# US 10,499,668 B2 · Nutritional products comprising human milk oligosaccharides and methods for manufacture thereof

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- Number: US 10,499,668 B2
- Document ID: US10499668B2
- Status: Granted
- Kind: B2
- Application number: 14/117,424
- Filed: 2012-05-10
- Published: 2014-09-04
- Granted: 2019-12-10
- Earliest claimed: 2011-05-13
- Assignees: Societe des Produits Nestle S.A.
- Applicants: Dominique Brassart; Gyula Dekany; Peter Erdmann; Andrea Schwarz; Norbert Sprenger
- Inventors: Dominique Brassart; Gyula Dekany; Peter Erdmann; Andrea Schwarz; Norbert Sprenger
- Web page: https://patentgrep.com/patent/US10499668B2

## CPC

- A23L 5/00: Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L 29/30: containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin (products from apiculture A23L21/20; artificial sweetening agents A23L27/30)
- A23L 33/00: Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L 33/40: {Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula}
- C07H 5/06: Aminosugars
- C07H 7/027: Keto-aldonic acids
- C07H 15/18: Acyclic radicals, substituted by carbocyclic rings
- A23L 7/00: Cereal-derived products; Malt products; Preparation or treatment thereof (preparation of malt for brewing C12C)
- A23L 33/125: containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates (indigestible substances A23L33/21)
- A23V 2002/00: Food compositions, function of food ingredients or processes for food or foodstuffs
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- A23V: INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION

## Abstract

A method for the manufacture an infant formula or an infant nutritional product comprising mixture of human milk oligosaccharides is disclosed. The method involves the catalytic hydrogenolysis of compounds of the general formula 1 and 2 with particular oligosaccharide profiles.

## Claims

### Claim 1

A method for manufacturing an infant nutritional product or infant formula comprising a mixture of human milk oligosaccharides (HMOs), the method comprising: subjecting a mixture of at least two compounds selected from the group consisting of compounds of general formula 1 and compounds of general formula 2 to catalytic hydrogenolysis to remove,
wherein R is a group removable by the catalytic hydrogenolysis,
R1 is independently a fucosyl or an H,
R2 is an N-acetyl-lactosaminyl group or a lacto-N-biosyl group,
R3 is an H or an N-acetyl-lactosaminyl group
R4 is independently a sialyl or an H,
and salts thereof,
at least one of R1 or R4 is not H in general formula 2, and
a quantity of the mixture of the at least two compounds selected from the group consisting of compounds of general formula 1 and compounds of general formula 2 are selected such that the mixture of human milk oligosaccharides (HMOs) has an oligosaccharide profile that is substantially similar to a milk selected from the group consisting of colostrum breast milk, transitional breast milk and mature breast milk.

### Claim 2 (depends on 1)

The method of claim 1, wherein the catalytic hydrogenolysis is carried out in at least one protic solvent in the presence of a hydrogenolysis catalyst.

### Claim 3 (depends on 1)

The method of claim 1, wherein the at least two compounds are selected from the group consisting of R-glycosides of 2′-fucosyllactose, 3-fucosyllactose, 2′,3-difucosyllactose, 3′-sialyllactose, 6′-sialyllactose, 3′-sialyl-3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LST-a, LST-b, LST-c, FLST-a, FLST-b, FLST-c, LNDFH-I, LNDFH-II, LNDFH-III, DS-LNT, FDS-LNT I and FDS-LNT II.

### Claim 4 (depends on 1)

The method of claim 1, wherein the infant nutritional product or infant formula is in a form selected from the group consisting of a liquid, a powder or a solid.

### Claim 5 (depends on 1)

The method of claim 1, comprising adding a component selected from the group consisting of pharmaceutically or nutritionally acceptable carriers, lactose, proteins, and fat to the mixture of human milk oligosaccharides.

### Claim 6 (depends on 1)

The method of claim 1, further comprising adding prebiotics to the mixture of human milk oligosaccharides.

### Claim 7 (depends on 1)

The method of claim 1, wherein the N-acetyl lactosaminyl group comprises a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups, and at least one N-acetyl-lactosaminyl or lacto-N-biosyl group can be substituted with a sialyl and/or fucosyl residue; and
R3 is N-acetyl-lactosaminyl group substituted with a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups, and at least one N-acetyl-lactosaminyl or lacto-N-biosyl group is substituted with a sialyl and/or fucosyl residue.

### Claim 8

A method for manufacturing an infant nutritional product or infant formula comprising a mixture of human milk oligosaccharides (HMOs), the method comprising: subjecting a mixture of at least two compounds selected from the group consisting of compounds of general formula 1 and general formula 2 to catalytic hydrogenolysis to remove R,
wherein R is a group removable by the catalytic hydrogenolysis,
R1 is independently a fucosyl or an H,
R2 is an N-acetyl-lactosaminyl group or a lacto-N-biosyl group,
R3 is an H or an N-acetyl-lactosaminyl group,
R4 is independently a sialyl or an H,
and salts thereof,
at least one of R1 or R4 is not H in general formula 2,
wherein the compounds of general formula 1 are selected from the group consisting of compounds of general formula 1a and compounds of general formula 1b,
R2a is an N-acetyl-lactosaminyl group substituted with a glycosyl residue comprising one N-acetyl-lactosaminyl and/or one lacto-N-biosyl group; at least one N-acetyl-lactosaminyl or lacto-N-biosyl group is substituted with a sialyl and/or fucosyl residue,
R3a is an H or an N-acetyl-lactosaminyl group substituted with a lacto-N-biosyl group; at least one N-acetyl-lactosaminyl or lacto-N-biosyl group is substituted with a sialyl and/or fucosyl residue,
R2b is a lacto-N-biosyl group substituted with sialyl and/or fucosyl residue, and
R3b is an H or an N-acetyl-lactosaminyl group substituted with one or two N-acetyl-lactosaminyl and/or one lacto-N-biosyl group; at least one N-acetyl-lactosaminyl or lacto-N-biosyl group is substituted with sialyl and/or fucosyl residue, and
a quantity of the mixture of the at least two compounds selected from the group consisting of compounds of general formula 1 and compounds of general formula 2 are selected such that the mixture of human milk oligosaccharides (HMOs) has an oligosaccharide profile that is substantially similar to a milk selected from the group consisting of colostrum breast milk, transitional breast milk and mature breast milk.

### Claim 9 (depends on 8)

The method of claim 8, wherein
the N-acetyl-lactosaminyl group in the glycosyl residue of R2a in general formula 1a is attached to the another N-acetyl-lactosaminyl group with 1-3 interglycosidic linkage,
the lacto-N-biosyl group in the glycosyl residue of R2a in general formula 1a is attached to the N-acetyl-lactosaminyl group with 1-3 interglycosidic linkage,
the lacto-N-biosyl group in the glycosyl residue of R3a in general formula 1a is attached to the N-acetyl-lactosaminyl group with 1-3 interglycosidic linkage,
the N-acetyl-lactosaminyl group in the glycosyl residue of R3b in general formula 1b is attached to the another N-acetyl-lactosaminyl group with 1-3 or 1-6 interglycosidic linkage, and/or
the lacto-N-biosyl group in the glycosyl residue of R3b in general formula 1b is attached to the N-acetyl-lactosaminyl group with 1-3 interglycosidic linkage.

### Claim 10 (depends on 8)

The method of claim 8, wherein general formula 1a represents the R-glycosides of lacto-N-neotetraose, para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-neohexaose, para-lacto-N-octaose and lacto-N-neooctaose substituted with one or more sialyl and/or fucosyl residue, and general formula 1b represents the R-glycosides of lacto-N-tetraose, lacto-N-hexaose, lacto-N-octaose, iso-lacto-N-octaose, lacto-N-decaose and lacto-N-neodecaose substituted with one or more sialyl and/or fucosyl residue.

### Claim 11

A method for manufacturing an infant nutritional product or infant formula comprising a mixture of human milk oligosaccharides (HMOs), the method comprising: subjecting a mixture of at least two compounds selected from the group consisting of compounds of general formula 1 and general formula 2 to catalytic hydrogenolysis to remove R,
wherein R is a group removable by the catalytic hydrogenolysis,
R1 is independently a fucosyl or an H,
R2 is an N-acetyl-lactosaminyl group or a lacto-N-biosyl group,
R3 is an H or an N-acetyl-lactosaminyl group,
R4 is independently a sialyl or an H,
and salts thereof,
at least one of R1 or R4 is not H in general formula 2,
the fucosyl residue attached to the N-acetyl-lactosaminyl and/or the lacto-N-biosyl group includes a linkage selected from the group consisting of
the galactose of the lacto-N-biosyl group with 1-2 interglycosidic linkage,
the N-acetyl-glucosamine of the lacto-N-biosyl group with 1-4 interglycosidic linkage, and
the N-acetyl-glucosamine of the N-acetyl-lactosaminyl group with 1-3 interglycosidic linkage; and
the sialyl residue attached to the N-acetyl-lactosaminyl and/or the lacto-N-biosyl group includes a linkage selected from the group consisting of
the galactose of the lacto-N-biosyl group with 2-3 interglycosidic linkage,
the N-acetyl-glucosamine of the lacto-N-biosyl group with 2-6 interglycosidic linkage, and
the galactose of the N-acetyl-lactosaminyl group with 2-6 interglycosidic linkage, and
a quantity of the mixture of the at least two compounds selected from the group consisting of compounds of general formula 1 and compounds of general formula 2 are selected such that the mixture of human milk oligosaccharides (HMOs) has an oligosaccharide profile that is substantially similar to a milk selected from the group consisting of colostrum breast milk, transitional breast milk and mature breast milk.

### Claim 12

A method for manufacturing an infant nutritional product or infant formula comprising a mixture of human milk oligosaccharides (HMOs), the method comprising: subjecting a mixture of at least two compounds selected from the group consisting of compounds of general formula 1 and compounds of general formula 2 to catalytic hydrogenolysis to remove R,
wherein the R-glycoside is a β-anomer,
R1 is independently a fucosyl or an H,
R2 is an N-acetyl-lactosaminyl group or a lacto-N-biosyl group,
R3 is an H or an N-acetyl-lactosaminyl group,
R4 is independently a sialyl or an H,
and salts thereof,
at least one of R1 or R4 is not H in general formula 2, and
a quantity of the mixture of the at least two compounds selected from the group consisting of compounds of general formula 1 and compounds of general formula 2 are selected such that the mixture of human milk oligosaccharides (HMOs) has an oligosaccharide profile that is substantially similar to a milk selected from the group consisting of colostrum breast milk, transitional breast milk and mature breast milk.

### Claim 13 (depends on 12)

The method of claim 12, wherein the N-acetyl lactosaminyl group comprises a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups, and at least one N-acetyl-lactosaminyl or lacto-N-biosyl group is substituted with a sialyl and/or fucosyl residue; and
R3 is N-acetyl-lactosaminyl group substituted with a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups, and at least one N-acetyl-lactosaminyl or lacto-N-biosyl group is substituted with one or more sialyl and/or fucosyl residue.

### Claim 14

A method for manufacturing an infant nutritional product or infant formula comprising a mixture of human milk oligosaccharides (HMOs), the method comprising: subjecting a mixture of at least two compounds selected from the group consisting of compounds of general formula 1 and compounds of general formula 2 to catalytic hydrogenolysis to remove R,
wherein R is a benzyl,
R1 is independently a fucosyl or an H,
R2 is an N-acetyl-lactosaminyl group or a lacto-N-biosyl group,
R3 is an H or an N-acetyl-lactosaminyl group,
R4 is independently a sialyl or an H,
and salts thereof,
at least one of R1 or R4 is not H in general formula 2, and
a quantity of the mixture of the at least two compounds selected from the group consisting of compounds of general formula 1 and compounds of general formula 2 are selected such that the mixture of human milk oligosaccharides (HMOs) has an oligosaccharide profile that is substantially similar to a milk selected from the group consisting of colostrum breast milk, transitional breast milk and mature breast milk.

### Claim 15 (depends on 14)

The method of claim 14, wherein the N-acetyl lactosaminyl group comprises a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups, and at least one N-acetyl-lactosaminyl or lacto-N-biosyl group can be substituted with a sialyl and/or fucosyl residue; and
R3 is N-acetyl-lactosaminyl group substituted with a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups, and at least one N-acetyl-lactosaminyl or lacto-N-biosyl group can be substituted with one or more sialyl and/or fucosyl residue.

### Claim 16

A method for manufacturing an infant nutritional product or infant formula comprising a mixture of human milk oligosaccharides (HMOs), the method comprising: subjecting a mixture of at least two compounds selected from the group consisting of compounds of general formula 1 and general formula 2 to catalytic hydrogenolysis to remove R,
wherein R is a group removable by the catalytic hydrogenolysis,
R1 is independently a fucosyl or an H,
R2 is an N-acetyl-lactosaminyl group or a lacto-N-biosyl group,
R3 is an H or an N-acetyl-lactosaminyl group,
R4 is independently a sialyl or an H,
and salts thereof,
at least one of R1 or R4 is not H in general formula 2, and
a quantity of the mixture of the at least two compounds selected from the group consisting of compounds of general formula 1 and compounds of general formula 2 are selected such that the mixture of human milk oligosaccharides (HMOs) has an oligosaccharide profile that is substantially similar to a milk selected from the group consisting of colostrum breast milk, transitional breast milk and mature breast milk,
wherein the catalytic hydrogenation leads to human milk oligosaccharides (HMOs) according to general formula 3 and general formula 4
wherein R1 is independently a fucosyl or an H,
R2 is an N-acetyl-lactosaminyl group or a lacto-N-biosyl group,
R3 is an H or an N-acetyl-lactosaminyl group,
R4 is independently a sialyl or an H,
and salts thereof, and
at least one of R1 or R4 is not H in general formula 4.

### Claim 17 (depends on 16)

The method of claim 16, wherein the HMOs are selected from the group consisting of: 2′-fucosyllactose, 3-fucosyllactose, 2′,3-difucosyllactose, 3′-sialyllactose, 6′-sialyllactose, 3′-sialyl-3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LST-a, LST-b, LST-c, FLST-a, FLST-b, FLST-c, LNDFH-I, LNDFH-II, LNDFH-III, DS-LNT, FDS-LNT I and FDS-LNT II.

### Claim 18 (depends on 16)

The method of claim 16, wherein the infant nutritional product or infant formula is in a form selected from the group consisting of a liquid, a powder and a solid.

### Claim 19 (depends on 16)

The method of claim 16, comprising adding a component selected from the group consisting of pharmaceutically or nutritionally acceptable carriers, lactose, proteins, and fat to the mixture of human milk oligosaccharides.

### Claim 20 (depends on 16)

The method of claim 16, further comprising adding prebiotics to the mixture of human milk oligosaccharides.

### Claim 21 (depends on 16)

The method of claim 16, wherein the HMOs according to general formula 3 are characterized by general formula 3a and general formula 3b
wherein
R2a is an N-acetyl-lactosaminyl group substituted with a glycosyl residue comprising one N-acetyl-lactosaminyl and/or one lacto-N-biosyl group; at least one N-acetyl-lactosaminyl or lacto-N-biosyl group is substituted with a sialyl and/or fucosyl residue,
R3a is an H or an N-acetyl-lactosaminyl group substituted with a lacto-N-biosyl group; at least one N-acetyl-lactosaminyl or lacto-N-biosyl group is substituted with a sialyl and/or fucosyl residue, and
R2b is lacto-N-biosyl group substituted with a sialyl and/or fucosyl residue.

### Claim 22 (depends on 21)

The method of claim 21, wherein
the N-acetyl-lactosaminyl group in the glycosyl residue of R2a in general formula 3a is attached to the another N-acetyl-lactosaminyl group with 1-3 interglycosidic linkage,
the lacto-N-biosyl group in the glycosyl residue of R2a in general formula 3a is attached to the N-acetyl-lactosaminyl group with 1-3 interglycosidic linkage,
the lacto-N-biosyl group in the glycosyl residue of R3a in general formula 3a is attached to the N-acetyl-lactosaminyl group with 1-3 interglycosidic linkage,
the N-acetyl-lactosaminyl group in the glycosyl residue of R3b in general formula 3b is attached to the another N-acetyl-lactosaminyl group with 1-3 or 1-6 interglycosidic linkage, and and/or
the lacto-N-biosyl group in the glycosyl residue of R3b in general formula 3b is attached to the N-acetyl-lactosaminyl group with 1-3 interglycosidic linkage.

### Claim 23 (depends on 21)

The method of claim 21, wherein general formula 3a represents lacto-N-neotetraose, para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-neohexaose, para-lacto-N-octaose and lacto-N-neooctaose optionally substituted with one or more sialyl and/or fucosyl residue, and general formula 3b represents lacto-N-tetraose, lacto-N-hexaose, lacto-N-octaose, iso-lacto-N-octaose, lacto-N-decaose and lacto-N-neodecaose optionally substituted with one or more sialyl and/or fucosyl residue.

### Claim 24 (depends on 21)

The method of claim 21, wherein
the fucosyl residue attached to the N-acetyl-lactosaminyl and/or the lacto-N-biosyl group includes a linkage selected from the group consisting of
the galactose of the lacto-N-biosyl group with 1-2 interglycosidic linkage,
the N-acetyl-glucosamine of the lacto-N-biosyl group with 1-4 interglycosidic linkage and
the N-acetyl-glucosamine of the N-acetyl-lactosaminyl group with 1-3 interglycosidic linkage,
the sialyl residue attached to the N-acetyl-lactosaminyl and/or the lacto-N-biosyl group includes a linkage selected from the group consisting of
the galactose of the lacto-N-biosyl group with 2-3 interglycosidic linkage,
the N-acetyl-glucosamine of the lacto-N-biosyl group with 2-6 interglycosidic linkage, and
the galactose of the N-acetyl-lactosaminyl group with 2-6 interglycosidic linkage.