Prebiotic composition and method for producing the same

Abstract

The present invention relates to a prebiotic composition comprising (i) enzymatically treated high-potency sweetener glycosides and oligosaccharides. In particular, the present invention relates to galactosylated and / or fructosylated and / or deglycosylated high-potency sweetener glycosides and (ii) oligosaccharides obtained during the same enzymatic reaction. Methods for using and preparing the composition are also described. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] The present invention relates to a sweetening prebiotic composition that has specific applications as a functional food ingredient for food products and as an incorporated material in prepared food products, or that can be used alone to add sweetness to food. [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 trend towards low-calorie or zero-calorie sweeteners. Several sweeteners, such as steviol glycoside and mogroside V, are classified as high-intensity sweeteners (HIS), reportedly approximately 150 and 400 times sweeter than sucrose, respectively. However, some HIS have a bitter or off-flavor, which diminishes their appeal to consumers.

[0003] Prebiotics are substrates selectively utilized by health-beneficial host microorganisms such as Lactobacillus or Bifidobacterium, and have found much increased applications in the food sector. Prebiotics can be non-digestible food ingredients that are selectively metabolized by colonic bacteria that contribute to improved health. Therefore, using prebiotics promotes beneficial changes within the commensal gut microbiota, which can help the viability of probiotics. Prebiotics have a holistic effect on the gut bacterial population and are distinctly different from many dietary fibers such as pectin, celluloses, and xylan, which are not selectively metabolized in the gut. The classification criteria for a prebiotic are that it must be resistant to gastric acidity, hydrolysis by mammalian enzymes, and absorption in the upper digestive tract, and must reach the colon in an appropriate amount to be fermented by the gut microbiota and selectively stimulate the growth and / or activity of gut bacteria associated with health and well-being. [Overview of the project] [Problems that the invention aims to solve]

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

[0005] According to a first aspect of the present invention, (i) Enzyme-treated high-intensity sweetener glycoside, (ii) Oligosaccharides and A prebiotic composition containing the following is provided.

[0006] According to related embodiments of the present invention, (i) Enzyme-treated high-intensity sweetener glycoside, (ii) Enzymatically synthesized (enzymatically synthesized) oligosaccharides A prebiotic composition containing the following is provided.

[0007] According to a further related aspect of the present invention, (i) Enzyme-treated high-intensity sweetener glycoside, (ii) Enzymatically synthesized (enzymatically synthesized) oligosaccharides A synthetic prebiotic composition containing the following is provided. [Brief explanation of the drawing]

[0008] [Figure 1]Figure 1A shows the HPLC-DAD profile and detection of the steviol glycoside described in Example 1, and Figure 1B shows the GC-FID profile and detection of the carbohydrate described in Example 2. [Figure 2] Figure 2A shows the HPLC-DAD profile for the detection of steviol glycoside described in Example 1, and Figure 2B shows the GC-FID profile for the detection of carbohydrate described in Example 2. [Figure 3] Figure 3A shows the HPLC-DAD profile for the detection of mogrosides described in Example 3, and Figure 3B shows the GC-FID profile for the detection of carbohydrates described in Example 3. [Figure 4] Figure 4A shows the HPLC-DAD profile for the detection of mogrosides described in Example 4, and Figure 4B shows the GC-FID profile for the detection of carbohydrates described in Example 4. [Figure 5] Figure 5 is a bar graph showing the results of the sensory evaluation of sweetness for the samples tested in Example 5 (each bar represents the average value, and the error bars extend to ± half-LSD). [Figure 6] Figure 6 is a bar graph showing the results of the sensory evaluation of the intensity of off-flavors for the samples tested in Example 5 (bars represent the average value, and error bars extend to ± half-LSD). [Figure 7] Figure 7 is a bar graph showing the results of the sensory evaluation of bitterness for the samples tested in Example 5 (each bar represents the average value, and the error bars extend to ± half-LSD). [Figure 8] Figure 8 is a bar graph showing the results of the sensory evaluation of licorice flavor for the samples tested in Example 5 (bars represent the average value, and error bars extend to ± half LSD). [Figure 9] Figure 9 is a bar graph showing the results of the sensory evaluation of the sweet aftertaste for the samples tested in Example 5 (bars represent the average value, and error bars extend to ± half LSD). [Figure 10]Figure 10 is a plot showing the HPLC-MS profile of steviol glycoside enzymatically treated with A. acculitus carbohydrase as described in Example 6. RA: rebaudioside A; ST: stevioside; RF: rebaudioside F; RC: rebaudioside C; Ru: rubusoside; Sb: steviol bioside. [Figure 11] Figure 11 is a plot showing the MALDI-TOF profile for steviol glycosides enzymatically treated with A. accuritas carbohydrase as described in Example 6. RA: Rebaudioside A; ST: Stevioside; RF: Rebaudioside F; RC: Rebaudioside C; Ru: Rubusoside; Sb: Steviol bioside; Fru: Fructose. [Figure 12] Figure 12 is a plot showing the HPLC-MS profile of 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: steviol bioside. [Figure 13] Figure 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: Steviol bioside; Gal: Galactose. [Figure 14] Figure 14 is a plot showing the HPLC-MS profile for mogrosides enzymatically treated with A. accuritas carbohydrase as described in Example 6. M: Mogroside.

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

Mode for Carrying Out the Invention

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

[0011] The high-intensity sweetener glycoside may be enzymatically treated by galactosylation and / or fructosylation and / or deglycosylation.

[0012] The above high-intensity sweetener glycoside may be selected from one or more (or combinations) of the following: steviol glycoside (e.g., rebaudioside A) or mogroside (e.g., mogroside V), or derivatives thereof.

[0013] Preferably, the enzyme is derived from a microorganism. The enzyme may also be derived from the genus Aspergillus. The above 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 nidulansl; or Aspergillus niger.

[0014] The resulting oligosaccharide may be any one of the following: fructooligosaccharide (FOS), galactooligosaccharide (GOS), α-galactooligosaccharide, β-glucooligosaccharide, xylooligosaccharide, or a combination thereof. Preferably, the resulting oligosaccharide is one or more of the following: galactooligosaccharide (GOS) or fructooligosaccharide (FOS).

[0015] The composition may contain a high-intensity sweetener glycoside that is galactosylated to about 5% and / or fructosylated to about 5% and / or deglycosylated to 5%. Preferably, the composition may contain a high-intensity sweetener glycoside that is galactosylated to about 2% and / or fructosylated to about 2% and / or deglycosylated to 2%. More preferably, the composition contains a high-intensity sweetener glycoside that is galactosylated to about 1.5% and / or fructosylated to about 1.5% and / or deglycosylated to 1.5%.

[0016] The above-mentioned high-intensity sweetener glycoside may be galactosylated and / or fructosylated and / or deglycosylated during oligosaccharide synthesis.

[0017] The above-mentioned high-intensity sweetener glycoside is preferably galactosylated and / or fructosylated and / or deglycosylated simultaneously with the synthesis of the oligosaccharide.

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

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

[0020] The above stevioside may include a mixture of steviosides having different modifications. For example, the composition may include a mixture of one or more of the following: (i) rebaudioside A, rebaudioside F, rebaudioside C, rubusoside, or stevioside having 1 unit of fructose; (ii) stevioside having 2 units of fructose or rebaudioside A or rebaudioside C having 1 unit of fructose; and (iii) stevioside having 2 units of fructose or rebaudioside A or rebaudioside C having 1 unit of fructose. Selectively, the composition comprises a mixture of one or more of the following: (i) rebaudioside A and rebaudioside C or stevioside having 1 unit of galactose; (ii) stevioside having 2 units of galactose or rebaudioside A or rebaudioside C having 1 unit of galactose; (iii) stevioside having 3 units of galactose or rebaudioside A or rebaudioside C having 2 units of galactose; and (iv) stevioside having 4 units of galactose or rebaudioside A or rebaudioside C having 2 units of galactose.

[0021] The above mogrosides may include mixtures of mogrosides having different modifications. For example, a mogroside may include a mixture of one or more mogrosides II, III, IV, V, or VI. Alternatively, a mogroside may include a mixture of one or more of the following: (i) mogroside V; (ii) mogroside IV, and (iii) mogroside III. Furthermore, a mogroside may include a mixture of one or more of the following: (i) mogroside III; (ii) mogroside IV; (iii) mogroside V; (iv) a mogroside having 1 unit of fructose; and (v) a mogroside having 2 units of fructose. Furthermore, the mogroside may also contain the following mixtures: (i) mogroside IV; (ii) mogroside having 1 unit of galactose; (iii) mogroside V having 2 units of galactose; and (iv) mogroside V having 3 units of galactose.

[0022] All embodiments of the prebiotic compositions described herein have been shown to form sweet, natural, and healthy fibers that are not digested in the human upper digestive tract, and therefore can be used as calorie-free or substantially calorie-free functional ingredients. These sweet fibers have been developed as promising bulk sugar substitutes, having a sucrose-like sweetness but being calorie-free or substantially calorie-free, while also improving microbiome diversity.

[0023] The components of this prebiotic composition were found to have the advantage of having fewer off-flavors (e.g., bitterness, sourness, moldy smell, saltiness, etc.), and were also found to be significantly sweeter than all other samples.

[0024] A second aspect of the present invention provides the use of prebiotic compositions described herein as low-calorie or calorie-free sweeteners. It will be apparent to those skilled in the art that such compositions may be incorporated into, or intended to be incorporated into, a range of food products, food supplements, or calorie-restricted prepared foods, or may be used on their own to provide sweetness.

[0025] In one embodiment, the composition may be in the form of a powder or granules, or optionally, may be packaged in a sachet or bottle so that consumers can add a desired amount of the composition to food products.

[0026] The term “foodstuffs” is intended to mean any material that can be safely consumed 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 coarse flours. This term encompasses products that require reconstitution before being cooked or consumed. This term also encompasses any food / nutrition supplement or medicine (such as vitamin tablets or antibiotic solutions).

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

[0028] According to a third aspect of the present invention, a method for producing a sweet prebiotic composition, a) A step of contacting a high-intensity sweetener glycoside with one or more enzymes effective in galactosylating and / or fructosyling and / or deglycosylating the high-intensity sweetener glycoside in the presence of different donors (such as sucrose and / or lactose), mainly disaccharides, b) A step of obtaining a high-intensity sweetener glycoside having different oligosaccharides during the galactosylation and / or fructosylation and / or deglycosylation of the high-intensity sweetener glycoside in order to form a sweet prebiotic composition. A method is provided that includes this.

[0029] The high-intensity sweetener glycoside in this method may be selected from one or more of the following: steviol glycoside (e.g., rebaudioside A), or mogroside (e.g., mogroside V), or derivatives thereof. Preferably, the high-intensity sweetener glycoside is selected from one or more of the following: steviol glycoside, or mogroside V, or derivatives thereof.

[0030] The high-intensity sweetener glycoside in this method may be galactosylated, fructosylated, and / or deglycosylated using β-galactosidase and one or more enzymes selected from a multi-enzyme complex containing a wide range of carbohydrases produced by Aspergillus species / strains, such as arabinase, cellulase, β-glucanase, hemicellulase, pectinase, and xylanase.

[0031] The oligosaccharide in this method may be a synthetic product and may be one or more of the following: fructooligosaccharide (FOS), galactooligosaccharide (GOS), β-glucooligosaccharide, α-galactooligosaccharide, and xylooligosaccharide, and combinations thereof. The synthetic oligosaccharide is preferably selected from one or more of galactooligosaccharide (GOS) or fructooligosaccharide (FOS).

[0032] The sweetening prebiotic composition in the method may contain a high-intensity sweetener glycoside that is galactosylated to about 5% and / or fructosylated to about 5% and / or deglycosylated to about 5%. Preferably, the composition in the method may contain a high-intensity sweetener glycoside that is galactosylated to about 2% and / or fructosylated to about 2% and / or deglycosylated by 2%. More preferably, the composition in the method contains a high-intensity sweetener glycoside that is galactosylated to about 1.5% and / or fructosylated to about 1.5% and / or deglycosylated by 1.5%.

[0033] The above-mentioned high-intensity sweetener glycoside may be galactosylated and / or fructosylated and / or deglycosylated in the presence of the above-mentioned enzyme and disaccharide (donor).

[0034] The composition of the said method may contain galactooligosaccharides or fructooligosaccharides.

[0035] The method may be used to produce the compositions described herein with reference to the first and second aspects of the present invention.

[0036] It will be apparent to those skilled in the art that some of the compositional features listed in relation to some aspects of the present invention are interchangeable with respect to the described compositions and methods, insofar as they are not incompatible.

[0037] Embodiments of the present invention are described herein by reference only. [Examples]

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

[0039] Example 1 - Production of enzymatically treated steviol glycoside 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 the Aspergillus carbohydrase complex and the Lactobacillus inulinase (R&D). The substrates were sucrose and steviol glycoside. The conditions used were 1.5% steviol glycoside and 60% sucrose. Purification was performed by yeast fermentation, and the drying process utilized freeze-drying and vacuum evaporation.

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

[0041] The best results were obtained using a microbial enzyme complex. This experiment suggested that mixing fructosylated and / or deglycosylated steviol glycosides with FOS generated during synthesis using a prepared commercial enzyme improved the flavor. Therefore, these results suggest that the above 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 treated steviol glycoside 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 and Bifidobacterium bifidum. The substrates were lactose and steviol glycosides. The conditions were 1.5% steviol glycoside and 40% lactose. Purification was performed by yeast fermentation, and the drying process was freeze-drying and rotary evaporator.

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

[0044] The best results were obtained by using β-galactosidase derived from the Aspergillus genus. These results indicate the potential of using commercially available enzymes to produce galactosylated and / or deglycosylated steviol glycosides and GOS obtained during synthesis. Therefore, these 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 treated 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 the carbohydrase complex from Aspergillus acuritas and inulinase (R&D) from Lactobacillus gasseri. The substrates were sucrose and steviol glycoside. The conditions used were 1.5% steviol glycoside and 60% sucrose. Purification was performed 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. Figure 3B shows the GC-FID profile for the detection of carbohydrates (trimethylsilyl oxime).

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

[0048] Example 4 - Production of enzymatically treated 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 and Bifidobacterium bifidum. The substrates used were lactose and mogrosides. The conditions used were 1.5% mogroside and 40% lactose. Purification was carried out using yeast fermentation, and the drying process was performed using freeze-drying and a rotary evaporator.

[0049] Figure 4A shows the HPLC-DAD profile for mogroside detection. Figure 4B shows the GC-FID profile for carbohydrate detection.

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

[0051] Example 5 - Sensory evaluation data of modified HIS A sensory panel trained at the Sensory Science Centre in Reading, UK, was employed for the sensory profiling of the above samples. The panel consisted of 10 members with 1 to 9 years of experience. A Quantitative Descriptive Analysis (QDA) profiling approach was used. The panel used the same vocabulary developed as an agreement for the tasting sessions. Such vocabulary included the term "licorice flavor," a characteristic taste of steviol glycoside. The panel was retrained over three separate tasting sessions at the start of the sample set. This retraining focused on ensuring that the panelists could reliably score sweetness against new concentrations at the sucrose reference position.

[0052] Scoring was performed in two independent groups in isolated sensory evaluation booths using an imaginary linear scale (0-100). However, to improve the distinction regarding sweetness, four sucrose samples were used as references. The mean values ​​for each of these samples, as agreed upon by the panel, are shown in Table 1 below.

[0053] [Table 1]

[0054] At the start of each scoring session, the panel tasted the four reference samples in order of increasing intensity to re-familiarize themselves with the positioning of these levels of sweetness on the linescale. The reference sample (10 mL) was served in a clear polystyrene cup (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 sessions, and similarly cleansed their palates again between each sample scoring session.

[0055] Samples labeled with randomly assigned 3-letter codes were presented in a sequential mono-product format (using a sequential monadic method) with a maximum of 6 samples per day, in an unbiased presentation order. The room's air conditioning was set to 23°C, and the samples were presented at 23-24°C (room temperature).

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

[0057] [Table 2]

[0058] Figures 5 to 9 illustrate sensory evaluation data for important tastes such as sweetness, off-flavors, bitterness, licorice flavor, or sweet aftertaste.

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

[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 using a Voyager DE-PRO mass spectrometer (Applied Biosystems) equipped with a nitrogen laser emitting at 337 nm for 3 ns and at a frequency of 3 Hz. Ions generated by laser desorption were introduced into a time-of-flight analyzer (1.3 m flight path) in linear positive ion mode using an acceleration voltage of 25 kV, a 94% grid voltage, a 0.075% ion guidewire voltage, and a delay time of 400 ns. Mass spectra were obtained over the m / z range of 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 the matrix. The sample was diluted 1:100 in water and then mixed with the matrix solution in a ratio of approximately 1:3. 1 μL 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+H]+ values ​​of des-Arg1 bradykinin, angiotensin I, Glu1-fibrinopeptide B, ACTH(1-17), ACTH(18-39), ACTH(7-38), and bovine insulin from Calibration Mixtures 1 and 2, Sequazyme Peptide Mass Standards Kits; Applied Biosystems.

[0063] Enzymatically treated steviol glycosides and mogrosides were separated and analyzed by LC-MS at a flow rate of 0.1 mL / min using a solvent gradient of acetonitrile and water (0.1% formic acid) on a C18 column (150 mm × 2.1 mm, 3.5 mm particle size, Thermo Fisher) at 25°C. All experiments were performed using a Finnigan Surveyor pump with a quaternary radiant system coupled to a Finnigan LCQ Deca ion trap mass spectrometer via an ESI interface. Sample injection (10 mL) was performed using a Finnigan Surveyor autosampler. All instruments (Thermo Fisher Scientific, San Jose, California, USA) and data acquisition were managed using 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 batch culture media with controlled pH and temperature. The effects of organic acid concentrations were compared with short-chain fructooligosaccharides (prebiotic-positive control; FUJIFILM Wako Chemicals, Germany) and carbohydrate-negative controls. Fructooligosaccharides and galactooligosaccharides produced by the same enzyme activity used to synthesize denatured MV and SG (1% w / v) were also tested together with undenatured MV and SG (0.2% w / v).

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

[0066] A sterile fermenter (20 mL working volume, Soham scientific, Ely, UK) was filled with peptone water (Oxoid, Basingstoke, UK) at 2 g / L ,

[0067] , ,

[0068] , , , , , , ; yeast extract (Oxoid, Basingstoke, UK) at 2 g / L -1 ; NaCl at 0.1 g / L -1 ; K2HPO4 at 0.04 g / L -1 ; KH2PO4 at 0.04 g / L -1 ; MgSO4.7H2O at 0.01 g / L -1 ; CaCl2.6H2O at 0.01 g / L -1 ; NaHCO3 at 2 g / L -1 ; hemin at 0.05 g / L -1 ; cysteine.HCl at 0.5 g / L -1 ; bile salts at 0.5 g / L -1 ; a pre-reduced (deoxygenated) sterile basal medium consisting of vitamin K1 at 10 μL; Tween 80 at 2 mL (Sigma Aldrich) was filled, and anaerobic N2 was injected to establish and maintain anaerobic conditions. Stirring was carried out using a magnetic stirrer. The carbohydrate to be tested (1% w / v) was added to the designated container, and immediately thereafter, it was inoculated with fecal slurry (10% v / v prepared in anaerobic phosphate-buffered saline) from one donor. All tests for one donor were carried out in parallel. The fermentation temperature was maintained at 37 °C by a circulating water bath. An automatic pH controller (Fermac 260; Electrolab, UK) maintained the pH of the culture broth within the range of 6.7 - 6.9 by adding 0.5 M NaOH and 0.5 M HCl as needed. Fermentation was carried out for 24 hours, and samples were withdrawn for organic acid analysis at 0 hours, 5 hours, 10 hours, and 24 hours. Table 4 below shows the results of the fermentation experiment.

[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). An Agilent HP 6890 gas chromatograph analyzer with an HP-5MS column (30 m × 0.25 mm) equipped with a flame ionization detector (FID) and a 0.25 μm coating (crosslinked (5%-phenyl)-methylpolysiloxane, Hewlett Packard, UK) was used for SCFA measurement. Helium was used as the carrier gas at a flow rate of 1.7 mL / min (head pressure 133 kPa). The oven was initially set to 63°C, followed by a temperature gradient of 15°C / min up to 190°C, where the oven was held for 3 minutes. A split ratio of 100:1 was used. The appearance of OA in chromatograms was confirmed based on the retention times of each commercially available OA standard (lactic acid, acetic acid, propionic acid, and butyric acid) (Sigma-Aldrich, UK).

[0069] Referring to Figures 10 to 17, overall, SG-GOS and SG-FOS showed denaturation of the steviol glycoside by up to 3 units to 4 or more units of lactose or fructose, respectively, through deglycosylation, galactosylation, and fructosylation. This behavior was also observed in MV-GOS and MV-FOS, in which case the mogroside underwent galactosylation of 3 galactose units and fructosylation of 2 fructose units.

[0070] SG-GOS fermented rapidly, as indicated by significant increases in lactic acid levels at 5 and 10 hours of fermentation, a behavior similar to that observed in prebiotic controls and GOS. Lactic acid is a fermentation intermediate that is rapidly utilized through cross-ingestion by other members of the gut microbiome. Lactic acid accumulates in the culture medium when the rate of production is greater than the rate of utilization, which is characteristic of the rapid gut microbiome fermentation rate observed during the sugar breakdown of oligosaccharides. The concentrations of acetic acid, propionic acid, and butyric acid were also significantly higher compared to the negative control, following a pattern similar to that observed in prebiotic controls and GOS.

[0071] In the SG-FOS culture medium, lactic acid accumulation was significantly lower compared to SG-GOS, and fermentation proceeded rapidly, as indicated by lactic acid accumulation at 5 and 10 hours of fermentation. This was also observed in the positive control, where levels were significantly lower than the prebiotic control but very similar to FOS, indicating a slower fermentation rate. The concentrations of acetic acid, propionic acid, and butyric acid were all significantly higher than the negative control and similar to those in FOS fermentation, but significantly lower than the prebiotic control in terms of acetic acid production.

[0072] In the MV-GOS culture medium, lactic acid accumulation was significantly lower compared to GOS and prebiotic controls, indicating less rapid fermentation. Acetic acid concentration was significantly higher than that of the negative control, but gradually increased over the fermentation period, following a similar pattern to that of the prebiotic control and GOS, albeit at lower levels. MV-GOS significantly increased propionic acid concentration, with levels significantly higher than those of 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 the same pattern as MV-GOS, with the exception of butyrate, which did not increase significantly.

[0074] Overall, the fermentation behavior of the synthesized compounds shows a very close resemblance to that of commercially available prebiotics. Their effects on the metabolic activity of the human gut microbiome are characteristic of oligosaccharide glycolysis. These compounds not only significantly increased acetic acid, but also significantly increased propionic acid and butyric acid, organic acids that play important roles in cholesterol production, appetite regulation, tight junction integrity, and immunomodulation.

[0075] The embodiments described above are not intended to limit the scope of protection provided by the claims, but rather to describe examples of ways in which the present invention may be carried out.

Claims

1. A method for producing a sweet prebiotic composition, comprising contacting one or more high-intensity sweetener glycosides selected from steviol glycosides and mogrosides with (i) a mixture of carbohydrases derived from Aspergillus species in the presence of sucrose, or (ii) β-galactosidase derived from Aspergillus species and / or Bifidobacterium bifidum in the presence of lactose, wherein the high-intensity sweetener glycoside is galactosylated and / or fructosylated and / or deglycosylated simultaneously with the synthesis of oligosaccharides.

2. The method according to claim 1, wherein the composition comprises a steviol glycoside, the steviol glycoside comprises rebaudioside A, or the mogroside comprises mogroside V.

3. The method according to claim 1 or claim 2, wherein the oligosaccharide is one or more of the following galactooligosaccharides (GOS) or fructooligosaccharides (FOS).

4. The method according to any one of claims 1 to 3, wherein the composition comprises a high-intensity sweetener glycoside that is galactosylated to 5% and / or fructosylated to 5%.

5. The method according to any one of claims 1 to 4, wherein the composition comprises a high-intensity sweetener glycoside that is galactosylated to 2% and / or fructosylated to 2%.

6. The method according to any one of claims 1 to 5, wherein the composition comprises a high-intensity sweetener glycoside that is galactosylated to 1.5% and / or fructosylated to 1.5%.

7. The method according to any one of claims 1 to 6, wherein the high-intensity sweetener glycoside comprises a steviol glycoside that has been modified with up to 3 units of lactose or fructose by galactosylation and / or fructosylation.

8. The method according to any one of claims 1 to 7, wherein the steviol glycoside is modified with up to four units of lactose or fructose by galactosylation and / or fructosylation.

9. The method according to claim 8, wherein the steviol glycoside is modified with four or more units of lactose or fructose by galactosylation and / or fructosylation.

10. The method according to any one of claims 1 to 6, wherein the mogroside is modified by galactosylation with up to 3 units of galactose.

11. The method according to any one of claims 1 to 6, wherein the mogroside is modified by fructosylation with up to two units of fructose.

12. The method according to any one of claims 1 to 9, wherein the steviol glycoside comprises a mixture of steviol glycosides having different modifications.

13. The method according to any one of claims 1 to 6 and 10 to 11, wherein the mogroside comprises a mixture of mogrosides having different modifications.

14. The method according to claim 13, wherein the mogroside comprises a mixture of one or more mogrosides II, mogroside III, mogroside IV, mogroside V, or mogroside VI.

15. The method according to claim 13, wherein the mogroside comprises a mixture of one or more of (i) mogroside V, (ii) mogroside IV, and (iii) mogroside III.

16. The method according to claim 13, 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.

17. The method according to claim 13, wherein the mogroside comprises a mixture of (i) mogroside IV, (ii) a mogroside having one unit of galactose, (iii) mogroside V having two units of galactose, and (iv) mogroside V having three units of galactose.

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