Sweetener iso-mogroside V

Abstract

Provided is the novel sweetener and sweetness enhancer iso-mogroside V, compositions comprising the sweetener/sweetness enhancer for use in consumables (food products and products place in the oral cavity including mouth wash and other dental hygiene products), and sweetened or sweetness enhanced food products comprising the novel sweetener/sweetness enhancer.

Claims

1. A consumable composition comprising 3-[(4-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-mogrol-24-O-β-D-glucopyranosyl-(1.fwdarw.2)-O-[β-D-glucopyranosyl-(1.fwdarw.6)]-β-D-glucopyranoside (iso-mogroside V), wherein the iso-mogroside V is present in a concentration of 10-200 ppm, and wherein the composition further comprises at least one excipient.

2. The composition of claim 1 wherein the iso-mogroside V is isosweet to 0.5% sucrose.

3. The composition of claim 1 wherein the composition further comprises at least one sweetener selected from the group consisting of: sucrose, fructose, glucose, high fructose corn syrup, xylose, arabinose, rhamnose, erythritol, xylitol, mannitol, sorbitol, inositol, AceK, aspartame, neotame, sucralose, saccharine, naringin dihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NDHC), rubusoside, rebaudioside A, stevioside, mogroside IV, siamenoside I, mogroside V, and trilobatin.

4. The composition of claim 1 wherein the composition is a water-based consumable selected from: beverage, water, aqueous beverage, enhanced/slightly sweetened water drink, mineral water, carbonated beverage, non-carbonated beverage, carbonated water, still water, soft drink, non-alcoholic drink, alcoholic drink, beer, wine, liquor, fruit drink, juice, fruit juice, vegetable juice, broth drink, coffee, tea, black tea, green tea, oolong tea, herbal tea, cacoa, tea-based drink, coffee-based drinks, cacao-based drink, syrup, frozen fruit, frozen fruit juice, water-based ice, fruit ice, sorbet, dressing, salad dressing, sauce, soup, and beverage botanical materials, or instant powder for reconstitution.

5. The composition of claim 1 wherein the composition is a solid dry consumable selected from: cereals, baked food products, biscuits, bread, breakfast cereal, cereal bar, energy bars/nutritional bars, granola, cakes, cookies, crackers, donuts, muffins, pastries, confectioneries, chewing gum, chocolate, fondant, hard candy, marshmallow, pressed tablets, snack foods, botanical materials (whole or ground), and instant powders for reconstitution.

6. The composition of claim 1 wherein the composition is a consumable selected from: a dairy product, a dairy-derived product and a dairy-alternative product selected from: milk, fluid milk, cultured milk product, cultured and noncultured dairy-based drink, cultured milk product cultured with lactobacillus, yoghurt, yoghurt-based beverage, smoothy, lassi, milk shake, acidified milk, acidified milk beverage, butter milk, kefir, milk-based beverages, milk/juice blend, fermented milk beverage, ice cream, dessert, sour cream, dip, salad dressing, cottage cheese, frozen yoghurt, soy milk, rice milk, soy drink, and rice milk drink.

7. A method of providing a sweetened consumable, the method comprising the step of: admixing 3-[(4-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-mogrol-24-O-β-D-glucopyranosyl-(1.fwdarw.2)-O-[β-D-glucopyranosyl-(1.fwdarw.6)]-β-D glucopyranoside (iso-mogroside V) with a consumable to provide a resultant total concentration of iso-mogroside V of 10-200 ppm in the consumable, and wherein the consumable comprises at least one excipient.

8. The method according to claim 7 wherein the consumable further comprises a sweetener.

9. The method according to claim 8 wherein the sweetener is selected from the group consisting of: sucrose, fructose, glucose, high fructose corn syrup, xylose, arabinose, rhamnose, erythritol, xylitol, mannitol, sorbitol, inositol, AceK, aspartame, neotame, sucralose, saccharine, naringin dihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NDHC), rubusoside, rebaudioside A, stevioside, mogroside IV, siamenoside I, mogroside V, and trilobtain.

10. The method according to claim 7 wherein the consumable is a water-based consumable.

11. The method according to claim 7 wherein the consumable is a solid dry consumable.

12. The method according to claim 7 wherein the consumable is a dairy product, a dairy-derived product or a dairy-alternative product.

13. An additive which when combined with a consumable composition when the additive is present in a concentration of 10-200 ppm, the additive comprising 3-[(4-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-mogrol-24-O-β-D-glucopyranosyl-(1.fwdarw.2)-O-[β-D-glucopyranosyl-(1.fwdarw.6)]-β-D-glucopyranoside (iso-mogroside V), and wherein the consumable composition further comprises at least one excipient.

14. The additive of claim 13 which at a concentration of 10 ppm is isosweet to 0.5% sucrose.

15. The additive of claim 13 wherein two glucose molecules are connected through a (1-4) glycosidic linkage instead of a (1-6) linkage.

16. The additive of claim 13 which further comprises at least one sweetener selected from the group consisting of: sucrose, fructose, glucose, high fructose corn syrup, xylose, arabinose, rhamnose, erythritol, xylitol, mannitol, sorbitol, inositol, AceK, aspartame, neotame, sucralose, saccharine, naringin dihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NDHC), rubusoside, rebaudioside A, stevioside, mogroside IV, siamenoside I, mogroside V, and trilobatin.

17. A consumable comprising the additive of claim 13.

18. The consumable of claim 17, wherein the consumable is a water-based consumable selected from: beverage, water, aqueous beverage, enhanced/slightly sweetened water drink, mineral water, carbonated beverage, non-carbonated beverage, carbonated water, still water, soft drink, non-alcoholic drink, alcoholic drink, beer, wine, liquor, fruit drink, juice, fruit juice, vegetable juice, broth drink, coffee, tea, black tea, green tea, oolong tea, herbal tea, cacoa, tea-based drink, coffee-based drinks, cacao-based drink, syrup, frozen fruit, frozen fruit juice, water-based ice, fruit ice, sorbet, dressing, salad dressing, sauce, soup, and beverage botanical materials, or instant powder for reconstitution.

19. The consumable of claim 17, wherein the consumable is selected from: cereals, baked food products, biscuits, bread, breakfast cereal, cereal bar, energy bars/nutritional bars, granola, cakes, cookies, crackers, donuts, muffins, pastries, confectioneries, chewing gum, chocolate, fondant, hard candy, marshmallow, pressed tablets, snack foods, botanical materials (whole or ground), and instant powders for reconstitution.

20. The consumable of claim 17 wherein the consumable is selected from: milk, fluid milk, cultured milk product, cultured and noncultured dairy-based drink, cultured milk product cultured with lactobacillus, yoghurt, yoghurt-based beverage, smoothy, lassi, milk shake, acidified milk, acidified milk beverage, butter milk, kefir, milk-based beverages, milk/juice blend, fermented milk beverage, ice cream, dessert, sour cream, dip, salad dressing, cottage cheese, frozen yoghurt, soy milk, rice milk, soy drink, and rice milk drink.

21. The composition of claim 1, wherein the iso-mogroside V is isolated or purified.

22. The method of claim 7, wherein the iso-mogroside V is isolated or purified.

23. The additive of claim 13, wherein the iso-mogroside V is isolated or purified.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is an HPLC profile and peak identity graph of the swindle extract described in Example 3.

(2) FIG. 2 is a DEPT spectra of Iso-mogroside V; and

(3) FIG. 3 is a DEPT spectra of Mogroside V.

Example 1a

Comparison of the Sweetness of Mogroside V and Iso-Mogroside V

(4) The intensity of sweet taste of iso-mogroside V when compared to mogroside V was determined in water using near-sweetness detection threshold concentrations. The direct comparison was carried out by a panel of 4 sweet sensitive panelists. Blind samples (identity unidentifiable by panelists) were randomly presented to panelists in 15 ml aliquots at ambient temperature. Sensory evaluation started with the lower sample concentration (10 ppm).

(5) The results are indicated in the table below.

(6) TABLE-US-00001 sample concentrations Taste of iso-mogroside V samples [ppm] compared to mogroside V 10 sweeter 20 sweeter

(7) At 10 ppm, two panelists indicated iso-mogroside V is sweeter than mogroside V. At 20 ppm level, all 4 panelists indicated iso-mogroside V is sweeter than mogroside V.

Example 1b

Comparison of the Sweetness Enhancement of Iso-Mogroside V at Near Threshold Concentration

(8) The sweetness enhancement properties of iso-mogroside V was determined using a sample having a concentration of 10 ppm iso-mogroside V, which is near the sweetness detection threshold, in 7% sucrose solution.

(9) The iso-mogroside V sample was directly compared to samples of 7%, 8% or 9% sucrose, and panelists were instructed to compare the sweetness intensity of the samples.

(10) The comparisons were carried out by a panel of 6 sweet sensitive panelists. All samples were presented to panelists in 15 ml aliquots at ambient temperature. Panelists compared the iso-mogroside V sample to each of the sucrose samples. The results are indicated below.

(11) Iso-mogroside V samples showed a sweetness at least equal to 8% sucrose. The majority of panelists (5 of 6) found that the sample was as sweet or sweeter as 8% sucrose, and 1 of 6 panelist found the iso-mogroside sample to be sweeter than 7% but less sweet than 8% sucrose.

(12) The sweetness threshold of iso-mogroside V in water was determined to be 10 ppm, isosweet to 0.5% sucrose (see example 2), therefore an enhancement effect of at least equal to 0.5% sucrose or higher was determined.

Example 2

Determination of the Sweetness Threshold of Iso-Mogroside V

(13) Iso-mogroside V was evaluated by 5 sweet sensitive panelists at 10 ppm in water for isointensity to sucrose solutions (0.5, 1.0 and 1.5% sucrose) using a paired comparison method. Samples were paired and tasted left to right with rinsing of the mouth (water) in-between. 20 ppm iso-mogroside V was evaluated as described but with 4 sweet sensitive panelists and 0.5, 1.0 and 1.5% sucrose. The results are indicated in the table below.

(14) TABLE-US-00002 Taste of iso-mogroside Iso-mogroside V V samples Sucrose [ppm] compared to sucrose [% wt/wt] 10 sweeter 0 10 isosweet 0.5 10 less sweet 1.0 20 sweeter 0.5 20 isosweet   1% 20 less sweet 1.5%

(15) 10 ppm iso-mogroside V was sweeter than water (0% sucrose), less sweet than 1% sucrose, and isosweet to 0.5% sucrose (barely sweet). The sweetness detection threshold concentration of iso-mogroside V in water is accordingly about 10 ppm. The 20 ppm of iso-mogroside V sample was sweeter than 0.5% sucrose and less sweet than 1.5% sugar, but was found to be isosweet to 1% sucrose.

(16) At the low concentration of 10 ppm iso-mogroside V is isosweet to 0.5% sucrose, showing its high potency sweetener characteristic equaling about 500 times the sweetness of sucrose.

Example 3

LC-MS Analysis of Swingle Extract

(17) The analysed sample was swingle extract. Sample was dissolved in MeOH at a concentration of 1% and filtered. LC-MS analysis was performed using a Waters Q-Tof micro mass spectrometer coupled with a Waters 2795 separation module. The HPLC conditions were as follows: Phenomenex Luna C18(2), 5 μm, 4.6×150 mm, 55% MeOH—H.sub.2O 30 min, 0.8 ml/min; analogue detector: ESA Corona and UV 210 nm.

(18) The HPLC profile and peak identities are shown in FIG. 1.

(19) LC-MS analysis of the swingle extract identified the major sweet mogroside (mogroside V) along with a few known minor analogues (11-oxo-mogroside V, siamenoside I, mogrosides IVa, and IVe). Further LC-MS analysis of the minor components indicated the presence of a previously unreported component which had nearly the same HPLC retention time and exactly the same molecular composition as the major sweet compound, mogroside V.

Example 4

Purification of Iso-Mogroside V

(20) Swingle extract (20 g) was applied to a column of Diaion HP-20 (600 g) (Mitsubishi Chemicals, Tokyo, Japan) and washed successively with 30, 50, 70% MeOH/water and 100% methanol, using 3000 ml for each washing step. Part of the 70% methanol fraction (2.23 g) was purified over a reversed phase C-18 column using a Biotage Flash chromatography.

(21) Further preparative HPLC purification (4 repetitions) of the fractions of interest employing a Phenomenex preparative column (Luna C18(2), 5 μm, 21.2×250 mm) afforded iso-mogroside V (8.0 mg) in a purity of about 95 to 98%.

Example 5

Structural Identification of Iso-Mogroside V

(22) Iso-mogroside V (1) was isolated as an amorphous solid with an [α].sup.20.sub.D −2.1 (c 0.57, MeOH) and a molecular formula of C.sub.60H.sub.102O.sub.29 determined from its positive ion high resolution ESI-TOF MS (at m/z 1287.6530 [M+H].sup.+).

(23) The aglycone part of iso-mogroside V (structural formula shown below) was identified as mogrol by analysis of 1D (.sup.1H, .sup.13C and DEPT), and 2D (COSY, TOCSY, HSQC and NOESY) NMR and further confirmed by the long-range connectivity observed in HMBC, the results of which are listed in the table below.

(24) ##STR00008##

(25) TABLE-US-00003 TABLE Aglycones of iso-mogroside V (1) and mogroside V (2) in CD.sub.3OD: .sup.1H and .sup.13C NMR data. number of carbon atom as shown in the structural formula above .sup.13C .sup.1H 1 27.4 1.49, 2.23 dd (2.85, 12.4) 2 29.8 1.93 (2H) 3 88.3 3.47 m 4 43.0 — 5 145.2 — 6 119.8 5.49 d (6.0) 7 25.3 1.81 dd (5.0, 12.5), 2.39 dd (6.8, 12.5) 8 44.8 1.67 d (7.6) 9 41.0 — 10 37.4 2.50 d (12.3) 11 79.5 3.86 12 41.2 1.82, 1.88 13 48.4 — 14 50.7 — 15 35.5 1.14, 1.21 16 29.6 1.33, 1.98 17 51.9 1.63 d (9.2) 18 17.3 0.89 s 19 26.3 1.11 s 20 37.6 1.46 m 21 19.5 0.98 d (6.3) 22 34.2 1.49, 1.56 23 30.1 1.85 24 93.5 3.40 25 70.4 — 26 26.5 1.12 s 27 24.0 1.15 s 28 28.1 1.08 s 29 26.4 1.19 s 30 20.2 0.89 s

(26) For the sugar parts of iso-mogroside V (1), the .sup.1H and .sup.13C NMR displayed five anomeric protons at [δ 4.77 d (J=7.2 Hz), 4.43 d (J=7.4 Hz), 4.42 (J=8.0 Hz), 4.31 (J=8.0 Hz), 4.29 (J=7.7 Hz) and carbons at (δ 106.3, 104.6, 104.5, 104.4, 104.2) (see figure and table below).

(27) The sequence of the oligosaccharide chains were established by a combination of COSY, TOCSY, HSQC, HMBC and NOESY. To facilitate the proton assignments, the five anomeric protons were consecutively labeled by the letters G-1 to G-5 from the lower field (see figure which shows iso-mogroside V and the results in the table below).

(28) ##STR00009##

(29) TABLE-US-00004 TABLE Sugar parts of iso-mogroside V (1) and mogroside V (2) in CD.sub.3OD: .sup.1H and .sup.13C NMR Data identities (G-1 to G5) of the sugar carbon atoms as indicated in the structural formula above, using conventional Iso-mogroside (1) Mogroside (2) numbering .sup.13C .sup.1H .sup.13C .sup.1H G-1 1 104.5 4.77 d (7.2) 104.6 4.78 d (7.7) 2 75.7 3.28 75.8 3.28 3 78.0 3.36 78.0 3.37 4 72.5 3.22 72.5 3.23 5 78.1 3.28 78.2 3.28 6 63.7 3.86 63.7 3.87 3.65 3.65 G-2 1 104.2 4.43 d (7.4) 104.3 4.44 d (7.2) 2 81.3 3.61 81.2 3.62 3 78.5 3.59 78.4 3.58 4 71.6 3.34 71.4 3.34 5 76.5 3.51 76.3 3.51 6 70.2 3.63 70.0 3.64 4.24 br.d (8.7) 4.24 br.d (8.7) G-3 1 104.6 4.42 d (8.0) 104.9 4.43 d (7.8) 2 75.8 3.21 75.7 3.20 3 77.8 3.36 77.7 3.36 4 71.5 3.27 71.5 3.28 5 78.2 3.29 78.1 3.25 6 62.6 3.86 62.8 3.86 3.65 3.67 G-4 1 106.3 4.31 d (8.0) 106.5 4.29 d (7.7) 2 75.5 3.26 75.3 3.21 3 76.6 3.48 78.8 3.37 4 80.9 3.55 71.7 3.28 5 77.8 3.36 77.4 3.41 6 62.1 3.83 (2H) 69.9 3.81 dd (12.0, 5.6) 4.06 dd (12.0, 1.7) G-5 1 104.4 4.29 d (7.7) 104.5 4.28 d (7.7) 2 75.3 3.21 75.3 3.21 3 78.2 3.36 78.2 3.36 4 71.6 3.29 71.7 3.29 5 78.3 3.27 78.2 3.26 6 62.8 3.85 62.8 3.86 3.66 3.67

(30) Starting from the anomeric protons of each sugar unit, all the hydrogens within each spin system were traced using COSY with the aid of TOCSY and NOESY. The individual spin-systems of each sugar can be discerned from the sub-spectra corresponding to the anomeric protons in the TOCSY (mixing time=120 ms). A NOESY experiment (mixing time=600 ms) in addition to the Nuclear Overhauser Effect (NOE) contacts across the glycosidic bonds also revealed the 1,3- and 1,5-diaxial relationship for the sugars of the pyranosyl rings, thus greatly facilitating the mapping of these spin systems. Information from COSY, TOCSY, and NOESY gave the complete assignment of all protons of the compound. On the basis of the assigned protons, the .sup.13C resonances of each sugar unit were identified by HSQC and further confirmed by HMBC. Interpretation of the COSY and TOCSY spectra revealed the presence of 5 glycosyl residues. Measurement of the magnitude of homonuclear .sup.1H-.sup.1H scalar couplings provided geometric information that allowed the glycosyl configuration corresponding to each isolated spin system to be identified. The magnitude of homonuclear .sup.1H-.sup.1H scalar couplings combined with the strong NOEs between H-1 and H-3, H-1 and H-5 in all the five glycosyl residues as well as the .sup.13C NMR data identified all the sugar components to be β-glucose.

(31) The linkages between the glycosyl residues were assigned by several complementary approaches. The initial assignment of the glycosyl linkages was based on NOE contacts between H-1 resonances and resonances of the aglyconic residues. The NOE contact between H-1 of G-1 (δ 4.77 ppm) and H-2 of G-2 (δ 3.61 ppm) was diagnostic for the 1.fwdarw.2 linkage between these two glycosyl residues. Similarly, the NOE contacts between H-1 of G-5 (δ 4.29 ppm) and both H-6 (δ 3.63, 4.24 ppm) of G-2 indicated the 1.fwdarw.6 connection. At the same time, a strong NOE was also observed between the H-1 of G-2 (δ 4.43 ppm) and H-24 of the aglycone, mogrol (δ 3.40 ppm). Thus, the two terminal glucose residues (G-1 and 0-5) were linked through a 2,6-branched glucose (G-2) to C-24 of mogrol. The NOE contact between H-1 of G-3 (δ 4.42 ppm) and H-4 of G-4 (δ 3.55 ppm) indicated that the two remaining glycosyl residues were linked via a 1.fwdarw.4 linkage and the disaccharide was connected to C-4 of the aglycone based on a strong NOE contact between H-1 of G-4 (δ 4.31 ppm) and H-3 of the aglycone (δ 3.47 ppm). However, due to the highly overlapping nature of the proton NMR of the glycosyl residues, to ensure accuracy, NOE should not be used as the sole source of data for the inter-sugar linkage. Therefore, the sugar linkage was further confirmed by HMBC. The linkage of the sugar units at C-24 was established from the following HMBC correlations: H-1 of G-1 (δ 4.77 ppm) and C-2 of G-2 (δ 81.3 ppm); H-1 of G-5 (δ 4.29 ppm) and C-6 of G-2 (δ 70.2 ppm). The attachment of the trisaccharide moiety to C-24 of the aglycone was confirmed by the long-range coupling between H-1 of G-2 (δ 4.43 ppm and C-24 of the mogrol (δ 93.5 ppm). The crosspeak between H-1 of G-3 (δ 4.42 ppm) and C-4 of G-4 (δ 80.9 ppm) confirmed the 1.fwdarw.4 linkage between the remaining two glucose units. The attachment site of the disaccharide was further confirmed from the long-range coupling between H-1 of G-4 (δ 4.31 ppm) to that of C-3 of mogrol (δ 88.3 ppm).

(32) The fragmentation patterns observed by ESI-MS/MS confirm the results of the above sugar sequence analysis.

(33) MS/MS analysis of the deprotonated molecular ion [M−H].sup.− (m/z 1285.6) gave a series of daughter ions (m/z 1223.9 [(M−H)−162].sup.−, m/z 961.8 [(M−H)−2×162].sup.−, m/z 799.7 [(M−H)−3×162].sup.−, m/z 637.6 [(M−H)−4×162].sup.−, and m/z 475.5 [mogrol, (M−H)−5×162].sup.− by subsequent loss of the terminal glucose residues.

(34) The formula below shows the key NOE contacts and HMBC long-ranged couplings for iso-mogroside V (1).

(35) ##STR00010##

(36) All the glycosyl residues were in the pyranose form as determined from their .sup.13C NMR data. The β-anomeric configurations were evident from their .sup.3J.sub.H1,H2 (7-8 Hz) coupling constants as well as from NOE information.

(37) As the compounds (iso-mogroside V, mogroside V) are derived from a natural botanical source, the glucose residues all have D-configuration.

(38) Thus, the structure of iso-mogroside V (1) was established as 3-[(4-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-mogrol-24-O-β-D-glucopyranosyl-(1.fwdarw.2)-O-[β-D-glucopyranosyl-(1.fwdarw.6)]-β-D-glucopyranoside.

(39) As comparison, the detailed NMR analyses were also carried out on the major sweet component, mogroside V (2). The completed NMR assignment was achieved by a combination of COSY, TOCSY, NOESY, HSQC and HMBC (see summary of the .sup.1H and .sup.13C NMR data from these methods in the respective tables above).

(40) A direct comparison of the DEPT (Distortionless Enhancement by Polarization Transfer) spectra of iso-mogroside V (1) and mogroside V (2) clearly showed the difference between the two isomers, see results in FIG. 2 and FIG. 3.

(41) FIG. 3 shows the complete spectrum from 6 to 150 ppm, and FIG. 2 shows an enlargement of the sugar part from 60 to 85 ppm. The x-axis of both figures gives the δ ppm, the y axis of both figures gives the relative intensity.

(42) Looking at the enlargement of the field, the free, non-glycosylated C-6 of the glucose residues appeared in the region of δ 60-64 ppm in the .sup.13C NMR spectra as expected.

(43) For mogroside V (2), two C-6 of the glucose residues were found in the downfield region at around δ 70 ppm indicating that two of the five glucose residues were glycosylated at C-6 position. However, for iso-mogroside V (1), only one 1,6-glycosylated C-6 of a glucose residue was found (compare the figure at 70 ppm).

(44) Furthermore, another .sup.13C resonance was found at around δ 81 ppm indicating that one of the terminal glucose residues was, instead of the 1,6-glycosidic bond as in mogroside V, attached elsewhere to the ring (the linkage was identified as 1,4-glycosidic by further analysis employing 2D NMR).

(45) Accordingly, iso-mogroside V was identified as an isomer of mogroside V wherein the difference of the two isomers is the linkage between the G-3 and G-4 glucose residues (iso-mogroside V: 1,4-β-glycosidic, mogroside V: 1,6-β-glycosidic). Both compounds displayed almost identical HPLC retention times and identical MS/MS fragmentation patterns.