Prebiotic compositions and production methods thereof

Abstract

To provide: prebiotic compositions, or compositions comprising a prebiotic component, which can impart a sweet taste; and production methods thereof.SOLUTION: The present invention provides prebiotic compositions comprising: (i) an 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 production methods of the compositions are also described.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a sweet prebiotic composition which has particular application as a functional food ingredient for foodstuffs and as an incorporated ingredient in deli products, or used alone to sweeten foods. [Background technology]

[0002] The global sweetener market is currently dominated by sugar and is expected to reach $112 billion by 2022. There is a growing movement towards low-calorie or non-calorie sweeteners. Some sweeteners, such as steviol glycosides and mogroside V, are classified as high-intensity sweeteners (HIS) and reportedly have approximately 150 and 400 times the sweetness of sucrose, respectively. However, some HIS have a bitter or off-taste that makes them less attractive to consumers.

[0003] Prebiotics are substrates selectively utilized by host microorganisms that provide health benefits, such as lactobacilli or bifidobacteria, and are finding much increased application in the food sector. Prebiotics can be non-digestible food ingredients that are selectively metabolized by colonic bacteria that contribute to improved health. Thus, the use of prebiotics promotes beneficial changes in the indigenous gut flora, which can aid in the survival of probiotics. Prebiotics have a global effect on the gut bacterial population and are distinct from many dietary fibers, such as pectins, celluloses, xylans, etc., that are not selectively metabolized in the gut. The criteria for classification as a prebiotic are that it must be resistant to gastric acidity, hydrolysis by mammalian enzymes, and absorption in the upper gastrointestinal tract, and must reach the colon in adequate amounts to be fermented by the gut microflora and selectively stimulate the growth and / or activity of gut bacteria associated with health and wellness. Summary of the Invention [Problem to be solved by the invention]

[0004] It is an object of the present invention to provide a prebiotic composition, i.e. a composition comprising a prebiotic component, that can impart a sweet taste. In particular, it is an object of the present invention to provide a prebiotic composition, i.e. a composition comprising a prebiotic component, that can impart a sweet taste with reduced bitterness and / or undesirable aftertaste. It is a further object of the present invention to provide a prebiotic composition, i.e. a composition comprising a prebiotic component, that is not digested in the upper gastrointestinal tract of humans or animals and therefore can be used as a non-caloric or substantially non-caloric functional ingredient that can improve the diversity of the gut microbiome. [Means for solving the problem]

[0005] According to a first aspect of the present invention, (i) an enzyme-treated high-intensity sweetener glycoside; (ii) oligosaccharides; A prebiotic composition is provided comprising:

[0006] According to a related aspect of the invention, (i) an enzyme-treated high-intensity sweetener glycoside; (ii) enzymatically synthesized (enzymatically synthesized) oligosaccharides; A prebiotic composition is provided comprising:

[0007] According to a further related aspect of the invention, (i) an enzyme-treated high-intensity sweetener glycoside; (ii) enzymatically synthesized (enzymatically synthesized) oligosaccharides; A synthetic prebiotic composition is provided comprising: [Brief description of the drawings]

[0008] [Figure 1]FIG. 1A shows the HPLC-DAD profile and detection of steviol glycosides as described in Example 1, and FIG. 1B shows the GC-FID profile and detection of carbohydrates as described in Example 2. [Diagram 2] FIG. 2A shows the HPLC-DAD profile for the detection of steviol glycosides as described in Example 1, and FIG. 2B shows the GC-FID profile for the detection of carbohydrates as described in Example 2. [Diagram 3] FIG. 3A shows the HPLC-DAD profile for the detection of mogrosides as described in Example 3, and FIG. 3B shows the GC-FID profile for the detection of carbohydrates as described in Example 3. [Figure 4] FIG. 4A shows the HPLC-DAD profile for the detection of mogrosides as described in Example 4, and FIG. 4B shows the GC-FID profile for the detection of carbohydrates as described in Example 4. [Diagram 5] FIG. 5 is a bar graph showing sensory test results for sweetness for the samples tested in Example 5 (bars represent mean values, error bars extend ±half LSD). [Figure 6] FIG. 6 is a bar graph showing the sensory test results for off-taste intensity for the samples tested in Example 5 (bars represent average values, error bars extend ±half LSD). [Figure 7] FIG. 7 is a bar graph showing sensory test results for bitterness for the samples tested in Example 5 (bars represent average values, error bars extend ±half LSD). [Figure 8] FIG. 8 is a bar graph showing the sensory test results for licorice taste for the samples tested in Example 5 (bars represent mean values, error bars extend ±half LSD). [Figure 9] FIG. 9 is a bar graph showing sensory results for sweet aftertaste for the samples tested in Example 5 (bars represent average values, error bars extend ±half LSD). [Figure 10]10 is a plot showing the HPLC-MS profile for steviol glycosides enzymatically treated with A. aculeatus carbohydrase as described in Example 6. RA: rebaudioside A; ST: stevioside; RF: rebaudioside F; RC: rebaudioside C; Ru: rubusoside; Sb: steviolbioside. [Figure 11] 11 is a plot showing the MALDI-TOF profile for steviol glycosides enzymatically treated with A. aculeatus carbohydrase as described in Example 6. RA: rebaudioside A; ST: stevioside; RF: rebaudioside F; RC: rebaudioside C; Ru: rubusoside; Sb: steviolbioside; Fru: fructose. [Figure 12] 12 is a plot showing the HPLC-MS profile for steviol glycosides enzymatically treated with β-galactosidase as described in Example 6. RA: rebaudioside A; ST: stevioside; RF: rebaudioside F; RC: rebaudioside C; Ru: rubusoside; Sb: steviolbioside. [Figure 13] 13 is a plot showing the MALDI-TOF profile for steviol glycosides enzymatically treated with β-galactosidase as described in Example 6. RA: rebaudioside A; ST: stevioside; RF: rebaudioside F; RC: rebaudioside C; Ru: rubusoside; Sb: steviolbioside; Gal: galactose. [Figure 14] 14 is a plot showing the HPLC-MS profile for mogrosides enzymatically treated with A. aculeatus carbohydrase as described in Example 6. M: mogroside.

[0009] [Figure 15] 15 is a plot showing the MALDI-TOF profile for mogrosides enzymatically treated with A. aculeatus carbohydrase. M: mogroside; Fru: fructose. [Figure 16] 16 is a plot showing the HPLC-MS profile for mogrosides enzymatically treated with β-galactosidase as described in Example 6. M: mogroside. [Figure 17] 17 is a plot showing the MALDI-TOF profile for mogrosides enzymatically treated with β-galactosidase as described in Example 6. M: mogroside; Gal: galactose. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The terms "synthetic" and "synthesized" are intended to mean products that do not exist in nature or are not produced in nature. The term, of course, encompasses naturally occurring precursor compositions and products that are "man-made" using natural products, such as naturally occurring enzymes.

[0011] The intense sweetener glycosides may be enzymatically modified by galactosylation and / or fructosylation and / or deglycosylation.

[0012] The intense sweetener glycoside may be selected from one or more (or a combination) of the following: a steviol glycoside (e.g., rebaudioside A) or a mogroside (e.g., mogroside V), or a derivative thereof.

[0013] Preferably, the enzyme is of microbial origin. The enzyme may be from the genus Aspergillus. The enzyme may be derived from one or more of the following species of the genus Aspergillus: Aspergillus officinalis; Aspergillus aculeatus; Aspergillus awamori; Aspergillus carbonarius; Aspergillus cellulosae; Aspergillus oryzae; Aspergillus flavus; Aspergillus japonicas; Aspergillus nidulans; or Aspergillus niger.

[0014] The resulting oligosaccharides may be one of the following: fructooligosaccharides (FOS), galactooligosaccharides (GOS), α-galactooligosaccharides, β-glucooligosaccharides, xylooligosaccharides and combinations thereof. It is preferred that the resulting oligosaccharides are one or more of the following: galactooligosaccharides (GOS) or fructooligosaccharides (FOS).

[0015] The composition may comprise about 5% galactosylated and / or about 5% fructosylated and / or about 5% deglycosylated high-intensity sweetener glycosides. Preferably, the composition may comprise about 2% galactosylated and / or about 2% fructosylated and / or about 2% deglycosylated high-intensity sweetener glycosides. More preferably, the composition comprises about 1.5% galactosylated and / or about 1.5% fructosylated and / or about 1.5% deglycosylated high-intensity sweetener glycosides.

[0016] The intense sweetener glycosides may be galactosylated and / or fructosylated and / or deglycosylated during oligosaccharide synthesis.

[0017] The intense sweetener glycosides will preferably be galactosylated and / or fructosylated and / or deglycosylated simultaneously with the synthesis of the oligosaccharides.

[0018] In one embodiment, the high intensity sweetener glycoside comprises a steviol glycoside modified by galactosylation and / or fructosylation and / or deglycosylation with up to about 3 units of lactose or fructose. In another embodiment, the high intensity sweetener glycoside comprises a steviol glycoside modified by galactosylation and / or fructosylation and / or deglycosylation with up to about 4 units of lactose or fructose. In another embodiment, the high intensity sweetener glycoside comprises a steviol glycoside modified by galactosylation and / or fructosylation and / or deglycosylation with about 4 or more units of lactose or fructose.

[0019] In one embodiment, the high intensity sweetener glycoside comprises a mogroside modified by galactosylation with up to about 3 units of galactose. In another embodiment, the high intensity sweetener glycoside comprises a mogroside modified by fructosylation with up to about 2 units of fructose. In another embodiment, the high intensity sweetener glycoside comprises a mogroside modified by fructosylation with about 2 or more units of fructose.

[0020] The stevioside may comprise a mixture of steviosides with different modifications. For example, the composition may comprise a mixture of one or more of the following: (i) Rebaudioside A, Rebaudioside F, Rebaudioside C, rubusoside or stevioside with one unit of fructose; (ii) Stevioside with two units of fructose or Rebaudioside A or Rebaudioside C with one unit of fructose; and (iii) Stevioside with two units of fructose or Rebaudioside A or Rebaudioside C with one unit of fructose. Optionally, the composition comprises a mixture of one or more of the following: (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.

[0021] The mogroside may comprise a mixture of mogrosides with different modifications. For example, the mogroside may comprise a mixture of one or more of mogroside II, mogroside III, mogroside IV, mogroside V, or mogroside VI. Alternatively, the mogroside may comprise a mixture of one or more of the following: (i) mogroside V; (ii) mogroside IV, and (iii) mogroside III. Further alternatively, the mogroside may comprise a mixture of one or more of the following: (i) mogroside III; (ii) mogroside IV; (iii) mogroside V; (iv) mogrosides with one unit of fructose; and (v) mogrosides with two units of fructose. Still further, the mogroside may include a mixture of: (i) mogroside IV; (ii) mogroside having one unit of galactose; (iii) mogroside V having two units of galactose; and (iv) mogroside V having three units of galactose.

[0022] All of the embodiments of the prebiotic compositions described herein have been shown to advantageously form sweet, natural, healthy fibers that are not digested in the upper human gastrointestinal tract and can therefore be used as non-caloric or substantially non-caloric functional ingredients. These sweet fibers have been developed as potential bulk sugar substitutes, with a sweetness similar to sucrose but with no or substantially no calories, while also advantageously improving microbiome diversity.

[0023] The prebiotic composition components were found to be significantly sweeter than all other samples with the advantage of having fewer off-tastes (eg, bitter, sour, musty, salty, etc.).

[0024] According to a second aspect of the present invention there is provided the use of the prebiotic composition hereinbefore described as a low or non-caloric sweet prebiotic. It will be apparent to the skilled artisan that the composition may be incorporated or intended for incorporation into a range of foodstuffs, food supplements or calorie restricted prepared food products or may be used on its own to provide sweetness.

[0025] In some embodiments, the composition may be in the form of a powder or granules, and may optionally be packaged in a sachet or bottle to allow the consumer to add a desired amount of the composition to a food product.

[0026] The term "foodstuff" is intended to mean any material that can be safely ingested by humans or animals, including, but not limited to, foods, beverages, cereals, bakery products, breaded and coated products (fried foods), dairy products, confectionery, snack foods, and meal products. The term encompasses products that require cooking or reconstitution before being eaten. The term also encompasses any food / nutritional supplement or medicine (such as vitamin tablets or antibiotic liquids).

[0027] It will be apparent to one skilled in the art that the modified high-potency sweetener glycosides may be incorporated into products by blending or mixing the glycosides with other ingredients, or the modified high-potency sweetener glycosides may be used to coat the product.

[0028] According to a third aspect of the present invention, there is provided a method for producing a sweet prebiotic composition comprising the steps of: a) contacting high-intensity sweetener glycosides with one or more enzymes effective to galactosylate and / or fructosylate and / or deglycosylate the high-intensity sweetener glycosides in the presence of different donors, primarily disaccharides such as sucrose and / or lactose; b) obtaining high-intensity sweetener glycosides with different oligosaccharides during galactosylation and / or fructosylation and / or deglycosylation of said high-intensity sweetener glycosides to form a sweet prebiotic composition; A method is provided comprising:

[0029] The high intensity sweetener glycoside in the method may be selected from one or more of the following: a steviol glycoside (such as rebaudioside A), or a mogroside (such as mogroside V), or a derivative thereof. Preferably, the high intensity sweetener glycoside is selected from one or more of the following: a steviol glycoside, or a mogroside V, or a derivative thereof.

[0030] The intense sweetener glycosides in the method may be galactosylated, fructosylated and / or deglycosylated using one or more enzymes selected from a multienzyme complex containing β-galactosidase and a broad range of carbohydrases produced by Aspergillus species / strains, such as arabinase, cellulase, β-glucanase, hemicellulase, pectinase and xylanase.

[0031] The oligosaccharides in the method may be synthetic and may be one or more of the following: fructooligosaccharides (FOS), galactooligosaccharides (GOS), β-gluco-oligosaccharides, α-galactooligosaccharides and xylo-oligosaccharides and combinations thereof. Preferably, the synthetic oligosaccharides are selected from one or more of galactooligosaccharides (GOS) or fructooligosaccharides (FOS).

[0032] The sweetened prebiotic composition of the method may comprise about 5% galactosylated and / or about 5% fructosylated and / or about 5% deglycosylated high-intensity sweetener glycosides. Preferably, the composition of the method may comprise about 2% galactosylated and / or about 2% fructosylated and / or about 2% deglycosylated high-intensity sweetener glycosides. More preferably, the composition of the method comprises about 1.5% galactosylated and / or about 1.5% fructosylated and / or about 1.5% deglycosylated high-intensity sweetener glycosides.

[0033] The intense sweetener glycosides may be galactosylated and / or fructosylated and / or deglycosylated in the presence of the enzyme and a disaccharide (donor).

[0034] The composition of the method may include a galactooligosaccharide or a fructooligosaccharide.

[0035] The process may be used to prepare the compositions described hereinbefore with reference to the first and second aspects of the invention.

[0036] It will be apparent to one skilled in the art that some of the composition features recited with respect to some of the embodiments of the present invention are interchangeable with respect to the compositions and methods described, unless incompatible.

[0037] Embodiments of the present invention will now be described, by way of example only. EXAMPLES

[0038] The aim of these experiments was to determine the sweetness and any off-taste intensities of several oligosaccharides and enzymatically treated high strength glycosides obtained during the same enzymatic reaction.

[0039] Example 1 – Production of Enzymatically Modified Steviol Glycosides and FOS This experiment aimed to investigate the possible yields and preferred enzymes for producing fructosylated and / or deglycosylated steviol glycosides and FOS during the same enzymatic reaction. The enzymes investigated were carbohydrase complex from Aspergillus and inulinase from Lactobacillus (R&D). The substrates were sucrose and steviol glycosides. The conditions used were 1.5% steviol glycosides, 60% sucrose, purification was by yeast fermentation, and the drying process utilized freeze-drying and vacuum evaporation.

[0040] FIG. 1A shows the HPLC-DAD profile and detection of steviol glycosides, and FIG. 1B shows the GC-FID profile of carbohydrates (trimethylsilyl oxime).

[0041] The best results were obtained using the microbial enzyme complex. The experiment suggested that the use of commercially available enzymes prepared fructosylated and / or deglycosylated steviol glycosides mixed with FOS produced during synthesis improves the flavor. Therefore, the results suggest that the mixture would be suitable for use as a prebiotic due to the high FOS concentration obtained during the enzymatic reaction.

[0042] Example 2 – Production of Enzymatically Modified Steviol Glycosides and GOS This experiment aimed to investigate the possible yields and preferred enzymes for producing galactosylated and / or deglycosylated steviol glycosides and GOS during the same enzymatic reaction. The enzymes investigated were β-galactosidases from Aspergillus sp. and Bifidobacterium bifidum. The substrates were lactose and steviol glycosides. The conditions were 1.5% steviol glycosides, 40% lactose. Purification was by yeast fermentation and the drying process was by freeze-drying and rotary evaporation.

[0043] Figure 2A shows the HPLC-DAD profile for the detection of steviol glycosides, and Figure 2B shows the GC-FID profile for the detection of carbohydrate (trimethylsilyl oxime).

[0044] The best results were obtained by using β-galactosidase from Aspergillus sp. This result shows the promise of using commercially available enzymes to produce galactosylated and / or deglycosylated steviol glycosides and GOS obtained during synthesis. Therefore, the results suggest that the high GOS concentration obtained during enzymatic synthesis would be suitable for use as a prebiotic.

[0045] Example 3 – Production of enzymatically modified mogrosides and FOS This experiment aimed to investigate the possible yields and preferred enzymes for producing fructosylated and / or deglycosylated mogrosides and FOS during the same enzymatic reaction. The enzymes investigated were carbohydrase complex from Aspergillus aculeatus and inulinase from Lactobacillus gasseri (R&D). The substrates were sucrose and steviol glycosides. The conditions used were 1.5% steviol glycosides, 60% sucrose, purification was by yeast fermentation, and the drying process utilized freeze-drying and vacuum evaporation.

[0046] Figure 3A shows the HPLC-DAD profile for the detection of mogrosides, and Figure 3B shows the GC-FID profile for the detection of carbohydrate (trimethylsilyloxime).

[0047] The best results were obtained using the microbial enzyme complex. This experiment suggested that the flavor of fructosylated and / or deglycosylated mogrosides could be improved by using the prepared commercial enzymes. This is believed to be the first report of fructosylated mogrosides. Therefore, the results suggest that fructosylated and / or deglycosylated mogrosides and FOS simultaneously obtained during the enzymatic reaction would provide a good prebiotic, mainly due to the high FOS concentration.

[0048] Example 4 – Production of enzymatically modified mogrosides and GOS This experiment aimed to investigate the possible yields and preferred enzymes for producing galactosylated and / or deglycosylated mogrosides and GOS during the same enzymatic reaction. The enzymes investigated were β-galactosidases from Aspergillus sp. and Bifidobacterium bifidum. The substrates used were lactose and mogrosides. The conditions used were 1.5% mogrosides, 40% lactose. Purification was performed using yeast fermentation and the drying process used freeze drying and rotary evaporation.

[0049] Figure 4A shows the HPLC-DAD profile for the detection of mogrosides, and Figure 4B shows the GC-FID profile for the detection of carbohydrates.

[0050] The best results were obtained with β-galactosidase from Aspergillus sp. This is believed to be the first report of galactosylated mogrosides. Therefore, the results suggest that galactosylated and / or deglycosylated mogrosides, when mixed with GOS simultaneously obtained during the enzymatic reaction, would provide a good prebiotic due to the high GOS concentration.

[0051] Example 5 - Sensory Test Data of Modified HIS A trained sensory panel from the Sensory Science Centre, Reading (UK) was recruited for the sensory profiling of the samples. There were 10 panelists with 1-9 years of experience. A QDA (Quantitative Descriptive Analysis) profiling approach was employed. The panel used the same vocabulary developed as consensus for the tasting session. Such vocabulary included the term "liquorice taste", which is the characteristic taste of steviol glycosides. The panel was retrained over three separate tasting sessions at the start of the sample set. This retraining was focused on ensuring that the panelists could reliably score sweetness for the new concentrations of sucrose standard positions.

[0052] Scoring was performed in duplicate and independently in a separate sensory booth using an arbitrary line scale (scale of 0 to 100). However, to improve sweetness discrimination, four sucrose samples were used as standards. The mean values ​​for each of these samples, as agreed by the panel, are shown in Table 1 below.

[0053] [Table 1]

[0054] At the beginning of each scoring session, the panel tasted the four reference samples in order of increasing intensity to reacclimate themselves to the positioning of these levels of sweetness on the line scale. Reference samples (10 mL) were served in clear polystyrene cups (30 mL). The panel then cleansed their palates with warm filtered tap water and low-salt crackers (Carr's water crackers) before beginning the sample tasting session, and again between each sample scoring session.

[0055] Samples labeled with random three-letter codes were presented in a sequential monadic manner in an unbiased presentation order, with a maximum of six samples per day. The room was air-conditioned and set at 23°C, and samples were served at 23–24°C (room temperature).

[0056] The panel used 15 characteristics to define the samples, as shown in Table 2 below. The average scores (0-100) for mogrosides (M) and steviol glycosides (SG) modified with two different glycosidase mixtures (F: Examples 1 and 3 and G: Examples 2 and 4) are shown.

[0057] [Table 2]

[0058] 5-9 graphically depict sensory data for important sweet, off-taste, bitter, licorice, or sweet aftertastes.

[0059] Table 3 below shows the sucrose equivalent and relative sweetness values ​​of the samples tested.

[0060] [Table 3]

[0061] Example 6 –Effects of MV-FOS, MV-GOS, SG-FOS and SG-GOS on the human gut microbiome Experiments were conducted to evaluate the effects of MV-FOS, MV-GOS, SG-FOS and SG-GOS (1% w / v) on the metabolic activity of the human gut microbiome.

[0062] 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 for 3 ns and 3 Hz frequency. Ions generated by laser desorption were introduced into the time-of-flight analyzer (1.3 m flight path) in linear positive ion mode using an accelerating voltage of 25 kV, 94% grid voltage, 0.075% ion guidewire voltage, and a delay time of 400 ns. Mass spectra were acquired 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, Bedfort, USA) was used as the matrix. Samples were diluted 1:100 in water and then mixed with matrix solution in an approximate ratio of 1:3. 1 μL of this solution was spotted onto a flat stainless steel sample plate and allowed to dry in air. External mass calibration was applied using calibration mixtures 1 and 2, monoisotopic [M+H]+ values ​​of des-Arg1 bradykinin, angiotensin I, Glu1-fibrinopeptide B, ACTH(1-17), ACTH(18-39), ACTH(7-38) and insulin (bovine) from Sequazyme Peptide Mass Standards Kits; Applied Biosystems.

[0063] Separation and analysis of enzymatically treated steviol glycosides and mogrosides by LC-MS was carried out at 25 °C using a C18 column (150 mm × 2.1 mm, 3.5 mm particle size, ThermoFisher) with a solvent gradient of acetonitrile and water (0.1% formic acid) at a flow rate of 0.1 mL / min. All experiments were performed with a Finnigan Surveyor pump equipped with a quaternary gradient system coupled to a Finnigan LCQ Deca ion trap mass spectrometer using an ESI interface. Sample injections (10 mL) were performed by a Finnigan Surveyor autosampler. All instruments (Thermo Fisher Scientific, San Jose, CA, USA) and data acquisition were managed by Xcalibur software (version 1.2; Thermo Fisher Scientific).

[0064] The effects of MV-FOS, MV-GOS, SG-FOS and SG-GOS (1% w / v) on the metabolic activity of the human gut microbiome were investigated in pH- and temperature-controlled batch cultures. The effects on the concentration of organic acids were compared with short-chain fructooligosaccharides (prebiotic positive control; FUJIFILM Wako Chemicals, Germany) and carbohydrate negative controls. Fructooligosaccharides and galactooligosaccharides produced by the same enzymatic activity used for the synthesis of modified MV and SG (1% w / v) were also tested together with native MV and SG (0.2% w / v).

[0065] Freshly voided fecal samples were obtained from five healthy adults without gastrointestinal disorders who had not taken antibiotics for 6 months prior to the study and had not taken prebiotics and / or probiotics for 6 weeks prior to the study.

[0066] A sterile fermenter (20 mL working volume, Soham Scientific, Ely, UK) was filled with 2 g L of peptone water (Oxoid, Basingstoke, UK). -1 ; yeast extract (Oxoid, Basingstoke, UK) 2gL -1 ;NaCl 0.1gL -1 ;K2HPO40.04gL -1 ;KH2PO40.04gL -1 ;MgSO4.7H2O 0.01gL -1 ;CaCl2.6H2O 0.01gL -1 ;NaHCO32gL -1 ;Hemin 0.05gL -1 ;Cysteine.HCl 0.5gL -1 ;Bile salts 0.5gL -1 Anaerobic conditions were established and maintained by filling with pre-reduced (deoxygenated) sterile basal medium consisting of: 10 μL of ethanol; 10 μL of vitamin K1; 2 mL of Tween 80 (Sigma Aldrich) and injecting with oxygen-free N2. Agitation was performed using a magnetic stirrer. The carbohydrate to be tested (1% w / v) was added to the designated vessel and immediately inoculated with fecal slurry (10% v / v prepared in anaerobic phosphate-buffered saline) from one donor. All tests on one donor were performed in parallel. Fermentation temperature was maintained at 37 °C by a circulating water bath. An automated pH controller (Fermac 260; Electrolab, UK) maintained the pH of the broth within the range of 6.7-6.9 by adding 0.5 M NaOH and 0.5 M HCl as required. Fermentations were run for 24 h and samples were drawn at 0, 5, 10 and 24 h for organic acid analysis. Table 4 below shows the results of the fermentation runs.

[0067] [Table 4]

[0068] Organic acid (OA) concentrations were determined by gas chromatography with a flame ionization detector (GC-FID) using 2-ethylbutyric acid as an internal standard based on the method described by Richardson et al. (1989). A gas chromatograph analyzer (Agilent / HP 6890) with a flame ionization detector (FID) and an HP-5MS column (30 m x 0.25 mm) with a 0.25 μm coating (crosslinked (5%-phenyl)-methylpolysiloxane, Hewlett Packard, UK) was used for SCFA measurements. Helium was used as the carrier gas at a flow rate of 1.7 mL / min (head pressure 133 KPa). The initial oven temperature was set at 63°C, followed by a temperature ramp of 15°C / min to 190°C, at which the oven was held for 3 min. 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).

[0069] 10-17, overall, SG-GOS and SG-FOS showed modification of steviol glycosides with up to 3 to 4 or more units of lactose or fructose by deglycosylation and galactosylation and fructosylation, respectively. This behavior was also found for MV-GOS and MV-FOS, where the mogrosides were galactosylated with 3 galactose units and fructosylated with 2 fructose units.

[0070] SG-GOS was rapidly fermented, as indicated by a significant increase in the level of lactate at 5 and 10 hours of fermentation, a behavior similar to that of the prebiotic control and GOS. Lactate is a fermentation intermediate that is rapidly utilized through cross-uptake by other members of the gut microbiome. Lactate accumulates in the broth when the rate of production is greater than the rate of utilization, a hallmark of the rapid gut microbiome fermentation rates observed during glycolysis of oligosaccharides. Concentrations of acetate, propionate, and butyrate were also significantly higher than the negative control, following a similar pattern to that observed with the prebiotic control and GOS.

[0071] In the SG-FOS cultures, lactate accumulation was significantly lower than in the SG-GOS and was rapidly fermented as indicated by lactate accumulation at 5 and 10 hours of fermentation, which was also observed for the positive control, where the levels were significantly lower than the prebiotic control but very similar to FOS, indicating a slower fermentation rate. Acetic, propionic and butyric acid concentrations were all significantly higher than the negative control and similar to those in the FOS fermentation, but significantly lower than the prebiotic control in terms of acetate production.

[0072] In MV-GOS cultures, lactate accumulation was significantly lower than in the GOS and prebiotic controls, indicating less rapid fermentation. Acetic acid concentrations were significantly higher than in the negative control, but increased gradually over the fermentation period, following a similar pattern to the prebiotic control and GOS, albeit at lower levels. MV-GOS significantly increased propionic acid concentrations, with levels significantly higher than in the prebiotic control and GOS. A significant increase in butyric acid was observed after 24 hours of fermentation, comparable to the increase in butyric acid for the prebiotic control and GOS.

[0073] The production of MV-FOS metabolites followed an identical pattern to MV-GOS, with the exception of butyrate, which was not significantly increased.

[0074] Overall, the fermentation behavior of the synthesized compounds shows a very close similarity to that of commercial prebiotics. Their impact on the metabolic activity of the human gut microbiome is characteristic of the glycolysis of oligosaccharides. The compounds significantly increased not only acetate but also propionate and butyrate, organic acids that have important roles in cholesterol production, appetite regulation, tight junction integrity and immune regulation.

[0075] The above embodiments are not intended to limit the scope of protection afforded by the claims, but rather to describe examples of how the invention may be practiced.

Claims

1. (i) a galactosylated and / or fructosylated high-intensity sweetener glycoside, (ii) Oligosaccharides and A prebiotic composition containing the following:

2. The composition according to claim 1, wherein the high-intensity sweetener glycoside is selected from one or more of the following: steviol glycoside, mogroside, or derivatives thereof.

3. The composition according to claim 2, wherein the steviol glycoside comprises rebaudioside A, or the mogroside comprises mogroside V.

4. The composition according to claim 1, wherein the oligosaccharide is one or more of the following galactooligosaccharides (GOS) or fructooligosaccharides (FOS).

5. The composition according to any one of claims 1 to 4, comprising a high-intensity sweetener glycoside that is galactosylated to about 5% and / or fructosylated to about 5%.

6. The composition according to any one of claims 1 to 5, wherein the composition comprises a high-intensity sweetener glycoside that is galactosylated to about 2% and / or fructosylated to about 2%.

7. The composition according to any one of claims 1 to 6, wherein the composition comprises a high-intensity sweetener glycoside that is galactosylated to about 1.5% and / or fructosylated to about 1.5%.

8. The composition according to any one of claims 1 to 7, wherein the high-intensity sweetener glycoside is galactosylated and / or fructosylated at the same time as the synthesis of the oligosaccharide.

9. The composition according to claim 8, wherein the high-intensity sweetener glycoside comprises a steviol glycoside that has been modified by galactosylation and / or fructosylation with up to 3 units of lactose or fructose.

10. The composition according to claim 8, wherein the high-intensity sweetener glycoside comprises a steviol glycoside that has been modified by galactosylation and / or fructosylation with up to 4 units of lactose or fructose.

11. The composition according to claim 8, wherein the high-intensity sweetener glycoside comprises a steviol glycoside that has been modified by galactosylation and / or fructosylation with about 4 or more units of lactose or fructose.

12. The composition according to claim 8, wherein the high-intensity sweetener glycoside comprises a mogroside that has been modified by galactosylation with up to 3 units of galactose.

13. The composition according to claim 8, wherein the high-intensity sweetener glycoside comprises a mogroside that has been modified by fructosylation with up to two units of fructose.

14. The composition according to any one of claims 3 to 11, wherein the stevioside comprises a mixture of steviol glycosides having different modifications.

15. The composition according to any one of claims 3 to 8 and 12 to 13, wherein the mogroside comprises a mixture of mogrosides having different modifications.

16. The composition according to claim 15, wherein the mogroside comprises a mixture of one or more mogrosides II, mogroside III, mogroside IV, mogroside V, or mogroside VI.

17. The composition according to claim 15, wherein the mogroside comprises a mixture of one or more of (i) mogroside V, (ii) mogroside IV, and (iii) mogroside III.

18. The composition according to claim 15, wherein the mogroside comprises a mixture of one or more of (i) mogroside III; (ii) mogroside IV, (iii) mogroside V, (iv) mogroside having one unit of fructose; and (v) mogroside having two units of fructose.

19. The composition according to claim 15, wherein the mogroside comprises a mixture of (i) mogroside IV, (ii) mogroside having one unit of galactose, (iii) mogroside V having two units of galactose, and (iv) mogroside V having three units of galactose.

20. Use of the composition according to any one of claims 1 to 19 as a low-calorie or calorie-free sweetening prebiotic.

21. Use of the composition according to any one of claims 1 to 19, wherein the composition is incorporated into or intended to be incorporated into a food product, a food supplement, or a calorie-restricted prepared food product.

22. Use of the composition according to claim 21, wherein the composition is in granular form and optionally placed in a small bag or bottle.

23. A method for producing a sweet-tasting prebiotic composition, A step of contacting a high-intensity sweetener glycoside with one or more enzymes effective in galactosylating and / or fructosyling the high-intensity sweetener glycoside in the presence of sucrose and / or lactose, in order to simultaneously produce different oligosaccharides. A method for providing this.

24. The method according to claim 23, wherein the high-intensity sweetener glycoside is selected from one or more of the following: steviol glycoside, mogroside, or derivatives thereof.

25. The method according to claim 23 or claim 24, wherein the high-intensity sweetener glycoside is galactosylated and / or fructosylated using one or more enzymes selected from a carbohydrase mixture obtained from Aspergillus species and β-galactosidase.

26. The method according to any one of claims 23 to 25, wherein the oligosaccharide synthesized is one or more of the following galactooligosaccharides (GOS) or fructooligosaccharides (FOS).

27. ​​The method according to any one of claims 23 to 26, wherein the sweetening prebiotic composition comprises a high-intensity sweetener glycoside that is galactosylated to about 5% and / or fructosylated to about 5%.

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