Rebaudioside M

Bioconversion of Stevioside to Rebaudioside E Using Glycosyltransferase UGTSL2.[Pubmed:33057971]

Appl Biochem Biotechnol. 2020 Oct 15. pii: 10.1007/s12010-020-03439-y.

Rebaudioside E, one of the minor components of steviol glycosides, was first isolated and identified from Stevia rebaudiana in 1977. It is a high-intensity sweetener that tastes about 150-200 times sweeter than sucrose and is also a precursor for biosynthesis of rebaudioside D and Rebaudioside M, the next-generation Stevia sweeteners. In this work, new unknown steviol glycosides were enzymatically synthesized from stevioside by coupling UDP-glucosyltransferase UGTSL2 from Solanum lycopersicum and sucrose synthase StSUS1 from Solanum tuberosum. Rebaudioside E was speculated to be the main product of glucosylation of the Glc(beta1-->C-19) residue of stevioside along with the formation of a (beta1-->2) linkage based on the analysis of the regioselectivity and stereoselectivity of UGTSL2, and verified afterwards by LC-MS/MS with standard. In a 20-ml bioconversion reaction of 20 g/l stevioside by UGTSL2 and StSUS1, 15.92 g/l rebaudioside E was produced for 24 h.

Rapid and Economic Determination of 13 Steviol Glycosides in Market-Available Food, Dietary Supplements, and Ingredients: Single-Laboratory Validation of an HPLC Method.[Pubmed:32790304]

J Agric Food Chem. 2020 Sep 16;68(37):10142-10148.

Steviol glycosides, obtained from leaves of Stevia rebaudiana Bertoni (stevia) or produced via bioconversion and biosynthesis, are diterpenes used by the food/dietary supplement industry as zero-calorie sweeteners derived from natural sources. JECFA 2017 is the most updated international standardized method but it runs for 80 min per sample with suboptimal separations on several critical pairs for its high-performance liquid chromatography-ultraviolet (HPLC-UV) determination. We developed and validated a rapid and economic HPLC-UV method using the superficially porous particle column to determine 13 steviol glycosides (stevioside, dulcoside A, rubusoside, steviobioside, and rebaudioside A-F, I, M, and N). Baseline separation with a minimum resolution of 1.5 for 13 steviol glycosides was achieved within only 14 min of separation time. The hydrocarbon stationary phase with additional steric interactions from the isobutyl side chains on the C18 ligand was shown to be an important contributor to chromatographic selectivity of several critical pairs of steviol glycosides. The method was proven to perform suitably on columns from three different manufacturers and two HPLC instruments. The method was further used to perform a single-lab validation on eight food and supplement products with multiple matrices. The results ranged from 0.05% w/w rebaudioside A for a hard-candy finished product to 100.8% w/w purity for a Rebaudioside M raw ingredient. The validation test results showed that the method was linear, suitable, specific, accurate, and precise. The method is therefore suitable to be considered as a new industrial standard for quality control analysis for stevia products.

Safety of the proposed amendment of the specifications for steviol glycosides (E 960) as a food additive: Rebaudioside M produced via enzyme-catalysed bioconversion of purified stevia leaf extract.[Pubmed:32626146]

EFSA J. 2019 Oct 28;17(10):e05867.

The EFSA Panel on Food Additives and Flavourings (FAF) provides a scientific opinion on the safety of the proposed amendment of the specifications for steviol glycosides (E 960) as a food additive, in particular related to Rebaudioside M produced via enzyme-catalysed bioconversion of purified stevia leaf extract. Rebaudioside M (95% on dry basis) is produced via enzymatic bioconversion of purified stevia leaf extract using uridine diphosphate (UDP)-glucosyltransferase and sucrose synthase enzymes produced by the genetically modified yeasts K. phaffii UGT-a and K. phaffii UGT-b, that facilitates the transfer of glucose to purified stevia leaf extract via glycosidic bonds. The Panel considered that the parental strain K. phaffii ATCC 20864 qualifies for the qualified presumption of safety (QPS) approach for safety assessment and, therefore, is considered to be safe for production purposes. The Panel concluded that there is no safety concern for Rebaudioside M produced via enzymatic bioconversion of purified stevia leaf extract using UDP-glucosyltransferase and sucrose synthase enzymes produced by the genetically modified yeasts K. phaffii UGT-a and K. phaffii UGT-b, to be used as a food additive. However, the Panel recommended that the European Commission considers establishing separate specifications for Rebaudioside M produced via enzymatic bioconversion of purified stevia leaf extract in Commission Regulation (EU) No 231/2012.

Highly sweet compounds of plant origin: From ethnobotanical observations to wide utilization.[Pubmed:31279071]

J Ethnopharmacol. 2019 Oct 28;243:112056.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnobotanical studies have been of very great importance in recognizing plants that contain substances that modulate the heterodimer T1R2-T1R3 sweet taste receptor, inclusive of Stevia rebaudiana (Asteraceae) and Siraitia grosvenorii (Cucurbitaceae). AIM OF THE REVIEW: In addition to reviewing relevant ethnobotanical literature, inclusive of original field work conducted, the authors have provided a progress report on the ultimate regulatory acceptance of highly sweet ent-kaurane (steviol) diterpene glycosides from S. rebaudiana leaves ("stevia") and cucurbitane triterpene glycosides (mogrosides) from the fruits of S. grosvenorii (popularly known as "monk fruit"). Despite their relatively high prices relative to that of sucrose, the steviol glycosides and mogrosides are of current great interest for further more extensive utilization on the market as sweet-tasting non-caloric food additives, due to increases in the rates of obesity and diabetes all over the world. Recent phytochemical work on the sweet principles of these two species is highlighted, including the important "next-generation" sweetener, Rebaudioside M, from S. rebaudiana. RESULTS: Initial observations on the ethnobotany of both S. rebaudiana and S. grosvenorii have proved crucial to indicating the presence of their sweet-tasting principles to the wider scientific community. CONCLUSIONS: Ethnobotanical observations have been pivotal in enabling the discovery of many sweet-tasting plant constituents, with those of S. rebaudiana and S. grosvenorii both being examples. Extractives prepared from these species are now commercially used widely in the U.S. as additives for the sweetening of foods and beverages.

A Novel Diterpene Glycoside with Nine Glucose Units from Stevia rebaudiana Bertoni.[Pubmed:28146121]

Biomolecules. 2017 Jan 31;7(1). pii: biom7010010.

Following our interest in new diterpene glycosides with better taste profiles than that of Rebaudioside M, we have recently isolated and characterized Rebaudioside IX-a novel steviol glycoside-from a commercially-supplied extract of Stevia rebaudiana Bertoni. This molecule contains a hexasaccharide group attached at C-13 of the central diterpene core, and contains three additional glucose units when compared with Rebaudioside M. Here we report the complete structure elucidation-based on extensive Nuclear Magnetic Resonance (NMR) analysis (1H, 13C, Correlation Spectroscopy (COSY), Heteronuclear Single Quantum Coherence-Distortionless Enhancement Polarization Transfer (HSQC-DEPT), Heteronuclear Multiple Bond Correlation (HMBC), 1D Total Correlation Spectroscopy (TOCSY), Nuclear Overhauser Effect Spectroscopy (NOESY)) and mass spectral data-of this novel diterpene glycoside with nine sugar moieties and containing a relatively rare 16 alpha-linked glycoside. A steviol glycoside bearing nine glucose units is unprecedented in the literature, and could have an impact on the natural sweetener catalog.

Microbial production of next-generation stevia sweeteners.[Pubmed:27923373]

Microb Cell Fact. 2016 Dec 7;15(1):207.

BACKGROUND: The glucosyltransferase UGT76G1 from Stevia rebaudiana is a chameleon enzyme in the targeted biosynthesis of the next-generation premium stevia sweeteners, rebaudioside D (Reb D) and Rebaudioside M (Reb M). These steviol glucosides carry five and six glucose units, respectively, and have low sweetness thresholds, high maximum sweet intensities and exhibit a greatly reduced lingering bitter taste compared to stevioside and rebaudioside A, the most abundant steviol glucosides in the leaves of Stevia rebaudiana. RESULTS: In the metabolic glycosylation grid leading to production of Reb D and Reb M, UGT76G1 was found to catalyze eight different reactions all involving 1,3-glucosylation of steviol C 13- and C 19-bound glucoses. Four of these reactions lead to Reb D and Reb M while the other four result in formation of side-products unwanted for production. In this work, side-product formation was reduced by targeted optimization of UGT76G1 towards 1,3 glucosylation of steviol glucosides that are already 1,2-diglucosylated. The optimization of UGT76G1 was based on homology modelling, which enabled identification of key target amino acids present in the substrate-binding pocket. These residues were then subjected to site-saturation mutagenesis and a mutant library containing a total of 1748 UGT76G1 variants was screened for increased accumulation of Reb D or M, as well as for decreased accumulation of side-products. This screen was performed in a Saccharomyces cerevisiae strain expressing all enzymes in the rebaudioside biosynthesis pathway except for UGT76G1. CONCLUSIONS: Screening of the mutant library identified mutations with positive impact on the accumulation of Reb D and Reb M. The effect of the introduced mutations on other reactions in the metabolic grid was characterized. This screen made it possible to identify variants, such as UGT76G1Thr146Gly and UGT76G1His155Leu, which diminished accumulation of unwanted side-products and gave increased specific accumulation of the desired Reb D or Reb M sweeteners. This improvement in a key enzyme of the Stevia sweetener biosynthesis pathway represents a significant step towards the commercial production of next-generation stevia sweeteners.

A New Diterpene Glycoside: 15alpha-Hydroxy-Rebaudioside M Isolated from Stevia rebaudiana.[Pubmed:26410999]

Nat Prod Commun. 2015 Jul;10(7):1159-61.

In a continued search for novel diterpenoid glycosides, we recently isolated and characterized a Rebaudioside M derivative with a hydroxyl group at position 15 in the central diterpene core from an extract of Stevia rebaudiana Bertoni. Here we report the complete structure elucidation of 15alpha-hydroxy-Rebaudioside M (2) on the basis of NMR (1H, 13C, COSY, HSQC-DEPT, HMBC, 1D TOCSY, NOESY) and mass spectral data. Steviol glycoside with a hydroxyl group at C-15 in the central diterpene core has not been previously reported.

Isolation and characterization of a novel rebaudioside M isomer from a bioconversion reaction of rebaudioside A and NMR comparison studies of rebaudioside M isolated from Stevia rebaudiana Bertoni and Stevia rebaudiana Morita.[Pubmed:24970220]

Biomolecules. 2014 Mar 31;4(2):374-89.

A minor product, Rebaudioside M2 (2), from the bioconversion reaction of rebaudioside A (4) to rebaudioside D (3), was isolated and the complete structure of the novel steviol glycoside was determined. Rebaudioside M2 (2) is considered an isomer of Rebaudioside M (1) and contains a relatively rare 1-->6 sugar linkage. It was isolated and characterized with NMR (1H, 13C, COSY, HSQC-DEPT, HMBC, 1D-TOCSY, and NOESY) and mass spectral data. Additionally, we emphasize the importance of 1D and 2D NMR techniques when identifying complex steviol glycosides. Numerous NMR spectroscopy studies of Rebaudioside M (1), rebaudioside D (3), and mixture of 1 and 3 led to the discovery that SG17 which was previously reported in literature, is a mixture of rebaudioside D (3), Rebaudioside M (1), and possibly other related steviol glycosides.

Minor diterpene glycosides from the leaves of Stevia rebaudiana.[Pubmed:24758242]

J Nat Prod. 2014 May 23;77(5):1231-5.

Two new diterpene glycosides in addition to five known glycosides have been isolated from a commercial extract of the leaves of Stevia rebaudiana. Compound 1 (rebaudioside KA) was shown to be 13-[(O-beta-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid 2-O-beta-d-glucopyranosyl-beta-d-glucopyranosyl ester and compound 2, 12-alpha-[(2-O-beta-d-glucopyranosyl-beta-d-glucopyranosyl)oxy]ent-kaur-16-en-19- oic acid beta-d-glucopyranosyl ester. Five additional known compounds were identified, rebaudioside E, Rebaudioside M, rebaudioside N, rebaudioside O, and stevioside, respectively. Enzymatic hydrolysis of stevioside afforded the known ent-kaurane aglycone 13-hydroxy-ent-kaur-16-en-19-oic acid (steviol) (3). The isolated metabolite 1 possesses the ent-kaurane aglycone steviol (3), while compound 2 represents the first example of the isomeric diterpene 12-alpha-hydroxy-ent-kaur-16-en-19-oic acid existing as a glycoside in S. rebaudiana. The structures of the isolated metabolites 1 and 2 were determined based on comprehensive 1D- and 2D-NMR (COSY, HSQC, and HMBC) studies. A high-quality crystal of compound 3 has formed, which allowed the acquisition of X-ray diffraction data that confirmed its structure. The structural similarities between the new metabolites and the commercially available stevioside sweeteners suggest the newly isolated metabolites should be examined for their organoleptic properties. Accordingly rebaudiosides E, M, N, O, and KA have been isolated in greater than gram quantities.

Development of Next Generation Stevia Sweetener: Rebaudioside M.[Pubmed:28234311]

Foods. 2014 Feb 27;3(1):162-175.

This work aims to review and showcase the unique properties of Rebaudioside M as a natural non-caloric potential sweetener in food and beverage products. To determine the potential of Rebaudioside M, isolated from Stevia rebaudiana Bertoni, as a high potency sweetener, we examined it with the Beidler Model. This model estimated that Rebaudioside M is 200-350 times more potent than sucrose. Numerous sensory evaluations of Rebaudioside M's taste attributes illustrated that this steviol glycoside possesses a clean, sweet taste with a slightly bitter or licorice aftertaste. The major reaction pathways in aqueous solutions (pH 2-8) for Rebaudioside M are similar to rebaudioside A. Herein we demonstrate that Rebaudioside M could be of great interest to the global food industry because it is well-suited for blending and is functional in a wide variety of food and beverage products.

Structural characterization of the degradation products of a minor natural sweet diterpene glycoside Rebaudioside M under acidic conditions.[Pubmed:24424316]

Int J Mol Sci. 2014 Jan 14;15(1):1014-25.

Degradation of Rebaudioside M, a minor sweet component of Stevia rebaudiana Bertoni, under conditions that simulated extreme pH and temperature conditions has been studied. Thus, Rebaudioside M was treated with 0.1 M phosphoric acid solution (pH 2.0) and 80 degrees C temperature for 24 h. Experimental results indicated that Rebaudioside M under low pH and higher temperature yielded three minor degradation compounds, whose structural characterization was performed on the basis of 1D (1H-, 13C-) & 2D (COSY, HSQC, HMBC) NMR, HRMS, MS/MS spectral data as well as enzymatic and acid hydrolysis studies.

In vitro metabolism of rebaudioside B, D, and M under anaerobic conditions: comparison with rebaudioside A.[Pubmed:24361573]

Regul Toxicol Pharmacol. 2014 Mar;68(2):259-68.

The hydrolysis of the steviol glycosides rebaudioside A, B, D, and M, as well as of steviolbioside (a metabolic intermediate) to steviol was evaluated in vitro using human fecal homogenates from healthy donors under anaerobic conditions. Incubation of each of the rebaudiosides resulted in rapid hydrolysis to steviol. Metabolism was complete within 24h, with the majority occurring within the first 8h. There were no clear differences in the rate or extent of metabolism of rebaudioside B, D, or M, relative to the comparative control rebaudioside A. The hydrolysis of samples containing 2.0mg/mL of each rebaudioside tended to take slightly longer than solutions containing 0.2mg/mL. There was no apparent gender differences in the amount of metabolism of any of the rebaudiosides, regardless of the concentrations tested. An intermediate in the hydrolysis of Rebaudioside M to steviol, steviolbioside, was also found to be rapidly degraded to steviol. The results demonstrate that rebaudiosides B, D, and M are metabolized to steviol in the same manner as rebaudioside A. These data support the use of toxicology data available on steviol, and on steviol glycosides metabolized to steviol (i.e., rebaudioside A) to substantiate the safety of rebaudiosides B, D, and M.