In vitro metabolism of rebaudioside B, D, and M under anaerobic conditions: Comparison with rebaudioside A

Purkayastha, Sidd; Pugh, George; Lynch, Barry; Roberts, Ashley; Kwok, David; Tarka, Stanley M.

doi:10.1016/j.yrtph.2013.12.004

Cited by 30 publications

(29 citation statements)

References 12 publications

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“…Rebaudioside D can be cleaved to steviol by the gastrointestinal microflora but it appears that the extent and rate of this cleavage may be lower than that seen with rebaudioside A. Similar complete microfloral cleavage of rebaudioside D to steviol by 24-hour incubation with in vitro cultures of human faecal microorganisms is also reported in the manuscript by Purkayastha et al (2014). The authors of both these papers attribute the apparent decrease in the rate and extent of metabolism of rebaudioside D to the difference in the substituent at R1.…”

Section: Absorption Distribution Metabolism and Excretionsupporting

confidence: 68%

“…Therefore, steviol glycosides entered the colon intact, where they were subjected to microbial degradation by members of the Bacteroidaceae family, resulting in the release of the aglycone steviol (Renwick and Tarka, 2008). Several in vitro studies mimicking the anaerobic conditions of the colon demonstrated the ability of the gut microbiota from mice, rats, hamsters and humans to hydrolyse steviol glycosides completely to steviol (Wingard et al, 1980;Hutapea et al, 1997;Gardana et al, 2003;Koyama et al, 2003b;Nikiforov et al, 2013;Purkayastha et al, 2014).…”

Section: Absorption Distribution Metabolism and Excretionmentioning

confidence: 99%

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Scientific opinion on the safety of the proposed amendment of the specifications for steviol glycosides (E 960) as a food additive

2015

EFS2

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Following a request from the European Commission, the EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) provides a scientific opinion regarding the safety of an amendment of the specifications for the food additive steviol glycosides (E 960). It was requested that rebaudioside M be added to the list of steviol glycosides as an authorised component of the mixture and that the minimum amount of 75% of stevioside and/or rebaudioside A be deleted. The ANS Panel received a dossier from the applicant and subsequently requested additional data. The Panel noted that for all stevioside and any rebaudioside following intestinal degradation, only steviol would be available in the plasma. The toxicological conclusions made by the ANS Panel in 2010 were also based on the conversion to steviol in the intestine and that steviol was the only compound systemically available. Further to this, the Panel considered that the toxicological studies performed with stevioside and rebaudioside A were relevant for assessing the safety of any steviol glycoside degraded in the intestine. The ANS Panel therefore concluded that extending the current specifications to include rebaudioside D and M as alternatives to rebaudioside A in the predominant components would be of no safety concern. The Panel also concluded that provided that the total amount of steviol glycosides (stevioside; rebaudioside, A, B, C, D, E, F and M; steviolbioside; rubusoside and dulcoside) were greater than 95% which are all converted to steviol and given that there was no evidence of absorption for intact glycosides at realistic use levels, the specific steviol glycosides (E 960) composition would not be of safety concern. Finally it was considered that the ADI of 4 mg/kg bw/day can also be applied where total steviol glycosides comprise more than 95% of the material.

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Section: Absorption Distribution Metabolism and Excretionsupporting

confidence: 68%

Section: Absorption Distribution Metabolism and Excretionmentioning

confidence: 99%

Scientific opinion on the safety of the proposed amendment of the specifications for steviol glycosides (E 960) as a food additive

2015

EFS2

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“…The safety of the steviol glucosides has been ascertained by studies of their catabolism and their use as food additives resulting in their general use in the US as well as in the EU [4–9]. Steviol glucosides consist of a diterpenoid steviol backbone decorated with one to three glucoses at the steviol C 13 -hydroxyl and/or C 19 -carboxylic acid positions (Fig.…”

Section: Introductionmentioning

confidence: 99%

Microbial production of next-generation stevia sweeteners

et al. 2016

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BackgroundThe 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.ResultsIn 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.ConclusionsScreening 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0609-1) contains supplementary material, which is available to authorized users.

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“…In contrast, elimination of administered steviol glycosides in humans, is primarily in the urine as steviol glucuronide with very small amounts of the unchanged glycoside or steviol (Kraemer and Maurer, 1994;Simonetti et al, 2004;Geuns et al, 2006Geuns et al, , 2007Wheeler et al, 2008). Recent studies using human fecal homogenates have demonstrated that regardless of the type of sugar moieties or linkages present on the steviol backbone, these are hydrolyzed to the common metabolite steviol (Purkayastha et al, 2014(Purkayastha et al, , 2015(Purkayastha et al, , 2016.…”

Section: Introductionmentioning

confidence: 99%

“…Although steviol glycosides share a common metabolic fate in both rats and humans (Wingard et al, 1980;Hutapea et al, 1997;Gardana et al, 2003;Koyama et al, 2003a;Geuns et al, 2007;Roberts and Renwick, 2008;Wheeler et al, 2008;Nikiforov et al, 2013;Purkayastha et al, 2014Purkayastha et al, , 2015Purkayastha et al, , 2016, some differences in pharmacokinetics have been noted. As evaluated by JECFA at their sixty-ninth meeting (JECFA, 2009), studies comparing the pharmacokinetics of steviol and steviol glucuronide following oral administration of steviol glycosides demonstrated substantial differences in the concentration of circulating plasma steviol and steviol glucuronide between rats and humans (Roberts and Renwick, 2008;Wheeler et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Chemical-specific adjustment factors (inter-species toxicokinetics) to establish the ADI for steviol glycosides

Roberts

Lynch

Rogerson

et al. 2016

Regulatory Toxicology and Pharmacology

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Abbreviations: λz, terminal elimination constant; ADI, acceptable daily intake; AUC0-last, area under the curve from time zero until the last sampling time point; AE, adverse events; AUC, area under the plasma concentration-time curve; AUC-0.75-inf, AUC from time zero to infinity; AUClast, AUC to the last quantifiable observation; BLQ, below the limit of quantification; BMI, body mass index; bw, body weight; Cmax, concentration maximum; CSAF, chemical-specific adjustment factor; EE, efficacy evaluable population; EKG, electrocardiogram; HBsAg, hepatitis B surface antigen; IQR, interquartile range; IRB, Institutional Review Board; JECFA, Joint FAO/WHO Expert Committee on Food Additives; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LLOQ, lower limit of quantification, LOQ, limit of quantification; NOAEL, noobserved-adverse-effect level; PP, per protocol population; SD, standard deviation; T1/2, half-life; Tmax, time to maximum concentration; WHO, World Health Organization * Corresponding author: Tel: +1-905-542-2900 E-mail address: ashley.roberts@intertek.com AbstractThe acceptable daily intake (ADI) of commercially available steviol glycosides is currently 4 mg/kg body weight (bw)/day, based on application of a 100-fold uncertainty factor to a noobserved-adverse-effect-level value from a chronic rat study. Within the 100-fold uncertainty factor is a 10-fold uncertainty factor to account for inter-species differences in toxicokinetics (4-fold) and toxicodynamics (2.5-fold). Single dose pharmacokinetics of stevioside were studied in rats (40 and 1,000 mg/kg bw) and in male human subjects (40 mg/kg bw) to generate a chemical-specific, inter-species toxicokinetic adjustment factor. Tmax values for steviol were at ~8 and ~20 hours after administration in rats and humans, respectively. Peak concentrations of steviol were similar in rats and humans, while steviol glucuronide concentrations were significantly higher in humans. Glucuronidation in rats was not saturated over the dose range 40-1,000 mg/kg bw. The AUC0-last for steviol was approximately 2.8-fold greater in humans compared to rats. Chemical-specific adjustment factors for extrapolating toxicokinetics from rat to human of 1 and 2.8 were established based on Cmax and AUC0-last data respectively. Because these factors are lower than the default value of 4.0, a higher ADI for steviol glycosides of between 6 to 16 mg/kg bw/d is justified.

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In vitro metabolism of rebaudioside B, D, and M under anaerobic conditions: Comparison with rebaudioside A

Cited by 30 publications

References 12 publications

Scientific opinion on the safety of the proposed amendment of the specifications for steviol glycosides (E 960) as a food additive

Scientific opinion on the safety of the proposed amendment of the specifications for steviol glycosides (E 960) as a food additive

Microbial production of next-generation stevia sweeteners

Chemical-specific adjustment factors (inter-species toxicokinetics) to establish the ADI for steviol glycosides

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