Fructo-oligosaccharides and method for preparation thereof

Abstract

The present disclosure relates to a composition comprising carbohydrates and to a method for the preparation thereof. The carbohydrates comprise short-chain fructo-oligosaccharides (ScFOS) GF2, GF3, GF4 and GF5, wherein the ScFOS comprise, relative to the total weight of ScFOS in the composition, at least 16 wt.% of GF4 and GF5.

Description

[0001] f.

[0002] BACKGROUND OF THE INVENTION

[0003] Fructo-oligosaccharides (FOS) are oligosaccharides that occur naturally in plants, such as vegetables, mushrooms or fruits (e.g., banana, onion, asparagus, burdock, garlic, honey, chicory root and the like). They are composed of linear chains of one glucose unit usually linked by a a(1— >2) bond to a series of fructose units usually linked by [3(1 — >2) bonds.

[0004] FOS possess several beneficial properties that make their use as food ingredient attractive. FOS have a low sweetness intensity; they are also less caloric than saccharose, glucose or fructose and they are considered as soluble dietary fiber. Furthermore, FOS have important beneficial physiological effects such as low carcinogenicity, improved mineral absorption and decreased levels of serum cholesterol, triacylglycerols and phospholipids. FOS are also a recognized class of prebiotics and have been shown to promote a positive impact on gut microbiota composition and metabolic activity. Notably, they act as nutrients for beneficial gut bacteria such as bifidobacteria and lactobacilli. These bacteria ferment FOS, leading to increased production of short chain fatty acids, including butyrate, which have various beneficial effects such as promoting gut barrier integrity, reducing inflammation, improving mineral absorption, reducing cholesterol levels, regulating blood sugar, and promoting immune function.

[0005] Therefore, FOS are increasingly included in food and beverages, infant foods, animal feed and dietary supplements. They are conveniently used for supplying non-sucrose sweetness, as a texturizer, and / or for their prebiotic properties.

[0006] FOS can be produced by three methods namely: (i) extraction from plants (ii) enzymatic production from inulin by endoinulinases and (iii) transfructosylation of sucrose by [3- fructofuranosidases. Their extraction from plants being costly and providing low yield, industrial preparation of FOS is typically carried out either by the hydrolytic action of endoinulinases on inulin or by transfructosylation of sucrose by [3-fructofuranosidases. In transfructosylation process, [3-fructofuranosidases break the linkages present in between sucrose molecules and transfer the fructose to receptor sucrose / FOS. FOS prepared by this method are of short chain length (DP<6) with a formula a-D-Glu-[p-D-Fru-]n, notably a-D-Glu(1— >2)-[[3-D-Fru(1— >2)-]n, where ‘n’ represents the fructosyl units which varies between 2 and 5 (GF series with a degree of polymerization of 3 to 6 and designated as GF2 to GF5). They are short-chain FOS (ScFOS). In production from inulin, indoinulinases hydrolyse the internal linkages of inulin to produce usually FOS with a DP of up to 10 with a formula a-D-Glu-[p-D-Fru-]n, notably a-D-Glu(1— >2)-[[3- D-Fru(1— >2)-]n, where ‘n’ ranges from 2 to 9 and side-products with formula [3-D-Fru-[[3-D-Fru- ]n, notably p-D-Fru(1— >2)-[[3-D-Fru(1— >2)-]n, where ‘n’ ranges between 1 and 9 (FF series with DP of 2 to 10 and designated as F2 to F9). Long-chain FOS or lcFOS have a DP>6.

[0007] Whilst everybody agrees on the prebiotic properties of FOS, some experts declare that GF2 is the most valuable FOS species, and others argue that FOS from inulin are the most beneficial FOS for gut microbiota. Thus, two kinds of FOS compositions are mostly commercialized. The first kind is a composition comprising mostly GF2, GF3 species and little amount of GF4. The second kind is a composition comprising various FOS including species with a wide scope of DP, including GF series and FF series.

[0008] Though the transfructosylation process may appear of great potential because it allows controlling chain length by modulating the reaction time, known transfructosylation processes produce large amount of by-products (unreacted sucrose, generated fructose and glucose) which increases the purification cost and decreases the product yield. In addition, FOS obtained by known transfructosylation processes tend to change color over time, becoming yellowish.

[0009] An enzymatic process, notably transfructosylation process, can occur by in vivo bioconversion using a living microorganism or by in vitro biocatalysis using an enzyme.

[0010] For bioconversion (or biotransformation), the enzyme transforming a substrate is present in a living biomass which was produced by fermentation. The step of bioconversion happens in a culture medium which comprises nutrients for microorganism growth. It also requires conditions compatible with a living microorganism (temperature, time, pH, osmotic condition...) and the substrate to be converted is usually strongly diluted in aqueous medium. Such conditions are not always compatible with the development of an industrial process (time, concentration of substrate, purification step...).

[0011] On the opposite, biocatalysis allows to work with conditions which will be optimized for the transformation of the substrate. For biocatalysis, the enzyme used can be isolated by purification, or it can be part of a mix of proteins (culture supernatant) or it can also be present inside an expression cell (whole cell sample). The reaction medium is not a culture medium. It is optimized for the reaction. Thus, high concentrations of substrate can be used, pH and temperature are optimized for the enzyme activity and not the growth of the microorganism. The substrate transformation by biocatalysis is reproductible, fast and with high yields and concentrations.

[0012] Therefore, there remains a need for a new process for preparing FOS via transfructosylation, in particular by biocatalysis, which allows decreasing the purification costs and increasing the product yield and for new compositions that remain stable over time (no coloration) and that exhibit advantageous properties, such as efficient prebiotic properties.

[0013] SUMMARY OF THE INVENTION

[0014] The invention relates to a composition comprising mainly carbohydrates, the carbohydrates comprising short-chain fructo-oligosaccharides (ScFOS) GF2, GF3, GF4 and GF5, wherein the ScFOS comprise, relative to the total weight of ScFOS in the composition, at least 16 wt.% of GF4 and GF5.

[0015] The present invention also relates to a method for preparing a composition as described herein, notably by biocatalysis, the method comprising a step a) of incubating an enzyme chosen from fructosyltransferase (Ftase) or [3-fructofuranosidase with saccharose to catalyze the synthesis of ScFOS and obtain a composition comprising ScFOS, monosaccharides and disaccharides. Further aspects of the invention are as disclosed herein and in the claims.

[0016] DEFINITIONS

[0017] As used herein, the term “oligosaccharides” refers to saccharides consisting of at least two and up to ten linked monosaccharide units, i.e. having a degree of polymerization of 2 to 10.

[0018] As used herein, the term “degree of polymerization” (DP) refers to the number of monosaccharide units in the oligosaccharides.

[0019] As used herein, the term “short-chain fructo-oligosaccharide” (ScFOS) refers to fructooligosaccharides having of degree of polymerization (DP) <6. ScFOS are represented by the following formula a-D-Glu-[p-D-Fru-]n, notably a-D-Glu(1— >2)-[[3-D-Fru(1— >2)-]n, where ‘n’ represents the number of fructosyl units which varies between 2 and 5 (GFn series with a degree of polymerization of 3 to 6 and designated as GF2 to GF5). Exemplary ScFOS include 1-kestose (GF2, having a DP of 3), 1-nystose (GF3, having a DP of 4), 1F- [3-fructofuranosyl nystose (GF4, having a DP of 5) and 1 ,1 ,1 ,1-Kestohexaose (GF5, having a DP of 6).

[0020] In other terms, ScFOS consist of linear [3-D-fructofuranosyl units in which the fructofuranosyl units (F) are bound to sucrose or notably GFn (1 F(1-p-D-fructofuranosyl)n-i sucrose).

[0021] As used herein, the term “carbohydrate” refers to saccharides, including monosaccharides (e.g. glucose, fructose), disaccharides (e.g. sucrose (also known as saccharose), lactose, maltose), oligosaccharides and polysaccharides.

[0022] Percentage by weight, percentage by mole and percentage by volume are respectively abbreviated herein as wt.%, mol.% and vol.%.

[0023] DESCRIPTION OF THE INVENTION

[0024] The present invention relates to a composition comprising mainly carbohydrates, the carbohydrates comprising short-chain fructo-oligosaccharides (ScFOS) GF2, GF3, GF4 and GF5, wherein the ScFOS comprise, relative to the total weight of ScFOS in the composition, at least 16 wt.% of GF4 and GF5. The composition of the invention comprises mainly carbohydrates. This means that the carbohydrates are the major components of the composition, i.e. they represent more than 50% by weight of the weight of the composition in a dried form (dry weight), in particular more than 75% by weight, notably more than 90%.

[0025] Further components may be present in the composition. In particular, the composition may further comprise water, organic solvents, salts, preservatives, denatured proteins and any combinations thereof. In other words, the composition of the invention can comprise, or consist of, carbohydrates and one or more components selected from the group consisting of water, organic solvent, salts, preservatives and denatured proteins.

[0026] The composition of the invention may comprise carbohydrates at a concentration of at least 30 g of carbohydrate for 100 g of composition, notably at least 50 g of carbohydrate, preferably at least 60 g or at least 62g of carbohydrate, more preferably at least 65g of carbohydrate for 100g of composition. The ScFOS of the composition may comprise at least 17 wt.% or at least 20 wt.% of GF4 and GF5, relative to the total weight of ScFOS in the composition.

[0027] Notably, the ScFOS of the composition may comprise at least 22 wt.% or at least 25 wt.% of GF4 and GF5, relative to the total weight of ScFOS in the composition. In some embodiments, the ScFOS comprises from 16 wt.% to 50 wt.%, or from 17 wt.% to 45 wt.%, or from 20 wt.% to 40 wt.% of GF4 and GF5, relative to the total weight of ScFOS in the composition.

[0028] The ScFOS of the composition may comprise at least 17 wt.% or at least 20 wt.% of GF4, relative to the total weight of ScFOS in the composition.

[0029] Notably, the ScFOS of the composition may comprise at least 22 wt.% or at least 26 wt.% of GF4, relative to the total weight of ScFOS in the composition. In some embodiments, the ScFOS comprises from 16 wt.% to 50 wt.%, or from 20 wt.% to 45 wt.%, or from 25 wt.% to 40 wt.% of GF4, relative to the total weight of ScFOS in the composition.

[0030] The ScFOS of the composition may comprise at least 4 wt.% or at least 5 wt.% of GF5, relative to the total weight of ScFOS in the composition.

[0031] Notably, the ScFOS of the composition may comprise at least 6 wt.% or at least 7 wt.% of GF5, relative to the total weight of ScFOS in the composition. In some embodiments, the ScFOS comprises from 4 wt.% to 20 wt.%, or from 5 wt.% to 15 wt.%, or from 6 wt.% to 12 wt.% of GF5, relative to the total weight of ScFOS in the composition.

[0032] The ScFOS of the composition may comprise less than 2 wt.% or less than 1 wt.% of GF6, relative to the total weight of ScFOS in the composition.

[0033] Notably, the ScFOS of the composition may not comprise GF6, or it comprises traces of GF6.

[0034] The ScFOS of the composition may further comprise GF3, in particular from 25 wt.% to 55 wt.% of GF3 relative to the total weight of ScFOS in the composition. In some embodiments, the ScFOS of the composition further comprise from 27 wt.% to 52 wt.% or from 29 wt.% to 50 wt.% of GF3 relative to the total weight of ScFOS in the composition.

[0035] In some embodiments, the ScFOS of the composition comprise, relative to the total weight of ScFOS in the composition, at least 60 wt.%, notably of at least 63 wt.% or at least 68 wt. % of GF3 and GF4, or of GF3, GF4 and GF5, when GF5 is present. In some embodiments, the ScFOS comprise from 60 wt.% to 90 wt.%, or from 63 wt.% to 85 wt.% or from 68 wt.% to 85 wt.%, relative to the total weight of ScFOS in the composition, of GF3 and GF4, or of GF3, GF4 and GF5, when GF5 is present.

[0036] In the composition of the invention, the amount in weight of GF3 is typically higher than the total amount in weight of (GF4 and GF5).

[0037] In the composition of the invention, the weight ratio of GF3 / (GF4 and GF5) may range from 3:1 to 1 :2, notably from 2.5:1 to 1 :1.5, in particular from 2.2:1 to 1 :1.2, more particularly from 2:1 to 1 :1.

[0038] The ScFOS of the composition may further comprise GF2, in particular from 10 wt.% to 42 wt.% of GF2 relative to the total weight of ScFOS in the composition. In some embodiments, the ScFOS of the composition further comprises from 12 wt.% to 40 wt.% or from 15 wt.% to 38 wt.% of GF2 relative to the total weight of ScFOS in the composition.

[0039] In the composition of the invention, the amount in weight of GF2 is typically lower than the total amount in weight of (GF4 and GF5).

[0040] In the composition of the invention, the weight ratio of GF2 / GF4 may range from 2:1 to 1 :2, in particular from 1.8:1 to 1 :2, or from 1.6:1 to 1 :1.9, more particularly from 1.5:1 to 1 :1.8 or from 1.4:1 to 1 :1.6, or even from 1.3:1 to 1 :1.3.

[0041] In the composition of the invention, the weight ratio of GF2 / (GF4 and GF5) may range from 2:1 to 1 :3, in particular from 1.5:1 to 1 :2.5, or from 1.3:1 to 1 :2, more particularly from 1.1 :1 to 1 :1.9 or from 1 :1 to 1 :1.8.

[0042] In the composition of the invention, the weight ratio of GF2 / GF3 / (GF4 and GF5) may range from 2:3:1 to 1 :2:3.

[0043] Typically, the ScFOS of the composition of the invention comprise GF2, GF3, GF4 and optionally GF5. GF2, GF3, GF4 and GF5 are ScFOS of the GF series.

[0044] The ScFOS of the composition of the invention comprise notably at least 90 wt.% of ScFOS of GF series relative to the total weight of ScFOS in the composition, notably at least 97 wt.% or at least 98 wt.%, or even at least 99 wt.% of ScFOS of GF series, relative to the total weight of ScFOS in the composition. Typically, the ScFOS of the composition of the invention comprise less than 5wt.% of ScFOS of FF series, relative to the total weight of ScFOS in the composition, notably less than 2 wt.% or even less than 1 wt.% of FOS of FF series, relative to the total weight of ScFOS in the composition. The ScFOS of FF series include notably F2, F3, F4, F5 and F6.

[0045] In some embodiments, the ScFOS of the composition do not comprise FOS of FF series. In other terms, the ScFOS of the composition do not comprise F2, F3, F4, F5 or F6.

[0046] The carbohydrates of the composition may also comprise IcFOS, typically less than 5wt.% of IcFOS relative to the total weight of carbohydrates in the composition, notably less than 2 wt.% or even less than 1wt.% of IcFOS relative to the total weight of carbohydrates in the composition.

[0047] Typically, the carbohydrates of the composition do not comprise IcFOS.

[0048] The carbohydrates of the composition may also comprise saccharose, glucose and / or fructose. Typically, the carbohydrates of the composition do not comprise polysaccharides.

[0049] The compositions of the present invention are typically produced from sucrose by transfructosylation with an enzyme with transfructosylating activity as disclosed herein below. Therefore, they typically do not comprise compounds of formula [3-D-Fru(1— >2)-[[3-D-Fru(1— >2)- ]n, where ‘n’ ranges between 1 and 9 (FF series with DP of 2 to 10 and designated as F2 to F9) and FOS with a formula a-D-Glu(1— >2)-[[3-D-Fru(1— >2)-]n, where ‘n’ ranges from 6 to 9 (GF series with DP of 7 to 10 and designated as GF6 to GF9). In particular, the composition of the invention is typically free from F3, F4 and F5 and / or typically free from GF6, GF7, GF8 and GF9. If present, compounds with formula p-D-Fru(1— >2)-[[3-D-Fru(1— >2)-]n, where ‘n’ ranges between 1 and 9 and / or FOS with formula a-D-Glu(1— >2)-[[3-D-Fru(1— >2)-]n, where ‘n’ ranges from 6 to 9 are only present in trace amount (lower than 5 wt.% relative to scFOS, in particular less than 2 wt.%, more particularly less than 1wt.%).

[0050] The compositions of the invention, such as obtained by enzymatic route, notably by biocatalysis, as disclosed herein below (without purification), typically comprise a total amount of ScFOS ranging from 52 wt.% to 67 wt.% relative to the total weight of carbohydrates in the composition, preferably a total amount of ScFOS ranging from 54 wt.% to 65 wt.% or from 55 wt.% to 63 wt.% relative to the total weight of carbohydrates in the composition. Such compositions typically further comprise fructose. Fructose is a side-product of the enzymatic reaction. However, the amount of fructose in the composition of the invention is typically low. In particular, the compositions comprise less than 3 wt.%, preferably less than 2 wt.%, of fructose relative to the total weight of carbohydrates in the composition. Preferably, such compositions comprise from 0.2 wt.% to 3 wt.% or from 0.5 wt.% to 2 wt.% or from 0.7 wt.% to 1.7 wt.% of fructose relative to the total weight of the carbohydrates in the composition.

[0051] The compositions of the invention, such as obtained by enzymatic route, notably by biocatalysis, as disclosed herein below (without purification), typically further comprise glucose, in particular from 25 wt.% to 45 wt.% of glucose relative to the total weight of carbohydrates in the composition, notably from 27 wt.% to 42 wt.% or from 28 wt.% to 40 wt.% relative to the total weight of carbohydrates in the composition. Glucose is also a side-product of the enzymatic reaction.

[0052] The compositions of the invention, such as obtained by enzymatic route, notably by biocatalysis, as disclosed herein below (without purification), typically further comprise saccharose, in particular less than 12 wt.% of saccharose relative to the total weight of the carbohydrates in the composition, preferably less than 10 wt.%, notably from 0.5 wt.% to 15 wt.% or from 1 wt.% to 12% or from 2 wt.% to 10 wt.% relative to the total weight of carbohydrates in the composition. Saccharose is the starting material.

[0053] The compositions of the invention may be further purified to increase the total amount of ScFOS in the composition, i.e. saccharose, fructose and glucose are mainly separated from ScFOS. Thus, in some other embodiments, the compositions of the invention comprise a total amount of ScFOS ranging from 90 wt.% to 100 wt.% relative to the total weight of carbohydrates in the composition, preferably from 92 wt.% to 99 wt.% or from 94 wt.% to 98 wt.% relative to the total weight of carbohydrates in the composition.

[0054] In some other embodiments, the ScFOS composition comprises a total amount of ScFOS ranging from 60 wt.% to 90 wt.% relative to the total weight of carbohydrates in the composition, preferably from 65 wt.% to 85 wt.% or from 70 wt.% to 85 wt.% relative to the total weight of carbohydrates in the composition. In addition to carbohydrates, the composition can also comprise water, organic solvent, salts, preservatives, and / or denatured proteins.

[0055] The compositions of the present invention are useful for the preparation of foodstuffs and beverages, infant foods, dietary supplement, pet food, or in animal feed.

[0056] Preparation methods

[0057] The compositions of the present invention can be prepared by enzymatic catalysis, notably by biocatalysis, preferably from saccharose, more preferably by transfructosylation of saccharose catalyzed by an enzyme with transfructosylating activity. Enzymes with transfructosylating activity are classified as fructosyltransferase (Ftases) or [3-fructofuranosidase (Ffase or invertase).

[0058] Typically, the method of the invention by biocatalysis is performed without use of a revivable microorganism, i.e. no revivable microorganism is used in any of steps of the method of the present invention.

[0059] In particular, the compositions of the present invention can be prepared by a method as described herein below. The present invention also relates to such a method and to compositions obtained or susceptible to be obtained by the disclosed method.

[0060] The method of the invention comprises the following steps: a) incubating an enzyme chosen from fructosyltransferase (Ftase) or [3-fructofuranosidase with saccharose to catalyze the synthesis of ScFOS and obtain a composition comprising ScFOS, monosaccharides and disaccharides; b) optionally denaturing the enzyme after step a) or, when the enzyme is an immobilized enzyme, filtrating the composition obtained after step a); c) optionally demineralizing and / or polishing the composition obtained after step b); d) optionally removing the monosaccharides and disaccharides from the composition obtained after step b) or c).

[0061] Step a)

[0062] The method of the invention comprises the step of incubating an enzyme chosen from fructosyltransferase (Ftase) or [3-fructofuranosidase with sucrose to catalyze the synthesis of ScFOS and obtain a composition comprising ScFOS. The Ftase useful in the present invention typically belongs to class EC 2.4.1 , more specifically the Ftase is a hexosyltransferase belonging to class EC 2.4.1 , notably classes EC 2.4.1.9, 2.4.1.99 or 2.4.1.100.

[0063] The p-fructofuranosidase useful in the present invention typically belong to class EC 3.2.1.26. In particular, the [3-fructofuranosidase can be a 2,1-fructan:2,1-fructan 1 - fructosyltransferase, an inulosucrase or a sucrose / sucrose fructosyltransferase.

[0064] The enzyme useful in the present invention is typically obtained from a culture of Aspergillus sp. In other terms the enzyme useful in the present invention is typically a FTase of Aspergillus sp.

[0065] The enzyme may be produced in yeasts, notably Pichia pastoris, Saccharomyces cerevisiae, or Yarrowia lipolytica. Preferably the yeast is modified so that it has no invertase activity.

[0066] The enzyme can be free or immobilized.

[0067] An enzyme suitable to perfom step (a) include SEQENZYM® FT that comes from the enzyme collection from Proteus by Seqens.

[0068] The incubation is typically performed in an aqueous solution. In particular, the incubation is not performed in a culture medium, i.e. a medium comprising a source of nitrogen, a source of carbon and minerals. The concentration of saccharose in the aqueous solution may be from 30 to 80°Brix, typically from 50 to 80°Brix, in particular from 62 to 77°Brix, more particularly from 65 to 75°Brix (one degree Brix corresponds to 1 gram of saccharose in 100 grams of solution).

[0069] The saccharose useful in the method of the invention can be cane sugar or beet sugar. It can be refined, unrefined, brown or unprocessed.

[0070] Step a) can be performed at a temperature from 40 to 65°C, notably from 45 to 60°C.

[0071] Step a) can be performed from 1 hour to 96 hours, preferably from 1 to 80 hours.

[0072] Step a) is typically performed under stirring.

[0073] Step a) is typically performed at a pH ranging from 4 to 9, in particular from 4.5 to 7, more particularly from 5 to 6,5. Step a) allows obtaining a composition comprising mainly carbohydrates and comprising a total amount of ScFOS ranging from 52 wt.% to 67 wt.% relative to the total weight of the carbohydrates in the composition. The composition may be as disclosed herein above.

[0074] Step b)

[0075] The method of the invention can further comprise the step of denaturating the enzyme after step a) or, when the enzyme is an immobilized enzyme, filtrating the composition obtained after step a).

[0076] The enzyme can be denatured by heat treatment such as pasteurization or sterilization. For instance, the composition obtained after step a) can be heated at a temperature ranging from 80 to 100°C for at least 3 minutes, preferably at least 5 minutes, notably less than 15 minutes. The sterilization is typically carried out at 121 to 135 ° C for 10-20 s.

[0077] Alternatively, the enzyme can be removed by filtration when the enzyme is immobilized.

[0078] The compositions obtained after step b) can then undergo purification steps, notably step c) and / or d).

[0079] Step c)

[0080] The method of the invention can further comprise the step demineralizing and / or polishing of the composition obtained after step b).

[0081] Demineralizing aims at removing mineral and organic salts from the composition.

[0082] The composition can be colored. Polishing aims at removing this color from the composition. It can be done with activated carbon.

[0083] Step c) allows purifying the composition. Such purification can be advised before step d), especially when chromatographic separation methods are used in step d).

[0084] Step d)

[0085] The method of the invention can further comprise the step of removing the monosaccharides and disaccharides from the composition obtained after step b) or c). The removal may be performed by well-known separation methods such as chromatographic separation methods or nanofiltration membrane separation methods. For example, Sephadex G-10 resin may be used to perform a chromatographic separation. When the separation is performed by nanofiltration, membranes with a molecular weight cut-off of 200Da or 300Da may be used. Typically, removal is performed by simulated moving bed chromatography (SMBC), notably by using the technique of sequential simulated moving bed (SSMB).

[0086] Step d) allows increasing the total amount of ScFOS in the composition: the monosaccharides and disaccharides (saccharose, fructose and glucose), or part thereof, are separated from the ScFOS.

[0087] Step d) may allow obtaining compositions comprising a total amount of ScFOS ranging from from 60 wt.% to 90 wt.% relative to the total weight of carbohydrates in the composition, preferably from 65 wt.% to 85 wt.% or from 70 wt.% to 85 wt.% relative to the total weight of carbohydrates in the composition or may allow obtaining compositions comprising from 90 wt.% to 100 wt.% relative to the total weight of carbohydrates in the composition, preferably from 92 wt.% to 99 wt.% or from 94 wt.% to 98 wt.% relative to the total weight of carbohydrates in the composition.

[0088] The method Of the invention can be performed as follows: step a) then b) then c) then d), or step a) then b) then d).

[0089] The method of the present invention allowing obtaining compositions with higher proportion of GF4 or GF4 and GF5 and consequently lower proportion of GF2 and saccharose compared to know methods. Purification of the compositions is resultantly easier. In addition, the compositions of the invention are stable overtime, no change of color is noticeable.

[0090] The method of the present invention is typically performed without use of yeast, notably of Saccharomyces cerevisiae, i.e. no yeast strain, notably a strain of Saccharomyces cerevisiae, is used in any of steps of the method of the present invention.

[0091] Embodiments of the present invention will now be described by way of the following examples which are provided for illustrative purposes only, and not intended to limit the scope of the disclosure.

[0092] The present invention also relates to a prebiotic composition comprising the composition of the present invention. In particular, the prebiotic composition is a prebiotic composition for butyrate-producing bacteria.

[0093] The prebiotic composition may be for used for maintaining and / or restoring the intestinal flora, for intestinal regulation, for stimulating the growth of bifidobacteria and / or lactobacilli and / or inhibiting the growth of at least one of bacteroides, Clostridia, coliforms, sulphate reducing bacteria, for prevention or improvement of diarrhea, for prevention or improvement of an inflammatory bowel disease or prevention of large intestine cancer.

[0094] The present invention also relates to a method for maintaining and / or restoring the intestinal flora, for intestinal regulation, for stimulating the growth of bifidobacteria and / or lactobacilli and / or for inhibiting the growth of at least one of bacteroides, Clostridia, coliforms, sulphate reducing bacteria, for prevention or improvement of diarrhea, for prevention or improvement of an inflammatory bowel disease or prevention of large intestine cancer, the method comprising the administration to a subject (e.g. humans) in need thereof of an effective amount of a prebiotic composition as disclosed herein.

[0095] The effective amount of the composition varies as a function of numerous parameters such as, for example, the weight, age, sex, the state of progress of the disorder to be treated and the sensitivity of the individual to be treated.

[0096] The prebiotic composition can be a food product or a drink.

[0097] The present invention also deals with a food product comprising the composition of the present invention.

[0098] Typically, the food product is in an administrable form, notably the administrable form is a pharmaceutical formulation, a nutritional formulation, a dietary supplement, a functional food, a beverage product, or a combination thereof.

[0099] EXAMPLES

[0100] Materials

[0101] Seqenzym® FT sold by Proteus by Seqens with an activity of 6000U / mL.

[0102] Example A

[0103] Method: • Weight sucrose solution 67°Brix,

[0104] • Add the amount of SEQENZYM® FT indicated in the table below (in w / w% compared to sucrose solution),

[0105] • Incubate at 50°C under continuous agitation for the time indicated in table 1 , • stop the reaction by heating at 95°C for at least 5 minutes.

[0106] Analysis process:

[0107] Samples are anlysed by HPLC according to the following method

[0108] • Column: Sugar-PakTM (Waters) + precolumn • Flow: 0.4 mL / min

[0109] • Mobile phase: ultrapure water with 50 mg / L EDTACa2+

[0110] • Rl detector: 35°C

[0111] • column temperature: 85°C

[0112] • Sample volume: 20 pL • Run time: 17 min

[0113] Results are provided in table 1 .

[0114]

[0115] Table 1: wt.% of ScFOS, monosaccharides and disaccharides in the compositions of the invention

[0116] Example B

[0117] Preparation Composition 8

[0118] A 2L double jacketed reactor was filled with deionized water (330 g). The water was heated at 50°C, then sucrose (770 g) was added in three portions under stirring. After complete dissolution of the sugar, Seqenzym® FT (1.267 g) was added and the reaction was stirred at 50°C for 70h. The FOS solution was quenched at 90°C. Deionized water (2750 g) was added to obtain a 20°Bx solution.

[0119] Preparation Composition 9 (comparative)

[0120] A sample of Nutraflora from Ingredion was diluted in deionized water, glucose and saccharose were added so as to obtain a solution of carbohydrates with concentration of 200g / L and with a composition of carbohydrate as described in Table 2 below.

[0121] Preparation Composition 10 (comparative)

[0122] A sample of Orafti Synergy 1 from Beneo was diluted in deionized water, glucose and saccharose were added so as to obtain a solution of carbohydrate at a concentration of 200g / L with a composition of carbohydrates as described in Table 2 below.

[0123] Analysis process: Samples are anlysed by HPLC / CAD according to the following method

[0124] • Column: Asahipak NH2P-50 4E 250*4.6mm; 5pmSugar-PakTM (Waters) + precolumn

[0125] • Flow: 1.0 mL / min

[0126] • Mobile phase: water / acetonitrile gradient

[0127] • CAD detector: 62.5psi, 80°C, filtre 5.0, power function 1.0, frequency 10.

[0128] • column temperature / injection temperature: 25°C / 8°C

[0129] • Sample volume: 10 pL • SAmple concentration 1.0mg / mL

[0130] • Run time: 28 min

[0131] Results are provided in table 2. a) Amount in wt.% relative to total weight of carbohydrates in the composition b) Amount in wt.% relative to the total weight of ScFOS Table 2: wt.% of carbohydrates in Compositions 8, 9 and 10

[0132] Prebiotic tests

[0133] The impact of composition 8 on the gut microbiota of nine healthy adult donors with no history of chronic disease and no antibiotic use at least four months preceding the experiment.

[0134] A negative control (not containing test product) and two comparative compositions 9 and 10 were included as reference conditions.

[0135] An assessment was made of effects on microbial metabolic activity targeting saccharolytic markers (SCFA and lactate) and proteolytic markers (ammonium).

[0136] Solutions of compositions 8, 9 and 10 were prepared in water at a concentration of 35g / L. They were subjected to dialysis to simulate absorption of small molecules taking place at the level of the small intestine (removal of mono- and disaccharides). Nitrogen was then bubbled into solutions.

[0137] Solutions were added to a carbohydrate-depleted background nutritional medium in a low volume reactor resulting in a final concentration of 3.5 g / L. Then, 10% (v / v) of fecal inoculum containing 7.5% (v / v) fecal material of healthy human donor was added to each reactor and served as the microbial source. Reactors were incubated for 6h, 24h and / or 48h at 37°C under continuous mild shaking (90rpm) and anaerobic atmosphere.

[0138] Samples were collected at the start of the experiment (Oh) at 6h, 24h and 48h. They were used to determine effects on microbial activity:

[0139] Butyrate concentration was evaluated based on a liquid-liquid extraction of sample followed by analysis with gas chromatography and detection with FID. Lactate concentration was evaluated using EnzytecTM kit (R-biopharm).

[0140] - Ammonium concentrations were evaluated by colorimetric analysis using the indophenol blue spectrophotometric method.

[0141] Results are presented in tables 3 to 5. *Statistically significant difference (p<0.05) within the time interval

[0142] Compositions 8, 9 and 10 are statistically different compared to negative control (p<0.05)

[0143] Table 3: Average butyrate production (mM)

[0144] Composition 8 leads to butyrate production which has various beneficial effects as illustrated in introduction.

[0145] Compositions 8, 9 and 10 are statistically different compared to negative control (p<0.05)

[0146] Table 4: Average lactate consumption (mM)

[0147] Composition 8 leads to higher lactate production at early stage of incubation than compositions 9 and 10 indicate a more pronounced prebiotic effect. Indeed, lactate are produced by lactic acid bacteria which are considered to be beneficial bacteria in the colon.

[0148] *Statistically significant difference (p<0.05) within the time interval

[0149] Compositions 8, 9 and 10 are statistically different compared to negative control (p<0.05)

[0150] Table 5: Average ammonium production (mg / L)

[0151] Composition 8 leads to low production of ammonium, a product of proteolytic degradation. Proteolytic fermentation results in the production of potentially toxic or carcinogenic compounds such as p-cresol and p-phenol. It indicates that composition 8 has improved prebiotic properties.

Claims

CLAIMS1. A composition comprising carbohydrates, the carbohydrates comprising short-chain fructo-oligosaccharides (ScFOS) GF2, GF3, GF4 and GF5, wherein the ScFOS comprise, relative to the total weight of ScFOS in the composition, at least 16 wt.% of GF4 and GF5.

2. The composition according to claim 1 , wherein the ScFOS further comprise from 25 wt.% to 55 wt.% of GF3 relative to the total weight of ScFOS in the composition.

3. The composition according to claim 1 or 2, wherein the weight ratio of GF2 / GF4 ranges from 2:1 to 1 :2, preferably from 1.3:1 to 1 :1.3.

4. The composition according to any one of claims 1 to 3, wherein the amount of GF3 in weight is higher than the amount of GF4 and GF5 in weight and the amount of GF4 and GF5 in weight is higher than the amount of GF2 in weight.

5. The composition according to any one of claims 1 to 4, wherein it comprises a total amount of ScFOS ranging from 52 wt.% to 67 wt.% relative to the total weight of carbohydrates in the composition.

6. The composition according to any one of claims 1 to 5, wherein the carbohydrates further comprise saccharose, in particular in an amount of less than 12 wt. % relative to the total weight of carbohydrates in the composition.

7. The composition according to any one of claims 1 to 4, wherein it comprises a total amount of ScFOS ranging from 90 wt.% to 100 wt.% relative to the total weight of carbohydrates in the composition.

8. The composition according to any one of claims 1 to 7 obtained by biocatalysis, preferably from saccharose.

9. A method for preparing a composition according to any one of claims 1 to 8, the method comprising a step a) of incubating an enzyme chosen from fructosyltransferase (FTase) or [3- fructofuranosidase with saccharose to catalyze the synthesis of ScFOS and obtain a composition comprising ScFOS, monosaccharides and disaccharides.

10. The method according to claim 9 wherein it further comprises a step b) of denaturing the enzyme once the incubation is terminated or, when the enzyme is an immobilized enzyme, filtrating the composition obtained after step a).11 . The method according to claim 9 or 10 wherein it further comprises the step of removing the monosaccharides and disaccharides from the composition obtained after step b) or c) to obtain a composition according to claim 7.

12. Use of composition according to any one of claims 1 to 8 in foodstuffs, infant food, dietary supplement, pet food, or in animal feed.

13. Prebiotic composition comprising a composition according to any one of claims 1 to 8.

14. Prebiotic composition according to claim 13 for use for maintaining and / or restoring the intestinal flora, for intestinal regulation, for stimulating the growth of bifidobacteria and / or lactobacilli and / or for inhibiting the growth of at least one of bacteroides, Clostridia, coliforms, sulphate reducing bacteria, for prevention or improvement of diarrhea, for prevention or improvement of an inflammatory bowel disease or prevention of large intestine cancer.

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