Process for modifying gluco-oligosaccharides with an enriching step

Abstract

The present invention relates to a bioconversion for elongating gluco-oligosaccharides with a glucosyl moiety. The present invention also relates to the elongated gluco-oligosaccharides obtained by the process. The elongated gluco-oligosaccharides obtained by the process can be used in edible products, for instance as a dietary fiber or a (low calorie) bulking agent, a prebiotic, in sugar replacement or in calorie reduction. The elongated gluco-oligosaccharides obtained by the process present a decreased digestibility compared to known gluco-oligosaccharides.

Description

PT-1884-WO-PCT PROCESS FOR MODIFYING GLUCO-OLIGOSACCHARIDES WITH AN ENRICHING STEP CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of European Application No. EP24196291.9, filed August 23, 2024, which is incorporated by reference herein in its entirety. SEQUENCE LISTING

[0002] The content of the Sequence Listing XML titled “PT-1884 Final” produced with WIPO Sequence Software Version 2.3.0 on 29 May 2024 is incorporated by reference in its entirety. FIELD OF INVENTION

[0003] The present invention relates to a bioconversion for elongating gluco-oligosaccharides with a glucosyl moiety. The present invention also relates to the elongated gluco- oligosaccharides obtained by the process. The elongated gluco-oligosaccharides obtained by the process can be used in edible products, for instance as a dietary fiber or a (low calorie) bulking agent, a prebiotic, in sugar replacement or in calorie reduction. The elongated gluco- oligosaccharides obtained by the process present a decreased digestibility compared to known gluco-oligosaccharides. BACKGROUND OF THE INVENTION

[0004] Oligo- and polysaccharides are widely used in the food industry. Hydrolysed plant- based starches are a source of glucose containing oligo- and polysaccharides. Hydrolysed starches include maltodextrins and glucose syrups.

[0005] Maltodextrins, sucrose and glucose syrups may be used as a texturiser or as a full calorie ingredient providing bulking functionality. Maltodextrins, sucrose and glucose syrups may also be used as a coating and for encapsulation. Maltodextrins, sucrose and glucose syrups are easily digested and are fully and rapidly absorbed by the gastrointestinal tract. The fast digestibility of the maltodextrins, sucrose and glucose results in a strong rise in blood glucose levels after consumption. Maltodextrins, sucrose and glucose syrups are therefore productsPT-1884-WO-PCT with a high glycemic index. Further, maltodextrins, sucrose and glucose syrups are full calorie carbohydrates.

[0006] There is an ongoing need in the food industry for bulking ingredients and carbohydrates that have a lower or slower digestibility and that are lower in calories. There is also an ongoing need in the food industry for bulking ingredients and carbohydrates that decrease the glycemic impact of the oligo- or polysaccharides used for this purpose. One suggestion has been to replace the existing polysaccharides and carbohydrates with naturally occurring alternatives with a lower calorie content. Another alternative is to modify existing oligo- and polysaccharides.

[0007] The present inventors have found a process for modifying oligo- or polysaccharides such as maltodextrins or glucose syrups by means of an enzymatic process followed by an enriching step, resulting in a carbohydrate with decreased digestibility. SUMMARY OF THE INVENTION

[0008] The present invention relates to a bioconversion for elongating gluco-oligosaccharides with a glucosyl moiety. The present invention also relates to the elongated gluco- oligosaccharides obtained by the process. The elongated gluco-oligosaccharides obtained by the process can be used in edible products, for instance as a dietary fiber or a (low calorie) bulking agent, a prebiotic, in sugar replacement or in calorie reduction. The elongated gluco- oligosaccharides obtained by the process present a decreased digestibility compared to known gluco-oligosaccharides.

[0009] The present invention thus provides in a first aspect a process for elongating gluco- oligosaccharides with at least one α-(1,2)-terminally linked glucosyl moiety, comprising the steps of: (a) providing a starting acceptor composition comprising gluco-oligosaccharides, wherein the gluco-oligosaccharides have a degree of polymerisation of 2, 3, 4, 5, 6 or more glucosyl moieties, wherein each oligosaccharide in the starting acceptor composition comprises at least one α-1,4-linkage, and wherein the starting acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation;PT-1884-WO-PCT (b) reacting the starting acceptor composition in an aqueous medium with (i) a polypeptide having maltose phosphorylase catalytic activity, (ii) a phosphate source and (iii) a polypeptide having kojibiose phosphorylase catalytic activity; (c) obtaining from step (b) a first syrup, wherein the first syrup comprises one or more elongated gluco-oligosaccharides wherein each elongated gluco-oligosaccharide comprises at least one α-1,2-terminally linked glucosyl moiety, optionally one or more α-1,2-non-terminally linked glucosyl moieties; (d) concentrating the first syrup to a dry substance concentration of from 70 weight % or less of dry substance; and (e) enriching the first syrup to form a second syrup, wherein the second syrup comprises a non-digestible oligosaccharide at from 70 weight % or more of dry substance and monosaccharides and disaccharides at from 15 weight % or less of dry substance of the total weight of the dry substance of the second syrup, wherein the non-digestible oligosaccharide comprises one or more elongated gluco-oligosaccharides, wherein each elongated gluco-oligosaccharide comprises at least one α-1,2-terminally linked glucosyl moiety and each elongated gluco-oligosaccharide has a degree of polymerisation of 3, 4, 5, 6 or 7.

[0010] The present invention results in the addition of one or more glucosyl moieties to the gluco-oligosaccharides in the starting (or a mixed) acceptor composition by introducing an α- 1,2-linkage between the glucosyl moiety and the gluco-oligosaccharide. The glucosyl moiety is terminally linked to the gluco-oligosaccharide, i.e., it is bound to the non-reducing end of the gluco-oligosaccharide. Optionally, one or more glucosyl moieties are non-terminally α - 1,2-linked to the acceptor gluco-oligosaccharide.

[0011] Without wishing to be bound to any theory, it is believed that the orientation of this linkage leads to steric hindrance of the digestive enzymes in the gastrointestinal tract. This is believed to be because enzymes are selective and all or a proportion of the enzymes attack an oligosaccharide or a polysaccharide at only one end of the chain. For example, exo-acting enzymes hydrolyse glycosidic linkages only from the non-reducing end of starch derivatives.

[0012] The use of polypeptide having kojibiose phosphorylase activity is known. For instance, US6066477 describes a kojibiose phosphorylase that catalyses the transfer reaction of a glucosyl group to a single monosaccharide or to a single oligosaccharide, using β-D-glucose-PT-1884-WO-PCT 1-phosphoric acid as a saccharide donor. The enzyme is obtainable from natural sources such as microorganisms of the genus Thermoanaerobium or by recombinant technology.

[0013] Chaen et al, Journal of Bioscience and bioengineering, Vol.92, No.2, 173-176, 2001, describe the enzymatic synthesis of oligosaccharides from L-sorbose, maltose and sucrose using kojibiose phosphorylase.

[0014] The inventors have advantageously found that using a starting acceptor composition which is a mixture of gluco-oligosaccharides having different degrees of polymerisation, in particular a hydrolysed starch, elongation of the gluco-oligosaccharides with (at least one) α- 1,2-linked glucosyl moiety can be obtained, resulting in a gluco-oligosaccharide that has decreased digestibility.

[0015] The inventors have advantageously found that the inclusion of an enriching step in the process results in a second syrup that advantageously comprises a non-digestible oligosaccharide at a high weight percent of dry substance, for example at from 70 weight % or more of dry substance.

[0016] This result is unexpected since it was uncertain whether the inclusion of an enriching step would result in a second syrup comprising a non-digestible oligosaccharide at a high weight percent of dry substance, no experiments involving an enriching step have been reported to date.

[0017] In a second aspect, the invention relates to a composition comprising gluco- oligosaccharides elongated with at least one α-(1,2)-terminally linked glucosyl residue.

[0018] In a third aspect, the invention relates to an edible product comprising the composition of the invention.

[0019] Advantageously, the present invention enables the production of tailor-made products with different oligosaccharide contents and different sugar content. Further advantageously, the present invention enables the production of products with short chains and a narrow molecular weight distribution which enables low viscosity and mouthfeel advantages in multiple applications.

[0020] Representative features of the present invention are set out in the following clauses, which stand alone or may be combined, in any combination, with one or more features disclosed in the text and / or figures of the specification.

[0021] The present invention is as set out in the following clauses:PT-1884-WO-PCT 1. A process for elongating gluco-oligosaccharides comprising the steps of: (a) providing a starting acceptor composition comprising gluco-oligosaccharides, wherein the gluco-oligosaccharides have a degree of polymerisation of 2, 3, 4, 5, 6 or more glucosyl moieties, wherein each gluco-oligosaccharide in the starting acceptor composition comprises at least one α-1,4-linkage, and wherein the starting acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation; (b) reacting the starting acceptor composition in an aqueous medium with (i) a polypeptide having maltose phosphorylase catalytic activity, (ii) a phosphate source and (iii) a polypeptide having kojibiose phosphorylase catalytic activity; (c) obtaining from step (b) a first syrup, wherein the first syrup comprises one or more elongated gluco-oligosaccharides wherein each elongated gluco-oligosaccharide comprises at least one α-1,2-terminally linked glucosyl moiety, optionally one or more α-1,2-non-terminally linked glucosyl moieties; (d) concentrating the first syrup to a dry substance concentration of from 70 weight % or less of dry substance; and (e) enriching the first syrup to form a second syrup, wherein the second syrup comprises a non-digestible oligosaccharide at from 70 weight % or more of dry substance and monosaccharides and disaccharides at from 15 weight % or less of dry substance, wherein the non-digestible oligosaccharide comprises one or more elongated gluco-oligosaccharides, wherein each elongated gluco-oligosaccharide comprises at least one α-1,2-terminally linked glucosyl moiety and each elongated gluco-oligosaccharide has a degree of polymerisation of 3, 4, 5, 6 or 7. 2. The process of clause 1, wherein the second syrup is formed from enriching the first syrup only without any further additions to or treatment of the first syrup, optionally except for deactivation and / or removal of the polypeptides prior to the enriching step (e) . 3. The process of clause 1 or clause 2, wherein the first syrup is concentrated to a dry substance concentration of from 70 weight % or less, or, 65 weight % or less of dry substance, or, from 60 weight % or less of dry substance, or, from 30 to 65 weight % of dry substance, or, from 40 to 60 weight % of dry substance of the total weight of the first syrup prior to concentration.PT-1884-WO-PCT 4. The process of any of clauses 1 to 3, wherein the second syrup comprises the non- digestible oligosaccharide at from 70 weight % or more of dry substance, or, from 75 weight % or more of dry substance, or, from 78 weight % or more or dry substance, or, from 80 weight % or more of dry substance, or, from 85 weight % or more of dry substance, or, from 90 weight % or more of dry substance, or, from 95 weight % or more of dry substance of the total weight of the second syrup, and / or, the second syrup comprises the monosaccharides and disaccharides at from 10 weight % or less of dry substance, or, 5 weight % or less of dry substance of the total weight of the second syrup. 5. The process of any of clauses 1 to 4, wherein enriching in step (e) is carried out by chromatography, or, membrane filtration, or, membrane filtration followed by chromatography, or, chromatography followed by membrane filtration; optionally, wherein membrane filtration is diafiltration; preferably wherein enriching happens after inactivation and / or removal of the polypeptides. 6. The process of any of clauses 1 to 5, wherein enriching the first syrup in step (e) comprises removal of at least partially the monosaccharides from the first syrup, and / or, at least partially the disaccharides from the first syrup when forming the second syrup, such that the second syrup has a total monosaccharides and disaccharides content of less than 15 weight % dry substance of the total weight of the dry substance of the second syrup. 7. The process of any of clauses 1 to 6, wherein enriching the first syrup in step (e) comprises the formation of a third syrup having at least 40 weight % dry substance of monosaccharides of the total weight of the dry substance of the third syrup. 8. The process of any of clauses 1 to 7, wherein enriching the first syrup in step (e) comprises the formation of a fourth syrup having at least 40 weight % dry substance of disaccharides comprising both maltose and kojibiose of the total weight of the dry substance of the fourth syrup.PT-1884-WO-PCT 9. The process of clause 8, wherein the fourth syrup obtained from step (e) is combined with the starting acceptor composition in step (b) to recycle the fourth syrup back into the process; optionally, the fourth syrup obtained from step (e) is filtered before being combined with the starting acceptor composition in step (b) to recycle the fourth syrup back into the process; optionally, wherein the fourth syrup is filtered by diafiltration. 10. The process of any of clauses 1 to 9, wherein the starting acceptor composition comprises at least three gluco-oligosaccharides having a different degree of polymerisation, or, wherein the starting acceptor composition comprises at least four gluco-oligosaccharides having a different degree of polymerisation, or, wherein the starting acceptor composition comprises at least five gluco-oligosaccharides having a different degree of polymerisation. 11. The process of any of clauses 1 to 10, wherein the starting acceptor composition comprises at least two different gluco-oligosaccharides selected from the group consisting of maltose, maltotriose, maltotetraose, maltopentaose and maltohexaose. 12. The process of any of clauses 1 to 11, wherein the starting acceptor composition comprises from 45 weight % dry substance or more, or, from 50 weight % dry substance or more, or, from 55 weight % dry substance or more, or, from 60 weight % dry substance or more, or, from 65 weight % dry substance or more, or, from 70 weight % dry substance or more, or, from 75 weight % dry substance or more, or, from 80 weight % dry substance or more, or, from 85 weight % dry substance or more, or, from 90 weight % dry substance or more, or, from 95 weight % dry substance or more of gluco-oligosaccharides having a degree of polymerisation of DP2 of the total weight of the dry substance of the starting acceptor composition. 13. The process of any of clauses 1 to 12, wherein the starting acceptor composition comprises less than 10 weight % dry substance, or, less than 5 weight % dry substance, or, less than 4 weight % dry substance of monosaccharides of the total weight of the dry substance of the starting acceptor composition.PT-1884-WO-PCT 14. The process of any of clauses 1 to 13, wherein the polypeptide having kojibiose phosphorylase activity is a recombinant kojibiose phosphorylase obtainable from a bacteria selected from the group consisting of Thermococcus barophilus, Pseudothermotoga thermarum, Thermoanaerobacter brockii, Thermoanaerobacterium thermosacharolyticum, Caldicellulosiruptor saccharolyticus, Pyrococcus sp., Palaeococcus pacificus and Thermofilum pendens. 15. The process of any of clauses 1 to 14, wherein the process further comprises a step of preparing a 1-glucosyl phosphate donor by placing maltose in an aqueous medium with the polypeptide having maltose phosphorylase catalytic activity, and at least phosphoric acid and / or a salt thereof. 16. The process of clause 15, wherein the 1-glucosyl phosphate donor is β-D-glucose-1- phosphate. 17. The process of any of clauses 1 to 16, wherein the polypeptide having maltose phosphorylase catalytic activity is a recombinant maltose phosphorylase obtainable from a bacteria selected from the group consisting of Lactobacillus acidophilus, Bacillus selenitireducens, Enterococcus faecalis, Lactobacillus brevis, Lactobacillus sanfranciscensis, Paenibacillus sp. and Bacillus sp. 18. The process of any of clauses 1 to 17, further comprising the step of refining the first syrup. 19. The process of clause 18, wherein the step of refining the first syrup includes one or more of heating, inactivation of enzyme present, ion exchanging, electrodialysis, evaporation, reverse osmosis, resin polishing, filtration, sterilize filtration, and / or activated carbon treatment. 20. The process of clause 18 or clause 19, wherein the step of refining the first syrup occurs before step (e); optionally, wherein the step of refining is ion exchanging and / or electrodialysis.PT-1884-WO-PCT 21. The process of any of clauses 1 to 20, wherein step (e) comprises enriching by subjecting the first syrup to a polypeptide having alpha-glucosidase catalytic activity; optionally, wherein the polypeptide having alpha-glucosidase catalytic activity is glucose amylase. 22. The process of any of clause 18 to 21, wherein the first syrup is subjected to a polypeptide having alpha-glucosidase catalytic activity prior to the step of refining the first syrup. 23. A composition comprising elongated gluco-oligosaccharides obtained or obtainable by the process according to any of clause 1 to 22. 24. A composition comprising elongated gluco-oligosaccharides with at least one α-1,2- terminally linked glucosyl moiety and optionally one or more α-1,2-non-terminally linked glucosyl moieties, wherein the elongated gluco-oligosaccharides have a degree of polymerisation of 3, 4, 5, 6 or more, and, wherein the composition comprises from 70 weight % or more dry substance of non-digestible oligosaccharide and from 15 weight % or less of dry substance of monosaccharides and disaccharides. 25. The composition of clause 23 or clause 24, wherein the composition comprises monosaccharides and disaccharides at from 10 weight % or less of dry substance of dry substance, or, from 5 weight % or less of dry substance of the total weight of the dry substance of the composition. 26. The composition of any of clauses 23 to 25, wherein the composition comprises a non- digestible oligosaccharide at from 70 weight % or more of dry substance, or, from 75 weight % or more of dry substance, or, from 78 weight % or more or dry substance, or, from 80 weight % or more of dry substance, or, from 85 weight % or more of dry substance, or, from 90 weight % or more of dry substance, or, from 95 weight % or more of dry substance of the total weight of the dry substance of the composition.PT-1884-WO-PCT 27. The composition of any of clauses 23 to 26, wherein the composition further comprises one or more of kojibiose, kojitriose, kojitetraose and kojipentaose. 28. The composition of any of clauses 23 to 27, comprising at least two different elongated gluco-oligosaccharides selected from:2-O-α-D-glucosyl-maltose (α-D-Glucopyranosyl-(1^2)-α-D-glucopyranosyl-(1^4)- D-glucopyranose); and / or,PT-1884-WO-PCT 2-O-α-D-glucosyl-maltotriose (α-D-Glucopyranosyl-(1^2)-α-D-glucopyranosyl- (1^4)-α-D-glucopyranosyl-(1^4)-D-glucopyranose); and / or,2-O-α-D-glucosyl-maltotetraose (α-D-Glucopyranosyl-(1^2)-α-D- glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-D- glucopyranose); and / or2-O-α-D-glucosyl-maltopentaose (α-D-Glucopyranosyl-(1^2)-α-D- glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-α-D- glucopyranosyl-(1^4)-D-glucopyranose); and / or,PT-1884-WO-PCT2-O-α-D-glucosyl-maltohexaose (α-D-Glucopyranosyl-(1^2)-α-D- glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-α-D- glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-D-glucopyranose). 29. A composition comprising and / or consisting of monosaccharides, disaccharides and gluco-oligosaccharides obtained or obtainable by the process according to any of clauses 1 to 22, wherein the composition comprises at least 40 weight % dry substance of monosaccharides and at most 60 weight % dry substance disaccharides comprising both maltose and kojibiose and gluco-oligosaccharides having a degree of polymerisation of 3, 4, 5, 6 or more and higher of the total dry substance of the composition. 30. The composition of clause 29 obtained or obtainable by the process of clause 7. 31. A composition comprising and / or consisting of monosaccharides, disaccharides and gluco-oligosaccharides obtained or obtainable by the process according to any of clauses 1 to 22, wherein the composition comprises at least 40 weight % dry substance disaccharides comprising both maltose and kojibiose, and, at least 60 weight % dry substance monosaccharides and gluco-oligosaccharides having a degree of polymerisation of 3, 4, 5, 6 or higher of the total dry substance of the composition. 32. The composition of clause 31 obtained or obtainable by the process of clause 8.PT-1884-WO-PCT 33. A foodstuff, supplements for specialized nutrition, pharmaceutical products, personal care and cosmetic products, pet foods, animal feed or aqua feed, comprising the composition according to any one of clauses 23 to 32. DETAILED DESCRIPTION

[0022] Embodiments of the invention are described below with reference to the accompanying drawings. The accompanying drawings illustrate various embodiments of systems, methods, and embodiments of various other aspects of the disclosure. Any person with ordinary skills in the art will appreciate that the illustrated element boundaries (e.g., boxes, groups of boxes, or other shapes) in the figures represent one example of the boundaries. It may be that in some examples one element may be designed as multiple elements or that multiple elements may be designed as one element. In some examples, an element shown as an internal component of one element may be implemented as an external component in another and vice versa. Furthermore, elements may not be drawn to scale. Non-limiting and non-exhaustive descriptions are described with reference to the following drawings. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating principles.

[0023] Embodiments of the present disclosure will be described more fully hereinafter with reference to the accompanying drawings in which like numerals represent like elements throughout the several figures, and in which example embodiments are shown. Embodiments of the claims may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0024] The words “comprising,” “having,” “containing,” and “including,” and other forms thereof, are intended to be equivalent in meaning and be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items or meant to be limited to only the listed item or items. It must also be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Although any systems and methods similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure, the preferred systems and methods are now described.

[0025] Some of the terms used to describe the present invention are set out below:PT-1884-WO-PCT

[0026] “Acceptor composition” refers to a starting acceptor composition, a mixed acceptor composition or a combination of a starting acceptor composition and a mixed acceptor composition.

[0027] “Dextrose equivalent” or “DE” refers to a measure of the total reducing sugars calculated as D-glucose on a dry weight basis. The approved method for determining DE is the Lane–Eynon titration method, which measures reduction of a copper sulfate solution. Unhydrolyzed starch has a DE value of zero, while the DE value of anhydrous D-glucose is 100. Glucose syrups range from 20 to 97 DE.

[0028] “Degree of polymerisation” or “DP” refers to the number of monomeric units in a polymer. For example, DP1 refers to monosaccharides such as but not limited to glucose, dextrose, fructose and galactose, whilst DP2 refers to disaccharides such as but not limited to sucrose, lactose, maltose and kojibiose.

[0029] “Elongated gluco-oligosaccharide” refers to a gluco-oligosaccharide that comprises at least one α-1,2-terminally linked glucosyl moiety.

[0030] “Enriching” refers to a method for producing a syrup comprising a non-digestible oligosaccharide at from 70 weight % or more of dry substance and monosaccharides and disaccharides together at from 15 weight % or less of dry substance of the total weight of the dry substance of the syrup, wherein the non-digestible oligosaccharide comprises one or more elongated gluco-oligosaccharides, wherein each elongated gluco-oligosaccharide comprises at least one α-1,2-terminally linked glucosyl moiety and each elongated gluco-oligosaccharide has a degree of polymerisation of 3, 4, 5, 6 or 7. Preferably, enriching is carried out by chromatography. Enriching happens after inactivation / denaturisation of the polypeptides / enzymes, which can be carried out by any known means, for instance heat treatment or pH adjustment. Enriching preferably happens after removal of the inactivated polypeptides / enzymes.

[0031] “First syrup” refers to a syrup comprising one or more elongated gluco- oligosaccharides wherein each elongated gluco-oligosaccharide comprises at least one α-1,2- terminally linked glucosyl moiety and optionally one or more α-1,2-non-terminally linked glucosyl moieties. The first syrup is produced by reacting a starting acceptor composition in an aqueous medium with (i) a polypeptide having maltose phosphorylase catalytic activity, (ii) a phosphate source and (iii) a polypeptide having kojibiose phosphorylase catalytic activity.PT-1884-WO-PCT

[0032] “Fructose syrup” refers to an aqueous solution of nutritive saccharides obtained from starch in which a portion of the dextrose (D-glucose) has been isomerized to fructose.

[0033] “Gluco-oligosaccharides” refers to oligosaccharides having two or more glucosyl moieties in the molecular chain. These glucosyl moieties can be bound to each other via α- (1,4)-linkages or via α-(1,6)-linkages. In the present invention, the gluco-oligosaccharides in the starting acceptor composition comprise at least one α-(1,4)-linkage.

[0034] “Glucose syrup” refers to an aqueous solution of nutritive saccharides obtained from edible starch having a DE of 20 or more.

[0035] “Kojibiose phosphorylase” or “KP” is a carbohydrate-processing phosphorolytic enzyme and is classified into glycoside hydrolase family 65 (GH65).

[0036] “Maltodextrin” refers to a dried product or aqueous solutions of saccharides obtained from edible starch having a DE of less than 20.

[0037] “Mixed acceptor composition” refers to an acceptor composition that comprises the starting acceptor composition and the fourth syrup. The fourth syrup may comprise at least 40 weight % dry substance of disaccharides of the total weight of the dry substance of the fourth syrup.

[0038] “Non-digestible oligosaccharide” refers to an edible oligosaccharide, or “dietary fiber”, that cannot be digested and absorbed in the small intestine of a human and / or animal digestive system or that is not broken down by the digestive enzymes of a human and / or animal, i.e., that are resistant to degradation by mammalian enzymes. In particular, the non-digestible oligosaccharide comprises one or more elongated gluco-oligosaccharides, wherein each elongated gluco-oligosaccharide comprises at least one α-1,2-terminally linked glucosyl moiety and each elongated gluco-oligosaccharide has a degree of polymerisation of 3, 4, 5, 6 or 7. The method of determining the composition of the non-digestible oligosaccharide is set out in Examples 1 and 2.

[0039] “Polypeptide” refers to a linear organic polymer consisting of a large number of amino- acid residues bonded together in a chain, forming part of or a whole protein molecule.

[0040] “Second syrup” refers to a syrup comprising a non-digestible oligosaccharide at from 70 weight % or more of dry substance and monosaccharides and disaccharides together at from 15 weight % or less of dry substance of the total weight of the dry substance of the second syrup, wherein the non-digestible oligosaccharide comprises one or more elongated gluco- oligosaccharides, wherein each elongated gluco-oligosaccharide comprises at least one α-1,2-PT-1884-WO-PCT terminally linked glucosyl moiety and each elongated gluco-oligosaccharide has a degree of polymerisation of 3, 4, 5, 6 or 7. Preferably, the second syrup is produced by enriching the first syrup.

[0041] “Starting acceptor composition” refers to composition comprising gluco- oligosaccharides, wherein the gluco-oligosaccharides have a degree of polymerisation of 2, 3, 4, 5, 6 or more glucosyl moieties, wherein each gluco-oligosaccharide in the starting acceptor composition comprises at least one α-1,4-linkage, and wherein the starting acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation. Optionally, the starting acceptor composition can be combined with a fourth syrup to form a mixed acceptor composition, wherein the fourth syrup comprises at least 40 weight % dry substance of disaccharides of the total weight of the dry substance of the fourth syrup.

[0042] “Third syrup” refers to a syrup comprising at least 40 weight % dry substance of monosaccharides of the total weight of the dry substance of the third syrup. Preferably, the third syrup is produced by enriching the first syrup.

[0043] “Fourth syrup” refers to a syrup comprising at least 40 weight % dry substance of disaccharides comprising both maltose and kojibiose of the total weight of the dry substance of the fourth syrup. Preferably, the fourth syrup is produced by enriching the first syrup. Further preferably, the fourth syrup is combined with the starting acceptor composition. Upon combining the fourth syrup with the starting acceptor composition, a mixed acceptor composition is produced. Process

[0044] In one aspect, the present invention relates to a process for elongating gluco- oligosaccharides with at least one α-(1,2)-terminally linked glucosyl moiety. As discussed above, the process comprises the steps of: (a) providing a starting acceptor composition comprising gluco-oligosaccharides, wherein the gluco-oligosaccharides have a degree of polymerisation of 2, 3, 4, 5, 6 or more glucosyl moieties, wherein each gluco-oligosaccharide in the starting acceptor composition comprises at least one α-1,4-linkage, and wherein the starting acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation;PT-1884-WO-PCT (b) reacting the starting acceptor composition in an aqueous medium with (i) a polypeptide having maltose phosphorylase catalytic activity, (ii) a phosphate source and (iii) a polypeptide having kojibiose phosphorylase catalytic activity; (c) obtaining from step (b) a first syrup, wherein the first syrup comprises one or more elongated gluco-oligosaccharides wherein each elongated gluco-oligosaccharide comprises at least one α-1,2-terminally linked glucosyl moiety, optionally one or more α-1,2-non-terminally linked glucosyl moieties; (d) concentrating the first syrup to a dry substance concentration of from 70 weight % or less of dry substance; and (e) enriching the first syrup to form a second syrup, wherein the second syrup comprises a non-digestible oligosaccharide at from 70 weight % or more of dry substance and monosaccharides and disaccharides at from 15 weight % or less of dry substance, wherein the non-digestible oligosaccharide comprises one or more elongated gluco- oligosaccharides, wherein each elongated gluco-oligosaccharide comprises at least one α-1,2-terminally linked glucosyl moiety and each elongated gluco-oligosaccharide has a degree of polymerisation of 3, 4, 5, 6 or 7. Starting acceptor composition

[0045] As described above, the process of the invention has as a starting material a starting acceptor composition comprising gluco-oligosaccharides having a degree of polymerisation of 2, 3, 4, 5, 6 or more glucosyl moieties, wherein each gluco-oligosaccharide in the starting acceptor composition comprises at least one α-(1,4)-linkage.

[0046] In some examples of the present invention, the starting acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation. For example, the starting acceptor composition may comprise a gluco-oligosaccharide having a degree of polymerisation of two (two glucosyl moieties) and a gluco-oligosaccharide having a degree of polymerisation of three (three glucosyl moieties). As a further example, the starting acceptor composition can be obtained by mixing two, three, four, five or more gluco- oligosaccharides having different degrees of polymerisation. In some examples of the present invention, the starting acceptor composition comprises at least three gluco-oligosaccharides having a different degree of polymerisation, or, the starting acceptor composition comprises at least four gluco-oligosaccharides having a different degree of polymerisation, or, the startingPT-1884-WO-PCT acceptor composition comprises at least five gluco-oligosaccharides having a different degree of polymerisation.

[0047] Alternatively, the starting acceptor composition of the invention can be obtained by partial hydrolysis of starch as will be described hereafter.

[0048] Preferably, the starting acceptor composition comprises at least two different gluco- oligosaccharides selected from the group consisting of maltose, maltotriose, maltotetraose, maltopentaose and maltohexaose. CAS numbers for these compounds are: Maltose: 69-79-4; Maltotriose 1109-28-0; Maltotetraose 34612-38-9; Maltopentaose 34620-76-3; Maltohexaose 34620-77-4.

[0049] In some examples of the present invention, the starting acceptor composition comprises from 45 weight % dry substance or more, preferably from 50 weight % dry substance or more, more preferably from 55 weight % dry substance or more, more preferably from 60 weight % dry substance or more, more preferably from 65 weight % dry substance or more, more preferably from 70 weight % dry substance or more, more preferably from 75 weight % dry substance or more, more preferably from 80 weight % dry substance or more, more preferably from 85 weight % dry substance or more, more preferably from 90 weight % dry substance or more, most preferably from 95 weight % dry substance or more of gluco- oligosaccharides having a degree of polymerisation of DP2 of the total weight of the dry substance of the starting acceptor composition.

[0050] In some examples of the present invention, the starting acceptor composition comprises less than 10 weight % dry substance, preferably less than 5 weight % dry substance, more preferably less than 4 weight % dry substance of monosaccharides of the total weight of the dry substance of the starting acceptor composition.

[0051] In some examples of the present invention, the starting acceptor composition comprises 5 weight % or less dry substance of monosaccharides of the total weight of the dry substance of the starting acceptor composition. Preferably, the starting acceptor composition comprises 5 weight % or less dry substance of dextrose of the total weight of the dry substance of the starting acceptor composition.

[0052] In some examples of the present invention, the starting acceptor composition comprises 20 weight % or more dry substance of disaccharides, more preferably the starting acceptor composition comprises 30 weight % or more dry substance of disaccharides, even more preferably the starting acceptor composition comprises 40 weight % or more dryPT-1884-WO-PCT substance of disaccharides, even more preferably the starting acceptor composition comprises 50 weight % or more dry substance of disaccharides, even more preferably the starting acceptor composition comprises 60 weight % or more dry substance of disaccharides, most preferably the starting acceptor composition comprises 65 weight % or more dry substances of disaccharides of the total weight of the dry substance of the starting acceptor composition. Preferably, the starting acceptor composition comprises 68 weight % dry substance of disaccharides of the total weight of the dry substance of the starting acceptor composition. Preferably, the starting acceptor composition comprises 80 weight % dry substance of disaccharides of the total weight of the dry substance of the starting acceptor composition. Preferably, the starting acceptor composition comprises 88 weight % dry substance of disaccharides of the total weight of the dry substance of the starting acceptor composition. Preferably, the starting acceptor composition comprises 97 weight % dry substance of disaccharides of the total weight of the dry substance of the starting acceptor composition. Preferably, the starting acceptor composition comprises 99.9 weight % dry substance of disaccharides of the total weight of the dry substance of the starting acceptor composition.

[0053] Preferably, the disaccharides comprise maltose. Preferably, the starting acceptor composition comprises 20 weight % or more dry substance of maltose, more preferably the starting acceptor composition comprises 30 weight % or more dry substance of maltose, even more preferably the starting acceptor composition comprises 40 weight % or more dry substance of maltose, even more preferably the starting acceptor composition comprises 50 weight % or more dry substance of maltose, even more preferably the starting acceptor composition comprises 60 weight % or more dry substance of maltose, most preferably the starting acceptor composition comprises 65 weight % or more dry substance of maltose of the total weight of the dry substance of the starting acceptor composition. Preferably, the starting acceptor composition comprises 68 weight % dry substance of maltose of the total weight of the dry substance of the starting acceptor composition. Preferably, the starting acceptor composition comprises 80 weight % dry substance of maltose of the total weight of the dry substance of the starting acceptor composition. Preferably, the starting acceptor composition comprises 88 weight % dry substance of maltose of the total weight of the dry substance of the starting acceptor composition. Preferably, the starting acceptor composition comprises 97 weight % dry substance of maltose of the total weight of the dry substance of the starting acceptor composition.PT-1884-WO-PCT

[0054] In some examples of the present invention, the starting acceptor composition comprises less than 1 weight % or more dry substance of kojibiose of the total weight of the dry substance of the starting acceptor composition.

[0055] Beside the gluco-oligosaccharides having a degree of polymerisation of 2, 3, 4, 5, 6 or 7 glucosyl moieties, the starting acceptor composition can comprise further oligo- or polysaccharides, for example oligo- or polysaccharides obtained by liquefaction, saccharification and refining of starch.

[0056] In some examples of the present invention, the starting acceptor composition is preferably a partially hydrolysed starch having a dextrose equivalent (DE) of less than 65, more preferably less than 60, more preferably less than 50, more preferably less than 45. Optionally, the starting acceptor composition is preferably a partially hydrolysed starch having a dextrose equivalent (DE) of at least 4, preferably at least 10. Further optionally, the partially hydrolysed starch is maltodextrin or glucose syrup. Preferably, the partially hydrolysed starch is maltodextrin or glucose syrup having a DE of less than 45 and a DE of at least 10.

[0057] The partially hydrolysed starch may be obtained by submitting starch to enzymatic or acid hydrolysis or a combination thereof, such as acid hydrolysis followed by enzyme hydrolysis.

[0058] When a partially hydrolysed starch is used as the starting acceptor composition, it may contain further gluco-oligosaccharides having α-1,6-linkages, such as the isoforms of maltotriose, maltotetraose, maltopentaose and maltohexaose and oligosaccharides containing mixed α-1,6 and α-1,4 linkages and oligosaccharides having glucopyranosyl branches through mixed α-1,6 and α-1,4 linkages.

[0059] In some examples of the present invention, the starting acceptor composition may also comprise gluco-oligosaccharides having a degree of polymerisation higher than 6, e.g., 7 or 8. Polypeptide having maltose phosphorylase catalytic activity

[0060] In some examples of the present invention, the polypeptide having maltose phosphorylase catalytic activity is a recombinant maltose phosphorylase obtainable from a bacteria selected from the group consisting of Lactobacillus acidophilus, Bacillus selenitireducens, Enterococcus faecalis, Lactobacillus brevis, Lactobacillus sanfranciscensis, Paenibacillus sp. and Bacillus sp.PT-1884-WO-PCT Phosphate source

[0061] In some examples of the present invention, the phosphate source is phosphoric acid, phosphate salts such as but not limited to sodium phosphate and / or mixtures thereof. Preferably, a mixture thereof includes monobasic sodium phosphate and dibasic sodium phosphate. Preferably, the pH of the phosphate source is 6.5. Preferably, the phosphate source is sodium phosphate having a pH of 6.5. Polypeptide having kojibiose phosphorylase activity

[0062] Polypeptides with kojibiose phosphorylase activity are known and are enzymes classified under E.C.2.4.1.230.

[0063] Kojibiose phosphorylase (KP) catalyses the reversible phosphorolysis of kojibiose (α- D-glucopyranosyl-(1→2)-D-glucopyranose) to β-D-glucose-1-phosphate (β-G1P or β-G1P) and D-glucose (Glc), with an inversion of the anomeric configuration. KP activity was first identified in a cell extract of Thermoanaerobacter brockii ATCC35047. In the presence of suitable acceptors, KP from this strain (Tb-KP) transfers a glucosyl moiety from β-G1P to the acceptors and synthesizes kojioligosaccharides (Takuo Yamamote et. al., Biosci. Biotechnol. Biochem., 75 (6), 1208-1210, 2011).

[0064] The polypeptides with kojibiose phosphorylase activity include any one of the polypeptides separated from natural resources such as cultures of microorganisms which produce the polypeptide, mutants thereof obtained by treating with mutagens, and those that are artificially synthesized by applying recombinant DNA and peptide-synthesizing technologies. Preferably, the polypeptide having kojibiose phosphorylase activity is a recombinant kojibiose phosphorylase.

[0065] The recombinant kojibiose phosphorylase is preferably a polypeptide obtainable from a bacteria selected from the group consisting of Thermococcus barophilus, Pseudothermotoga thermarum, Thermoanaerobacter brockii, Thermoanaerobacterium thermosacharolyticum, Caldicellulosiruptor saccharolyticus, Pyrococcus sp., Palaeococcus pacificus and Thermofilum pendens. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity are obtained or obtainable or derived or derivable from a microorganism selected from the group consisting of Caldicellulosiruptor saccharolyticus, Thermococcus barophilus, Thermophilum pendens Hrk 5, and / or Palaeococcus pacificus DY20341. Preferably, the polypeptides with kojibiose phosphorylase activity are obtained orPT-1884-WO-PCT obtainable or derived or derivable from Thermococcus barophilus or Caldicellulosiruptor saccharolyticus. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity are obtained or obtainable or derived or derivable from Thermophilum pendens, optionally wherein the Thermophilum pendens is Thermophilum pendens Hrk 5. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity are obtained or obtainable or derived or derivable from Palaeococcus pacificus, optionally wherein the Palaeococcus pacificus is Palaeococcus pacificus DY20341.

[0066] In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise residues selected from the group consisting of: residues 1-748 of SEQ ID NO:3, residues 1-746 of SEQ ID NO:4 and / or residues 1-704 of SEQ ID NO:5. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise residues 1-748 of SEQ ID NO:3. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise residues 1-746 of SEQ ID NO:4. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise residues 1-704 of SEQ ID NO:5. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise residues 1-748 of SEQ ID NO:3 or residues 1-746 of SEQ ID NO:4. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise residues 1-748 of SEQ ID NO:3 or residues 1-704 of SEQ ID NO:5. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise residues 1-746 of SEQ ID NO:4 or residues 1- 704 of SEQ ID NO:5.

[0067] In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity consist of the amino acid sequence set forth in SEQ ID NO:1. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or, 80 identity to the amino acid sequence set forth in set forth in any one of SEQ ID NO:1 and / or to the amino acid sequence of amino acid residues 1-756 of SEQ ID NO:1. All homologs, orthologs and variants of SEQ ID NO:1 can be used in the present invention. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity consist of thePT-1884-WO-PCT amino acid sequence set forth in SEQ ID NO:2. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or, 80 identity to the amino acid sequence set forth in set forth in any one of SEQ ID NO: 2 and / or to the amino acid sequence of amino acid residues 1-775 of SEQ ID NO:2. All homologs, orthologs and variants of SEQ ID NO:2 can be used in the present invention. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity consist of the amino acid sequence set forth in SEQ ID NO:3. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or, 80 identity to the amino acid sequence set forth in set forth in any one of SEQ ID NO: 3 and / or to the amino acid sequence of amino acid residues 1-748 of SEQ ID NO:3. All homologs, orthologs and variants of SEQ ID NO:3 can be used in the present invention. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity consist of the amino acid sequence set forth in SEQ ID NO:4. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or, 80 identity to the amino acid sequence set forth in set forth in any one of SEQ ID NO: 4 and / or to the amino acid sequence of amino acid residues 1-746 of SEQ ID NO:4. All homologs, orthologs and variants of SEQ ID NO:4 can be used in the present invention. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity consist of the amino acid sequence set forth in SEQ ID NO:5. In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or, 80 identity to the amino acid sequence set forth in set forth in any one of SEQ ID NO: 5 and / or to the amino acid sequence of amino acid residues 1-704 of SEQ ID NO:5. All homologs, orthologs and variants of SEQ ID NO:5 can be used in the present invention.

[0068] In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise an alanine residue at the amino acid position corresponding to position 387 of Thermococcus barophilus kojibiose phosphorylase (SEQ ID NO: 3). In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise a proline residue at the amino acid position corresponding to position 393 ofPT-1884-WO-PCT Thermococcus barophilus kojibiose phosphorylase (SEQ ID NO: 3). In some examples of the present invention, the polypeptides with kojibiose phosphorylase activity comprise a leucine residue at the amino acid position corresponding to position 394 of Thermococcus barophilus kojibiose phosphorylase (SEQ ID NO: 3). Preferably, the polypeptides with kojibiose phosphorylase activity comprise an alanine residue at the amino acid position corresponding to position 387 of Thermococcus barophilus kojibiose phosphorylase (SEQ ID NO: 3), a proline residue at the amino acid position corresponding to position 393 of Thermococcus barophilus kojibiose phosphorylase (SEQ ID NO: 3) and a leucine residue at the amino acid position corresponding to position 394 of Thermococcus barophilus kojibiose phosphorylase (SEQ ID NO: 3).

[0069] According to the present invention the polypeptides with kojibiose phosphorylase activity are able to elongate gluco-oligosaccharides with at least one α-(1,2)-terminally linked glucosyl moiety. Donor

[0070] The process of the present invention can comprise a step of preparing a donor. Optionally, the donor is 1-glucosyl phosphate. Further optionally, the 1-glucosyl phosphate donor is ß-D-glucose-1-phosphate.

[0071] In some examples of the present invention, the donor is prepared by placing an appropriate phosphorylase with a saccharide in the presence of a phosphate source in a process coupled reaction (described below).

[0072] In some examples of the present invention, ß-D-glucose-1-phosphate is commercially available and can be used as such. Process coupled reaction

[0073] In some examples of the present invention, the donor is prepared by placing an appropriate phosphorylase with a saccharide in the presence of a phosphate source at a pH of 6.3 to 6.5 and a temperature of from 50 to 60 °C, more preferably a temperature of 55 °C.

[0074] In some examples of the present invention, a 1-glucosyl phosphate donor can be prepared by placing maltose in an aqueous medium with the polypeptide having maltose phosphorylase catalytic activity and a phosphate source, wherein the phosphate source can be phosphoric acid and / or a salt thereof. If the starting acceptor composition is partiallyPT-1884-WO-PCT hydrolysed starch, the starting acceptor composition may already contain kojibiose, or kojibiose is generated in situ during the α-1,2-linkage-creating reactions.

[0075] The polypeptide comprising maltose phosphorylase activity is an enzyme that catalyses the reaction of maltose and inorganic phosphate to glucose and β-D-glucose-1- phosphate. It is classified under EC 2.4.1.8. In particular the polypeptide comprising maltose phosphorylase activity is a recombinant maltose phosphorylase, preferably a polypeptide obtainable from a bacteria selected from the group consisting of Lactobacillus acidophilus, Bacillus selenitireducens, Enterococcus faecalis, Lactobacillus brevis, Lactobacillus sanfranciscensis, Paenibacillus sp. and Bacillus sp.

[0076] An example is a maltose phosphorylase from Lactobaciluus acidophilus, such as described by Nakai, H., et al. (2009), The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel α‐glucosides through reverse phosphorolysis by maltose phosphorylase. The FEBS Journal, 276: 7353-7365.

[0077] This “coupled reaction” is represented in Figure 1. In Figure 1, MP represents maltose phosphorylase from Lactobacillus acidophilus (LaMP), Pi stands for phosphoric acid and / or salt, β-G1P is β-D-glucose-1-phosphate, and KP represents kojibiose phosphorylase.

[0078] In some examples of the present invention, the present invention comprises a step of preparing the 1-glucosyl phosphate donor by placing trehalose in an aqueous medium with a polypeptide having trehalose phosphorylase activity, phosphoric acid and / or a salt thereof. The polypeptide having trehalose phosphorylase activity is a polypeptide that catalyses the reaction of trehalose and inorganic phosphate to glucose and β-D-glucose-1-phosphate. It is classified under (EC 2.4.1.64). See for instance TRENDS in Biotechnology Vol.20 No.10 October 2002.

[0079] In some examples of the present invention, the present invention comprises a step of preparing the 1-glucosyl phosphate donor by placing sucrose in an aqueous medium with a polypeptide having sucrose phosphorylase activity, phosphoric acid and / or a salt thereof.

[0080] In some examples of the present invention, the present invention comprises a step of preparing the 1-glucosyl phosphate donor by placing lactose in an aqueous medium with a polypeptide having lactose phosphorylase activity, phosphoric acid and / or a salt thereof. First syrupPT-1884-WO-PCT

[0081] The first syrup comprises one or more elongated gluco-oligosaccharides wherein each elongated gluco-oligosaccharide comprises at least one α-1,2-terminally linked glucosyl moiety and optionally one or more α-1,2-non-terminally linked glucosyl moieties.

[0082] Optionally, the first syrup is concentrated until the first syrup has a dry substance concentration of from 70 weight % or less of dry substance, preferably from 65 weight % or less of dry substance, more preferably from 60 weight % or less of dry substance, more preferably from 30 to 65 weight % of dry substance, most preferably from 40 to 60 weight % of dry substance of the total weight of the first syrup prior to concentration. Concentration is carried out by any means known in the art, such as but not limited to, evaporation. Second syrup

[0083] The second syrup comprises a non-digestible oligosaccharide at from 70 weight % or more of dry substance and monosaccharides and disaccharides at from 15 weight % or less of dry substance, wherein the non-digestible oligosaccharide comprises one or more elongated gluco-oligosaccharides, wherein each elongated gluco-oligosaccharide comprises at least one α-1,2-terminally linked glucosyl moiety and each elongated gluco-oligosaccharide has a degree of polymerisation of 3, 4, 5, 6 or 7. The remaining weight percentage of the second syrup includes trisaccharides, tetrasaccharides and other digestible oligosaccharides.

[0084] Optionally, the second syrup comprises the non-digestible oligosaccharide at from 70 weight % or more of dry substance, preferably from 75 weight % or more of dry substance, preferably from 78 weight % or more of dry substance, more preferably from 80 weight % or more of dry substance, more preferably from 85 weight % or more of dry substance, more preferably from 90 weight % or more of dry substance, most preferably from 95 weight % or more of dry substance of the total weight of the dry substance of the second syrup. Optionally, the second syrup comprises the monosaccharides and disaccharides at from 10 weight % or less of dry substance, preferably 5 weight % or less of dry substance of the total weight of the dry substance of the second syrup. Process conditions

[0085] The process of the present invention is carried out by placing the starting acceptor composition, polypeptide having maltose phosphorylase catalytic activity, a phosphate source and a polypeptide having kojibiose phosphorylase catalytic activity in an aqueous medium. InPT-1884-WO-PCT general, this means that the process is carried out in an aqueous solution. The polypeptides can also be immobilised polypeptides.

[0086] Process conditions are those generally known in the field. For examples, the concentration of the starting acceptor composition in the aqueous solution is from 5 to 60 weight %, the pH of the reaction is from 4 to 7 (such as from 5 to 7); the molar ratio starting acceptor composition to donor composition is from 1:1 to 1:10; the temperature is from 40 to 70 °C; the concentration of the isolated polypeptide is from 0.05 to 20 Units / m. The concentration of the phosphate buffer is from 2.5 to 100 mM.

[0087] In some examples of the present invention, further polypeptides are included in the reaction. The further polypeptides can be included simultaneously or sequentially. Optionally, the polypeptides are alpha-amylase (including variations such as maltogenic alpha-amylase), beta-amylase, debranching enzymes (pullulanase or / and isoamylase), and / or, mixtures thereof.

[0088] In some examples of the present invention, the second syrup is formed from enriching the first syrup only without any further additions to or treatment of the first syrup, optionally except for denaturization / deactivation (and optionally removal) of the polypeptides / enzymes prior to the enriching step (e).

[0089] In some examples of the present invention, additional processing steps are carried out between step (c) and step (d). For example, steps such as enzyme denaturisation (via heat treatment or any other method known in the art), cooling the temperature of the first syrup to room temperature, cation and anion exchange and / or decolourisation of the first syrup. The additional processing steps are carried out by any method known in the art.

[0090] In some examples of the present invention, the first syrup is concentrated in step (d) to a dry substance concentration of from 70 weight % or less, preferably 65 weight % or less of dry substance, more preferably from 60 weight % or less of dry substance, more preferably from 30 to 65 weight % of dry substance, most preferably from 40 to 60 weight % of dry substance of the total weight of the first syrup. Optionally, the first syrup is concentrated by removal of water by evaporation and / or filtration. Optionally, filtration is membrane filtration.

[0091] In some examples of the present invention, enriching the first syrup in step (e) is carried out by chromatography, and / or, membrane filtration, optionally, wherein membrane filtration is diafiltration, which happens after inactivation (=denaturization) of the polypeptides / enzymes. Inactivation can be carried out by any known method in the art, preferably by heat treatment or by pH adjustment. Preferably, enriching the first syrup in stepPT-1884-WO-PCT (e) is carried out by chromatography after inactivation of the polypeptides / enzymes. Preferably, enriching the first syrup in step (e) is carried out by membrane filtration, which can optionally be diafiltration, after inactivation of the polypeptides / enzymes. In some examples, one or more chromatographic steps and / or one or more membrane filtration steps are carried out after inactivation of the polypeptides / enzymes. In some examples, after inactivation of the polypeptides / enzymes, membrane filtration is followed by chromatography, or, chromatography is followed by membrane filtration; optionally, wherein membrane filtration is diafiltration. A skilled person would select the resin and operation conditions (such as temperature, pressure, dry substance and amount of water needed) for the chromatography according to well-known methods.

[0092] In some examples of the present invention, enriching the first syrup in step (e) forms the second syrup as well as a third and / or a fourth syrup. A skilled person would select the resin and operation conditions (such as temperature, pressure, dry substance and amount of water needed) for the chromatography according to well-known screening methods.

[0093] In some examples of the present invention, enriching the first syrup in step (e) is carried out in one step. The step is a three-fraction chromatography performed on the first syrup, wherein the second syrup, third syrup and fourth syrup are separately formed by the three- fraction chromatography. Optionally, the fourth syrup is subjected to diafiltration.

[0094] In some examples of the present invention, enriching the first syrup in step (e) is carried out in two steps. The first step is a two-fraction chromatography performed on the first syrup, wherein the second syrup and a blend comprising the third syrup and fourth syrup are formed by the two-fraction chromatography. The second step is a two-fraction chromatography performed on the blend comprising the third syrup and fourth syrup, wherein the third syrup and fourth syrup are formed by the two-fraction chromatography. A skilled person would select the resin and operation conditions (such as temperature, pressure, dry substance and amount of water needed) for the chromatography according to well-known screening methods.

[0095] In some examples of the present invention, enriching the first syrup in step (e) is carried out in two steps. The first step is a two-fraction chromatography performed on the first syrup, wherein the second syrup and a blend comprising the third syrup and fourth syrup are formed by the two-fraction chromatography. The second step is diafiltration performed on the blend comprising the third syrup and fourth syrup, wherein the third syrup and fourth syrup are formed by the two-fraction chromatography. A skilled person would select the resin for thePT-1884-WO-PCT chromatography and the membrane for the diafiltration and the operation conditions (such as temperature, pressure, dry substance and amount of water needed) according to well-known screening methods.

[0096] In some examples of the present invention, enriching the first syrup in step (e) comprises removal of at least partially the monosaccharides from the first syrup, and / or, at least partially the disaccharides from the first syrup when forming the second syrup, such that the second syrup has a total monosaccharides and disaccharides content of less than 15 weight % dry substance, more preferably less than 10 weight % dry substance of the total weight of the dry substance of the second syrup. This advantageously results in the second syrup having a low sugar content, which results in the second syrup being suitable for use in formulations, such as food or beverage products, requiring sugar reduction. Optionally, the monosaccharides and / or disaccharides obtained are removed from the second syrup by chromatography and / or diafiltration and combined with the starting acceptor composition in step (b) to recycle all or a proportion of the monosaccharides and / or disaccharides back into the process. Preferably, only the disaccharides are combined with the starting acceptor composition in step (b) to recycle all or a proportion of the disaccharides back into the process. By combining the disaccharides with the starting acceptor composition, a mixed acceptor composition is formed.

[0097] The recycling of the monosaccharides and disaccharides from the fourth syrup back into the starting acceptor composition improves the overall yield of the non-digestible oligosaccharide in the second syrup. This is because less new substrate is required owing to disaccharides being recycled.

[0098] In some examples of the present invention, enriching the first syrup in step (e) comprises the formation of a third syrup having at least 40 weight % dry substance of monosaccharides of the weight of the total dry substance of the third syrup, and / or the formation of a fourth syrup having at least 40 weight % dry substance of disaccharides comprising both maltose and kojibiose of the total weight of the dry substance of the fourth syrup. Optionally, the fourth syrup obtained from step (e) is combined with the starting acceptor composition in step (b) to recycle the fourth syrup back into the process. Optionally, the fourth syrup obtained from step (e) is filtered before being combined with the starting acceptor composition in step (b) to recycle the fourth syrup back into the process. Optionally, the fourth syrup is filtered by diafiltration. By combining the fourth syrup obtained from step (e) with the starting acceptor composition, a mixed acceptor composition is formed. Optionally,PT-1884-WO-PCT the third syrup is further processed by crystallization, hydrogenation, fermentation, enzymatic conversion, polycondensation, epimerization and / or combinations thereof.

[0099] In some examples of the present invention, the process further comprises the step of refining the first syrup. Optionally, wherein the step of refining the first syrup includes one or more of heating, inactivation of enzyme / polypeptide present, ion exchanging, electrodialysis, evaporation, reverse osmosis, resin polishing, filtration, sterilize filtration, and / or activated carbon treatment. Further optionally, wherein the step of refining the first syrup occurs before step (e). Further optionally, wherein the step of refining is ion exchanging and / or electrodialysis.

[0100] In some examples of the present invention, step (e) comprises enriching by subjecting the first syrup to a polypeptide having alpha-glucosidase catalytic activity. Optionally, wherein the polypeptide having alpha-glucosidase catalytic activity is glucose amylase. Further optionally, wherein the first syrup is subjected to the polypeptide having alpha-glucosidase catalytic activity prior to the step of refining the first syrup.

[0101] In some examples of the present invention, the process can further comprise a step of mixing the third syrup and / or the fourth syrup with one or more of flavours, flavour modulators, colourants, anticaking agents, emulsifiers, sweeteners, high intensity sweeteners, polyols, oligosaccharide, or any combination thereof.

[0102] In some examples of the present invention, the process can further comprise a step of purifying the first syrup and / or second syrup after deactivation of the polypeptides / enzymes, such as by fermentation, enzymatic transformation, filtration, microfiltration, decolorization / deodorization using active carbon or porous polymeric resins (like styrene divinyl benzene resins), demineralization using ion exchange resins, and / or one or more combinations thereof. Composition comprising elongated gluco-oligosaccharides

[0103] In another aspect of the present invention, the present invention relates to a composition comprising elongated gluco-oligosaccharides obtained or obtainable by the processes described above. In some examples of the present invention, the composition comprising elongated gluco-oligosaccharides is the second syrup.

[0104] In some examples of the present invention, the composition comprises elongated gluco-oligosaccharides with at least one α-1,2-terminally linked glucosyl moiety and optionallyPT-1884-WO-PCT one or more α-1,2-non-terminally linked glucosyl moieties, wherein the elongated gluco- oligosaccharides have a degree of polymerisation of 3, 4, 5, 6 or more, and, wherein the composition comprises from 70 weight % or more dry substance of non-digestible oligosaccharide and from 15 weight % or less of dry substance of monosaccharides and disaccharides.

[0105] In some examples of the present invention, the composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation.

[0106] In some examples of the present invention, each gluco-oligosaccharide in the composition comprises at least one α-(1,4)-linkage.

[0107] In some examples of the present invention, two or three glucosyl moieties are linked to the gluco-oligosaccharide, wherein the first glucosyl moiety is linked to the terminal non- reducing end of the gluco-oligosaccharide and the second glucosyl moiety to the previously attached glucosyl moiety.

[0108] In some examples of the present invention, the composition comprises monosaccharides and disaccharides at from 10 weight % or less of dry substance, preferably from 5 weight % or less of dry substance.

[0109] In some examples of the present invention, the composition comprises a non-digestible oligosaccharide at from 70 weight % or more of dry substance, preferably from 75 weight % or more of dry substance, preferably from 78 weight % or more or dry substance, more preferably from 80 weight % or more of dry substance, more preferably from 85 weight % or more of dry substance, more preferably from 90 weight % or more of dry substance, most preferably from 95 weight % or more of dry substance.

[0110] In some examples of the present invention, the composition further comprises at least two different elongated gluco-oligosaccharides selected from:PT-1884-WO-PCT2-O-α-D-glucosyl-maltose (α-D-Glucopyranosyl-(1^2)-α-D-glucopyranosyl-(1^4)- D-glucopyranose); and / or,2-O-α-D-glucosyl-maltotriose (α-D-Glucopyranosyl-(1^2)-α-D-glucopyranosyl- (1^4)-α-D-glucopyranosyl-(1^4)-D-glucopyranose); and / or,PT-1884-WO-PCT2-O-α-D-glucosyl-maltotetraose (α-D-Glucopyranosyl-(1^2)-α-D- glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-D- glucopyranose); and / or2-O-α-D-glucosyl-maltopentaose (α-D-Glucopyranosyl-(1^2)-α-D- glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-α-D- glucopyranosyl-(1^4)-D-glucopyranose); and / or,PT-1884-WO-PCT2-O-α-D-glucosyl-maltohexaose (α-D-Glucopyranosyl-(1^2)-α-D- glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-α-D- glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-D-glucopyranose).

[0111] In particular, the composition of the invention is characterized by elongated gluco- oligosaccharides comprising saccharide linkages selected from α-(1,2)-; α-(1,4)-and other saccharide linkages; wherein the amount of α-(1,4)-linkages is more than 50 % and the amount of α-(1,2)-linkages is less than 50 % of all saccharide linkages, excluding linkages from kojibiose and kojioligosaccharides.

[0112] The ratio of α-(1,4)-linkages and α-(1,2)-linkages can be determined by high performance anion exchange chromatography (HPAEC), and / or NMR techniques and / or structural analysis by methylation.

[0113] The composition may further contain kojibiose and kojioligosaccharides. These compounds also contribute to decreased digestibility of the composition of the invention.

[0114] Kojibiose and kojioligosaccharides are in particular characterized by the following formulas:PT-1884-WO-PCT Kojibiose (α-D-Glucopyranosyl-(1 →2)-D-glucopyranose)Kojitriose (α-D-Glucopyranosyl-(1 →2)-α-D-glucopyranosyl-(1 →2)-D- glucopyranose)Kojitetraose (α-D-Glucopyranosyl-(1 →2)-α-D-glucopyranosyl-(1 →2)-α-D- glucopyranosyl-(1 →2)-D-glucopyranose)Kojipentaose (α-D-Glucopyranosyl-(1 →2)-α-D-glucopyranosyl-(1 →2)-α-D- glucopyranosyl-(1 →2)-α-D-glucopyranosyl-(1 →2)-D-glucopyranose) Digestibility

[0115] Digestibility of the product can be shown by in-vitro digestion tests using in a first stage an alpha-amylase, followed by a digestion stage using a glucoamylase, an alpha- glucosidase or intestinal animal enzymes. Both stages can be combined to one digestionPT-1884-WO-PCT reaction using alpha-amylase and a glucoamylase or an alpha-glucosidase and intestinal animal enzymes.

[0116] Other advantageous properties of the product besides the lower calories compared to sucrose, are a high-water solubility, viscosities comparable to commercial glucose syrups and maltodextrins, lower than existing commercial bulking agents (such an inulin and resistant dextrin) and a lower relative sweetness than sucrose. Composition comprising monosaccharides

[0117] In another aspect, the present invention relates to a composition comprising monosaccharides obtained or obtainable by the processes described above. The composition comprising monosaccharides is the third syrup.

[0118] In some examples of the present invention, the composition (third syrup) comprises at least 40 weight % dry substance of monosaccharides of the total weight of the dry substance of the composition (third syrup). In some examples of the present invention, the composition (third syrup) comprises at least 40 weight % dry substance of monosaccharides and at most 60 weight % dry substance disaccharides comprising both maltose and kojibiose and gluco- oligosaccharides having a degree of polymerisation of 3, 4, 5, 6 or more and higher of the total weight of the dry substance of the composition (third syrup).

[0119] Optionally, the composition (third syrup) comprises at least 85 weight % dry substance of monosaccharides, preferably at least 90 weight % dry substance of monosaccharides, more preferably at least 95 weight % dry substance of monosaccharides, most preferably at least 99.8 weight % dry substance of monosaccharides of the total weight of the dry substance of the composition (third syrup).

[0120] Optionally, the composition (third syrup) comprises at most 50 weight % dry substance disaccharides, preferably at most 40 weight % dry substance disaccharides of the total weight of the dry substance of the composition (third syrup). Composition comprising disaccharides

[0121] In another aspect, the present invention relates to a composition comprising disaccharides obtained or obtainable by the processes described above. The composition comprising disaccharides is the fourth syrup. Optionally, the disaccharides include both maltose and kojibiose.PT-1884-WO-PCT

[0122] In some examples of the present invention, the composition (fourth syrup) comprises at least 40 weight % dry substance disaccharides comprising both maltose and kojibiose of the total weight of the dry substance of the composition (fourth syrup). In some examples of the present invention, the composition (fourth syrup) comprises at least 40 weight % dry substance disaccharides comprising both maltose and kojibiose, and, at most 60 weight % dry substance monosaccharides and gluco-oligosaccharides having a degree of polymerisation of 3, 4, 5, 6 or higher of the total weight of the dry substance of the composition (fourth syrup).

[0123] Optionally, the composition (fourth syrup) comprises at least 50 weight % dry substance disaccharides, preferably at least 60 weight % dry substance disaccharides, more preferably at least 70 weight % dry substance disaccharides of the total weight of the dry substance of the composition (fourth syrup). Use of the compositions obtained

[0124] In some examples of the present invention, the composition can be used in the form of a syrup or it can be dried down to be used in the form of a powder.

[0125] In some examples of the present invention, the composition can be used in foodstuffs for human consumption. In particular it can be used as a sweetener, sugar replacer, (low calorie) bulking agent, oligosaccharides, low calorie carbohydrate composition, prebiotic composition, low glycemic carbohydrate composition, a starch retrograding inhibiting agent, a crystallization inhibiting agent, an osmotic pressure regulator or a water activity regulator. In particular it can be used for calorie and / or sugar reduction and / or fiber enrichment.

[0126] In some examples of the present invention, the composition can be used in foodstuffs in combination with other bulking agents, fillers, soluble oligosaccharides, resistant starch, polydextrose, dextrins, resistant maltodextrin, inulin, or insoluble oligosaccharides. The combination can boost the oligosaccharide content of the foodstuff, enhance physiological benefits from consumption of the foodstuff, reduce the calorie content, reduce the sugars content, and / or enhance the nutritional profile of the foodstuff.

[0127] In some examples of the present invention, the composition can also be used in foodstuffs in combination with sweeteners, including high intensity sweeteners (such as sucralose, saccharin, aspartame, acesulfame K, brazzein, mogrosides (such as those extracted from Luo Han Guo fruit, including mogroside V) and steviol glycosides (such as those extracted from the stevia plant, including rebaudiosides and stevioside, or those obtained byPT-1884-WO-PCT fermentation or bioconversion), low calorie sweeteners (such as polyols, including erythritol, sorbitol, xylitol, lactitol, maltitol, mannitol, isomalt, etc.) and conventional caloric sweeteners.

[0128] Foodstuffs, in which the composition of the invention can be incorporated, are not limited and include: - bakery products, both yeast-raised (including donuts, sweet doughs, breads, brioche etc.) or chemically leavened (including cookies, cookie crisps, biscuits, muffins, cakes, brownies etc.); - breakfast cereals (including extruded cereals, puffed cereals etc.); - dairy products, for instance yogurt, yogurt drinks, dairy milk drinks, smoothies, ice cream, shakes, cottage cheese, dairy-based dressing, and dairy-based desserts (including mousse, puddings etc.) and the like; - confectionery products, for instance hard candies, fondants, nougats, marshmallows, gelatin jelly candies or gummies or jelly beans, jellies, chocolate, liquorice, chewing gum, caramels, toffees, chews, mints, tableted confections, and processed fruit snacks, chocolates, chocolate coatings, and icings (including frosting and glazes) and the like; - snacks, including puffed, extruded or sheeted or texturized snacks, for instance chips / crisps, crackers; - cereal bars, power bars, protein bars, meal replacement bars, granola bars, nutrition bars and the like; - fruit-based jams and jellies (or other gelatin-containing desserts); - fat-based fillings for sweet or savoury foods; - beverages, for instance carbonated beverages, fruit juices, concentrated juice mixes or syrups for dilution, clear-flavoured waters, protein shakes, and beverage dry mixes for dissolution; - and others: soups, syrups, sauces, dressings, edible films, coffee creamers, processed meats and fish, infant and toddler food, sport drinks and food, dietary supplements, clinical nutritional composition.

[0129] In some examples of the present invention, the composition can be used in feed or petfood (dry or moist) for animal consumption. For instance, it can be used as prebiotic composition to improve animal digestive health and / or to reduce calories and / or sugar content.

[0130] In some examples of the present invention, the composition can be used in personal care products, in particular cosmetics (for instance skin creams) and oral care products (forPT-1884-WO-PCT instance toothpaste). For instance, it can be used as personal care composition to improve the microbiome found on the skin. Or, for instance, it can be used in toothpaste or similar oral care compositions to improve the microbiome found in the mouth.

[0131] In some examples of the present invention, the composition may be utilized as a prebiotic and may also be coupled with a probiotic delivery system, for instance used in combination with one or more probiotics in a foodstuff. By "prebiotic" it is meant a food ingredient that beneficially affects the subject (human or animal) by selectively stimulating the growth and / or activity of one or a limited number of bacteria in the gastro-intestinal tract, particularly the colon, and thus improves the digestive health of the host. By "probiotic" it is meant living microbiological dietary supplements that provide beneficial effects to the subject through their passage and function in the digestive tract.

[0132] In some examples of the present invention, the composition may be utilized as a prebiotic and may also be coupled with a postbiotic delivery system, for instance used in combination with one or more postbiotics in a foodstuff. By "postbiotic" (also known as metabiotics, biogenics, or simply metabolites) it is meant soluble factors (metabolic products or byproducts), secreted by live bacteria, or released after bacterial lysis providing physiological benefits to the host. These are typically bioactive compounds the probiotic bacteria produce when they consume prebiotics (such as dietary fibers).

[0133] Foodstuffs of the present invention can also be used to help control the blood glucose concentration (glycemia) in humans and animals, for instance that suffer from insulin resistance, pre-diabetes or diabetes. When the foodstuff is consumed, the slowly digestible and / or digestion resistant components in the foodstuff coming from the composition of the invention can cause a more moderate relative glycemic response in the bloodstream, which can be beneficial for subjects with impaired glucose and / or insulin metabolism.

[0134] The foodstuff can also be a nutraceutical, a dietary supplement, or a functional, fortified, and / or enriched food providing health benefits to the consumer. DRAWINGS

[0135] Figure 1 provides an overview of the coupled reaction using maltose phosphorylase (MP) and kojibiose phosphorylase (KP).

[0136] Figure 2 provides an example of a chromatogram.PT-1884-WO-PCT EXAMPLES

[0137] The following are non-limiting examples that discuss, with reference to tables and figures, the advantages of the present invention. The examples set forth herein are merely examples among other possible examples. Enzyme production (CsKP, LaMP) Gene cloning and transformation

[0138] The gene encoding the Caldicellulosiruptor saccharolyticus kojibiose phosphorylase (CsKP, UniProt identifier A4XGP2) was codon optimized for E. Coli and synthesized. The Lactobacillus acidophilus maltose phosphorylase (LaMP, UniProt identifier Q5FI04) was codon optimized for E. Coli and synthesized (Life Technologies, Merelbeke, Belgium). Sequences were subsequently subcloned into a pET21 vector at NheI and XhoI restriction sites, consequently introducing a C-terminal His6-tag. The plasmid was transformed in E. Coli BL21(DE3) electrocompetent cells. DNA sequences of the resulting Caldicellulosiruptor saccharolyticus kojibiose phosphorylase gene (CsKP) (SEQ ID NO: 1) and the Lactobacillus acidophilus maltose phosphorylase gene (LaMP) (SEQ ID NO: 6) were expressed into the respective enzymes Caldicellulosiruptor saccharolyticus kojibiose phosphorylase (SEQ ID NO: 1) and Lactobacillus acidophilus maltose phosphorylase (SEQ ID NO: 6) and recovered. Enzyme expression and recovery

[0139] An overnight culture was inoculated (2 %) in 500 mL LB-Lennox medium containing 100 μg / mL ampicillin in a 2-L shake flask and incubated at 37 °C with continuous shaking at 200 rpm. The cultures were grown to OD6000.6, and expression of both enzymes (in pET21a) was induced by adding isopropyl β-D-1-thio-galactopyranoside to a final concentration of 0.1 mM. Gene expression of CsKP took place for 16 hours at 30 °C. Gene expression of LaMP took place for 16 hours at 37 °C. The cultures were then centrifuged (15 min, 9000 rpm), and the cell pellets were frozen at − 20 °C for at least 4 hours.

[0140] To extract the enzymes, the cell pellets were thawed and dissolved in 10 mL lysis buffer consisting of 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg / mL lysozyme, 10 mM imidazole and 50 mM phosphate buffered saline (PBS), pH 7.4. This suspension wasPT-1884-WO-PCT incubated on ice for 30 min and sonicated three times for 3 min (Branson Sonifier 250, level 3, 50 % duty cycle).

[0141] The cell debris was removed by centrifugation at 9000 rpm for 1 hour at 4 °C. The resulting cell extract was further purified by means of heat treatment. After an incubation of 1 hour at 60 °C, the heat-treated protein solutions were centrifuged two times for 30 min at 9000 rpm and the clear enzyme solution was filtered over a sterile 0.22 μm ø PES membrane filter (Millex® Syringe Filters 0.22 micrometre polyethersulfone, Merck-Millipore) to remove any suspended solids and stored at 4̊C.

[0142] The resulting cell extract was further purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography as described by the supplier (MCLab, San Francisco, USA), after which the buffer was exchanged to 50 mM 2-morpholinoethanesulfonic acid (MES, pH 6.5) in a 30-kDa Amicon Ultra centrifugal filter (Merck-Millipore, Burlington, Massachusetts, USA). Protein concentration could be measured with a Nanodrop ND-1000 (Thermo Scientific, Rockford, USA) using the molecular weight and extinction coefficients calculated with the ProtParam tool on the ExPASy server (http: / / web.expasy.org / protparam / ). Molecular weight and purity were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 10 % gel). Determination of enzyme activity

[0143] Activity of the purified enzymes was routinely determined by phosphorolysis of kojibiose and maltose for kojibiose phosphorylase (CsKP) and maltose phosphorylase (LaMP), respectively. To enable quantification of the enzyme activity, a standard curve was made in the range of 0-500 microM glucose. The glucose standard curve and glucose released by phosphorolysis of kojibiose and maltose was quantified with a colorimetric enzymatic coupled assay using glucose oxidase and peroxidase (GOD-POD). Glucose Oxidase (GOD) converts glucose into gluconolacton, producing hydrogen peroxide. The peroxide is subsequently reduced by peroxidase (POD), using ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) as electron donor. The oxidized ABTSH has a green colour, which can be measured at 420 nm.

[0144] Activity measurements were performed on 1-mL scale with a reaction mixture containing 100 mM kojibiose or maltose in 50 mM sodium phosphate (NaH2PO4 / Na2HPO4) buffer, pH 6.5 at 55 ˚C and shaken at 800 rpm.25-microL samples were taken at set timepointsPT-1884-WO-PCT (0, 1, 2, 4, 6, 8 and 10 min), and inactivated in 25 microL 0.2 M NaOAc buffer pH 2.5. Next, 200 microL GOD-POD reagent (173 microL POD solution (40 mg / mL in 0.2 M acetate buffer pH 4.5), 50 mg ABTS, 45.26 mg GOD, 0.2 M acetate buffer pH 4.5 to 100 mL) was added,and incubated at 37̊ C for 30 min. Finally, absorbance was measured with a spectrophotometerat 420 nm. Enzyme activity (Units) was calculated as the rate of glucose release per unit of time, in micromol glucose per second (1 U = 1 micromol / s). Example 1: Analysis of the effect of the concentration of dextrose in the acceptor composition

[0145] In this non-limiting example of the present invention, the effect of the dextrose concentration in the acceptor composition on the amount of non-digestible oligosaccharide present in the first syrup was analysed.

[0146] In this non-limiting example, five acceptor compositions were separately prepared in five separate Falcon test tubes. Four of the five acceptor compositions were obtained by combining a crude kojibiose composition (obtainable from any oligosaccharide supplier) with dextrose (obtainable from any fine chemicals or food ingredients supplier).

[0147] The five acceptor compositions produced were called AC1, AC2, AC3, AC4 and AC5. The compositions of the five acceptor compositions are set out in Table 1.

[0148] AC1 is a starting acceptor composition that simulates a starting acceptor composition wherein no fourth syrup has been added. AC2 is the crude kojibiose that, like AC1, is a starting acceptor composition that simulates a starting acceptor composition wherein no fourth syrup has been added. AC3, AC4 and AC5 represent mixed acceptor compositions that have been obtained by the enrichment of AC2 with dextrose.PT-1884-WO-PCT Table 1: Compositions of the acceptor compositions AC1, AC2, AC3, AC4 and AC5 (each component of the composition is present in terms of weight % of the total weight of the dry substance of the acceptor composition). Where the total weight % does not add to 100, the remainder is impurities. %tw / tw

[0149] Once the desired composition of the acceptor compositions had been achieved, each of the acceptor composition were separately mixed with demineralised water and sodium phosphate buffer to provide an acceptor composition at 33-34 % dry substance. Each of the five acceptor compositions when mixed with the demineralised water and sodium phosphate buffer had a pH of 6.5 and a sodium phosphate buffer concentration of 10 millimolar.

[0150] Each of the acceptor compositions, after mixing with the demineralised water and sodium phosphate buffer, were separately combined with 4.1 CsKP Units / g dry substance and 2.4 LaMP Units / g dry substance to form a first syrup corresponding to AC1, AC2, AC3, AC4 and AC5 in each of the separate falcon test tubes. The CsKP enzyme was added only once at the beginning of the reaction, whereas the LaMP enzyme was renewed every 24 hours by adding 2.4 Units / g dry substance each 24 hours to each separate falcon test tube.

[0151] The Falcon test tubes were then placed in an Eppendorf Thermomixer at 55 °C. At 96 hours, two 0.7 ml samples of the first syrup for each of the acceptor composition were separately transferred to two separate 2 ml Eppendorf vials. The 2 ml Eppendorf vials were placed in an Eppendorf Thermomixer at 100 °C for 10 minutes with 650 rpm stirring. By placing the 2 ml Eppendorf vials in the Eppendorf Thermomixer at these conditions, the enzyme present in the 2 ml Eppendorf vials was inactivated. Inactivated first and second samples of the first syrup were formed. The inactivated first and second samples of the first syrup were optionally frozen prior to further treatment.PT-1884-WO-PCT

[0152] The inactivated first sample of the first syrup then underwent digestion with glucoamylase. The inactivated second sample of the first syrup did not. The glucoamylase used for the glucoamylase treatment was Dextrozyme Peak from Novo (GA242). The buffer sodium acetate / acetic acid buffer at a concentration of 0.2 M and pH of 4.5 was used. For the glucoamylase digestion, the inactivated first sample was first diluted with water until the inactivated first sample had a concentration of 3-35 % dry substance. The sodium acetate / acetic acid buffer was then added to the inactivated first sample until the buffer was present at a concentration of 0.05 M and the inactivated first sample had a pH of 4.5. Then the inactivated first sample was heated to 50 °C. Once these conditions were achieved, the conditions were maintained for 24 hours. At 24 hours, the enzyme in the inactivated first sample was then deactivated by firstly heating the inactivated first sample to 100°C for 10 minutes, followed by cooling the inactivated first sample to room temperature. The inactivated first sample was then labelled and stored in a freezer to form a frozen inactivated first sample. The inactivated second sample underwent the same treatment as the inactivated first sample, except that there was no digestion with glucoamylase. The inactivated second sample was labelled and stored in a freezer to form a frozen inactivated second sample.

[0153] To prepare the frozen inactivated samples for HPLC (high pressure liquid chromatography), the frozen inactivated first sample and frozen inactivated second sample were defrosted and then diluted by the addition of water until each sample contained from 5 to 10 % dry substance. Then 1.5 ml of each sample was separately passed through a 0.3 g mixed bed BioRad resin whilst being exposed to a discontinuous vortex for 20° and 10° swirl. Each of the samples were then separately filtered over a 0.45 µm Sartorius disposable filter and injected into a high-pressure liquid chromatography (HPLC) column. The conditions of the HPLC analysis are set out in Table 2 and an example of a typical chromatogram is shown in Figure 2.PT-1884-WO-PCT Table 2: The conditions of the HPLC analysis.

[0154] The chromatography results of each sample is shown in Table 3 with respect to dextrose concentration in the acceptor composition. The % of dextrose after 96 hours in Table 3 corresponds to the relative percentage area of dextrose from the HPLC results of the inactivated second sample. The % of non-digestible oligosaccharides after 96 hours in Table 3 corresponds to the relative area of the oligosaccharides from the HPLC results of the inactivated first sample. The % of dextrose in the acceptor composition in Table 3 was determined by HPLC using the same conditions as set out above. Table 3: The effect of the initial amount of dextrose on the amount of dextrose and oligosaccharides after 96 hours determined by HPLC (wherein the results are shown in terms of weight % of the total weight of the composition). Where the total weight % does not add to 100, the remainder is impurities.

[0155] From Tables 1 and 3, it can be observed that the initial concentration of dextrose in the acceptor composition should not exceed 5 weight % of the total weight of the dry substance ofPT-1884-WO-PCT the acceptor composition in order to achieve an amount of non-digestible oligosaccharides in the first syrup that is equivalent to the amount of non-digestible oligosaccharides in the first syrup when the acceptor composition is not combined with a fourth syrup (AC1). Example 2: Analysis of the effect of the concentration of kojibiose in the acceptor composition

[0156] In this non-limiting example of the present invention, the effect of the kojibiose concentration in the acceptor composition on the amount of non-digestible oligosaccharide present in the first syrup was analysed.

[0157] In this non-limiting example, five acceptor compositions were separately prepared in five separate Falcon test tubes. Four of the five acceptor compositions were obtained by combining crude kojibiose (obtainable from any oligosaccharide supplier) with kojibiose and / or dextrose (obtainable from any fine chemicals or food ingredients supplier).

[0158] The five acceptor compositions produced were called AC1, AC2, AC6, AC7 and AC8. The compositions of the five acceptor compositions are set out in Table 4. AC1 and AC2 are the same as in Example 1. AC6, AC7 and AC8 represent mixed acceptor compositions that have been enriched with kojibiose. Table 4: Composition of the acceptor compositions AC1, AC2, AC6, AC7 and AC8 (each component of the composition is present in terms of weight % of the total weight of the dry substance of the composition). Where the total weight % does not add to 100, the remainder is impurities. ) %tw / tw(

[0159] Once the desired composition of the acceptor compositions had been achieved, each of the acceptor compositions were separately mixed with demineralised water and sodiumPT-1884-WO-PCT phosphate buffer to provide a acceptor composition at 33-34 % dry substance. Each of the five acceptor compositions when mixed with the demineralised water and sodium phosphate buffer had a pH of 6.5 and a sodium phosphate buffer concentration of 10 millimolar.

[0160] Each of the acceptor compositions, after mixing with the demineralised water and sodium phosphate buffer, were separately combined with 4.1 CsKP Units / g dry substance and 2.4 LaMP Units / g dry substance to form a first syrup corresponding to AC1, AC2, AC6, AC7 and AC8 in each of the separate falcon test tubes. The CsKP enzyme was added only once at the beginning of the reaction, whereas the LaMP enzyme was renewed every 24 hours by adding 2.4 Units / g dry substance each 24 hours to each separate falcon test tube.

[0161] The Falcon test tubes were then placed in an Eppendorf Thermomixer at 55 °C. At 96 hours, two 0.7 ml samples of the first syrup for each of the acceptor composition were separately transferred to two separate 2 ml Eppendorf vials. The 2 ml Eppendorf vials were placed in an Eppendorf Thermomixer at 100 °C for 10 minutes with 650 rpm stirring. By placing the 2 ml Eppendorf vials in the Eppendorf Thermomixer at these conditions, the enzyme present in the 2 ml Eppendorf vials was inactivated. Inactivated first and second samples of the first syrups were formed. The inactivated first and second samples of the first syrup were optionally frozen prior to further treatment.

[0162] The inactivated first sample of the first syrup then underwent digestion with glucoamylase. The inactivated second sample of the first syrup did not. The glucoamylase used for the glucoamylase treatment was Dextrozyme Peak from Novo (GA242). The buffer sodium acetate / acetic acid buffer at a concentration of 0.2 M and pH of 4.5 was used. For the glucoamylase digestion, the inactivated first sample was first diluted with water until the inactivated first sample had a concentration of 3-35 % dry substance. The sodium acetate / acetic acid buffer was then added until the buffer was present at a concentration of 0.05 M and the inactivated first sample had a pH of 4.5. Then the inactivated first sample was heated to 50 °C. Once these conditions were achieved, the conditions were maintained for 24 hours. At 24 hours, the enzyme in the inactivated first sample was then deactivated by firstly heating the inactivated first sample to 100 °C for 10 minutes, followed by cooling the inactivated first sample to room temperature. The inactivated first sample was then separately labelled and stored in a freezer to form a frozen inactivated first sample. The inactivated second sample underwent the same treatment as the inactivated first sample, except that there was no digestion with glucoamylase.PT-1884-WO-PCT The inactivated second sample was labelled and stored in a freezer to form a frozen inactivated second sample.

[0163] To prepare the frozen inactivated samples for HPLC (high pressure liquid chromatography), the frozen inactivated first sample and frozen inactivated second sample were defrosted and then diluted by the addition of water until each sample contained from 5 to 10 % dry substance. Then 1.5 ml of the sample was separately passed through a 0.3 g mixed bed BioRad resin whilst being exposed to a discontinuous vortex for 20° and 10° swirl. Each of the samples were then separately filtered over a 0.45 µm Sartorius disposable filter and injected into a high-pressure liquid chromatography (HPLC) column. The particular conditions of the HPLC analysis are set out in Table 2 and an example of a typical chromatogram is shown in Figure 2.

[0164] The chromatography results of each sample are shown in Table 5 with respect to kojibiose concentration in the acceptor composition.

[0165] The % of dextrose after 96 hours in Table 4 corresponds to the relative percentage area of dextrose from the HPLC results of the inactivated second sample. The % of non-digestible oligosaccharides after 96 hours in Table 4 corresponds to the relative area of the oligosaccharides from the HPLC results of the inactivated first sample. The % of dextrose in the acceptor composition was determined by HPLC using the same conditions as set out above.PT-1884-WO-PCT Table 5: The effect of the initial amount of disaccharides (maltose and kojibiose) on the amount of dextrose and non-digestible oligosaccharides after 96 hours determined by HPLC (wherein the results are shown in terms of weight % of the total weight of the dry substance of the composition). Where the total weight % does not add to 100, the remainder is impurities.

[0166] From Table 5, it can be observed that after 96 hours, AC2, AC6, AC7 and AC8 contain a significantly higher amount of non-digestible oligosaccharides than AC1. AC1 is a starting acceptor composition that does not contain kojibiose, whereas AC2, AC6, AC7 and AC8 contain kojibiose. It can be concluded that kojibiose is a suitable starting material.

[0167] Table 5 additionally shows that the the amount of dextrose after 96 hours of reaction is higher in the case of AC1 than in the cases of AC2, AC6, AC7 and AC8. The formation of a high percentage of dextrose present after 96 hours is one explanation of why AC1 results in the formation of less non-digestible oligosaccharide compared to the other compositions. The formation of dextrose and the non-digestible oligosaccharide is in equilibrium and, without wishing to be bound by theory, it could be assumed that when one of the products of the reaction reaches a certain threshold, equilibrium is reached and the reaction does not progress any further. Example 3: Formation and analysis of the syrups obtained by enrichment

[0168] In this non-limiting example of the present invention, the composition of different syrups obtained by enrichment were analysed.

[0169] In this non-limiting example, a starting acceptor composition was produced by diluting maltose (in syrup form) in demineralized water until the starting acceptor composition had a dry substance of 35 % of the total weight of the acceptor composition.PT-1884-WO-PCT

[0170] The starting acceptor composition produced was called AC9. The composition of AC9 is set out in Table 6. Table 6: Composition of AC9. Each component of the composition is present in terms of weight % of the total dry substance of the composition. Where the total weight % does not add to 100, the remainder is impurities. ) %tw / tw(

[0171] Once the desired composition of the starting acceptor composition had been achieved, the starting acceptor composition was then mixed with demineralised water and a sodium phosphate buffer to provide a starting acceptor composition at 34-35 % dry substance. The starting acceptor composition when mixed with the sodium phosphate buffer had a pH of 6.5 and a sodium phosphate buffer concentration of 10 millimolar.

[0172] The starting acceptor composition, after mixing with the demineralised water and sodium phosphate buffer, was combined with 4.1 CsKP Units / g dry substance and 2.4 LaMP Units / g dry substance over a 168 hour period to form a first syrup. The CsKP enzyme was added only once at the beginning of the reaction, whereas the LaMP enzyme was renewed every 24 hours by adding 2.4 Units / g dry substance each 24 hours. The mixture of starting acceptor composition, demineralized water, sodium phosphate buffer and enzymes was stirred at 30 rpm and the temperature maintained constant at 55 °C over the 168 hour period.

[0173] Every 24 hours, two 0.7 ml samples of the first syrup were taken from the reactor and transferred to two separate 2 ml Eppendorf vials. The 2 ml Eppendorf vials were placed in an Eppendorf Thermomixer at 100 °C for 10 minutes with 650 rpm stirring. By placing the 2 ml Eppendorf vials in the Eppendorf Thermomixer at these conditions, the enzyme present in the 2 ml Eppendorf vials was inactivated. An inactivated first and second sample of the first syrup were formed every 24 hours until 168 hours was achieved.

[0174] The inactivated first samples then separately underwent digestion with glucoamylase. The inactivated second samples did not undergo digestion with glucoamylase.PT-1884-WO-PCT

[0175] The glucoamylase used was Dextrozyme Peak from Novo (GA242). The buffer used was sodium acetate / acetic acid buffer at a concentration of 0.2 M and pH of 4.5. For the glucoamylase digestion, each of the inactivated first samples were diluted with water until the inactivated first samples had a dry substance concentration of 3-35 % dry substance. The sodium acetate / acetic acid buffer was then added until the buffer had a concentration of 0.05 M and the inactivated first samples had a pH of 4.5. Then the inactivated first samples were heated to 50 °C. Once these conditions were achieved, the conditions were maintained for 24 hours. At 24 hours, the inactivated first samples were heated to 100 °C for 10 minutes and then cooled to room temperature. The inactivated second samples underwent all the following preceding treatments as the inactivated first samples, except the addition of glucoamylase. The inactivated first and second samples were then stored in a freezer to form a frozen inactivated first sample and a frozen inactivated second sample.

[0176] To prepare the frozen inactivated first samples and the frozen inactivated second samples for chromatography, the samples were defrosted. The defrosted samples were then separately diluted by the addition of water until each sample contained from 5-10 % dry substance. Then 1.5 ml of each sample was separately passed through a 0.3 g mixed bed BioRad resin whilst being exposed to a discontinuous vortex for 20° and 10° swirl. Each sample was then separately filtered over a 0.45 µm Sartorius disposable filter and injected into a high- pressure liquid chromatography (HPLC) column. The particular conditions of the HPLC analysis are set out in Table 2 and an example of a typical chromatogram is shown in Figure 2.

[0177] Determination of the composition of the acceptor composition and of the different first syrups was obtained by using the HPLC results of the corresponding inactivated first sample and inactivated second sample with the following equations: - % Dextrose = % Dextrose without glucoamylase treatment. - % Maltose = % Disaccharides without glucoamylase treatment - % disaccharides after glucoamylase treatment. - % Kojibiose = % Disaccharides after glucoamylase treatment. - % Digestible oligosaccharides (DP3) = % oligosaccharides with a degree of polymersation equal to three without glucoamylase treatment - % oligosaccharides with a degree of polymersation equal to three after glucoamylase treatment.PT-1884-WO-PCT - % Digestible oligosaccharides (DP≥4) = % oligosaccharides with a degree of polymersation equal or higher than four without glucoamylase treatment - % oligosaccharides with a degree of polymersation equal or higher than four with glucoamylase treatment. - % Non-digestible oligosaccharides (DP3) = % oligosaccharides with a degree of polymersation equal to three after glucoamylase treatment. - %Non-digestible oligosaccharides (DP≥4) = % oligosaccharides with a degree of polymersation equal or higher than four after glucoamylase treatment.

[0178] The composition of the first syrup after the different time intervals is given in Table 7. The first syrups are called AC10-1, AC10-2, AC10-3, AC10-4 and AC10-7 and correspond to the composition of the first syrup after 24 hours, 48 hours, 72 hours, 94 hours and 168 hours respectively. Table 7: The composition of AC10-1, AC10-2, AC10-3, AC10-4 and AC10-7 (each component of the composition is present in terms of weight % of the total weight of composition). Where the total weight % does not add to 100, the remainder is impurities. ) %tw / tw(

[0179] As shown in Table 7, after 4 days of reaction the amount of non-digestible oligosaccharides and the amount of dextrose reaches a maximum. The results shown in Table 7 (pilot scale) are in-line with the results shown in Table 3 (laboratory scale).

[0180] After the 168 hours incubation, the first syrup (AC10-7) then underwent heat treatment at 100 °C for 10 minutes followed by cooling to a temperature of 50 °C. The first syrup was then passed through an ultrafiltration unit equipped with a 20.000 Dalton membrane, whereinPT-1884-WO-PCT the ultrafiltration unit is operating at a pressure of 2.5 bars. Upon passing through the ultrafiltration unit, a second syrup was formed. The second syrup was present in the permeate obtained from the ultrafiltration unit, whilst the retentate contained the denatured enzymes (CsKP and LaMP).

[0181] The second syrup underwent ion exchange treatment: cation exchange treatment on a Lanxess S2568 resin followed with an anion exchange treatment on a Lanxess S4468 to form an ion-exchange treated second syrup. The ion exchange columns were fed with the second syrup, where the second syrup had been diluted to have a dry substance concentration of 35 % weight per total weight of the second syrup. The temperature of the treatment was 50 °C, the ratio of syrup volume on resin volume was 1 and the flow rate through the resin was 10 L / h. The ion exchange treatment was done in a such way that the conductivity of the syrup coming out of the anion exchange resin was below 25 µS / cm.

[0182] The ion-exchange treated second syrup was then concentrated under vacuum to form a concentrated second syrup. The ion-exchange treated second syrup was concentrated under vacuum until the concentrated second syrup had a dry substance 48-50 % of the total weight of the ion-exchange treated second syrup. The concentrated second syrup can optionally be frozen until further treatment.

[0183] The concentrated second syrup then underwent fractionation in a 6-column Improved Simulated Moving Bed (ISMB) system. The resin used was a DiaionTMUBK 530 from Resindion s.r.l which consisted of sulphonated copolymer of styrene and divinylbenzene. The ISMB chromatographic system enabled the separation of the concentrated second syrup into three fractions called (1) second syrup rich in non-digestible oligosaccharides, (2) third syrup rich in monosaccharides and (3) fourth syrup rich in disaccharides.

[0184] Two samples were taken from each of the three fractions. The first sample of each fraction underwent digestion with a glucoamylase enzyme whereas the second sample of each fraction did not. Both samples were treated in the same way, except that the second samples were not mixed with the glucoamylase enzyme.

[0185] The glucoamylase used was Dextrozyme Peak from Novo (GA242). The buffer used was sodium acetate / acetic acid buffer at a concentration of 0.2 M and pH of 4.5. For the glucoamylase digestion, each of the samples were diluted with water until the samples had a dry substance concentration of 3-35 % dry substance. The sodium acetate / acetic acid buffer was then added until the buffer had a concentration of 0.05 M and the samples had a pH of 4.5.PT-1884-WO-PCT Then the samples were heated to 50 °C. Once these conditions were achieved, the conditions were maintained for 24 hours. At 24 hours, the samples were heated to 100 °C for 10 minutes and then cooled to room temperature. The samples that did not undergo digestion with glucoamylase underwent all the following treatments as the other samples, except the addition of glucoamylase. The samples were then stored in a freezer to form frozen samples.

[0186] To prepare the frozen samples for chromatography, the samples were defrosted. The defrosted samples were then separately diluted by the addition of water until each sample contained from 5-10 % dry substance. Then 1.5 ml of each sample was separately passed through a 0.3 g mixed bed BioRad resin whilst being exposed to a discontinuous vortex for 20° and 10° swirl. Each sample was then separately filtered over a 0.45 µm Sartorius disposable filter and injected into a high-pressure liquid chromatography (HPLC) column. The particular conditions of the HPLC analysis are set out in Table 2 and an example of a typical chromatogram is shown in Figure 2.

[0187] Determination of the different components of each syrup was obtained by the combination of the HPLC results of the corresponding samples for each syrup, the equations used are: - % Dextrose = % Dextrose without glucoamylase treatment. - % Maltose = % Disaccharides without glucoamylase treatment - % disaccharides after glucoamylase treatment. - % Kojibiose = % Disaccharides after glucoamylase treatment. - % Digestible oligosaccharides (DP3) = % oligosaccharides with a degree of polymersation equal to three without glucoamylase treatment - % oligosaccharides with a degree of polymersation equal to three after glucoamylase treatment. - % Digestible oligosaccharides (DP≥4) = % oligosaccharides with a degree of polymersation equal or higher than four without glucoamylase treatment - % oligosaccharides with a degree of polymersation equal or higher than four with glucoamylase treatment. - % Non-digestible oligosaccharides (DP3) = % oligosaccharides with a degree of polymersation equal to three after glucoamylase treatment. - %Non-digestible oligosaccharides (DP≥4) = % oligosaccharides with a degree of polymersation equal or higher than four after glucoamylase treatment.PT-1884-WO-PCT

[0188] The composition of the second syrup, third syrup and fourth syrup are shown in Table 8. Table 8: The composition of the second syrup rich in non-digestible oligosaccharides (called second syrup in Table 8), third syrup rich in monosaccharides (called third syrup in Table 8) and fourth syrup rich in disaccharides (called fourth syrup in Table 8). Each component of the composition is present in terms of weight % of the total weight of each syrup. Where the total weight % does not add to 100, the remainder is impurities. ) %tw / tw(As shown in Table 8, the second syrup is rich in non- digestible oligosaccharides, the third syrup is rich in monosaccharides and the fourth syrup is rich in disaccharides. Example 4: Analysis of a diafiltration step on the fourth syrup

[0189] In this non- limiting example of the present invention, the effect of including a diafiltration step on the fourth syrup was analysed.

[0190] In this non-limiting example, the fourth syrup that was produced in example 3 was further treated with diafiltration step. The composition of the fourth syrup is provided in Table 9.PT-1884-WO-PCT Table 9: The composition of the fourth syrup. The composition is presented in terms of weight % of the total weight of the fourth syrup.

[0191] The fourth syrup was treated with diafiltration using a pilot unit from Koch membrane systems. The equipment had an internal circulation loop. The membrane used was an NFW (TFC 300-500 Da) from Synder filtration, USA. The membrane had a filtration surface of 7.6 m2. The filtration temperature was maintained at between 40 and 45 °C and the filtration pressure was fixed at 30 bars. The fourth syrup, prior to diafiltration, had a dry substance of 23 % of the total weight of the fourth syrup. The retentate flow recycle was maintained at 5 m3 / h and the permeate flow was maintained at 50 L / h.m2.

[0192] The diafiltration took place in 6 successive diafiltration steps. Each step consisted of adding demineralized water in a quantity equivalent to the half of the mass of the permeate and in removing the same quantity of permeate. The resulting diafiltration factor was therefore three.

[0193] The composition of the fourth syrup post diafiltration is shown in Table 10.

[0194] At the end of the diafiltration operation, the retentate was added to a starting acceptor composition to form a mixed acceptor composition. The composition of the starting acceptor composition used, and the mixed acceptor composition formed (AC11) is shown in Table 10. Table 10: The composition of the fourth syrup after diafiltration, the starting acceptor composition used in this non-limiting example and the composition of the mixed acceptor composition formed (AC11).

[0195] AC11 was then mixed with demineralised water and sodium phosphate buffer to provide an acceptor composition at 33-34 % dry substance. The acceptor composition whenPT-1884-WO-PCT mixed with the demineralised water and sodium phosphate buffer had a pH of 6.5 and a sodium phosphate buffer concentration of 10 millimolar.

[0196] AC11, after mixing with the demineralised water and sodium phosphate buffer, was combined with 4.1 CsKP Units / g dry substance and 2.4 LaMP Units / g dry substance and underwent the same reaction conditions, sampling, and analytics as AC10 in example 3 to form AC11-7 (the syrup after 168 hours reaction). The composition of AC11-7 is shown in Table 11. Table 11: The composition of AC11.The values are shown as weight % of the total weight of dry substance of the composition. Where the composition does not add to 100 %, the remainder is unavoidable impurities. ) %tw / tw(

[0197] From comparing the compositions of AC10-7 and AC11-7, it can be observed that the amount of non-digestible oligosaccharides is comparable. The diafiltration treatment of the fourth syrup enables the amount of dextrose in the fourth syrup to be reduced and for the fourth syrup to be incorporated into the acceptor composition.

[0198] The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be utilized for realizing the invention in diverse forms thereof.

[0199] Although certain example aspects of the invention have been described, the scope of the appended claims is not intended to be limited solely to these examples. The claims are to be construed literally, purposively, and / or to encompass equivalents. SEQUENCE LISTINGSPT-1884-WO-PCT SEQ ID NO: 1 SEQ 1: Amino acid sequence of the kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus (strain DSM 8903). (UniProt identifier A4XGP2). The C-terminal His6-tag and linker sequence are underlined. MKLSEREWLIEQDKLEASGKFETCFALTNGYIGIRGINEEVFCEETPGTYIAGVFDKST AQVTELVNLPNPIGLRIYINREFLNPLKCEILEFKRVLDLKQGILYRKLRLKDVKGRIT TIEGFRFVSMNNKNLIVQKYDVVCENYSAVLNVESFIDATTVNSKDVPNDRVKHYEI DKKKDFADGIYLGITTKDKKYKVGIASSTKVLLNNQRCYFNRFTKDLGYIITENFEVE AKQGERYEIEKLTVLVSSREKNVGDVFETCTNKLKEFETKSAEKLLFEHIEEYKRLW DVANIDIVGDEVANKSVKFNIFHLISMANPEDEHVSLGAKGLHGEGYKGHVFWDTEI FMLPFYIYTNPAAAKAMLMYRYNLLDAARENARKNGYKGAQFPWESADTGEEETP KWGYDYLGNPVRIWTGDIEYHISADIAYAVMNYVRATDDIDFLLNYGSEIIIETARFW ASICKYNKEKGRYEINDVIGPDEFHEHCNNNAYTNYLAKWNLLKASELCNLLLEKY PKYFEKLSKKINLSDEEPFVWQEIASKIYIPYHPDKKLIEQFEGYFNLKDFVIKEYDQN NMPVWPEGVELDKLNNYQLIKQADVVMLLYLLGEEFDDQTKKINYDYYEKRTMHK SSLSPSIYALMGVRVGETNRAYINFMRTALTDLEDNQGNTHLGIHAASLGGTWQAL VFGFGGISIEKDDVLSVNPWLPEKWESLKFSIWWKGNLLDFKITKDNVEVKKRVEKG NVKLKIKGQEAIILEHHHHHH SEQ ID NO: 2 SEQ 2: Amino acid sequence of the kojibiose phosphorylase from Thermoanaerobacter brockii (UniProt identifier Q8L163). The C-terminal His6-tag and linker sequence are underlined. MVKHMFLEDVNNLISDDKWLIFQNEYNTEVNPRYETLFTLTNGYMGVRGTFEEGSE GERSGNFIAGIFDKSDAQVREIVNAQNWLRIKLYVEGEELSLDKCQLIEFKRILDMKK GILFRSMLIKDSKDRITRIEGYRFISRSDLHRSAIKLFVTPVNYSGVVGIESIIDGTVLNS ADSPKHRVKHLKVADNSSLNKSGVYLETATIDDDIRIATGSAVRLYHYEDKEKNNIA KFKRFLPLGEMSIEYFEFDGTENKTVVIDKFIITYTSRDVKKGLLKSTVEKELFAFAGE GIDKELQRHIEVYEELWSVADINIEGDEEADKALRFNIFHLMSSVNENDPMVSIAAKA LHGEGYKGHVFWDTEIFMLPFFIYVHPKAAKTLLMYRYNMLDAARKNAALNGYKGPT-1884-WO-PCT AQYPWESADTGEEETPKWGFDYMGNPVRIWTGDLEHHITADIAFAVWEYFRATEDI EFMLNYGAEVIFETARFWVSRCEYVKELDRYEINNVIGPDEFHEHVDNNAYTDYLA KWNIKKGLELINMLKEKYPEHYHAISNKKCLTNEEMEKWKEVEEKIYIPYDKDKKLI EQFEGYFDKKDYVIDKFDENNMPIWPEGVDITKLGDTQLIKQADVVMLMLLLGEEF DEETKRINYEYYEKRTMHKSSLGPSMYAIMGLKVGDHKNAYQSFMRSANVDLVDN QGNTKEGLHAASAGGTWQVVVFGFGGMEIDKEGALNINSWLPEKWDKLSYKVFW KGNLIEVIVTKQEVTVKKLKGKGNIKVKVKGKELTIELEHHHHHH SEQ ID NO: 3 SEQ 3: Amino acid sequence of the kojibiose phosphorylase from Thermococcus barophilus (strain DSM 11836) (UniProt identifier F0LIH8). The C-terminal His6-tag and linker sequence are underlined. MKFHFSFKEYDPKAEAVYGTILTLGNGYIGIRGEIELEPTIYGTTIAGVYDYAPYFYRE IVNAPRVIGLQIFFNGEPISLSTQKILKYERELNIEDATLKTLIAIETQSRTRIEYESIRIVH GKIKNLILLKFKIKANEDGMLTIISPIKTNVVNPSYRNEIMVKHLNVMQMEDRENEIY AEVETLDGRYRIGIASSLISGTKAKRAVIKSADGIAEILTLSVKKNKTYEFIKYITILSSK TPTPNLREAVLQKLQIAKNSCFSRLYEEHKDYWKEIWKRAKIEIEGDKNAENGLNFSI FHLIQSMPIDSRISLTARGIHGFGYRGHIFWDTEIYALPFFMAVFPEKAREMLMYRYN NLNAARENAKMNGYSGAQFPWESADDGYEATPSVIPLDMMGKKVVKIYTGEEEHH ITADIAYAVELYYKFTGDEEFMFRYGLEIILETARFWASRVEYDEKKGYVIKKVIGPD EYHEHVDNSFFTNLMARYNLLLAVKYFKIARRSGGEWSKTLKRINITEKEVQRWLEI AEKIYIPRQKNGVFEEFEGYFDLSDYTLDPYGLGEKRLPEEIRRNLRKTKIIKQADVIA AQYLLKDQFDLETIRKNFDYYIIRTTHASSLSMPPYAIVASWLDYEDLAYDYFMKCA FIDLHNLYGNTQDGFHLATAGGVWQIIFRGLCGIDINNEGVEINPKLPKKWKAVRLR FFFKKALLSLEVRKDSVRVKLLKGEEVKIRAFGRNAVVKRGKETILHKLEHHHHHH SEQ ID NO: 4 SEQ 4: Amino acid sequence of the kojibiose phosphorylase from Paleococcus pacificus DY20341 (UniProt identifier A0A075LVI8). The C-terminal His6-tag and linker sequence are underlined.PT-1884-WO-PCT MKYNFKFKEYTPKEEAVYGTVLTLGNGHIGIRGEIELEPTIYGTTIAGVYDYAPYFYR EIVNAPRVIGIQAFFNGEPLSLSTQRLLKYERELNIEDATLKTALCLETQNGTNIEYESI RIVHGKRKNIVLLKFAIKASEDGILTIISPIKTDVANPSYRDDITVRHIDVEELKAHEDY IYAGTRTLDGKYKIGIASSLVSKSKAKRSFLKNAGGISEILTLNVEKGKSYEFTKYITIV SSESPEVKEKAIRELQDAKELGFEALYTEHKNYWQEVWNLAKVEIEGDKEAERGLA FSLFHLIQSAPINDKISLTARGIHGFGYRGHVFWDTDIYALPFFMAVFPKKARDMLKY RYNNLDAARENARLNGYEGAQFPWESADDGYEATPPLIPLDIVGKEAVRIYTGEEEH HITADVAYAVELYYRFTKDEEFMSKYGLEIILEAARFWASRVEYESDKGYVIEKVIGP DEYHEHINNSFFTNLMAKYNLLLAVRYFEKAKKLGGTWHETVQRIGVSEEEVKTWT EIAEKIYLPRQIGGVFEEFDGYFELADYTVDPYGLGEKRLPEEVRRNLRKTRLIKQAD VIAAQYLLKEQFDLETIRKNFDYYIVRTTHASSLSMPTYSIIASWLGYDDLAYDYFMK CAFIDLNNIYGNTQDGFHLATAGGVWQIFFRGFCGIDVKDDIVEIAPRLPQKWTSVK MKFFFKGALVELELKNDEVKAKVINDRRVKIRAFGKESSLGPGDEVVLRGLEHHHH HH SEQ ID NO: 5 SEQ 5: Amino acid sequence of the kojibiose phosphorylase from Thermofilum pendens Hrk 5. (UniProt identifier A1RZC3). The C-terminal His6-tag and linker sequence are underlined. MKKYAFSDRERPIRKIATLTTLSNGFLSVRGDPETAPSEYGTLVAGVYSYTPIFYREL VNLPRITPVYVELDGVPMLPVQGSNEFLLDAEGGTLSYKAILGSSLGELEYESLRLTH KKFKGIFALRYRLASRNAEGRLCIKHPIELDTLNVSSPPEVKVKLYKVEEVSAEGSSPS LSVRTADNAYRVLYALIVRGSPAEPKSYYTGKEVGSCYCVDVKPGSVVEGEKVVIV ALSKEELEKFRGIATSESFGGLVSSHVGFWRGLWGRVGFRLYGDSALEDALVFNAFH LLQLYNEGGGEFMLPARGLHGYGYRGHVFWDSDTYSLPFYLLLEPEAARKILEYRC RCLGAAREYASSTGFRGARYPWEGVDDCREATPVEVPLDLEGSRKAFIETGRLEQHI TADVAYAVDMYYEYTGDEEFMERCGLRIIFETARFWASRVELGGDGYYHIRGVIGP DEYHVGVDDSFYTNVMARYNLVLGAKYYALSQSKPGWLRVAVEEGVSREEAEGW LEVAGRVRVPCEPGGLCEEFEGYFKLKDLEVSNCFGDSCAKGLDVGSTRLVKQADV VAGLFLLRRFFDRRVLEGNYEYYLRRTTHASSLSLPMYAAMAAYLGRVEEALALLRPT-1884-WO-PCT KAASTDLEDTYGNLEDGFHVAAAAGSWMALLLGFLGLEPRGGKLVAEPRLPEGLG VELNVWFRGKLHRVEARGSEYRITELEHHHHHH SEQ ID NO: 6 SEQ 6: Amino acid sequence of the maltose phosphorylase from Lactobacillus acidophilus (UniPoint identifier: Q5FI04. MKRIFEIDPWKVITHKFDPKDKRLQESMTAIGNDYMGMRGNFEEGYSGDSLQGTYL AGVWFPDKTVVGWWKNGYPKYFGKTPNAPSFIGIGINVNGEKVDLAKVKFSDFELS LDMHQGLLSRSFIYEGKDVKVKLEFERFLHIVQKEAALIKVKATVLEGHAKIDFDSTL DGTVVNEDSNYGDRFWIPLGEDKDEKTIQVKTKKNPYDVPQFTVLLKEALRNNGVA VNGEVTTEDAKLSERFSVELDEGQSYELEKDVIVVTSRDVEEKDQAAVANNLMSKL QTKSFEDNLADHTEAWKKRWETSDVEISGDDAAQQGIRFNICQLFMTYYGEDKRLN VGPKGFTGEKYGGATYWDTEAFIVPMYLAVTKPSVTRALLQYRHDQLPGAYHNAK EQGLPGALFPMVTFNGIECHNEWEITFEEIHRNADIPHAIAMYTDYTGDDSYVKNEG MDVLVGTARFWAARVHWSKMRNKYVMHGVTGPNEYENNVNNNWFTNTMARWL LKYTLERLPLATKEAQERVRVTDEEKAKWQDIVDNMYLPEDEDLGIFLQQDDFLDK DIRPVTEIEDQRPINQHWSWDKILRSPFIKQADVLQGIYFFDDQYTMDQKEKNFDFYE PLTVHESSLSPCIYSIMAAELGKKEKAVELYQRTARLDLDNYNNDTVDGLHITSMSG SWLAIVQGFAGMRYDHDQLKFNPFVPDGWDHYSFKINYRGRLIEVYVDHDECKITL LSGDDLEVMVHDNKLDLKEGKTKCLKALEHHHHHH

Claims

PT-1884-WO-PCT CLAIMS 1. A process for elongating gluco-oligosaccharides comprising the steps of: (a) providing a starting acceptor composition comprising gluco-oligosaccharides, wherein the gluco-oligosaccharides have a degree of polymerisation of 2, 3, 4, 5, 6 or more glucosyl moieties, wherein each gluco-oligosaccharide in the starting acceptor composition comprises at least one α-1,4-linkage, and wherein the starting acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation; (b) reacting the starting acceptor composition in an aqueous medium with (i) a polypeptide having maltose phosphorylase catalytic activity, (ii) a phosphate source and (iii) a polypeptide having kojibiose phosphorylase catalytic activity; (c) obtaining from step (b) a first syrup, wherein the first syrup comprises one or more elongated gluco-oligosaccharides wherein each elongated gluco-oligosaccharide comprises at least one α-1,2-terminally linked glucosyl moiety, optionally one or more α-1,2-non-terminally linked glucosyl moieties; (d) concentrating the first syrup to a dry substance concentration of from 70 weight % or less of dry substance; and (e) enriching the first syrup to form a second syrup, wherein the second syrup comprises a non-digestible oligosaccharide at from 70 weight % or more of dry substance and monosaccharides and disaccharides at from 15 weight % or less of dry substance, wherein the non-digestible oligosaccharide comprises one or more elongated gluco-oligosaccharides, wherein each elongated gluco-oligosaccharide comprises at least one α-1,2-terminally linked glucosyl moiety and each elongated gluco-oligosaccharide has a degree of polymerisation of 3, 4, 5, 6 or 7.

2. Process of claim 1, wherein enriching the first syrup in step (e) comprises the formation of a fourth syrup having at least 40 weight % dry substance of disaccharides comprising both maltose and kojibiose of the total weight of the dry substance of the fourth syrup;PT-1884-WO-PCT wherein the fourth syrup obtained from step (e) is combined with the starting acceptor composition in step (b) to recycle the fourth syrup back into the process.

3. The process of claim 1 or 2, wherein the second syrup comprises the non-digestible oligosaccharide at from 70 weight % or more of dry substance, or, from 75 weight % or more of dry substance, or, from 78 weight % or more or dry substance, and / or, the second syrup comprises the monosaccharides and disaccharides at from 10 weight % or less of dry substance, or, 5 weight % or less of dry substance of the total weight of the second syrup.

4. The process of any one of claims 1 to 3, wherein enriching the first syrup in step (e) comprises removal of at least partially the monosaccharides from the first syrup, and / or, at least partially the disaccharides from the first syrup when forming the second syrup, such that the second syrup has a total monosaccharides and disaccharides content of less than 15 weight % dry substance of the total weight of the dry substance of the second syrup.

5. The process of any one of claims 1 to 4, wherein enriching the first syrup in step (e) comprises the formation of a third syrup having at least 40 weight % dry substance of monosaccharides of the total weight of the dry substance of the third syrup.

6. The process of any one of claims 1 to 5, wherein the starting acceptor composition comprises from 50 weight % dry substance or more, or, from 75 weight % dry substance or more, or, from 85 weight % dry substance or more of gluco-oligosaccharides having a degree of polymerisation of DP2 of the total weight of the dry substance of the starting acceptor composition.

7. The process of any of claims 1 to 6, wherein the polypeptide having kojibiose phosphorylase activity is a recombinant kojibiose phosphorylase obtainable from a bacteria selected from the group consisting of Thermococcus barophilus, Pseudothermotoga thermarum, Thermoanaerobacter brockii, Thermoanaerobacterium thermosacharolyticum, Caldicellulosiruptor saccharolyticus, Pyrococcus sp., Palaeococcus pacificus and Thermofilum pendens; and / or, wherein the polypeptide having maltose phosphorylase catalytic activity is a recombinant maltose phosphorylase obtainable from a bacteria selected from the group consisting ofPT-1884-WO-PCT Lactobacillus acidophilus, Bacillus selenitireducens, Enterococcus faecalis, Lactobacillus brevis, Lactobacillus sanfranciscensis, Paenibacillus sp. and Bacillus sp.

8. The process of any of claims 1 to 7, further comprising the step of refining the first syrup; preferably wherein the step of refining the first syrup includes one or more of heating, inactivation of enzyme present, ion exchanging, electrodialysis, evaporation, reverse osmosis, resin polishing, filtration, sterilize filtration, and / or activated carbon treatment; more preferably wherein the step of refining the first syrup occurs before step (e); optionally, wherein the step of refining is ion exchanging and / or electrodialysis.

9. The process of any of claims 1 to 8, wherein step (e) comprises enriching by subjecting the first syrup to a polypeptide having alpha-glucosidase catalytic activity; optionally, wherein the polypeptide having alpha-glucosidase catalytic activity is glucose amylase.

10. The process of any of claims 1 to 9, wherein the first syrup is subjected to a polypeptide having alpha-glucosidase catalytic activity prior to the step of refining the first syrup.

11. A composition comprising elongated gluco-oligosaccharides obtained or obtainable by the process according to any of claims 1 to 10.

12. A composition comprising elongated gluco-oligosaccharides with at least one α-1,2- terminally linked glucosyl moiety and optionally one or more α-1,2-non-terminally linked glucosyl moieties, wherein the elongated gluco-oligosaccharides have a degree of polymerisation of 3, 4, 5, 6 or more, and, wherein the composition comprises from 70 weight % or more dry substance of non-digestible oligosaccharide and from 15 weight % or less of dry substance of monosaccharides and disaccharides.

13. The composition of claim 11 or claim 12, wherein the composition comprises monosaccharides and disaccharides at from 10 weight % or less of dry substance of dry substance, or, from 5 weight % or less of dry substance of the total weight of the dry substance of the composition.

14. The composition of any one of claims 11 to 13, wherein the composition comprises a non- digestible oligosaccharide at from 70 weight % or more of dry substance, or, from 75 weightPT-1884-WO-PCT % or more of dry substance, or, from 78 weight % or more or dry substance of the total weight of the dry substance of the composition.

15. The composition of any one of claims 11 to 14, wherein the composition further comprises one or more of kojibiose, kojitriose, kojitetraose and kojipentaose.

16. The composition of any one of claims 11 to 15 comprising at least two different elongated gluco-oligosaccharides selected from:2-O-α-D-glucosyl-maltose (α-D-Glucopyranosyl-(1^2)-α-D-glucopyranosyl-(1^4)- D-glucopyranose); and / or,PT-1884-WO-PCT2-O-α-D-glucosyl-maltotriose (α-D-Glucopyranosyl-(1^2)-α-D-glucopyranosyl- (1^4)-α-D-glucopyranosyl-(1^4)-D-glucopyranose); and / or,2-O-α-D-glucosyl-maltotetraose (α-D-Glucopyranosyl-(1^2)-α-D- glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-D- glucopyranose); and / orPT-1884-WO-PCT2-O-α-D-glucosyl-maltopentaose (α-D-Glucopyranosyl-(1^2)-α-D- glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-α-D- glucopyranosyl-(1^4)-D-glucopyranose); and / or,2-O-α-D-glucosyl-maltohexaose (α-D-Glucopyranosyl-(1^2)-α-D- glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-α-D- glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-D-glucopyranose).

17. A composition comprising and / or consisting of monosaccharides, disaccharides and gluco-oligosaccharides obtained or obtainable by the process according to any of claims 1 to 10, wherein the composition comprises at least 40 weight % dry substance of monosaccharides and at most 60 weight % dry substance disaccharides comprising both maltose and kojibiose and gluco-oligosaccharides having a degree of polymerisation of 3, 4, 5, 6 or more and higher of the total dry substance of the composition;PT-1884-WO-PCT optionally, wherein the composition is obtained or obtainable by the process of claim 5.

18. A composition comprising and / or consisting of monosaccharides, disaccharides and gluco-oligosaccharides obtained or obtainable by the process according to any of claims 1 to 9, wherein the composition comprises at least 40 weight % dry substance disaccharides comprising both maltose and kojibiose, and, at most 60 weight % dry substance monosaccharides and gluco-oligosaccharides having a degree of polymerisation of 3, 4, 5, 6 or higher of the total dry substance of the composition; optionally, wherein the composition is obtained or obtainable by the process of claim 2.

19. A foodstuff, supplements for specialized nutrition, pharmaceutical products, personal care and cosmetic products, pet foods, animal feed or aqua feed, comprising the composition according to any one of claims 11 to 18.

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