Prebiotic Compositions And Methods Of Production Thereof

Abstract

The present invention relates to a prebiotic composition comprising: (i) a enzymatically modified high intensity sweetener glycoside; and an oligosaccharide. In particular, the invention relates to a galactosylated and/or fructosylated and/or deglycosylated high intensity sweetener glycoside; and (ii) an oligosaccharide obtained during the same enzymatic reaction. Uses and methods of producing the composition are also described.

Claims

1. A prebiotic composition comprising: (i) an enzymatically modified high intensity sweetener glycoside; and (ii) an oligosaccharide.

2. The prebiotic composition as claimed in claim 1, wherein the high intensity sweetener glycoside is enzymatically modified by galactosylation and/or fructosylation and/or deglycosylation.

3. The composition as claimed in claim 1, wherein the high intensity sweetener glycoside is selected from one or more of the following: Steviol glycosides or Mogroside, or derivatives thereof.

4. The composition as claimed in claim 4, wherein the Steviol glycoside comprises Rebaudioside A or the Mogroside comprises Mogroside V.

5. The composition as claimed in either claim 1 or 2, wherein the oligosaccharide is one or more of the following: galactooligosaccharides (GOS) or fructooligosaccharides (FOS).

6. The composition as claimed in any preceding claim, wherein the composition comprises up to about 5% galactosylated and/or up to about 5% fructosylated and/or up to 5% deglycosylated high intensity sweetener glycoside.

7. The composition as claimed in any preceding claim, wherein the composition comprises up to about 2% galactosylated and/or up to about 2% fructosylated and/or up to 2% deglycosylated high intensity sweetener glycoside.

8. The composition as claimed in any preceding claim, wherein the composition comprises up to about 1.5% galactosylated and/or up to about 1.5% fructosylated and/or up to 1.5% deglycosylated high intensity sweetener glycoside.

9. The composition as claimed in any preceding claim, wherein the high intensity sweetener glycoside has been galactosylated and/or fructosylated and/or deglycosylated simultaneously with the synthesis of oligosaccharides.

10. The composition as claimed in claim 9, wherein the high intensity sweetener glycoside comprises steviol glycosides which are modified by up to about 3 units of lactose or fructose by galactosylation and/or fructosylation and/or deglycosylation.

11. The composition as claimed in claim 10, wherein the high intensity sweetener glycoside comprises steviol glycosides which are modified by up to about 4 units of lactose or fructose by galactosylation and/or fructosylation and/or deglycosylation.

12. The composition as claimed in claim 9, wherein the high intensity sweetener glycoside comprises steviol glycosides which are modified by about 4 or more units of lactose or fructose by galactosylation and/or fructosylation and/or deglycosylation.

13. The composition as claimed in claim 9, wherein the high intensity sweetener glycoside comprises mogrosides which are modified by up to about 3 units of galactose by galactosylation.

14. The composition as claimed in claim 9, wherein the high intensity sweetener glycoside comprises mogrosides which are modified by up to about 2 units of fructose by fructosylation.

15. The composition as claimed in any one of claims 4 to 9 and 10 to 12, wherein the steviosides comprise a mixture of steviosides having different modifications.

16. The compositions as claimed in claim 15, wherein the composition comprises a mixture of one or more of: (i) rebaudioside A, rebaudioside F, rebaudioside C, rubusoside or stevioside with 1 unit of fructose; (ii) stevioside with 2 units of fructose or rebaudioside A or rebaudioside C with 1 unit of fructose; and (iii) stevioside with 2 units of fructose or rebaudioside A or rebaudioside C with 1 unit of fructose.

17. The composition as claimed in claim 15, wherein the composition comprises a mixture of one or more of: (i) rebaudioside A and rebaudioside C or stevioside with 1 unit of galactose; (ii) stevioside with 2 units of galactose or rebaudioside A or rebaudioside C with 1 unit of galactose; (iii) stevioside with 3 units of galactose or rebaudioside A or rebaudioside C with 2 units of galactose; and (iv) stevioside with 4 units of galactose or rebaudioside A or rebaudioside C with 2 units of galactose.

18. The composition as claimed in any one of claims 4 to 9 and 13 to 14, wherein the mogrosides comprises a mixture of mogrosides having different modifications.

19. The composition as claimed in claim 18, wherein the mogrosides comprise a mixture of one or more of mogroside II, mogroside III, mogrosid IV, mogroside V or mogroside VI.

20. The composition as claimed in claim 18, wherein the mogrosides comprise a mixture of one or more of: (i) mogroside V; (ii) mogrosid IV, and (iii) mogroside III.

21. The composition as claimed in claim 18, wherein the mogrosides comprise a mixture of one or more of: (i) mogroside III; (ii) mogrosid IV; (iii) mogroside V; (iv) mogroside with 1 unit of fructose; and (v) mogroside with 2 units of fructose.

22. The composition as claimed in claim 18, wherein the mogrosides comprises a mixture of: (i) mogroside IV; (ii) mogroside with 1 unit of galacose; (iii) mogroside V with 2 units of galacose; and (iv) mogroside V with 3 units of galacose.

23. Use of the composition as claimed in any preceding claim, as a low calorie or calorie free sweet prebiotic.

24. Use of the composition as claimed in any one of claims 1 to 22, wherein the composition is incorporated, or is for incorporation, in or on, a foodstuff, a food supplement or a calorie restricted meal replacement product.

25. Use of the composition as claimed in claim 24, wherein the composition is in a granular form, and optionally, included in a sachet or jar.

26. A method for producing a sweetened prebiotic composition, the method comprising: contacting a high intensity sweetener glycoside with one or more enzymes effective to galactosylate and/or fructosylate and/or deglyccosylate the high intensity sweetener glycoside in the presence of sucrose and/or lactose so as to produce simultaneouly different oligosaccharides

27. The method of claim 26, wherein the high intensity sweetener glycoside is selected from one or more of the following: Steviol glycosides or Mogroside, or derivatives thereof.

28. The method of either claim 26 or 27, wherein the high intensity sweetener glycoside is galactosylated and/or fructosylated and/or deglycosylated using one or more enzymes selected from: carbohydrase mixtures obtained from Aspergillus sp. and -galactosidases.

30. The method as claimed in one of claims 26 to 28, wherein the oligosaccharide synthesized is one or more of the following: galactooligosaccharides (GOS) or fructooligosaccharides (FOS).

31. The method as claimed in any one of claims 26 to 30, wherein the sweetened prebiotic composition comprises up to about 5% galactosylated and/or up to about 5% fructosylated and/or up to about 5% deglycosylated high intensity sweetener glycoside.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0043] Embodiments of the present invention will now be described, by way of examples only.

[0044] FIG. 1A shows the HPLC-DAD profile and the detection of Steviol glycosides described in Example 1, whereas FIG. 1B shows the GC-FID profile and the detection of carbohydrates as described in Example 2;

[0045] FIG. 2A shows the HPLC-DAD profile for the detection of Steviol glycosides as described in Example 1, whereas FIG. 2B shows the GC-FID profile for detection of carbohydrates as described in Example 2;

[0046] FIG. 3A shows the HPLC-DAD profile for the detection of Mogrosides as described in Example 3, whereas FIG. 3B shows the GC-FID profile for the detection of carbohydrates as described in Example 3;

[0047] FIG. 4A shows the HPLC-DAD profile for the detection of Mogrosides as described in Example 4, wherein FIG. 4B shows the GC-FID profile for the detection of carbohydrates as described in Example 4;

[0048] FIG. 5 is a bar chart showing sensory test results for sweet taste (bars represent mean values, error bars extend+/half LSD) for the samples tested in Example 5;

[0049] FIG. 6 is a bar chart showing sensory test results for the strength of off flavours (bars represent mean values, error bars extend+/half LSD) for the samples tested in Example 5;

[0050] FIG. 7 is a bar chart showing sensory test results for bitter taste (bars represent mean values, error bars extend+/half LSD) for the samples tested in Example 5;

[0051] FIG. 8 is a bar chart showing sensory test results for liquorice taste (bars represent mean values, error bars extend+/half LSD) for the samples tested in Example 5;

[0052] FIG. 9 is a bar chart showing sensory test results for sweet aftertaste (bars represent mean values, error bars extend+/half LSD) for the samples tested in Example 5;

[0053] FIG. 10 is a plot showing the HPLC-MS profile for enzymatically modified steviol glycosides with A. acualetus carbohydrases as described in Example 6. RA: rebaudioside A; ST: stevioside; RF: rebaudioside F; RC: rebaudioside C; Ru: Rubusoside; Sb: Steviolbioside;

[0054] FIG. 11 is a plot showing the MALDI-TOF profile for enzymatically modified steviol glycosides with A. acualetus carbohydrases as described in Example 6. RA: rebaudioside A; ST: stevioside; RF: rebaudioside F; RC: rebaudioside C; Ru: Rubusoside; Sb: Steviolbioside.; Fru: fructose;

[0055] FIG. 12 is a plot showing the HPLC-MS profile for enzymatically modified steviol glycosides with -galactosidases as described in Example 6. RA: rebaudioside A; ST: stevioside; RF: rebaudioside F; RC: rebaudioside C; Ru: Rubusoside; Sb: Steviolbioside;

[0056] FIG. 13 is a plot showing the MALDI-TOF profile for enzymatically modified steviol glycosides with -galactosidases as described in Example 6. RA: rebaudioside A; ST: stevioside; RF: rebaudioside F; RC: rebaudioside C; Ru: Rubusoside; Sb: Steviolbioside.; Gal: galactose;

[0057] FIG. 14 is a plot showing the HPLC-MS profile for enzymatically modified mogrosides with A. acualetus carbohydrases as described in Example 6. M: mogrosides.

[0058] FIG. 15 is a plot showing the MALDI-TOF profile for enzymatically modified mogrosides with A. acualetus carbohydrases. M: mogrosides; Fru: fructose;

[0059] FIG. 16 is a plot showing the HPLC-MS profile for enzymatically modified mogrosides with -galactosidases as described in Example 6. M: mogrosides; and

[0060] FIG. 17 is a plot showing the MALDI-TOF profile for enzymatically modified mogrosides with -galactosidases as described in Example 6. M: mogrosides; Gal: galactose.

[0061] The aim of these experiments was to determine the intensity of sweetness and any off flavours of a number of oligosaccharides and enzymatically modified high intensity glycosides, obtained during the same enzymatic reaction.

EXAMPLE 1

Enzymatically Modified Steviol Glycosides and FOS Production

[0062] This experiment sought to investigate the potential yield and preferred enzymes for producing, fructosylated and/or deglycosylated steviol glycosides and FOS during the same enzymatic reaction. The enzymes investigated were carbohydrases complexes from Aspergillus and Inulinase from Lactobacillus (R&D). The substrate was sucrose and steviol glycosides. The conditions used were 1.5% steviol glycosides, 60% sucrose, purification was by means of yeast fermentation and the drying process utilised lyophilisation and vacuum evaporation.

[0063] FIG. 1A shows the HPLC-DAD profile and the detection of Steviol glycosides, whereas FIG. 1B shows the GC-FID profile of the carbohydrates (trimethylsilyl oxime).

[0064] The best results were obtained utilising microbial enzymes complexes. The experiment suggested that the use of the commercial enzyme provided improved taste when the fructosylated and/or deglycosylated steviol glycosides was mixed with FOS generated during synthesis. The results therefore suggest that the mixture would be suitable for use as a prebiotic due to the high FOS concentration obtained during the enzymatic reaction.

EXAMPLE 2

Enzymatically Modified Steviol Glycosides and GOS Production

[0065] This experiment sought to investigate the potential yield and preferred enzymes for producing galactosylated and/or deglycosylated steviol glycosides, and GOS during the same enzymatic reaction. The enzymes investigated were -galactosidases from Aspergillus and Bifidobacterium bifidum. The substrates were lactose and steviol glycosides. The conditions were 1.5% steviol glycosides, 40% lactose. Purification was by means of yeast fermentation and the drying process was lyophilisation and rotovapor.

[0066] FIG. 2A shows the HPLC-DAD profile for the detection of Steviol glycosides. FIG. 2B shows the GC-FID profile for detection of carbohydrates (trimethylsilyl oxime).

[0067] The best results were obtained by using the -galactosidases from Aspergillus. The results show the potential for the use of commercial enzymes to produce galactosylated and/or deglycosylated steviol glycosides and GOS obtained during the synthesis. The results therefore suggest that the high GOS concentration obtained during the enzymatic syntheis would be suitable for use as a prebiotic.

EXAMPLE 3

Enzymatically Modified Mogrosides and FOS Production

[0068] This experiment sought to investigate the potential yield and preferred enzymes for producing fructosylated and/or deglycosylated mogrosides, and FOS, during the same enzymatic reaction. The enzymes investigated were carbohydrases complexes from Aspergillus acuelatus and Inulinase from Lactobacillus gasseri (R&D). The substrate was sucrose and steviol glycosides. The conditions used were 1.5% steviol glycosides, 60% sucrose, purification was by means of yeast fermentation and the drying process utilised lyophilisation and vacuum evaporation.

[0069] FIG. 3A shows the HPLC-DAD profile for the detection of Mogrosides. FIG. 3B shows the GC-FID profile for the detection of carbohydrates (trimethylsilyl oxime).

[0070] The best results were obtained utilising microbial enzymes complexes. The experiment suggested that the use of the commercial enzyme provided improved taste when the fructosylated and/or deglycosylated mogrosides. It is believed that this is the first report of fructosylated mogrosides. The results therefore suggest that fructosylated and/or deglycosylated mogrosides and FOS, obtained simultaneously during the enzymatic reaction, would provide a good prebiotic mainly due to the high FOS concentration.

EXAMPLE 4

Enzymatically Modified Mogrosides and GOS Production

[0071] This experiment sought to investigate the potential yield and preferred enzymes for producing during the same enzymatic reaction, galactosylated and/or deglycosylated mogrosides and GOS. The enzymes investigated were -galactosidases from Aspergillus and Bifodabcaterium. bifidum. The substrate used was lactose and mogrosides. The conditions used were 1.5% mogrosides, 40% lactose. Purification was performed using yeast fermentation and the drying process used lyophilisation and rotovapor.

[0072] FIG. 4A shows the HPLC-DAD profile for the detection of Mogrosides. FIG. 4B shows the GC-FID profile for the detection of carbohydrates.

[0073] The best results were obtained with were -galactosidases from Aspergillus. It is believed that this is the first report of galactosylated mogrosides. The results therefore suggest that galactosylated and/or deglycosylated mogrosides, mixed with GOS, obtained simultaneously during the enzymatic reaction, would provide a good prebiotic due to the high GOS concentration.

EXAMPLE 5

Sensory Test Data of Modified HIS

[0074] A trained sensory panel at the Reading Sensory Science Centre (UK) were employed for sensory profiling of the samples. There were 10 panellists with between 1 and 9 years' experience. A QDA (quantitative descriptive analysis) profiling approach was taken. The panel used the same vocabulary that they had developed as a consensus for the tasting sessions including the term liquorice flavour which is characteristic note of steviol glycosides. The panel were retrained at the start of the sample set over 3 separate tasting sessions. This re-training focused on ensuring that they could reliably score sweetness relative to the new concentration of sucrose standard positions.

[0075] Rating was carried out independently using unstructured lines scales (scaled 0-100), in duplicate, in isolated sensory booths. However, in order to improve discrimination for sweetness, the four sucrose samples were used as standards and the mean values for each of these samples, as agreed by the panel, are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Standard Sucrose Concentration Mean Rating Number (% w/v) (0-100) 1 2.0 10 2 4.0 35 3 6.0 75 4 8.0 100

[0076] At the start of each scoring session the panel tasted the four reference samples in order of increasing strength to re-familiarise themselves with the positioning of these levels of sweetness on the line scale. The reference samples (10 mL) were served in transparent polystyrene cups (30 mL). They then palate cleansed with warm filtered tap water and low salt crackers (Carr's water crackers) before commencing the sample tasting session, and again between each sample scoring session.

[0077] Samples, labelled with random 3 digit codes, were presented in a balanced presentation order in a monadic sequential manner with a maximum of 6 samples per day. Samples were served at 23-24 C. (room temperature) with air conditioning of the room set to 23 C.

[0078] The panel used 15 attributes to define samples, as shown in Table 2 below, where mean scores (0-100) for mogrosides (M) and steviol glycosides (SG) modified using two different glycosidase mixtures (F: Example 1 and 3 and G: Example 2 and 4) as shown.

TABLE-US-00002 TABLE 2 Sample Fisher's Signif- LSD icant MF MG SGF SGG Value (p) Sweet Taste .sup.28.6 .sup.c .sup.65.8 .sup.b .sup.91.1 .sup.a .sup.25.5 .sup.c 10.9 <.0001 Overall Off .sup.15.5 .sup.b .sup.35.3 .sup.a .sup.33.1 .sup.a .sup.19.1 .sup.b 9.3 0.0005 Flavour Bitter Taste 7.8 .sup.bc .sup.14.1 .sup.a 11.9 .sup.ab 6.6 .sup.bc 5.8 0.0027 Liquorice .sup.2.1 .sup.b .sup.13.2 .sup.a .sup.12.0 .sup.a .sup.1.8 .sup.b 5.8 <.0001 Flavour Cardboard 4.0 .sup.ab .sup.5.4 .sup.a .sup.0.6 .sup.c .sup.1.0 .sup.c 2.9 0.0145 (Stale) CandyFloss 1.1 5.1 6.6 2.6 5.5 0.2289 Sour/ 1.0 2.4 2.2 4.2 3.6 0.6943 RancidDairy Metallic Taste 5.9 5.4 4.3 3.2 4.7 0.5841 Salty Taste 1.9 2.1 2.7 3.7 3.5 0.8737 CrustyBread 1.1 0.4 0.7 0.4 1.5 0.7387 Flavour Perfume 0.5 0.0 0.0 0.0 0.6 0.4434 Sweet .sup.20.2 .sup.c .sup.43.2 .sup.b .sup.61.8 .sup.a .sup.12.0 .sup.c 11.4 <.0001 Aftertaste Bitter 8.4 9.0 8.7 6.0 4.8 0.1170 Aftertaste Liquorice .sup.2.2 .sup.b .sup.8.3 .sup.a .sup.8.2 .sup.a .sup.1.5 .sup.b 4.5 0.0009 Aftereffect Cooling 0.1 2.1 0.6 0.2 1.8 0.2241 Aftereffect

[0079] FIGS. 5 to 9 illustrate the sensory test data for the important sweet, off flavor, bitter, liquorice, or sweet aftertaste.

[0080] Table 3 below shows the equivalent sucrose and relative sweetness values of the samples tested.

TABLE-US-00003 TABLE 3 Equivalent Sucrose Relative Sweetness (%) to x % saccharide (sucrose = 100) (RS) Sample Mean Min Max Mean Min Max MF 0.9 0.4 1.4 18 8 28 MG 1.8 1.3 2.3 36 25 46 SGF 2.4 2.2 2.6 48 43 52 SGG 0.8 0.5 1.2 17 10 24

EXAMPLE 6

Impact of MV-FOS, MV-GOS, SG-FOS and SG-GOS on the Human Gut Microbiome

[0081] Experiments were conducted to assess the impact of MV-FOS, MV-GOS, SG-FOS and SG-GOS (1% w/v) on the metabolic activity of the human gut microbiome.

[0082] Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) spectra were recorded by using a Voyager DE-PRO mass spectrometer (Applied Biosystems) equipped with a nitrogen laser emitting at 337 nm with a 3 ns, and 3 Hz frequency. Ions generated by laser desorption were introduced into a time of flight analyzer (1.3 m flight path) with an acceleration voltage of 25 kV, 94% grid voltage, 0.075% ion guide wire voltage, and a delay time of 400 ns in the linear positive ion mode. Mass spectra were obtained over the m/z range 100-5000. 2,5-Dihydroxybenzoic acid (>98%, Fluka) at a concentration of 10 mg/ml in water (Milli-Q water, Millipore, Bedfor, USA) was used as matrix. Samples were diluted 1:100 in water and then, mixed with the matrix solution at a ratio of approximately 1:3. 1 L of this solution was spotted onto a flat stainless-steel sample plate and dried in air. External mass calibration was applied using the monoisotopic [M.sub.+H].sub.+ values of of des-Arg1 Bradykinin, Angiotensin I, Glu1-Fibrino-peptide B, ACTH (1-17), ACTH (18-39), ACTH (7-38) and Insuline (Bovine). of the Calibration Mixtures 1 and 2, Sequazyme Peptide Mass Standards Kits; Applied Biosystems.

[0083] Separation and analysis of enzymatically modified steviol glycosides and mogrosides by LC-MS was performed at 25 C. on a C18 column (150 mm2.1 mm, 3.5 mm particle size, ThermoFisher) at a flow rate of 0.1 mL/min with a solvent gradient of acetonitrile and water (0.1% formic acid). All experiments were carried out on a Finnigan Surveyor pump with quaternary gradient system coupled to a Finnigan LCQ Deca ion trap mass spectrometer using an ESI interface. Sample injections (10 mL) were carried out by a Finnigan Surveyor autosampler. All instruments (Thermo Fisher Scientific, San Jose, Calif., USA), and data acquisition were managed by Xcalibur software (1.2 version; Thermo Fisher Scientific).

[0084] The impact of MV-FOS, MV-GOS, SG-FOS and SG-GOS (1% w/v) on the metabolic activity of the human gut microbiome was investigated in pH and temperature-controlled batch cultures. Impact on the concentration of organic acids was compared to short chain fructooligosaccharides (prebiotics positive control; FUJIFILM Wako Chemicals, Germany), and a carbohydrate negative control. Fructooligosaccharides and galactooligosaccharides produced by the activity the same enzymes used for the synthesis of modified MV and SG (1% w/v) were also tested as well as non-modified MV and SG (0.2% w/v).

[0085] Freshly voided faecal samples were obtained from five healthy adults, free from gastrointestinal disorders who had not taken antibiotics for 6 months prior to the study and prebiotics and/or probiotics for 6 weeks prior to the study.

[0086] Sterile fermenters (20 mL working volume, Soham scientific, Ely, UK) were filled with pre-reduced sterile basal media consisting of: peptone water (Oxoid, Basingstoke, UK) 2 g L.sup.1; yeast extract (Oxoid, Basingstoke, UK) 2 g L.sup.1; NaCl 0.1 g L.sup.1; K.sub.2HPO.sub.4 0.04 g L.sup.1; KH.sub.2PO.sub.4 0.04 g L.sup.1; MgSO.sub.4.7H.sub.2O 0.01 g L.sup.1; CaCl.sub.2.6H.sub.2O 0.01 g L.sup.1; NaHCO.sub.3 2 g L.sup.1; haemin 0.05 g L.sup.1; cysteine.HCl 0.5 g L.sup.1; bile salts 0.5 g L.sup.1, vitamin K1 10 L; Tween 80 2 mL (Sigma Aldrich) and sparged with oxygen-free N.sub.2 to establish and maintain anaerobic conditions. Stirring was achieved using magnetic stirrers. Test carbohydrates (1% w/v) were added in designated vessels just prior to inoculation with the faecal slurry from a single donor (10% v/v prepared in anaerobic phosphate buffered saline). All tests for a single donor were carried out in parallel. Fermentation temperature was maintained at 37 C. by means of a circulating water bath. Automated pH controllers (Fermac 260; Electrolab UK) kept culture pH within a range of 6.7 and 6.9 by adding 0.5 M NaOH and 0.5 M HCl as required. Fermentations were run for a period of 24 h and samples were withdrawn at 0, 5, 10, and 24 h for organic acid analysis. Table 4 below shows the results of the fermentation runs.

TABLE-US-00004 TABLE 4 Short-chain fatty acids and lactate concentration after 5, 10 and 24 h of fermentation with human faecal samples Time SFCA Concentration (mM) point Negative SG Positive SFCA (h) Control SG-GOS MV-GOS MV-FOS CONTROL Control ACETATE 0 0.73 0.01 0.73 0.01 0.73 0.01 0.73 0.01 0.73 0.01 0.73 0.01.sup. 5 .sup.4.05 0.30.sup.ab 22.36 0.03.sup.c 8.56 0.01.sup.abc 8.77 0.01.sup.abc 3.04 0.00.sup.a 19.83 1.57.sup.bc 10 8.15 0.35.sup.abc 37.97 0.11.sup.f 20.32 0.15.sup.cde 21.53 0.07.sup.de .sup.4.57 0.14.sup.ab 43.89 0.10.sup.f 24 12.99 0.16.sup.ab 40.03 2.46.sup.def 30.78 0.12.sup.cde 29.56 0.28.sup.cd 13.89 5.86.sup.ab 43.60 0.17.sup.efg PROPIONATE 0 0.15 0.00 0.15 0.00 0.15 0.00 0.15 0.00 0.15 0.00 0.15 0.00.sup. 5 .sup.1.39 0.07.sup.ab .sup.1.95 0.01.sup.ab .sup.7.16 0.09.sup.d .sup.6.32 0.02.sup.cd .sup.0.98 0.01.sup.ab 2.30 0.29.sup.ab 10 1.86 0.12.sup.abc .sup.4.31 0.02.sup.bcd 9.49 0.17.sup.f 9.71 0.09.sup.f .sup.1.35 0.06.sup.ab 5.84 0.01.sup.de 24 2.53 0.10.sup.abc 13.12 0.57.sup.de 14.19 0.08.sup.de 13.08 0.16.sup.de .sup.3.70 2.20.sup.abc 6.78 3.50.sup.bc BUTYRATE 0 0.11 0.00 0.11 0.00 0.11 0.00 0.11 0.00 0.11 0.00 0.11 0.00.sup. 5 0.45 0.00.sup.abc 0.80 0.01.sup.cde 0.48 0.00.sup.abc .sup.0.55 0.01.sup.abcd .sup.0.23 0.01.sup.ab .sup.0.69 0.17.sup.bcde 10 .sup.1.36 0.01.sup.ab 2.49 0.03.sup.abc 2.43 0.02.sup.abc .sup.3.02 0.14.sup.abcd 0.68 0.01.sup.a .sup.2.89 0.01.sup.abcd 24 2.45 0.00.sup.abc .sup.5.41 1.20.sup.abcd .sup.7.27 0.00.sup.cd .sup.6.89 0.03.sup.bcd .sup.2.18 0.97.sup.abc .sup.6.56 0.69.sup.bcd LACTATE 0 0.11 0.00 0.11 0.00 0.11 0.00 0.11 0.00 0.11 0.00 0.11 0.00.sup. 5 0.12 0.00.sup.a 10.21 0.82.sup.d .sup.4.86 0.19.sup.bc .sup.2.74 0.08.sup.ab 0.18 0.01.sup.a 8.95 1.07.sup.cd 10 0.03 0.00.sup.a 13.09 0.36.sup.de .sup.3.35 0.03.sup.ab 0.94 0.09.sup.a 0.09 0.02.sup.a 20.90 0.19.sup.e 24 0.00 0.00.sup.a .sup.0.26 0.37.sup.ab 0.00 0.00.sup.a 0.00 0.00.sup.a 0.00 0.00.sup.a 3.75 5.30.sup.ab TOTAL 0 1.15 0.25 1.15 0.25 1.15 0.25 1.15 0.25 1.15 0.25 1.15 0.25.sup. 5 6.22 1.47.sup.a 36.33 8.38.sup.a 21.23 3.74.sup.a 18.57 3.55.sup.a 4.56 1.10.sup.a 31.90 7.46.sup.a 10 12.03 2.92.sup.abc 58.51 13.84.sup.ef 36.41 7.42.sup.cde 36.32 7.96.sup.cde 7.37 1.79.sup.a 73.92 16.46.sup.f 24 20.55 4.54.sup.ab .sup.63.77 14.35.sup.cd .sup.59.41 10.96.sup.cd .sup.55.29 10.57.sup.bc 21.97 4.91.sup.ab 82.23 15.85.sup.cd ACETATE/ 0 4.77 4.77 4.77 4.77 4.77 4.77 PROPIONATE 5 2.90 11.49 1.20 1.39 3.11 8.63 RATIO 10 4.38 8.80 2.14 2.22 3.69 7.51 24 5.14 3.05 2.17 2.26 3.75 1.97 Time SFCA Concentration (mM) point FOS MV GOS SFCA (h) CONTROL CONTROL CONTROL SG-FOS ACETATE 0 0.73 0.01 0.73 0.01.sup. 0.73 0.01 0.73 0.01 5 8.86 3.99.sup.abc 3.47 0.01.sup.a 24.51 0.12.sup.c 10.20 7.15.sup.abc 10 .sup.16.93 0.06.sup.bcd 6.44 1.16.sup.ab 43.09 0.30.sup.f .sup.15.59 4.28.sup.bcd 24 22.47 0.06.sup.bc 9.25 0.12.sup.ab 56.11 0.24.sup.g 21.93 0.15.sup.bc PROPIONATE 0 0.15 0.00 0.15 0.00.sup. 0.15 0.00 0.15 0.00 5 .sup.4.58 0.51.sup.bcd 1.35 0.01.sup.ab 3.17 0.02.sup.abc .sup.4.63 0.04.sup.bcd 10 8.10 0.00.sup.ef 2.34 1.19.sup.abc .sup.4.85 0.07.sup.cd 6.94 1.20.sup.def 24 .sup.9.18 0.10.sup.cd 1,82 0.05.sup.ab 11.89 0.27.sup.de .sup.8.66 0.04.sup.bcd BUTYRATE 0 0.11 0.00 0.11 0.00.sup. 0.11 0.00 0.11 0.00 5 .sup.0.57 0.13.sup.abcd .sup.0.53 0.02.sup.abcd .sup.1.04 0.05.sup.de 0.50 0.11.sup.abc 10 .sup.5.95 0.02.sup.d 1.66 1.15.sup.abc .sup.4.74 0.03.sup.cd .sup.2.59 0.80.sup.abcd 24 .sup.7.24 0.08.sup.cd 1.60 0.01.sup.ab 13.10 0.05.sup.e .sup.6.14 0.04.sup.bcd LACTATE 0 0.11 0.00 0.11 0.00.sup. 0.11 0.00 0.11 0.00 5 .sup.2.46 2.20.sup.ab 0.19 0.02.sup.a 10.29 0.11.sup.d .sup.3.23 2.17.sup.ab 10 0.73 0.03.sup.a 0.00 0.00.sup.a 15.35 0.34.sup.d 1.52 0.01.sup.a 24 0.00 0.00.sup.a 0.00 0.00.sup.a .sup.5.93 0.10.sup.b 0.20 0.01.sup.a TOTAL 0 1.15 0.25 1.15 0.25.sup. 1.15 0.25 1.15 0.25 5 16.67 3.32.sup.a 5.79 1.25.sup.a 39.26 9.10.sup.a 18.81 3.78.sup.a 10 .sup.32.87 6.25.sup.bcd 10.93 2.34.sup.ab 68.65 15.63.sup.f 27.76 5.64.sup.abc 24 42.72 8.03.sup.abc 13.99 3.27.sup.a .sup.91.12 19.66.sup.d 43.40 7.61.sup.abc ACETATE/ 0 4.77 4.77 4.77 4.77 PROPIONATE 5 1.93 2.57 7.72 2.20 RATIO 10 2.09 2.75 8.88 2.25 24 2.45 5.08 4.72 2.53

[0087] Organic acid (OA) concentrations were determined by gas chromatography equipped with flame ionisation detector (GC-FID) based on the method described by Richardson et al (1989) using 2-ethyl butyric acid as an internal standard. A gas chromatograph analyser (Agilent/HP 6890) equipped with a Flame Ionization Detector (FID) and an HP-5MS column (30 m0.25 mm) with a 0.25 m coating (Crosslinked (5%-Phenyl)-methylpolysiloxane, Hewlett Packard, UK) was used for SOFA measurements. Helium was used as carrier gas at a flow rate of 1.7 mL/min (head pressure 133 KPa). Oven initial temperature was set at 63 C., followed by a temperature ramp of 15 C./min to 190 C. and held constant for 3 minutes. A split ratio of 100:1 was used. The appearance of OA in the chromatograms was confirmed based on the retention times of the respective commercial OA standards (Lactic acid, Acetic acid, Propionic acid and Butyric acid) (Sigma-Aldrich, UK)

[0088] With reference to FIGS. 10 to 17, in general, SG-GOS and SG-FOS showed and modification of the steviol glycosides by the desglycosilation and galactosylation and fructosylation, respectively up to 3 to 4 more units of lactose or fructose. This behaviour was also found for MV-GOS and MV-FOS, in this case the the mogrosides were up to galacosylated by 3 galactose units and fructosylated by 2 fructose units.

[0089] SG-GOS was fermented rapidly as indicated by significant increases in the levels of lactate at 5 and 10 h of fermentation, behaving in a similar manner to the prebiotic control and GOS. Lactate is a fermentation intermediate, that is rapidly utilised through cross-feeding by other members of the gut microbiome. Lactate accumulates in culture when the rate of production is higher compared to the rate of utilisation and it is characteristic of rapid gut microbiome fermentation rates observed during the saccharolysis of oligosaccharides. Acetate, propionate and butyrate concentrations were also significantly higher compared to the negative control and followed similar patterns to those observed by the prebiotic control and GOS.

[0090] In the SG-FOS cultures, lactate accumulation was significantly lower compared to SG-GOS was fermented rapidly as was indicated by the accumulation of lactate at 5 and 10 h of fermentation, also observed with the positive control and levels were significantly lower compared to the prebiotic control but very similar to FOS, indicating slower fermentation rates. Acetate, propionate and butyrate concentrations were all significantly higher compared to the negative control and very similar to those in the FOS fermentations but significantly lower to the prebiotic control in terms of acetate formation. In the MV-GOS cultures lactate accumulation was significantly lower compared to GOS and the prebiotic control, indicating less rapid fermentation. Acetate concentrations were significantly higher compared to the negative control and gradually increased over the fermentation period, following similar patterns to the prebiotic control and GOS, albeit at lower levels. MV-GOS significantly increased propionate concentrations, with levels being significantly higher compared to the prebiotic control and GOS. Significant increases in butyrate were observed after 24 h fermentation and were comparable to those with the prebiotic control and GOS.

[0091] MV-FOS metabolite formation followed identical patterns to MV-GOS with the exception of butyrate which did not increase significantly.

[0092] Overall, the fermentation behaviour of the compounds synthesised shows very close similarities to that of commercially available prebiotics. Their impact on the metabolic activity of the human gut microbiome is characteristic of oligosaccharide saccharolysis. They all increased significantly acetate but also propionate and butyrate, organic acids with important role in cholesterolgenesis, appetite regulation, tight junction integrity and immunomodulation.

[0093] The forgoing embodiments are not intended to limit the scope of the protection afforded by the claims, but rather to describe examples of how the invention may be put into practice.