Safety evaluation of steviol glycoside preparations, including rebaudioside AM, obtained by enzymatic bioconversion of highly purified stevioside and/or rebaudioside A stevia leaf extracts
2021; Wiley; Volume: 19; Issue: 8 Linguagem: Inglês
10.2903/j.efsa.2021.6691
ISSN1831-4732
AutoresMaged Younes, Gabriele Aquilina, Laurence Castle, Karl‐Heinz Engel, Paul Fowler, Maria José Frutos Fernandez, Peter Fürst, Rainer Gürtler, Ursula Gundert‐Remy, Trine Husøy, Melania Manco, Wim Mennes, Sabina Passamonti, Peter Moldéus, Romina Shah, Ine Waalkens‐Berendsen, Detlef Wölfle, Matthew Wright, José Manuel Barat Baviera, Gisela Degen, Jean‐Charles Leblanc, Lieve Herman, Alessandra Giarola, Jaime Aguilera, Giorgia Vianello, Laurence Castle,
Tópico(s)Effects and risks of endocrine disrupting chemicals
ResumoEFSA JournalVolume 19, Issue 8 e06691 Scientific OpinionOpen Access Safety evaluation of steviol glycoside preparations, including rebaudioside AM, obtained by enzymatic bioconversion of highly purified stevioside and/or rebaudioside A stevia leaf extracts EFSA Panel on Food Additives and Flavourings (FAF), Corresponding Author fip@efsa.europa.eu Correspondence:fip@efsa.europa.euSearch for more papers by this authorMaged Younes, Search for more papers by this authorGabriele Aquilina, Search for more papers by this authorLaurence Castle, Search for more papers by this authorKarl-Heinz Engel, Search for more papers by this authorPaul Fowler, Search for more papers by this authorMaria Jose Frutos Fernandez, Search for more papers by this authorPeter Fürst, Search for more papers by this authorRainer Gürtler, Search for more papers by this authorUrsula Gundert-Remy, Search for more papers by this authorTrine Husøy, Search for more papers by this authorMelania Manco, Search for more papers by this authorWim Mennes, Search for more papers by this authorSabina Passamonti, Search for more papers by this authorPeter Moldeus, Search for more papers by this authorRomina Shah, Search for more papers by this authorIne Waalkens-Berendsen, Search for more papers by this authorDetlef Wölfle, Search for more papers by this authorMatthew Wright, Search for more papers by this authorJosé Manuel Barat Baviera, Search for more papers by this authorGisela Degen, Search for more papers by this authorJean-Charles Leblanc, Search for more papers by this authorLieve Herman, Search for more papers by this authorAlessandra Giarola, Search for more papers by this authorJaime Aguilera, Search for more papers by this authorGiorgia Vianello, Search for more papers by this authorLaurence Castle, Search for more papers by this author EFSA Panel on Food Additives and Flavourings (FAF), Corresponding Author fip@efsa.europa.eu Correspondence:fip@efsa.europa.euSearch for more papers by this authorMaged Younes, Search for more papers by this authorGabriele Aquilina, Search for more papers by this authorLaurence Castle, Search for more papers by this authorKarl-Heinz Engel, Search for more papers by this authorPaul Fowler, Search for more papers by this authorMaria Jose Frutos Fernandez, Search for more papers by this authorPeter Fürst, Search for more papers by this authorRainer Gürtler, Search for more papers by this authorUrsula Gundert-Remy, Search for more papers by this authorTrine Husøy, Search for more papers by this authorMelania Manco, Search for more papers by this authorWim Mennes, Search for more papers by this authorSabina Passamonti, Search for more papers by this authorPeter Moldeus, Search for more papers by this authorRomina Shah, Search for more papers by this authorIne Waalkens-Berendsen, Search for more papers by this authorDetlef Wölfle, Search for more papers by this authorMatthew Wright, Search for more papers by this authorJosé Manuel Barat Baviera, Search for more papers by this authorGisela Degen, Search for more papers by this authorJean-Charles Leblanc, Search for more papers by this authorLieve Herman, Search for more papers by this authorAlessandra Giarola, Search for more papers by this authorJaime Aguilera, Search for more papers by this authorGiorgia Vianello, Search for more papers by this authorLaurence Castle, Search for more papers by this author First published: 03 August 2021 https://doi.org/10.2903/j.efsa.2021.6691 Requestor: European Commission Question number: EFSA-Q-2019-00499 Panel members: Gabriele Aquilina, Laurence Castle, Karl-Heinz Engel, Paul Fowler, Maria Jose Frutos Fernandez, Peter Fürst, Rainer Gürtler, Ursula Gundert-Remy, Trine Husøy, Melania Manco, Wim Mennes, Sabina Passamonti, Peter Moldeus, Romina Shah, Ine Waalkens-Berendsen, Detlef Wölfle, Matthew Wright and Maged Younes. Note: The full opinion will be published in accordance with Article 12(3) of Regulation (EC) No 1331/2008 once the decision on confidentiality will be received from the European Commission. Declarations of interest: The declarations of interest of all scientific experts active in EFSA's work are available at https://ess.efsa.europa.eu/doi/doiweb/doisearch. Adopted: 22 June 2021 AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract The EFSA Panel on Food Additives and Flavourings (FAF) provides a scientific opinion on the safety of steviol glycoside preparations, including rebaudioside AM, obtained by enzymatic bioconversion of highly purified stevioside and/or rebaudioside A stevia leaf extracts. These steviol glycoside preparations are produced via enzymatic bioconversion of highly purified stevioside and/or rebaudioside A extracts obtained from stevia plant using two UDP-glucosyltransferases and one sucrose synthase enzymes produced by the genetically modified strains of E. coli K-12 that facilitate the transfer of glucose to purified stevia leaf extracts via glycosidic bonds. The Panel considered that the parental strain is a derivative of E. coli K-12 which is well characterised and its safety has been documented; therefore, it is considered to be safe for production purposes. The Panel concluded that there is no safety concern for steviol glycoside preparations, including rebaudioside AM, obtained by enzymatic bioconversion of highly purified stevioside and/or rebaudioside A stevia leaf extracts using UDP-glucosyltransferases and sucrose synthase enzymes produced by the genetically modified strains of E. coli K-12, to be used as a food additive. The Panel recommends the European Commission to consider the proposal of establishing separate specifications for steviol glycoside preparations, including rebaudioside AM, obtained by enzymatic bioconversion of highly purified stevioside and/or rebaudioside A stevia leaf extracts in Commission Regulation (EU) No 231/2012. Summary Following a request from the European Commission to the European Food Safety Authority (EFSA), the Panel on Food Additives and Flavourings (FAF) was asked to provide a scientific opinion on the safety of steviol glycoside preparations, including rebaudioside AM, obtained by enzymatic bioconversion of highly purified stevioside and/or rebaudioside A stevia leaf extracts, in accordance with Regulation (EC) No 1331/2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings. The present evaluation is based on the data on steviol glycosides in a newly submitted dossier by the applicant and additional information submitted by the applicant during the assessment process in response to requests by EFSA. These steviol glycoside preparations (≥ 95% total steviol glycosides, determined primarily as the sum of rebaudioside A, rebaudioside D, rebaudioside M and/or stevioside and rebaudioside AM) are produced via enzymatic bioconversion of highly purified stevioside and/or rebaudioside A extracts obtained from stevia plant using two UDP-glucosyltransferases and one sucrose synthase enzymes produced by the genetically modified strains of E. coli K-12, that facilitate the transfer of glucose to purified stevia leaf extracts via glycosidic bonds. The Panel considered that the proposed manufacturing process applied to the production of the steviol glycosides preparations subject of the present evaluation, involves enzymatic bioconversion steps of purified stevia leaf extracts. This process may result in impurities different from those that may be present in steviol glycosides (E 960) obtained from water extraction of the leaves of the Stevia rebaudiana followed by recrystallisation. In this respect, the Panel noted the proposal by the applicant to include a new entry in the applicable legislation, 'steviol glycosides produced via enzymatic bioconversion of steviol glycosides from Stevia plant'. The Panel considered that the parental strain is a derivative of E. coli K-12 which is well characterised and its safety has been documented; therefore, it is considered to be safe for production purposes. The recipient strain contains several genetic modifications which do not raise a safety concern. Since no viable cells nor their DNA remained in the final product, this manufacturing process does not pose a safety concern. The in vitro studies demonstrated that human digestive enzymes are not capable of hydrolysing β-glycosidic bonds of steviol glycosides and the intestinal microflora of humans (and rats) is able to hydrolyse steviol glycosides to steviol (EFSA ANS Panel, 2010; EFSA FAF Panel, 2019). The in vitro anaerobic metabolism of rebaudioside AM was investigated in pooled human faecal homogenates (Documentation provided to EFSA n. 3). The Panel agreed with the authors that the metabolism of rebaudioside AM produced by enzymatic bioconversion indicated its rapid deglycosylation to steviol as final metabolite. These findings are consistent with results from similar studies conducted in the same test system and experimental conditions with a mixture of steviol glycosides, including rebaudioside AM, previously assessed by the FAF Panel for the evaluation of another proposed amendment of the specifications for steviol glycosides (E 960) (EFSA FAF Panel, 2020). The metabolic fate of steviol glycosides, including rebaudioside AM, leads to the aglycone which is absorbed. Given the similarities in metabolic fate of steviol glycosides, a read-across with regard to toxicity was considered applicable considering the availability of toxicity studies on other previously evaluated steviol glycosides (EFSA ANS Panel, 2010). In the scientific opinion of the safety assessment of 60 steviol glycosides (EFSA FAF Panel 2020), the Panel concluded that the ADI of 4 mg/kg bw per day, expressed as steviol equivalents, can apply to all those 60 listed steviol glycosides. Considering also that no concern was identified from the manufacturing process of steviol glycoside preparations, including rebaudioside AM, obtained by enzymatic bioconversion of highly purified stevioside and/or rebaudioside A stevia leaf extracts, no additional toxicological data were required. The Panel further confirmed that the ADI of 4 mg/kg bw per day, expressed as steviol equivalents, also applies to the steviol glycoside preparations obtained by enzymatic bionconversion described in the present application. 1 Introduction The present scientific opinion deals with the safety evaluation of steviol glycoside preparations, including rebaudioside AM, obtained by enzymatic reaction of highly purified stevioside and/or rebaudioside A stevia leaf extracts. 1.1 Background and Terms of Reference as provided by the European Commission 1.1.1 Background The use of food additives is regulated under the European Parliament and Council Regulation (EC) No 1333/2008 on food additives.11 Regulation (EC) No 1333/2008 of the European Parliament and of the Council of 16 December 2008 on food additives. OJ L 354, 31.12.2008. Only food additives that are included in the Union list, in particular in Annex II to that regulation, may be placed on the market and used as in foods under the conditions of use specified therein. Moreover, food additives shall comply with the specifications as referred to in Article 14 of that Regulation and laid down in Commission Regulations (EU) No 231/201322 Commission Regulation (EU) No 231/2012 of 9 March 2012 laying down specifications for food additives listed in Annexes II and III to Regulation (EC) no 1333/2008 of the European Parliament and of the Council. OJ L 83, 22.3.2012. Steviol glycosides (E 960) is an authorised food additive in the European Union for use in several food categories and specifications have been adopted for it. Presently, the section definition in the specifications describes two main phases of the manufacturing process, i.e. water extraction followed by preliminary purification to yield a steviol glycoside primary extraction and recrystallization of the steviol glycosides resulting in a final product containing not less than 95% of the of 11 identified steviol glycosides - stevioside, rebaudiosides A, B, C, D, E, F, and M, steviolbioside, rubusoside, and dulcoside in any combination and ratio. The European Commission received an application requesting an amendment of the specifications in order to allow a further processing step – an enzymatic conversion of the highly purified rebaudioside A and/or stevioside from stevia leaf extract to minor glycosides that are present in the leaf, including rebaudioside AM that is not listed in the existing specifications. 1.1.2 Terms of Reference The European Commission requests the European Food Safety Authority to provide a scientific opinion as regards a proposed amendment of the specifications of the food additive Steviol glycosides (E 960) in accordance with Regulation (EC) No 1331/2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings.33 Regulation (EC) No 1331/2008 of the European Parliament and of the Council of 16 December 2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings. OJ L 354, 31.12.2008. 1.1.3 Interpretation of terms of reference The Panel noted that the original application submitted to the European Food Safety Authority (EFSA) for evaluation was in support of an amendment of the existing specifications for the food additive steviol glycosides (E 960), as laid down in Regulation (EC) No 231/2012, with respect to the definition of the food additive. The original proposed amendment was aimed at i) broadening the existing definition in order to capture the enzymatic bioconversion steps applied to the manufacturing of steviol glycoside preparations rich in selected steviol glycosides and ii) adding rebaudioside AM to the list of steviol glycosides detailed in the EU specifications for E 960 and contributing to the purity assay (Documentation provided to EFSA n. 1). In the course of the assessment, the applicant however has revised its original proposal for amending the existing EU specifications for the food additive E 960, requesting the creation of a new entry in the applicable legislation, e.g. E 960c(ii), for the food additive 'steviol glycosides produced via enzymatic bioconversion of steviol glycosides from Stevia plant' (Documentation provided to EFSA n. 8). In the present scientific opinion, the Panel has evaluated the latest proposal submitted by the applicant. 1.1.4 Information on existing authorisations and evaluations Steviol glycosides (E 960) from water extraction of the leaves of the Stevia rebaudiana Bertoni plant and described as 'not less than 95% steviolbioside, rubusoside, dulcoside A, stevioside, rebaudiosides A, B, C, D, E, F and M on the dried basis, in any combination and ratio' is an authorised food additive in the EU according to Regulation (EC) No 1333/2008 on food additives and specifications have been defined in Commission Regulation (EU) No 231/2012. The safety of steviol glycosides as a food additive was evaluated by EFSA in 2010 and an acceptable daily intake (ADI) of 4 mg/kg body weight (bw) per day, expressed as steviol equivalents, based on application of a 100-fold uncertainty factor to the no observed adverse effect level (NOAEL) from a 2-year carcinogenicity study in the rat was established (EFSA ANS Panel, 2010). Following a subsequent EFSA assessment in 2015 (EFSA ANS Panel, 2015a), rebaudioside D and M were included in the specifications for steviol glycosides (E 960). Rebaudioside A is also authorised as an EU flavouring substance ([FL-no: 16.113]) and was assessed within Flavouring Group Evaluation 310 (FGE.310), considering the toxicological dataset available for E 960 (EFSA CEF Panel, 2011). In 2019, the EFSA FAF Panel performed a safety evaluation of a modification of the specifications following a new production process of steviol glycosides (E 960) (EFSA FAF Panel, 2019). The FAF Panel concluded that there is no safety concern for rebaudioside M produced via enzymatic bioconversion, however 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. In 2020, the FAF Panel evaluated an application to amend the existing EU specifications for steviol glycosides to allow for the inclusion of 60 steviol glycosides identified in S. rebaudiana Bertoni leaves, including both 'major' and 'minor' steviol glycosides, that may comprise the assay value of not less than 95% total steviol glycosides. The Panel concluded that the overall metabolic fate of these steviol glycosides is the same and therefore it would be acceptable to use a read across approach for the safety assessment of the 60 steviol glycosides and the ADI of 4 mg/kg bw per day would apply to all those steviol glycosides. That list of 60 steviol glycosides included the three that are enriched in the current application; namely rebaudioside D and rebaudioside M (being among the 11 in the existing EU specifications) along with rebaudioside AM (being one of the additional 49 considered in the 2020 EFSA opinion). However, the Panel noted at that time that the proposed change from 11 to 60 specified steviol glycosides, while maintaining an assay value of not less than 95% as proposed by the applicant, would allow less pure preparations of the food additive onto the market. According to the proposed change in specifications, there would remain a small but not insignificant fraction of the additive that was undefined and therefore could be not evaluated by the Panel. Therefore, while inclusion of the 60 steviol glycosides in the specifications for steviol glycoside (E 960) would not be of safety concern, the FAF Panel could not conclude on the safety of the proposed amendment to the specifications of steviol glycosides (E 960) as food additive if the purity assay value of not less than 95% for the total content of steviol glycosides was maintained (EFSA FAF Panel, 2020). The Joint FAO/WHO Expert Committee on Food Additives (JECFA) established an ADI for steviol glycosides of 4 mg/kg bw per day, expressed as steviol (JECFA, 2009). In 2016, JECFA confirmed that rebaudioside A from multiple gene donors expressed in Yarrowia lipolytica is included in the ADI of 4 mg/kg bw, expressed as steviol (JECFA, 2016). JECFA has prepared new specifications for rebaudioside A from Multiple Gene Donors Expressed in Yarrowia lipolytica for the yeast derived product, recognising that it was manufactured by a distinctly different, biosynthetic process compared with stevia leaf–derived products (JECFA, 2016). In 2017, JECFA revised the specifications for 'Steviol Glycosides from Stevia rebaudiana Bertoni' that consist of a mixture of compounds containing a steviol backbone conjugated to any number or combination of the principal sugar moieties (glucose, rhamnose, xylose, fructose and deoxyglucose) in any of the orientations occurring in the leaves of S. rebaudiana Bertoni, provided that the total percentage of steviol glycosides is not less than 95% (JECFA, 2017). These specifications have been superseded in 2019 by new tentative JECFA specifications adopted jointly with a framework approach based on the different methods of production applied to the manufacturing of steviol glycosides, i.e. water extraction, fermentation, bioconversion and glucosylation (JECFA, 2019). The framework adopted in 2019 has been subsequently amended by JECFA at its 91st meeting in February 2021.44 http://www.fao.org/3/cb3689en/cb3689en.pdf Specifications of steviol glycosides were also revised at the same meeting and the existing assay for steviol glycosides was replaced by an HPLC–UV-MS method utilising external reference standards (JECFA, 2021). 2 Data and methodologies 2.1 Data The present evaluation is based on the data submitted in the original application dossier (Documentation provided to EFSA N. 1) and additional information submitted by the applicant during the assessment process following requests by EFSA (Documentation provided to EFSA n. 4 and 6). In addition, in June 2020 the applicant spontaneously submitted additional information (Documentation provided to EFSA n. 5). Following the evaluation of additional data sent to EFSA on 05 March 2021 (Documentation provided to EFSA n. 7), the applicant was invited to provide clarification during a technical hearing held at the 25th meeting of the WG Food Additives Applications.55 https://www.efsa.europa.eu/sites/default/files/wgs/food-ingredients-and-packaging/FAA_wg.pdf As a follow-up to the technical hearing, a further request for additional information was issued by EFSA and data in response were received on 09 June 2021 (Documentation provided to EFSA n. 8). 2.2 Methodologies This opinion was formulated following the principles described in the EFSA Guidance of the Scientific Committee on transparency with regard to scientific aspects of risk assessment (EFSA Scientific Committee, 2009) and following the relevant existing Guidance documents from the EFSA Scientific Committee. The current 'Guidance for submission for food additive evaluation' (EFSA ANS Panel, 2012), 'Guidance on the risk assessment of genetically modified microorganisms and their products intended for food and feed use' (EFSA GMO Panel, 2011) and the 'Statement on the characterisation of microorganisms used for the production of food enzymes' (EFSA CEP Panel, 2019) have been followed by the FAF Panel for evaluating the proposed enzymatic bioconversion of highly purified stevioside and/or rebaudioside A stevia leaf extracts to maximise the production of selected steviol glycosides, such as rebaudioside M, rebaudioside D and rebaudioside AM. 3 Assessment 3.1 Technical data 3.1.1 Identity of the substances The applicant proposes an enzymatic bioconversion of highly purified steviol glycosides rebaudioside A and/or stevioside extracts obtained from stevia plant using UDP-glucosyltransferases (EC 2.4.1.17) and sucrose synthase (EC 2.4.1.13) enzymes, derived from genetically modified strains of Escherichia coli K-12. This bioconversion will be applied to maximise the production of selected steviol glycosides, such as rebaudioside M, rebaudioside D and rebaudioside AM, which are naturally present only in low concentrations in the stevia leaf extract. The proposed enzymatic reaction is a stepwise conversion of the starting steviol glycosides extracts, as shown in Figure 1 below. When the starting material is the highly purified stevioside extract obtained from stevia plant, it is first converted to the 'intermediate' steviol glycoside rebaudioside E and then to the 'end' steviol glycoside rebaudioside AM (an isomer of rebaudioside D). Whereas, when the starting material is the highly purified rebaudioside A extract obtained from stevia plant, it is first converted to the 'intermediate' steviol glycoside rebaudioside D and then to the 'end' steviol glycoside rebaudioside M. Figure 1Open in figure viewerPowerPoint Enzymatic bioconversion of highly purified stevioside and/or rebaudioside A stevia leaf extracts to selected steviol glycosides, as provided by the applicant (Documentation provided to EFSA n: 1)66 The Panel noted that the correct names of the enzymes are UDP-glucosyltransferases. Upon request for clarifications on the chemical composition of the steviol glycosides preparations resulting from the enzymatic bioconversion of highly purified stevioside and/or rebaudioside A extracts obtained from stevia plant, the applicant indicated that the different duration of the enzymatic reaction on the two substrates (rebaudioside A and/or stevioside stevia leaf extracts) drives the formation to three main mixtures of selected steviol glycosides (Documentation provided to EFSA n. 1, 4 and 6): Steviol glycosides with a high content of rebaudioside M: such a blend is produced via enzymatic bioconversion of highly purified rebaudioside A stevia leaf extract with a sufficiently long enzymatic reaction time (up to 40 h) to get most of the 'intermediate' glycoside rebaudioside D converted to the 'end' glycoside rebaudioside M. The reaction yields a steviol glycosides preparation with at least 94% of rebaudioside M and the remaining components are residuals of the starting material (e.g. unreacted rebaudioside A) and rebaudioside D. Steviol glycosides with a high content of rebaudioside D: such a blend is produced via enzymatic bioconversion of highly purified rebaudioside A stevia leaf extract with a shorter enzymatic reaction time (up to 30 h) to get more 'intermediate' glycoside rebaudioside D by stopping its conversion to the 'end' glycoside rebaudioside M. The reaction yields a steviol glycosides preparation with at least 90% of rebaudioside D and the remaining components are residuals of the starting material (e.g. unreacted rebaudioside A) and rebaudioside M. Steviol glycosides with a high content of rebaudioside AM: such a blend is produced via enzymatic bioconversion of highly purified stevioside stevia leaf extract with a sufficiently long enzymatic reaction time (between 24 and 48 h) to get most of the 'intermediate' glycoside rebaudioside E converted to the 'end' glycoside rebaudioside AM. The reaction yields a steviol glycosides preparation with at least 97% of rebaudioside AM and the remaining components are residuals of the starting material (e.g. unreacted stevioside) and rebaudioside E. The applicant measured the steviol glycosides distribution in the three steviol glycosides preparations by HPLC77 For steviol glycosides preparations with high content of rebaudioside AM, a modified JECFA 2017 gradient HPLC method (JECFA, 2017) was used. ; five non-consecutive batches per each preparation were analysed. In addition, the applicant explained that such steviol glycosides preparations obtained by enzymatic bioconversion can also contain small fractions of other minor steviol glycosides that were present in the starting raw materials. For instance, the HPLC chromatogram of highly purified stevioside stevia leaf extract, used as the starting material, showed that 94.5% was accounted for stevioside and there were also small amounts of other steviol glycosides such as rebaudioside A (3.7%), steviolbioside (1.4%) and rubusoside (0.1%). Therefore, these minor steviol glycosides may be present in the final steviol glycoside preparations obtained by enzymatic bioconversion of highly purified stevia leaf extracts (Documentation provided to EFSA n. 6). The Panel noted that rebaudioside AM is currently not listed among the 11 steviol glycosides that should comprise the 95% of the assay for steviol glycosides (E 960) as per Regulation (EU) No 231/2012. The following information on rebaudioside AM was provided by the applicant (Documentation provided to EFSA n. 1): Chemical name: 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy]kaur-16-en-18-oic acid, 2-O-β-d-glucopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl ester. Synonyms: Rebaudioside AM, Reb AM, Stevia Reb AM CAS No: 2222580-26-7 Chemical formula: C50H80O28 Molecular weight: 1,129.15 According to the applicant, all steviol glycosides are glycosylated derivatives of the aglycone steviol. They share the same backbone structure and differ only in terms of type and number of glycoside units in positions R1 and R2. Figure 2Open in figure viewerPowerPoint Structures for steviol glycosides According to the applicant, the steviol glycosides preparations obtained by enzymatic bioconversion of highly purified stevioside and/or rebaudioside A stevia leaf extracts are white to yellow powders and they are approximately between 150 and 350 times sweeter than sucrose (at 5% sucrose equivalency). The steviol glycosides preparations obtained by enzymatic bioconversion of highly purified stevioside and/or rebaudioside A stevia leaf extracts are described as freely soluble (rebaudioside AM) to slightly soluble in water (rebaudioside M and D) (Documentation provided to EFSA n. 1). 3.1.2 Proposed specifications The initial proposal of the applicant for the present application was to amend the existing definition of E 960, as laid down in EC Regulation 231/2012, in order to capture this enzymatic bioconversion for manufacturing steviol glycoside preparations rich in selected steviol glycosides and to add rebaudioside AM to the list of authorised steviol glycosides which may account for the purity assay of E 960 (Documentation provided to EFSA n. 1). However, during the assessment the applicant reconsidered its original amendment and submitted a revised proposal, as outlined in Table 1 (Documentation provided to EFSA n. 8). Table 1. Specifications for steviol glycosides produced via enzymatic bioconversion of steviol glycosides from stevia plant as proposed by the applicant (Documentation provided to EFSA n. 8) E 960c(ii) Steviol glycosides produced via enzymatic bioconversion of steviol glycosides from stevia plant Synonyms Definition Steviol glycosides produced via en
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