoflotein Whey powder 80 comill P o ingedient Feed for young piglets Whey powder Solcomill P 0.50 f % Disialo ingredient 0.10 I % ‘ Feed for young piglets In combination with blood lasma | Basic feed 91.52 % ‘l I Potato protein 0.50 I % Whe owder 5.88 Soycomill P Disialo ingredient 0.10 % Blocfiplasma 1.50 % 2012/050857 Example 15: Effect of Di—Sia—GOS enriched fraction on in viva pathogen adhesion.
A neonatal rat model of necrotizing enterocolitis (NEG) can be used to objectively evaluate the protective effects of a di-Sia-GOS-enriched fraction by the clinical behaviour of the animals and the macroscopic appearance of the gut. als and Methods: Neonatal rats are delivered at term and assigned either to a l group (A) consisting of breastfeeding and no stress factors, to a NEC group (B) in which NEC is induced by gavage feeding + hypoxia + oral lipopolysaccharide (4 mg/kg/day once daily for the first 2 days of life), or to a d NEC group (C) in which NEC is induced by gavage feeding + hypoxia + oral lipopolysaccharide (4 mg/kg/day once daily for the first 2 days of life) plus di-Sia—GOS. Clinical status is assessed on day 4 using a clinical sickness score (general appearance, response to touch, natural activity, body Colour; 0-3 for each variable). Neonatal rats are sacrificed at 4 ent time points: day 1, day 2, day 3, and day 4. At sacrifice, a macroscopic assessment of the gut is performed using a scoring system based on: colour (0-2), consistency (0-2) and degree of dilatation (0-2). The ed gut is stained with haematoxylin/eosin, and evaluated microscopically by 2 independent blinded scorers, ing a consultant histopathologist. The histology results can be used to te the macroscopic gut assessment. Results are compared by ANOVA and linear regression analysis.
REFERENCES R. , M.E. Giorgi, and RM. de Lederkremer, The trans—sialidase from Trypartosoma cruzi efficiently transfers (a2a8)-linked N-glycolylneuraminic acid to terminal B-galactosyl units. Carbohydr. Res. 842 (2007) 2465-2469.
S. Asakuma, M. i, K. Kimura, Y. be, T. Nakamura, M. i, I. Arai, Y. Sanai, and T. Urashima, Sialyl oligosaccharides of human colostrum: Changes in concentration during the first three days of lactation, Biosc. Biotechnol. m. 71 (2007) 1447-1451.
L. Coulier, J. Timmermans, R. Bas, R. van den Dool, I. Haaksman, B.
Klarenbeek, T. Slaghek, and W. van Dongen, In-depth characterization of prebiotic galacto-oligosaccharides by a combination of analytical techniques, J.
Agr. Food Chem. 57 (2009) 8488—8495.
C.T.M. n, Structural analysis of soy bean polysaccharides and transgalactosylation products from lactose, PhD thesis Utrecht University (1999).
C.T.M. Fransen, K.M.J. Van Laere, A.A.C. van Wijk, L.P. Bri'ill, M. Dignum, J.E. Thomas—Oates, J. Haverkamp, HA. Schols, A.G.J. Voragen, J.P.
Kamerling, and J.F.G. Vliegenthart, oc-D-Glcp—(le1)-B-D-Galp-containing oligosaccharides, novel ts from lactose by the action of B-galactosidase, Carbohydr. Res. 814 (1998) 101-114.
AK. Goulas, PG. Kapasakalidis, H.R. Sinclair, RA. Rastall, and AS, Grandison, Purification of oligosaccharides by nanofiltration, J. Membr. Sc. 209 (2002) 821-385. 2012/050857 GO. Hansson, JF rs, H. Karlsson, and I. Carlstedt, Analysis of sialic acid-containing mucin oligosaccharides from porcine small intestine by high- temperature gas chromatography-mass spectrometry of their dimethylamides, Carbohydr. Res. 221 (1991) 179-189.
T. Idota, H. Kawakami, Y. Murakami, and M. Sugawara, Inhibition of cholera toxin by human milk fractions and sialyllactose, Biosci. Biotech. Biochem. 59 (1995) 417-419.
B. Neubacher, D. Schmidt, P. Ziegelmuller, and J. Thiem, Preparation of sialylated oligosaccharides ing recombinant trans-sialidase from Trypanosoma cruzi. Org. Biomol. Chem. 8 (2005) 1551-1556.
G. Paris, ML. Cremona, M.F. Amaya, A. Buschiazzo, S. Giambiagi, A.C.C.
Frasch, and PM. Alzari, g molecular function of trypanosomal ases: single point mutations can change substrate specificity and increase hydrolytic activity, Glycobiology 11 (2001) 805-311.
R. Schauer and J.P. Kamerling, The chemistry and biology of Trypanosomal trans-sialidases: virulence factors in Chagas disease and ng sickness, ChemBioChem 12 (2011) 2246-2264.
P. r, J.P. Doom, M. Chuenkova, LD. Manger, and M.E.A. Pereira, Enzymatic characterization of B-D-galactoside oc-2,3-trans-sialidase from osoma. cruzi, J. Biol. Chem. 268 (1993) 9886-9891.
PM. Simon, PL. Goods, A. Mobasseri. and D. Zopf, Inhibition of Helicobacter pylori binding to intestinal epithelial cells by sialic acid-containing oligosaccharides, Infection and Immunity 65 (1997) 750-757.
S. Thurl, M. Munzert, J. Henker, G. Boehm, B. Muller-Werner, J. Jelinek, and B. Stahl, Variation of human milk oligosaccharides in relation to milk groups and lactational s, British J. Nutr. 104 (2010) 1261—127 1.
WE. Turnbull, J.A. on, KP. Ravindranathan Kartha, S. Schenkman, and RA. Field, Observations on chemical and enzymatic approaches to O(~2,3- sialylated octyi B-lactoside, Tetrahedron 58 (2002) 32073216.
H. van Halbeek, L. d, JFG Vliegenthart, A.-M. Fiat, and P. Jollés, A BBQ-MHZ 1H-NMR study of three oligosaccharides isolated from cow in, Biochim. Biophys. Acta 623 (1980) 295-800.
S. Yanahira, T. Kobayashi, T. Suguri, M. Nakakoshi, S. Miura, H. Ishikawa, and I. Nakajima, Formation of oligosaccharides from lactose by Bacillus circulans B-galactosidase, Biosc. Biotech. m. 59 (1995) 1021-1026.

Claims (38)

What is claimed is:
1. A method for providing a composition sing sialic acid containing oligosaccharides, comprising the steps of : a) providing a source of non-digestible galactooligosaccharides (GOS) containing at least two terminally bonded B-linked galactose residues; b) providing a sialic acid donor having (a2-3)-sialylated O-glycans; c) ting said GOS with said sialic acid donor in the presence of an enzyme having trans-sialidase activity in an enzyme reaction mixture; and 10 d) obtaining from said enzyme reaction e a fraction comprising at least 5 percent by weight of disialylated galactooligosaccharides (di-Sia-GOS) based on the dry matter.
2. Method according to claim 1, wherein said source of 15 galactooligosaccharide is obtained by enzymatic treatment of lactose or a mixture of lactose and trehalose with B-galactosidase (EC 3.2.1.28).
3. Method according to claim 1 or 2, n said di-Sia-GOS have a degree of polymerization (DP) within the range of DP4 to DPlO.
4. Method according to claim 3, n the degree of polymerization (DP) is within the range of DP5 to DP8.
5. Method according to any one of the preceding claims, wherein said 25 sialic acid donor is a naturally occurring compound.
6. Method according to claim 5, wherein said sialic acid donor is a lly occurring compound selected from the group consisting of sialic acids bonded to accharides, polysaccharides, polysialic acids, glycoproteins and 80 glycolipids.
7. Method ing to claim 5 or 6, wherein said sialic acid donor is selected from the group ting of glycosylated Whey proteins and casein, and fragments of the same.
8. Method according to claim 7, wherein said sialic acid donor is glycomacropeptide from in (GMP).
9. Method according to claim 5 or 6, whereinsaid sialic acid donor is 10 selected from the group consisting of glycosylated mucus proteins, and fragments of the same.
10. Method according to claim 9, wherein said sialic acid donor is pig small intestinal glycoprotein (PSMG).
11. Method according to claim 9, wherein said sialic acid donor is obtained from mucin, optionally followed by tration of Sia-containing mucin polymers. 20
12. Method according to any one of the preceding claims, n said enzyme having trans-sialidase activity is encoded by a gene product from microorganisms of the Trypanosoma genus.
13. Method according to claim 12, wherein said microorganism is 25 Trypanosoma cruzi or Trypanosoma congolense.
14. Method according to any one of the preceding claims, wherein said enzyme is recombinantly produced.
15. Method according to any one of the preceding claims, wherein the pH of the enzyme reaction mixture ranges between 4 and 6.
16. Method according to claim 10, wherein the pH is of the enzyme reaction mixture ranges between 4.8 and 5.8.
17. Method according to any one of the ing claims, wherein step d) comprises a separation technique based on a difference in charge between components to be ted.
18. Method according to claim 17, n the separation technique is anion exchange tography.
19. Method according to any one of claims 1-17, wherein step d) 15 comprises a separation technique based on a difference in size between components to be separated.
20. Method according to claim 19, wherein the separation technique is size exclusion chromatography.
21. A ition comprising at least 5 percent by weight of disialylated galactooligosaccharides, obtainable by a method according to any one of claims 1-20. 25
22. Composition according to claim 21, comprising at least 7wt%,.
23. Composition according to claim 22, comprising at least 10wt% of disialylated galactooligosaccharides.
24. Composition according to any one of claims 21-23, comprising one or more di-Sia-GOS species selected from the group consisting of Neu5Ac(0L2-3)Gal([3 l-4)Glc(ocl— 1B)Gal(3-20L)Neu5Ac (0L2-3)Gal(B 1-2) [Neu5Ac(0L2-3)Gal(fi 1-6)] Glc (oc2-3)Gal([3 1-3) [Neu5Ac(0L2—8)Gal({3 1-6)] Glc, Neu5Ac(a2-3)Gal([3 1-4) {Neu5Ac(0L2-8)Gal(B 1-6)]Glc Neu5Ac(d2—3)Gal(B 1-2) [Neu5Ac(oc2-3)Gal(B 1-4)] Glc Neu5Ac(0L2—3)Gal(B l-4)Gal([3 1-4)GlC(OL1- lB)Gal(3—20L)Neu5Ac Neu5Ac(0L2-3)Gal([3 c(ocl- 1B)Gal(4- 1B)Gal(3-20L)Neu5Ac 10 (0L2—3)Gal([3 1-4)Gal([3 l—4)Gal({31—4)Glc(or1- 1B)Gal(3-20c)Neu5Ac Neu5Ac(oc2-3)Gal(B 1-4)Gal([3 1-4) Glc(ocl— 1B)Gal(4- lfi)Gal(3-20L)Neu5Ac (oc2-3)Gal([3 1-4)Glc(a1- 1f3)Gal(4- 1B)Gal(4- 1B)Gal(3-2oc)Neu5Ac Neu5Ac(0L2-3)Gal(B l-4)Gal([3 1—4)Gal(B1-4)Gal([3 1-4)Glc(a1- 1B)Gal(3- 20L)Neu5Ac 15 Neu5Ac(0L2-3)Gal([3 1—4)Gal([3 1-4)Gal(B1-4)Glc(ocl- IB)Ga1(4- 1B)Gal(3- 2a)Neu5Ac Neu5Ac(oc2-3)Gal([3 l-4)Gal(fi 1—4)Glc(oc1— (4— 1(3)Ga1(4- 1L3)Gal(3- 20L)Neu5Ac Neu5Ac(0L2-3)Gal([3 1-4)Glc(a1- 1fi)Gal(4- IB)Gal(4- 1B)Gal(4— 1l3)Gal(3- 20 2a)Neu5Ac
25. Composition according to any one of claims 21-24 for use as nutritional, pharmaceutical or eutical additive. 25
26. A nutritional, pharmaceutical or nutraceutical ve comprising the composition according to any one of claims 21-24.
27. Nutritional product comprising a composition according to any one of claims 21-24.
28. Nutritional product according to claim 27, wherein said product is an infant formula.
29. Animal feed product comprising a composition according to any one of claims 21-24.
30. Animal feed product according to claim 29, wherein the feed product is formulated for piglets or calves. 10
31. A method for providing an infant formula, comprising isolating a fraction comprising at least 5 percent by weight of disialylated galactooligosaccharides (di-Sia-GOS) based on the dry matter according to a method of any one of claims 1-20, and formulating said fraction into an infant formula together with a protein source, a fat source and a carbohydrate source.
32. Use of a composition according to any one of claims 21-24 in the manufacture of a medicament for treating or preventing an infection by a pathogen in a subject in need thereof. 20
33. Use ing to claim 32, wherein the subject is a human subject.
34. Use ing to claim 33, wherein the human subject is a m 25
35. Use according to any one of claims 32-34, wherein the pathogen is a bacterium or protozoan.
36. Use according to claim 35, wherein the pathogen is eba histolytica.
37. Use according to any one of claims 32-84, for treating or preventing amebiasis or necrotizing colitis (NEC).
38. Method according to claim 1, substantially as herein described with reference to any one of the Examples and/0r
NZ625977A 2011-12-07 2012-12-06 Methods for providing sialylated oligosaccharides NZ625977B2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
NL2007931A NL2007931C2 (en) 2011-12-07 2011-12-07 Methods for providing sialylated oligosaccharides and products obtainable thereby.
NL2007931 2011-12-07
PCT/NL2012/050857 WO2013085384A1 (en) 2011-12-07 2012-12-06 Methods for providing sialylated oligosaccharides

Publications (2)

Publication Number Publication Date
NZ625977A NZ625977A (en) 2016-01-29
NZ625977B2 true NZ625977B2 (en) 2016-05-03

Family

ID=

Similar Documents

Publication Publication Date Title
US20170080002A1 (en) Methods for providing sialylated oligosaccharides
Autran et al. Sialylated galacto-oligosaccharides and 2′-fucosyllactose reduce necrotising enterocolitis in neonatal rats
Kobata Structures and application of oligosaccharides in human milk
US8202842B2 (en) Inhibition of cholera toxins by galatooligosaccharides (GOS)
US20140248415A1 (en) Nutritional products comprising human milk oligosaccharides and methods for manufacture thereof
EP2183984B1 (en) Sialic acid producing bacteria
AU2006201A (en) Oligosaccharide mixture
US20150320780A1 (en) Mixture of fucosylate lactoses
Hennet et al. Decoding breast milk oligosaccharides
Kelly et al. Nutritional influences on interactions between bacteria and the small intestinal mucosa
WO2013190530A1 (en) Modified galactooligosaccharides
JP2023073244A (en) Intestinal environment improvement composition and method for manufacturing the same
US20150182549A1 (en) Method for enzymatic glycosylation of oligosaccharides from mammalian animal milk
US20200253232A1 (en) Composition and uses thereof
NZ625977B2 (en) Methods for providing sialylated oligosaccharides
US20220273733A1 (en) Nutritive compositions with bioactive proteins
WO2018048305A1 (en) Prebiotic branched galacto-oligosaccharides (gos)
Quinn et al. Dairy-Derived Oligosaccharides: Their Influence on Host-Microbe Interactions in the Gastrointestinal Tract of Infants
Verma et al. International Journal of Carbohydrate Research
Divya et al. Impact Of Human Milk Oligosaccharide (HMO) In Neonates