Process for modifying gluco-oligosaccharides with kojibiose phosphorylases

Abstract

The present invention relates to an isolated polypeptide comprising kojibiose phosphorylase activity (E.C. 2.4.1.230), wherein the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose, and alpha-1,2-glucosyl-maltose as a product respectively. The present invention also relates to an enzymatic process for elongating gluco-oligosaccharides with a glucosyl moiety, wherein the enzymatic process uses the isolated polypeptide of the present invention.

Description

PT-2001-WO-PCT PROCESS FOR MODIFYING GLUCO-OLIGOSACCHARIDES WITH KOJIBIOSE PHOSPHORYLASES CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of European Application No. 24196289.3, filed August 23, 2024, which is incorporated by reference herein in its entirety. SEQUENCE LISTING

[0002] The content of the Sequence Listing XML titled “V1_3 PT-2001 Final V3.xml” 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 an isolated polypeptide comprising kojibiose phosphorylase activity (E.C. 2.4.1.230), wherein the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose and alpha-1,2-glucosyl-maltose as a product respectively, and wherein the activity of the polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate. The present invention also relates to an enzymatic process for elongating gluco-oligosaccharides with a glucosyl moiety, wherein the enzymatic process uses the isolated polypeptide of the present invention. 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 and glucose syrups may be used as a texturiser or as a full calorie ingredient providing bulking functionality. Maltodextrins and glucose syrups may also be usedPT-2001-WO-PCT as a coating and for encapsulation. Maltodextrins and glucose syrups are easily digested and are fully and rapidly absorbed by the gastrointestinal tract. The fast digestibility results in a strong rise in blood glucose levels after consumption. Maltodextrins and glucose syrups are therefore products with a high glycemic index. Further, maltodextrins 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 using an isolated polypeptide comprising kojibiose phosphorylase activity (E.C.2.4.1.230) wherein the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose, and alpha-1,2-glucosyl-maltose as a product respectively, and wherein the activity of the polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate. Use of the isolated polypeptide advantageously results in a carbohydrate with decreased digestibility. SUMMARY OF THE INVENTION

[0008] The present invention relates to an isolated polypeptide comprising kojibiose phosphorylase activity (E.C. 2.4.1.230), wherein the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose, and alpha-1,2-glucosyl-maltose as a product respectively, and wherein the activity of the polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate. The present invention also relates to an enzymatic process for elongating gluco-oligosaccharides with a glucosyl moiety, wherein the enzymatic process uses the isolated polypeptide of the present invention. 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 caloriePT-2001-WO-PCT reduction. The elongated gluco-oligosaccharides obtained by the process present a decreased digestibility compared to known gluco-oligosaccharides.

[0009] The present invention provides in a first aspect an isolated polypeptide comprising kojibiose phosphorylase activity (E.C.2.4.1.230), wherein the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose, and alpha-1,2-glucosyl-maltose as a product respectively, and wherein the activity of the polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate.

[0010] The inventors found that use of an isolated polypeptide that is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose, and alpha-1,2-glucosyl-maltose as a product respectively, wherein the activity of the isolated polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate greatly increases the conversion rate of a polymer having a degree of polymerisation of three or more into higher fibers. Without wishing to be bound by theory, it is believed that this effect results from glucose being less likely to compete in the reaction – the higher relative activity of the isolated polypeptide towards kojibiose and maltose than to glucose compared to known enzymes results in a relatively lower preference of glucose in the reaction.

[0011] In a second aspect, the invention relates to a process for elongating gluco- oligosaccharides with at least one α-(1,2)-terminally linked glucosyl moiety, comprising the steps of: a. providing an acceptor composition comprising gluco-oligosaccharides having a degree of polymerisation of 2, 3, 4, 5 or 6 glucosyl moieties, wherein each gluco- oligosaccharide comprises at least one α-(1,4)-linkage and wherein the acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation; and b. placing the acceptor composition in fluid contact with the above isolated polypeptide in the presence of a 1-glucosyl phosphate donor.

[0012] The process results in the addition of one or more glucosyl moieties to the acceptor gluco-oligosaccharide 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., itPT-2001-WO-PCT 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.

[0013] 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.

[0014] The use of isolated polypeptides having kojibiose phosphorylase activity are 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-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.

[0015] 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.

[0016] The inventors have advantageously found that using an acceptor composition which is a mixture of gluco-oligosaccharides having different degrees of polymerisation, in particular a hydrolysed starch, with newly identified isolated polypeptides having kojibiose phosphorylase activity, (wherein the activity of the polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate), elongation of the gluco- oligosaccharides with (at least one) α-1,2-linked glucosyl moiety can be obtained, resulting in a gluco-oligosaccharide composition that has decreased digestibility (i.e. increased fiber content), compared to when using previously known polypeptides having kojibiose phosphorylase activity.

[0017] In a third 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 fourth aspect, the invention relates to an edible product comprising the composition of the invention.

[0019] 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.

[0020] The present invention is as set out in the following clauses: 1. An isolated polypeptide comprising kojibiose phosphorylase activity (E.C.2.4.1.230),PT-2001-WO-PCT Wherein the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose, and alpha- 1,2-glucosyl-maltose as a product respectively, and wherein the activity of the polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate. 2. The isolated polypeptide according to clause 1 wherein the isolated polypeptide comprises: 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). 3. The isolated polypeptide according to clause 1 or clause 2, wherein the isolated polypeptide is obtained or obtainable from a microorganism selected from the group consisting of Thermococcus barophilus, Thermophilum pendens , and / or Palaeococcus pacificus . 4. The isolated polypeptide according to clause 3, wherein the isolated polypeptide comprises the amino acid 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. 5. A process for elongating gluco-oligosaccharides with at least one α-(1,2)- terminally linked glucosyl moiety, comprising the steps of: a. providing an acceptor composition comprising gluco-oligosaccharides having a degree of polymerisation of 2, 3, 4, 5 or 6 glucosyl moieties, wherein each gluco- oligosaccharide comprises at least one α-(1,4)-linkage and wherein the acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation; and b. placing the acceptor composition in fluid contact with an isolated polypeptide according to clauses 1 to 4 in the presence of a 1-glucosyl phosphate donor.PT-2001-WO-PCT 6. The process according to clause 5, wherein the acceptor composition comprises at least 3, preferably at least 4 gluco-oligosaccharides having a different degree of polymerisation. 7. The process according to clause 5 or clause 6, wherein the acceptor composition is a partially hydrolysed starch having a dextrose equivalent (DE) of less than 60, preferably less than 50, more preferably less than 45. 8. The process according to clause 7, wherein the partially hydrolysed starch is a maltodextrin or glucose syrup. 9. The process according to any of clauses 5 to 8, wherein the acceptor composition comprises at least two different gluco-oligosaccharides selected from the group consisting of maltose, maltotriose, maltotetraose, maltopentaose and maltohexaose. 10. The process according to any of clauses 5 to 9, wherein the 1-glucosyl phosphate donor is β-d-glucose-1-phosphate. 11. The process according to any of clauses 5 to 10, wherein the process further comprises a step of preparing the 1-glucosyl phosphate donor by placing maltose in fluid contact with a polypeptide comprising maltose phosphorylase activity and phosphoric acid and / or a salt thereof. 12. The process according to clause 11, wherein the polypeptide comprising maltose phosphorylase activity is a recombinant maltose phosphorylase, preferably obtained or obtainable from a bacterium selected from the group consisting of Lactobacillus acidophilus, Bacillus selenitireducens, Enterococcus faecalis, Lactobacillus brevis, Lactobacillus sanfranciscensis, Paenibacillus sp. And Bacillus sp. 13. A composition comprising elongated gluco-oligosaccharides obtained or obtainable by the process according to any of clauses 5 to 12, comprising at least 40 wt.% of elongated gluco- oligosaccharides based on the total weight of the composition as dry substance.PT-2001-WO-PCT 14. A composition comprising at least 40 wt.% gluco-oligosaccharides elongated with at least one α-(1,2)-terminally linked glucosyl moiety based on the total weight of the composition as dry substance, wherein the gluco-oligosaccharides have a degree of polymerisation of 2, 3, 4, 5, 6 or 7; and wherein the composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation and wherein each gluco-oligosaccharide comprises at least one α-(1,4)-linkage. 15. The composition according to clause 13 or 14, wherein the elongated gluco- oligosaccharides comprise 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 the linkages from kojibiose and kojioligosaccharides. 16. The composition according to any of clauses 13 to 15, further containing one or more of kojibiose, kojitriose, kojitetraose and kojipentaose. 17. The composition according to any of clauses 13 to 16, 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-glucopyranosePT-2001-WO-PCT2-O-α-D-glucosyl-maltotriose α-D-Glucopyranosyl-(1^2)-α-D-glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-D- glucopyranose2-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-glucopyranose2-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-glucopyranosePT-2001-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 18. A foodstuff, pet food, feed or personal care product comprising the composition according to any of clauses 13 to 17. DETAILED DESCRIPTION

[0021] 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.

[0022] 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. EmbodimentsPT-2001-WO-PCT of the claims may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0023] 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.

[0024] Some of the terms used to describe the present invention are set out below:

[0025] “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.

[0026] “Elongated gluco-oligosaccharide” refers to a gluco-oligosaccharide that comprises at least one α-(1,2)-terminally linked glucosyl moiety.

[0027] “Fructose syrup” refers to an aqueous solution of nutritive saccharides obtained from edible starch in which a portion of the dextrose (D-glucose) has been isomerized to fructose.

[0028] “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 acceptor composition comprise at least one α-(1,4)-linkage.

[0029] “Glucose syrup” refers to an aqueous solution of nutritive saccharides obtained from edible starch having a DE of 20 or more.

[0030] “Kojibiose phosphorylase” or “KP” is a carbohydrate-processing phosphorolytic enzyme and is classified into the glycoside hydrolase family 65 (GH65). Kojibiose phosphorylase is known by the IUBMB Enzyme Nomenclature number EC 2.4.1.230. Kojibiose phosphorylase can be used to catalyse the reaction between a saccharide such as maltose and inorganic phosphate to glucose and β-D-glucose-1-phosphate.PT-2001-WO-PCT

[0031] “Maltodextrin” refers to a dried product or aqueous solutions of saccharides obtained from edible starch having a DE of less than 20.

[0032] “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 a protein molecule. Isolated polypeptide

[0033] In one aspect, the present invention relates to an isolated polypeptide. In particular, the present invention relates to an isolated polypeptide comprising kojibiose phosphorylase activity (E.C. 2.4.1.230), wherein the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose, and alpha-1,2-glucosyl-maltose as a product respectively, and wherein the activity of the polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate. Preferably, the activity of the polypeptide towards kojibiose and maltose is higher as compared to its activity towards glucose as an acceptor substrate.

[0034] In some examples of the present invention, the isolated polypeptide comprising kojibiose phosphorylase activity is obtained or obtainable or derived or derivable from a microorganism selected from the group consisting of Thermococcus barophilus, Thermophilum pendens, and / or Palaeococcus pacificus. In some examples of the present invention, the isolated polypeptide comprising kojibiose phosphorylase activity is obtained or obtainable or derived or derivable from a microorganism selected from the group consisting of Thermococcus barophilus, Thermophilum pendens Hrk 5, and / or Palaeococcus pacificus DY20341. In some examples of the present invention, the isolated polypeptide comprising kojibiose phosphorylase activity is obtained or obtainable or derived or derivable from Thermococcus barophilus. In some examples of the present invention, the isolated polypeptide comprising kojibiose phosphorylase activity is obtained or obtainable or derived or derivable from Thermophilum pendens, optionally wherein the Thermophilum pendens is Thermophilum pendens Hrk 5. In some example of the present invention, the isolated polypeptide comprising kojibiose phosphorylase activity is obtained or obtainable or derived or derivable from Palaeococcus pacificus, optionally wherein the Palaeococcus pacificus is Palaeococcus pacificus DY20341.PT-2001-WO-PCT

[0035] In some examples of the present invention, the isolated polypeptide comprises 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 isolated polypeptide comprises residues 1-748 of SEQ ID NO:3. In some examples of the present invention, the isolated polypeptide comprises residues 1-746 of SEQ ID NO:4. In some examples of the present invention, the isolated polypeptide comprises residues 1-704 of SEQ ID NO:5. In some examples of the present invention, the isolated polypeptide comprises 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 isolated polypeptide comprises 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 isolated polypeptide comprises residues 1-746 of SEQ ID NO:4 or residues 1-704 of SEQ ID NO:5.

[0036] In some examples of the present invention, the isolated polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In some examples of the present invention, the isolated polypeptide consists of the amino acid sequence set forth in SEQ ID NO:3. In some examples of the present invention, the isolated polypeptide comprises 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 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 isolated polypeptide consists of the amino acid sequence set forth in SEQ ID NO:4. In some examples of the present invention, the isolated polypeptide comprises 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 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 isolated polypeptide consists of the amino acid sequence set forth in SEQ ID NO:5. In some examples of the present invention, the isolated polypeptide comprises 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 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.PT-2001-WO-PCT

[0037] Without wishing to be bound by theory, the inventors have discovered that kojibiose phosphorylases with an alanine residue at an amino acid position corresponding to position 387 of a KP derived from T. barophilus (TbarKP) (SEQ ID NO: 3), a proline residue at an amino acid position corresponding to position 393 of TbarKP (SEQ ID NO: 3) and / or a leucine residue at an amino acid position corresponding to position 394 of TbarKP (SEQ ID NO: 3) have a much higher activity towards maltose compared to glucose when compared to kojibiose phosphorylases which contain, for example, other residues at amino acid positions corresponding to position 387, 393 and / or 394 of TbarKP (e.g. residues E400, G405 and Y406 shown in SEQ ID NO:1, or residues E418, G423 and F424 shown in SEQ ID NO:2 which are the amino acid positions for the KPs derived from C. Saccharolyticus and T. Brockii, respectively that correspond to positions 387, 393 and 394 of TbarKP). The residues of a KP derived from P. pacificus (PpKP) (SEQ ID NO:4) corresponding to amino acids at position 387, 393 and 394 of TbarKP (SEQ ID NO:3) are A385, P391, and L392. The residues of a KP derived from T. pendens (TpKP) (SEQ ID NO:5) corresponding to amino acids at position 387, 393 and 394 of TbarKP (SEQ ID NO:3) are A375, P381, and L382. The residues corresponding to amino acids at position 387, 393 and 394 of TbarKP (SEQ ID NO:3) of a given sequence can be determined by routine methods in the art, e.g. using a sequence alignment tool such as a Clustal Omega with default parameters e.g. as outlined in Sievers et al (Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega) Molecular Systems Biology 7:539 (2011).

[0038] In some examples of the present invention, the isolated polypeptide comprises 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 isolated polypeptide comprises a proline residue at the amino acid position corresponding to position 393 of Thermococcus barophilus kojibiose phosphorylase (SEQ ID NO: 3). In some examples of the present invention, the isolated polypeptide comprises a leucine residue at the amino acid position corresponding to position 394 of Thermococcus barophilus kojibiose phosphorylase (SEQ ID NO: 3). Preferably, the isolated polypeptide comprises 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 phosphorylasePT-2001-WO-PCT (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). Process

[0039] In a second 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 an acceptor composition comprising gluco-oligosaccharides having a degree of polymerisation of 2, 3, 4, 5 or 6 glucosyl moieties, wherein each gluco- oligosaccharide comprises at least one α-(1,4)-linkage and wherein the acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation; and b. placing the acceptor composition in fluid contact with an isolated polypeptide as described above in the presence of a 1-glucosyl phosphate donor. Acceptor composition

[0040] As described above, the process of the invention has as a starting material an acceptor composition comprising gluco-oligosaccharides having a degree of polymerisation of 2, 3, 4, 5 or 6 glucosyl moieties, wherein each gluco-oligosaccharide comprises at least one α-(1,4)- linkage and wherein the acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation.

[0041] The acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation. For instance, the 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).

[0042] The acceptor composition can be obtained by mixing 2, 3, 4 or more gluco- oligosaccharides having different degrees of polymerisation.

[0043] Alternatively, the acceptor composition of the invention can be obtained by partial hydrolysis of starch as will be described hereafter.

[0044] Preferably, the 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;PT-2001-WO-PCT Maltotriose 1109-28-0; Maltotetraose 34612-38-9; Maltopentaose 34620-76-3; Maltohexaose 34620-77-4.

[0045] Beside the gluco-oligosaccharides having a degree of polymerisation of 2, 3, 4, 5 or 6 glucosyl moieties, the acceptor composition can comprise further oligo- or polysaccharides, for instance those present in an acceptor composition obtained by partial hydrolysis of starch.

[0046] In some examples of the present invention, the acceptor composition is preferably a partially hydrolysed starch having a dextrose equivalent (DE) of less than 60, more preferably less than 50, more preferably less than 45. Optionally, the 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.

[0047] 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.

[0048] When a partially hydrolysed starch is used as the 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.

[0049] In some examples of the present invention, the acceptor composition may also comprise gluco-oligosaccharides having a degree of polymerisation higher than 6, e.g., 7 or 8. Polypeptide

[0050] The isolated polypeptide is as described above under the heading “isolated polypeptide”. Donor

[0051] 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.PT-2001-WO-PCT

[0052] 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).

[0053] In some examples of the present invention, ß-D-glucose-1-phosphate is commercially available and can be used as such. Process coupled reaction

[0054] 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. Optionally, the phosphate source is phosphoric acid and / or phosphate salts.

[0055] In some examples of the present invention, a 1-glucosyl phosphate donor can be prepared by placing maltose in fluid contact with a 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 acceptor composition is partially hydrolysed starch, the acceptor composition may already contain the maltose, or the maltose is generated in situ during the α- 1,2-linkage-creating reactions (i.e., from glucose and ß-glucose phosphate by maltose phosphorylase).

[0056] 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 Bacterium selected from the group consisting of Lactobacillus acidophilus, Bacillus selenitireducens, Enterococcus faecalis, Lactobacillus brevis, Lactobacillus sanfranciscensis, Paenibacillus sp. and Bacillus sp.

[0057] 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.

[0058] 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.PT-2001-WO-PCT

[0059] In some examples of the present invention, the present invention comprises a step of preparing the 1-glucosyl phosphate donor by placing trehalose in fluid contact 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.

[0060] In some examples of the present invention, the present invention comprises a step of preparing the 1-glucosyl phosphate donor by placing sucrose in fluid contact with a polypeptide having sucrose phosphorylase activity, phosphoric acid and / or a salt thereof. Process conditions

[0061] The process of the present invention is carried out by placing the acceptor composition, and donor in fluid contact with the isolated polypeptide. In general, this means that the process is carried out in an aqueous solution. The isolated polypeptide can also be an immobilised isolated polypeptide.

[0062] Process conditions are those generally known in the field. For example, the concentration of the acceptor composition in the aqueous solution is from 5 to 60 wt.%, the pH of the reaction is from 4 to 7 (such as from 5 to 7); the molar ratio 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 / mL.

[0063] 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), 4-alpha- glucanotransferase, transglucosidase, and / or, mixtures thereof.

[0064] In some examples of the present invention, the process can further comprise a step of purifying the composition obtained such as by filtration and / or microfiltration, decolorization / deodorization using active carbon or porous polymeric resins (like styrene divinyl benzene resins) and / or demineralization using ion exchange resins. Also, nano- or ultrafiltration and / or chromatographic separation steps can be carried out after deactivation of the enzymes to fractionate the composition or to eliminate undesired components from the composition.PT-2001-WO-PCT Composition

[0065] 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.

[0066] In some examples of the present invention, the composition comprises at least 40 wt.% of elongated gluco-oligosaccharides based on the total weight of the composition as dry substance.

[0067] In some examples of the present invention, the composition comprises at least 40 wt.% gluco-oligosaccharides elongated with at least one α-(1,2)-terminally linked glucosyl moiety based on the total weight of the composition as dry substance, wherein the gluco- oligosaccharides have a degree of polymerisation of 2, 3, 4, 5, 6 or 7; and wherein the composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation and wherein each gluco-oligosaccharide comprises at least one α-(1,4)-linkage.

[0068] In some examples of the present invention, the composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation.

[0069] In some examples of the present invention, each gluco-oligosaccharide in the composition comprises at least one α-(1,4)-linkage. Preferably, the α-(1,2)-terminally linked glucosyl moiety is adjacent to an α-(1,4)-linkage.

[0070] 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.

[0071] In some examples of the present invention, the composition further comprises at least two different elongated gluco-oligosaccharides selected from:PT-2001-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-2001-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-2001-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).

[0072] In particular, the composition of the invention is characterised 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.

[0073] 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 and / or gas chromatography mass spectrometer (GC-MS).

[0074] The composition may further contain kojibiose and kojioligosaccharides. These compounds also contribute to decreased digestibility of the composition of the invention.

[0075] Kojibiose and kojioligosaccharides are in particular characterised by the following formulas:PT-2001-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)

[0076] In particular, the composition of the invention comprises at least 20 wt. % elongated gluco-oligosaccharides and at least 10 wt.% kojioligosaccharides based on the total weight of the composition as a dry substance.

[0077] Preferably, the composition of the invention comprisesPT-2001-WO-PCT - one or more elongated gluco-oligosaccharides having a degree of polymerisation of 3, 4 or 5, wherein each elongated gluco-oligosaccharide comprises at least one α-1,2- terminally linked glucosyl moiety and at least one α-(1,4)-linkage, in an amount of from 25 wt.% or more, preferably from 30 wt.% or more, more preferably from 35 wt.% or more, even more preferably from 40 wt.% or more, even more preferably from 45 wt.% or more, most preferably from 50 wt.% or more of the total weight of dry substance of the composition; and / or - kojitriose, kojitetratose and kojipentaose in an amount of from 8 wt.% or more, preferably from 9 wt.% or more, more preferably from 10 wt.% or more, even more preferably from 11 wt.% or more, most preferably from 12 wt.% or more of the total weight of dry substance of the composition; and / or; - kojibiose in an amount of from 11 wt.% or less, preferably from 10 wt.% or less, more preferably from 9 wt.% or less, most preferably 8 wt.% or less of the total weight of dry substance of the composition.

[0078] Preferably, the composition of the invention comprises - one or more elongated gluco-oligosaccharides having a degree of polymerisation of 3, 4 or 5, wherein each elongated gluco-oligosaccharide comprises at least one α-1,2- terminally linked glucosyl moiety and at least one α-(1,4)-linkage, in an amount of from 30 wt.% to 70 wt.%, preferably from 40 wt.% to 65 wt.%; most preferably from 50 wt.% to 60 wt.% of the total weight of dry substance of the composition; and / or - kojitriose, kojitetratose and kojipentaose in an amount of from 8 wt.% to 25 wt.%, preferably from 10 wt.% to 20 wt.%, most preferably from 12 wt.% to 15 wt.% of the total weight of dry substance of the composition; and / or; - kojibiose in an amount of from 1 to 11 wt.%, preferably from 3 wt.% to 10 wt.%, most preferably from 5 to 9 wt.% of the total weight of dry substance of the composition. Digestibility

[0079] Digestibility of the composition 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 digestion reaction using alpha-amylase and a glucoamylase or an alpha-glucosidase and intestinal animal enzymes.PT-2001-WO-PCT

[0080] Other advantageous properties of the composition besides the lower calories compared to sucrose, are a high water solubility, viscosities comparable to commercial glucose syrups and maltodextrins and a lower relative sweetness than sucrose. Use of the composition obtained

[0081] 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.

[0082] 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, fiber, 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.

[0083] In some examples of the present invention, the composition can be used in foodstuffs in combination with other bulking agents, fillers, soluble fibers, resistant starch, polydextrose, dextrins, resistant maltodextrin, inulin, or insoluble fibers. The combination can boost the fiber 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.

[0084] 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 by fermentation or bioconversion), low calorie sweeteners (such as polyols, including erythritol, sorbitol, xylitol, lactitol, maltitol, mannitol, isomalt, etc.) and conventional caloric sweeteners (such as sucrose, glucose syrups, fructose syrups, dextrose, high fructose corn syrup, maltose, lactose, etc.).

[0085] Foodstuffs, in which the composition of the invention can be incorporated, are not limited and include:PT-2001-WO-PCT - 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.

[0086] 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.

[0087] 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 (for 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.PT-2001-WO-PCT

[0088] 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.

[0089] 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 fiber).

[0090] 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.

[0091] 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

[0092] Figure 1 provides an overview of the coupled reaction using maltose phosphorylase (MP) and kojibiose phosphorylase (KP). EXAMPLES

[0093] The following are non-limiting examples that discuss, with reference to tables, the advantages of the present invention. The examples set forth herein are merely examples among other possible examples.PT-2001-WO-PCT Enzyme productionGene cloning and transformation

[0094] The genes encoding the amino acid sequences for the kojibiose phosphorylases (KP) from C. saccharolyticus (CsKP, UniProt identifier A4XGP2), T. brockii (TbKP, UniProt identifier Q8L163), T. barophilus (TbarKP, UniProt identifier F0LIH8), P. pacificus (PpKP, UniProt identifier A0A075LVI8), and T. pendens (TpKP, UniProt identifier A1RZC3) were obtained from the database CAZy (database of Carbohydrate-Active enZYmes). The genes were codon optimized for E. coli and synthesised (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. Enzyme expression and recovery

[0095] 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 all KPs took place for 16 hours at 30 °C. The cultures were then centrifuged (15 min, 9000 rpm), and the cell pellets were frozen at -20 °C for at least 4 hours.

[0096] 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 and 50 mM 2-morpholinoethanesulfonic acid (MES) pH 6.5. This suspension was incubated on ice for 30 min and sonicated three times for 3 min (Branson Sonifier 250, level 3, 50% duty cycle).

[0097] Some of the enzymes were expressed as insoluble but active inclusion bodies, and therefore the crude cell lysates were not further purified. Potential background activity in the crude cell extract was inhibited by means of heat treatment. After an incubation of 1 hour at 60 ̊C, the heat-treated protein solutions were stored at 4 ̊C.

[0098] Molecular weight and expression level were verified by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE; 10% gel).PT-2001-WO-PCT Determination of enzyme activity

[0099] Activity of the enzymes was routinely determined by phosphorolysis of kojibiose and maltose. To enable quantification of the enzyme activity, a standard curve was made in the range of 0-500 µM 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.

[0100] 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 µL samples were taken at set timepoints (0, 1, 2, 4, 6, 8 and 10 min), and inactivated in 25 microL 0.2 M NaOAc buffer pH 2.5. Next, 200 µL GOD-POD reagent (173 µL 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 37 ˚C for 30 min. Finally, absorbance was measured with a spectrophotometer at 420 nm. Enzyme activity (Units) was calculated as the rate of glucose release per unit of time, in µmol glucose per second (1 U = 1 µmol / s). Example 1: Relative acceptor activity of kojibiose phosphorylases

[0101] In this non-limiting example of the present invention, the relative acceptor ability of known and novel KPs was measured.

[0102] In initial experiments, the evaluation of several acceptor substrates for all KPs took place. From literature it is known that TbKP is capable of using glucose, kojibiose and maltose, amongst others, as an acceptor substrate. However, this was merely indicated by qualitative thin layer chromatography (TLC) results (Nakada, T. et al. (2003) ‘Kojioligosaccharides: Application of Kojibiose Phosphorylase on the Formation of Various Kojioligosaccharides’, in Oligosaccharides in Food and Agriculture, pp. 104–117), in which the authors propose a seemingly equal activity towards the above mentioned acceptor substrates. Later, a KP from C. saccharolyticus (CsKP) showed an apparent preference for longer kojioligosaccharides, indicated by its shifted product equilibrium towards higher DPs in comparison with TbKPPT-2001-WO-PCT (Yamamoto, T. et al. (2011) ‘Enzymatic Properties of Recombinant Kojibiose Phosphorylase from Caldicellulosiruptor saccharolyticus ATCC43494’, Bioscience, Biotechnology, and Biochemistry, 75(6), pp. 1208–1210. doi: 10.1271 / bbb.110116). However, no data regarding CsKP’s behaviour towards maltose or malto-oligosacharides is known to date. In order to evaluate the behavior of known and novel KPs towards several acceptor substrates, reactions were set up to assess their glucosylation rate relative to glucose.

[0103] Reactions were performed on a 1 mL scale, starting from 50 mM of the respective acceptor substrate, 200 mM β-G1P, in 50 mM MES buffer at pH 6.5, 55 ˚C and 800 rpm. The enzyme was added in a sufficient amount to obtain a linear curve of product formation. The chosen acceptor substrates were glucose (glc), kojibiose (K2), and maltose (M2). Glucose and maltose (M2) were purchased from Sigma-Aldrich, kojibiose (Beerens, K. et al. (2017) ‘Biocatalytic synthesis of the rare sugar kojibiose: process scale-up and application testing’, Journal of Agricultural and Food Chemistry, p. acs.jafc.7b02258. doi: 10.1021 / acs.jafc.7b02258) and β-G1P (Van Der Borght, J., Desmet, T. and Soetaert, W. (2010) ‘Enzymatic production of β-D-glucose-1-phosphate from trehalose’, Biotechnology Journal, 5, pp. 986–993. doi: 10.1002 / biot.201000203) were produced in-house. Samples (50 µL) were taken at the beginning (t = 0 min) and at given timepoints (t = 1, 2, 4, 6, 8, 10 min), inactivated in 100 mM NaOH (50 µL) and further diluted to a 200-fold dilution for high performance anion exchange chromatography (HPAEC) analysis.

[0104] All HPAEC analysis with integrated pulsed amperometric detection (HPAEC-IPAD) was performed on a CarboPac PA203x150mm column, using the ICS-6000 Dionex system (Thermo Fisher Scientific) and a flow rate of 0.5 mL / min. In order to separate the respective carbohydrates, a 40 min protocol was used. During the first 4 min, an isocratic elution with 80 % eluent A (ddH20) and 20 % eluent B (100 mM NaOH) was used. Between 4 min and 8 min, eluent B was linearly increased from 20 % to 100 %, and maintained at 100 % until 10 min. Between 10 min and 30 min, eluent C (1 M NaOAc and 100 mM NaOH) was increased linearly from 0 % to 10 %. Between 30 min and 30.5 min, eluent C was linearly increased from 10 % to 30 %, and maintained at 30 % until 33 min. Next, between 33 min and 33.5 min, eluent C and B were decreased linearly to 0 % and 20 %, respectively. Thereby, the initial isocratic elution with 80 % eluent A and 20 % eluent B was restored and maintained until 40 min. If needed, standards of 20 μM were run to enable detection of the known compounds.PT-2001-WO-PCT

[0105] The slope of the increase in product peak area as a result of acceptor substrate glucosylation by the respective KPs was determined for each acceptor substrate. Consequently, the slope of product peak increase was used as an approximate of the acceptor activity. For each KP, its activity (slope) on glucose was set as 100 %, and the activities towards kojibiose and maltose were expressed relative to glucose. The results of which are shown in Table 1. The products formed by glucosylation of glucose, kojibiose and maltose by KP are kojibiose, kojitriose and α-1,2-glucosyl-maltose (G-M2), respectively (Nakada, T. et al. (2003) ‘Kojioligosaccharides: Application of Kojibiose Phosphorylase on the Formation of Various Kojioligosaccharides’, in Oligosaccharides in Food and Agriculture, pp.104–117). Table 1: The relative acceptor activity of known (TbKP, CsKP) and novel (TbarKP, PpKP, TpKP) KPs.

[0106] Surprisingly, all KPs show a higher activity towards the non-native acceptor substrate maltose, with respect to the native acceptor substrate glucose. This counterintuitive and quantitative result has not been reported to date. In addition, it became clear that the novel KPs (TbarKP, PpKP, TpKP) show a significantly increased preference for maltose (and kojibiose) with respect to the known enzymes (TbKP and CsKP). This finding is of particular importance when employing these enzymes in glucosylation processes using acceptor substrate mixtures containing malto-oligosaccharides and glucose, when the glucose glucosylation products are undesired by-products. With these novel enzymes (TbarKP, PpKP, TpKP), more maltose and presumably also malto-oligosaccharides will be glucosylated in the presence of glucose in the reaction mixture, compared to known KPs (TbKP and CsKP).PT-2001-WO-PCT EXAMPLE 2 - Coupled reaction – comparison of with TbarKP

[0107] In a next step, the performance of was evaluated in a coupled reaction using a commercial gluco-oligosaccharide mixture containing M2, M3 and M4, amongst others (30DE syrup:). This 30DE syrup contains about 12 wt.% DP2, about 19 wt.% DP3, about 7 wt.% DP4, about 20 wt.% DP5, about 11 wt.% DP6 and about 21 wt.% DP11 (w / w % dry solids). The remaining weight percentage to reach 100 wt.% on dry solids of the 30De syrup contains other saccharides as impurities. As such, it was evaluated whether its beneficial acceptor preference as illustrated in Example 1 also leads to a more desired product composition from an industrially relevant substrate. An identical reaction was performed with CsKP to enable their comparison. For the in situ production of β-G1P, maltose phosphorylase from Lactobacillus acidophilus (LaMP) was added. MP uses inorganic phosphate to perform phosphorolysis of the maltose present in the reaction mixture, leading to the production of β- G1P (Hüwel et al., 1997). Next, KP can use the released β-G1P as a donor substrate to elongate the gluco-oligosaccharides present in the reaction mixture.

[0108] In that respect, a reaction starting from 150 g / L 30DE syrup was evaluated. The reaction was performed on a 1 mL-scale at 55 ˚C, 50 mM MES buffer pH 6.5, 800 rpm, and 0.2 U / mL LaMP, 5 U / mL glucose isomerase (Sweetzyme IT extra) and 0.1 U / mL CsKP or TbarKP were added, respectively. Samples (50 µL) were taken, inactivated in a 100 °C heat block for 10 min and further diluted 20-fold in 200 mM NaOAc pH 4.0 and then 400-fold in H2O for high performance anion exchange chromatography (HPAEC) analysis following the abovementioned protocol of Example 1.

[0109] The 20-fold diluted sample (pH 4.0) was supplemented with 5 U / mL highly pure amyloglucosidase (Megazyme) and the mixture was incubated at 55 ˚C for 1 h. This enzyme performs hydrolysis of a-1,4-linked gluco-oligosaccharides, starting from the non-reducing end. Hereby all unmodified malto-oligosaccharide substrates are hydrolysed into glucose, enabling the quantification of reaction products. Additionally, the formation of glucosyl- maltotriose (DP4) can be quantified, as it initially shows large overlap with the corresponding maltotriose substrate during HPAEC-analysis. In a similar fashion to the previous reactions, also here the formation of gluco-oligosaccharides comprising an a-(1,4)-linkage elongated with at least one α-(1,2)-terminally linked glucosyl moiety ( a1,2-MDx (DP3-5)) and kojioligosaccharides (K2-K5) in the reaction mixture was detected (see Table 1). ThePT-2001-WO-PCT remaining weight percentage to reach 100 wt.% on dry solids contains other saccharides such as glucose, maltose, and maltotriose. Table 2: conversion of starting material (30DE syrup) into elongated gluco-oligosaccharides

[0110] This example shows that TbarKP’s beneficial acceptor preference as illustrated in Example 1 also leads to a more desired product composition from an industrially relevant substrate.

[0111] 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.

[0112] 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 LISTINGS 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 AKQGERYEIEKLTVLVSSREKNVGDVFETCTNKLKEFETKSAEKLLFEHIEEYKRLWPT-2001-WO-PCT 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 LHGEGYKGHVFWDTEIFMLPFFIYVHPKAAKTLLMYRYNMLDAARKNAALNGYKG AQYPWESADTGEEETPKWGFDYMGNPVRIWTGDLEHHITADIAFAVWEYFRATEDI EFMLNYGAEVIFETARFWVSRCEYVKELDRYEINNVIGPDEFHEHVDNNAYTDYLA KWNIKKGLELINMLKEKYPEHYHAISNKKCLTNEEMEKWKEVEEKIYIPYDKDKKLI EQFEGYFDKKDYVIDKFDENNMPIWPEGVDITKLGDTQLIKQADVVMLMLLLGEEF DEETKRINYEYYEKRTMHKSSLGPSMYAIMGLKVGDHKNAYQSFMRSANVDLVDN QGNTKEGLHAASAGGTWQVVVFGFGGMEIDKEGALNINSWLPEKWDKLSYKVFW KGNLIEVIVTKQEVTVKKLKGKGNIKVKVKGKELTIELEHHHHHH SEQ ID NO: 3PT-2001-WO-PCT 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. MKYNFKFKEYTPKEEAVYGTVLTLGNGHIGIRGEIELEPTIYGTTIAGVYDYAPYFYR EIVNAPRVIGIQAFFNGEPLSLSTQRLLKYERELNIEDATLKTALCLETQNGTNIEYESI RIVHGKRKNIVLLKFAIKASEDGILTIISPIKTDVANPSYRDDITVRHIDVEELKAHEDY IYAGTRTLDGKYKIGIASSLVSKSKAKRSFLKNAGGISEILTLNVEKGKSYEFTKYITIV SSESPEVKEKAIRELQDAKELGFEALYTEHKNYWQEVWNLAKVEIEGDKEAERGLA FSLFHLIQSAPINDKISLTARGIHGFGYRGHVFWDTDIYALPFFMAVFPKKARDMLKY RYNNLDAARENARLNGYEGAQFPWESADDGYEATPPLIPLDIVGKEAVRIYTGEEEH HITADVAYAVELYYRFTKDEEFMSKYGLEIILEAARFWASRVEYESDKGYVIEKVIGPPT-2001-WO-PCT 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 VAGLFLLRRFFDRRVLEGNYEYYLRRTTHASSLSLPMYAAMAAYLGRVEEALALLR KAASTDLEDTYGNLEDGFHVAAAAGSWMALLLGFLGLEPRGGKLVAEPRLPEGLG VELNVWFRGKLHRVEARGSEYRITELEHHHHHH

Claims

PT-2001-WO-PCT CLAIMS 1. A process for elongating gluco-oligosaccharides with at least one α-(1,2)- terminally linked glucosyl moiety, comprising the steps of: a. providing an acceptor composition comprising gluco-oligosaccharides having a degree of polymerisation of 2, 3, 4, 5 or 6 glucosyl moieties, wherein each gluco- oligosaccharide comprises at least one α-(1,4)-linkage and wherein the acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation; and b. placing the acceptor composition in fluid contact with an isolated polypeptide in the presence of a 1-glucosyl phosphate donor, wherein the isolated polypeptide comprises kojibiose phosphorylase activity (E.C.2.4.1.230), wherein the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose, and alpha- 1,2-glucosyl-maltose as a product respectively, and wherein the activity of the polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate.

2. Process according to claim 1, wherein the activity of the polypeptide towards kojibiose and maltose is higher as compared to its activity towards glucose as an acceptor substrate.

3. Process according to claim 1 or 2, wherein the isolated polypeptide comprises: 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).

4. Process according to any one of claims 1 to 3, wherein the isolated polypeptide is obtained or obtainable from a microorganism selected from the group consisting of Thermococcus barophilus, Thermophilum pendens , and / or Palaeococcus pacificus.PT-2001-WO-PCT 5. Process according to claim 4, wherein the isolated polypeptide comprises the amino acid 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.

6. The process according to any one of claims 1 to 5, wherein the acceptor composition comprises at least 3, preferably at least 4 gluco-oligosaccharides having a different degree of polymerisation.

7. The process according to any one of claims 1 to 6, wherein the acceptor composition is a partially hydrolysed starch having a dextrose equivalent (DE) of less than 60, preferably less than 50, more preferably less than 45; optionally, wherein the partially hydrolysed starch is a maltodextrin or glucose syrup.

8. The process according to any of claims 1 to 7, wherein the acceptor composition comprises at least two different gluco-oligosaccharides selected from the group consisting of maltose, maltotriose, maltotetraose, maltopentaose and maltohexaose; and / or, wherein the 1-glucosyl phosphate donor is β-d-glucose-1-phosphate.

9. The process according to any of claims 1 to 8, wherein the process further comprises a step of preparing the 1-glucosyl phosphate donor by placing maltose in fluid contact with a polypeptide comprising maltose phosphorylase activity and phosphoric acid and / or a salt thereof; optionally, wherein the polypeptide comprising maltose phosphorylase activity is a recombinant maltose phosphorylase, preferably obtained or obtainable from a bacterium selected from the group consisting of Lactobacillus acidophilus, Bacillus selenitireducens, Enterococcus faecalis, Lactobacillus brevis, Lactobacillus sanfranciscensis, Paenibacillus sp. And Bacillus sp.

10. A composition comprising elongated gluco-oligosaccharides obtained or obtainable by the process according to any of claims 1 to 9, comprising at least 40 wt.% of elongated gluco- oligosaccharides based on the total weight of the composition as dry substance.PT-2001-WO-PCT 11. A composition comprising - one or more elongated gluco-oligosaccharides having a degree of polymerisation of 3, 4 or 5, wherein each elongated gluco-oligosaccharide comprises at least one α-1,2- terminally linked glucosyl moiety and at least one α-(1,4)-linkage, in an amount of from 25 wt.% or more, preferably from 30 wt.% or more, more preferably from 35 wt.% or more, even more preferably from 40 wt.% or more, even more preferably from 45 wt.% or more, most preferably from 50 wt.% or more of the total weight of dry substance of the composition; and / or - kojitriose, kojitetratose and kojipentaose in an amount of from 8 wt.% or more, preferably from 9 wt.% or more, more preferably from 10 wt.% or more, even more preferably from 11 wt.% or more, most preferably from 12 wt.% or more of the total weight of dry substance of the composition; and / or - kojibiose in an amount of from 11 wt.% or less, preferably from 10 wt.% or less, more preferably from 9 wt.% or less, most preferably 8 wt.% or less of the total weight of dry substance of the composition.

12. The composition according to claim 11 comprising - the one or more elongated gluco-oligosaccharides having a degree of polymerisation of 3, 4 or 5, wherein each elongated gluco-oligosaccharide comprises at least one α-1,2- terminally linked glucosyl moiety and at least one α-(1,4)-linkage, in an amount of from 30 wt.% to 70 wt.%, preferably from 40 wt.% to 65 wt.%; most preferably from 50 wt.% to 60 wt.% of the total weight of dry substance of the composition; and / or - kojitriose, kojitetratose and kojipentaose in an amount of from 8 wt.% to 25 wt.%, preferably from 10 wt.% to 20 wt.%, most preferably from 12 wt.% to 15 wt.% of the total weight of dry substance of the composition; and / or - kojibiose in an amount of from 1 to 11 wt.%, preferably from 3 wt.% to 10 wt.%, most preferably from 5 to 9 wt.% of the total weight of dry substance of the composition.

13. A composition comprising at least 40 wt.% gluco-oligosaccharides elongated with at least one α-(1,2)-terminally linked glucosyl moiety based on the total weight of the composition as dry substance, wherein the gluco-oligosaccharides have a degree of polymerisation of 2, 3, 4, 5, 6 or 7; and wherein the composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation and wherein each gluco-oligosaccharide comprises at least one α-(1,4)-linkage.PT-2001-WO-PCT 14. The composition according to any one of claims 10 to 13, wherein the elongated gluco- oligosaccharides comprise 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 the linkages from kojibiose and kojioligosaccharides.

15. The composition according to any of claims 10 to 14, further containing one or more of kojibiose, kojitriose, kojitetraose and kojipentaose.

16. The composition according to any of claims 10 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-glucopyranose2-O-α-D-glucosyl-maltotriose α-D-Glucopyranosyl-(1^2)-α-D-glucopyranosyl-(1^4)-α-D-glucopyranosyl-(1^4)-D- glucopyranosePT-2001-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-glucopyranose2-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-glucopyranose2-O-α-D-glucosyl-maltohexaosePT-2001-WO-PCT 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 foodstuff, pet food, feed or personal care product comprising the composition according to any of claims 10 to 16.

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