Safety evaluation of glucosylated steviol glycosides as a food additive in different food categories
2022; Wiley; Volume: 20; Issue: 2 Linguagem: Inglês
10.2903/j.efsa.2022.7066
ISSN1831-4732
AutoresMaged Younes, Gabriele Aquilina, Karl–Heinz Engel, Paul J Fowler, María José Frutos Fernández, Peter Fürst, Rainer Gürtler, Ursula Gundert‐Remy, Trine Husøy, Melania Manco, Wim Mennes, Peter Moldéus, Sabina Passamonti, Romina Shah, Ine Waalkens‐Berendsen, Detlef Wölfle, Matthew Wright, José Manuel Barat Baviera, Gisela Degen, Lieve Herman, Jean‐Charles Leblanc, Jaime Aguilera, Alessandra Giarola, Ana María Rincón, Camilla Smeraldi, Giorgia Vianello, Laurence Castle,
Tópico(s)Effects and risks of endocrine disrupting chemicals
ResumoEFSA JournalVolume 20, Issue 2 e07066 Scientific OpinionOpen Access Safety evaluation of glucosylated steviol glycosides as a food additive in different food categories EFSA Panel on Food Additives and Flavourings (FAF), Corresponding Author EFSA Panel on Food Additives and Flavourings (FAF) fip@efsa.europa.eu Correspondence:fip@efsa.europa.euSearch for more papers by this authorMaged Younes, Maged YounesSearch for more papers by this authorGabriele Aquilina, Gabriele AquilinaSearch for more papers by this authorKarl-Heinz Engel, Karl-Heinz EngelSearch for more papers by this authorPaul J Fowler, Paul J FowlerSearch for more papers by this authorMaria Jose Frutos Fernandez, Maria Jose Frutos FernandezSearch for more papers by this authorPeter Fürst, Peter FürstSearch for more papers by this authorRainer Gürtler, Rainer GürtlerSearch for more papers by this authorUrsula Gundert-Remy, Ursula Gundert-RemySearch for more papers by this authorTrine Husøy, Trine HusøySearch for more papers by this authorMelania Manco, Melania MancoSearch for more papers by this authorWim Mennes, Wim MennesSearch for more papers by this authorPeter Moldeus, Peter MoldeusSearch for more papers by this authorSabina Passamonti, Sabina PassamontiSearch for more papers by this authorRomina Shah, Romina ShahSearch for more papers by this authorIne Waalkens-Berendsen, Ine Waalkens-BerendsenSearch for more papers by this authorDetlef Wölfle, Detlef WölfleSearch for more papers by this authorMatthew Wright, Matthew WrightSearch for more papers by this authorJose Manuel Barat, Jose Manuel BaratSearch for more papers by this authorGisela Degen, Gisela DegenSearch for more papers by this authorLieve Herman, Lieve HermanSearch for more papers by this authorJean-Charles Leblanc, Jean-Charles LeblancSearch for more papers by this authorJaime Aguilera, Jaime AguileraSearch for more papers by this authorAlessandra Giarola, Alessandra GiarolaSearch for more papers by this authorAna Maria Rincon, Ana Maria RinconSearch for more papers by this authorCamilla Smeraldi, Camilla SmeraldiSearch for more papers by this authorGiorgia Vianello, Giorgia VianelloSearch for more papers by this authorLaurence Castle, Laurence CastleSearch for more papers by this author EFSA Panel on Food Additives and Flavourings (FAF), Corresponding Author EFSA Panel on Food Additives and Flavourings (FAF) fip@efsa.europa.eu Correspondence:fip@efsa.europa.euSearch for more papers by this authorMaged Younes, Maged YounesSearch for more papers by this authorGabriele Aquilina, Gabriele AquilinaSearch for more papers by this authorKarl-Heinz Engel, Karl-Heinz EngelSearch for more papers by this authorPaul J Fowler, Paul J FowlerSearch for more papers by this authorMaria Jose Frutos Fernandez, Maria Jose Frutos FernandezSearch for more papers by this authorPeter Fürst, Peter FürstSearch for more papers by this authorRainer Gürtler, Rainer GürtlerSearch for more papers by this authorUrsula Gundert-Remy, Ursula Gundert-RemySearch for more papers by this authorTrine Husøy, Trine HusøySearch for more papers by this authorMelania Manco, Melania MancoSearch for more papers by this authorWim Mennes, Wim MennesSearch for more papers by this authorPeter Moldeus, Peter MoldeusSearch for more papers by this authorSabina Passamonti, Sabina PassamontiSearch for more papers by this authorRomina Shah, Romina ShahSearch for more papers by this authorIne Waalkens-Berendsen, Ine Waalkens-BerendsenSearch for more papers by this authorDetlef Wölfle, Detlef WölfleSearch for more papers by this authorMatthew Wright, Matthew WrightSearch for more papers by this authorJose Manuel Barat, Jose Manuel BaratSearch for more papers by this authorGisela Degen, Gisela DegenSearch for more papers by this authorLieve Herman, Lieve HermanSearch for more papers by this authorJean-Charles Leblanc, Jean-Charles LeblancSearch for more papers by this authorJaime Aguilera, Jaime AguileraSearch for more papers by this authorAlessandra Giarola, Alessandra GiarolaSearch for more papers by this authorAna Maria Rincon, Ana Maria RinconSearch for more papers by this authorCamilla Smeraldi, Camilla SmeraldiSearch for more papers by this authorGiorgia Vianello, Giorgia VianelloSearch for more papers by this authorLaurence Castle, Laurence CastleSearch for more papers by this author First published: 09 February 2022 https://doi.org/10.2903/j.efsa.2022.7066 Requestor: European Commission Question number: EFSA-Q-2019-00062 Panel members: Gabriele Aquilina, Laurence Castle, Karl-Heinz Engel, Paul J 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. Declarations of interest: The declarations of interest of all scientific experts active in EFSA's workare available at https://ess.efsa.europa.eu/doi/doiweb/doisearch. Adopted: 15 December 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 onFacebookTwitterLinked InRedditWechat Abstract The EFSA Panel on Food Additive and Flavourings (FAF) assessed the safety of glucosylated steviol glycosides proposed for use as a new food additive in different food categories. Glucosylated steviol glycosides consist of a mixture of glucosylated steviol glycosides, containing 1–20 additional glucose units bound to the parent steviol glycosides. Glucosylated steviol glycosides consist of not less than 95% (on dry, dextrin-free, basis) of total steviol glycosides, comprised of glucosylated and parent steviol glycosides. Glucosylated steviol glycosides are produced via enzymatic bioconversion using cyclomaltodextrin glucanotransferase (CGTase) (EC 2.4.1.19), derived from a non-genetically modified strain of Anoxybacillus caldiproteolyticus, that catalyses the transfer of glucose from starch to steviol glycosides mixtures isolated from the dried leaves of Stevia Rebaudiana. The Panel considered that the metabolism of glucosylated steviol glycosides is sufficiently similar to the already authorised steviol glycosides, and thus, the toxicological data previously assessed by the ANS Panel for steviol glycosides (E 960) were considered to support their safety as food additive. The existing acceptable daily intake (ADI) for steviol glycosides (E 960) of 4 mg/kg body weight (bw) per day expressed as steviol can also be applied to glucosylated steviol glycosides. The Panel concluded that there is no safety concern for the use of glucosylated steviol glycosides as a new food additive at the proposed use and use levels. The Panel recommended some modifications to the specifications proposed by the applicant for glucosylated steviol glycosides with respect to the assay, the definition of the proposed new food additive and the proposed maximum limits for arsenic. 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 safety in use of glucosylated steviol glycosides as a food additive in different food categories, 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 either spontaneously or following request from EFSA. The EFSA Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS Panel) had previously evaluated the safety of glucosylated steviol glycosides to be used as a food additive and concluded that the submitted data were insufficient to assess its safety (EFSA ANS Panel, 2018). 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) (expressed as steviol equivalents) per day was established. Glucosylated steviol glycosides' preparations were described by the applicant as a mixture of glucosylated steviol glycosides, containing 1–20 additional glucose units bound to the parent steviol glycoside via α-(1–4) linkages. The Panel noted that the definition of the food additive in the proposed specifications does not include any value for the additional glucose units and that the mixtures on which data have been provided contain, on average, only three or four additional glucose units. The Panel made a conservative assumption that the GSG may contain up to 10 additional glucose units on average in its evaluation and considered that information on the number of additional glucose units (1–20) should be added to the proposed definition of the food additive in the specifications. Glucosylated steviol glycosides' preparations consist of not less than 95% of the total steviol glycosides comprised of glucosylated steviol glycosides and parent steviol glycosides on the dried, dextrin-free, basis. The glucosylated fraction of the total steviol glycosides is between 80% and 92% w/w and the parent steviol glycosides between 5% and 15% w/w. Typical components of glucosylated steviol glycosides are presented in Appendix A as proposed by the applicant. The Panel considered that rather than having a non-definite list of components, as proposed by the applicant, the assay value should cover all steviol glycosides falling under the definition of E 960a along with their glucosylated derivatives. The proposed use and use levels for glucosylated steviol glycosides are the same as those for the already authorised steviol glycosides (E 960a–960c). Taking into account that glucosylation of the steviol glycosides results in higher molecular weights, the glucosylated steviol glycosides have a lower steviol equivalency compared to steviol glycosides E 960a. Therefore, a larger quantity of glucosylated steviol glycosides would be permitted to be added to food to give the same steviol equivalent value. Consequently, the resulting exposure to potential impurities, including toxic elements, has taken into account this higher addition levels (on a mass basis and not on a steviol equivalent basis). With the exception of arsenic, for which the lowest calculated margin of safety (MOS)/margin of exposure (MOE) was considered to be insufficient, for the other toxic elements (cadmium, mercury, lead) proposed for inclusion in the specifications the maximum limits do not give rise to safety concerns. The Panel noted that the proposed glucosylated steviol glycosides' preparations may contain up to 20 glucose moieties per molecule of steviol glycoside and this could lead to an additional exposure to glucose from a sweetener which is proposed to have a technological function of replacing sugars in food. Based on a worst-case assumption of an average of 10 additional glucose units added to the steviol glycosides, the Panel estimated that the proposed uses and use level of the new food additive glucosylated steviol glycosides would lead to an additional glucose intake of up to 0.4 g/day for toddlers and 1.2 g/day for adults. Glucosylated steviol glycosides are prepared in two production stages. Steviol glycosides mixtures – enriched individual steviol glycosides (e.g. high Rebaudioside A or Rebaudioside D content (> 95%)) or mixtures enriched in two or more individual steviol glycosides – are isolated from dried leaves of S. rebaudiana by hot water extraction and different purifications steps. The purified steviol glycosides extract undergoes enzymatic treatment to achieve glucosylation via α-(1-4) linkages of the steviol glycosides. A glucose donor, such as tapioca starch (extracted from Cassava roots), is treated with the enzyme CGTase (EC 2.4.1.19), derived from a non-genetically modified strain of Anoxybacillus caldiproteolyticus. CGTase (EC 2.4.1.19) is also used to catalyse the intermolecular glucosylation whereby the transfer occurs of α-glucosyl units from the starch onto the 4-hydroxy position of a glucosyl moiety on the steviol glycoside (trans-α-1,4-glucosylation). The enzyme is inactivated by heating and removed by treatment with activated carbon. The dextrin by-product can be removed from the mixture. The Panel noted that the safety of the enzyme CGTase (EC 2.4.1.19) derived from a non-genetically modified strain of Anoxybacillus caldiproteolyticus and intended to be used in the preparation of glucosylated steviol glycosides has been assessed by the EFSA CEP Panel. Based on the data provided, and the removal of food enzyme total organic solids (TOS) during the manufacture of modified steviol glycosides, the CEP Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use (EFSA CEP Panel, 2022). Glucosylated steviol glycosides contain α-oriented glycosidic bonds which are hydrolysed by digestive enzymes (i.e. salivary and/or pancreatic α-amylase) resulting in the parent steviol glycosides. Therefore, the glucosylated steviol glycosides share the same metabolic fate as steviol glycosides extracted from S. rebaudiana Bertoni, i.e. they undergo hydrolysis forming steviol as demonstrated in an in vitro study submitted in support of the current application. The results of this study with human faecal homogenates showed an extensive microbial degradation of mono- to tetra-glucosylated steviol glycosides to steviol at similar rates as Rebaudioside A. Overall, the Panel considered that the metabolism of glucosylated steviol glycosides is sufficiently similar to the already authorised steviol glycosides, and thus, the toxicological data previously assessed by the ANS Panel for steviol glycosides (E 960a) were considered to support their safety as food additive. Therefore, no additional toxicological data were required. The existing ADI for steviol glycosides (E 960a) of 4 mg/kg bw per day expressed as steviol can also be applied to glucosylated steviol glycosides. The Panel concluded that there is no safety concern for the use of glucosylated steviol glycosides as a new food additive at the proposed use and use levels. The Panel recommended that the specifications proposed by the applicant for glucosylated steviol glycosides would include the following modifications: – The assay value of more than 95% for total steviol glycosides, comprised of glucosylated and parent steviol glycosides, on the dried, dextrin-free, basis should be limited to those 11 named steviol glycosides that are included in the current definition of E 960a along with their glucosylated derivatives (1–20 added glucose units). – The description of the food enzyme used in the manufacturing process should be aligned to the description given in the CEP Panel opinion, i.e. 'Cyclomaltodextrin glucanotransferase (EC 2.4.1.19) derived from a non-GMO strain of Anoxybacillus caldiproteolyticus St-88'. – The proposed maximum limit for arsenic should be lowered. 1 Introduction The present scientific opinion deals with the safety of glucosylated steviol glycosides proposed for use as a new food additive in different food categories. 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. In 2017 the European Commission received a request for authorisation of the use of Glucosylated steviol glycosides as a sweetener in several food categories. Glucosylated steviol glycosides are manufactured by adding glucose units (between 1 to 10 additional units) to steviol glycosides extracted from the leaves of Stevia rebaudiana. This is achieved using enzymes that transfer glucose units from a starch source to steviol glycosides, and results in the production of a mixture of glucosylated (~80–92%) and non-modified parent steviol glycosides (~5–15%). The request was assessed by EFSA which concluded that the submitted data were insufficient to assess the safety of the glucosylated steviol glycoside preparations to be used as new food additives (EFSA ANS Panel, 2018). In January 2019 the European Commission received an updated application containing additional information, which according to the applicant, should address the concerns and questions raised in the EFSA's scientific opinion (EFSA ANS Panel, 2018). 1.1.2 Terms of Reference The Commission requests the European Food Safety Authority (EFSA) to provide a scientific opinion on the safety in use of glucosylated steviol glycosides as food additive in different food categories in accordance with the Regulation (EC) No 1331/2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings.22 Regulation (EC) No 1331/2008 of the European Parliament and of the Council of 16 December establishing a common authorisation procedure for food additives, food enzymes and food flavourings. OJ L 354, 31.12.2008. 1.2 Information on existing authorisations and evaluations An application for the proposed use of glucosylated steviol glycosides as a new food additive had been previously evaluated by the former EFSA ANS Panel in 2018. At that time, the ANS Panel concluded that the data submitted in support of the new application were insufficient to assess its safety (EFSA ANS Panel, 2018). Steviol glycosides from Stevia (E 960a)33 Since receipt of the present mandate, Regulation (EC) No 1333/2008 was amended by Commission Regulation (EU) 2021/1156 of 13 July 2021 replacing the entry for E 960 (Steviol glycosides) with two separate entries for 'steviol glycosides from Stevia (E 960a)' and 'enzymatically produced steviol glycosides (E 960c)'. Accordingly, in the present opinion, the Panel has replaced reference to E 960 with E 960a or E 960c, whenever appropriate. are an authorised food additive in the EU according to Regulation (EC) No 1333/2008 on food additives. The food additive is obtained from water extraction of the leaves of the Stevia rebaudiana Bertoni plant and according to the specifications defined in Commission Regulation (EU) No 231/201244 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.. It is described as containing not less than 95% of the identified 11 related steviol glycosides steviolbioside, rubusoside, dulcoside A, stevioside, rebaudiosides A, B, C, D, E, F and M on the dried basis, in any combination and ratio. 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 and a no observed adverse effect level (NOAEL) from a 2-year carcinogenicity study in the rat was established (EFSA ANS Panel, 2010). Following the EFSA assessment in 2015 (EFSA ANS Panel, 2015a), rebaudiosides D and M were included in the specifications for steviol glycosides (E 960a). The latest exposure assessment to steviol glycosides (E 960) was carried out by the ANS Panel in 2015 (EFSA ANS Panel, 2015b). A new entry for 'enzymatically produced steviol glycosides (E 960c)' was added to Annex II to Regulation (EC) No 1333/2008 as amended by Commission Regulation (EU) 2021/1156 of 13 July 202155 Commission Regulation (EU) 2021/1156 of 13 July 2021 amending Annex II to Regulation (EC) No 1333/2008 of the European Parliament and of the Council and the Annex to Commission Regulation (EU) No 231/2012 as regards steviol glycosides (E 960) and rebaudioside M produced via enzyme modification of steviol glycosides from Stevia. OJ L 249, 14.7.2021, p. 87–98.. This amendment to the Regulation is based on the conclusions from EFSA on the safety of a proposed amendment of the specifications of the food additive steviol glycosides (E 960) concerning rebaudioside M produced by enzyme modification of steviol glycosides, using UDP-glucosyltransferase and sucrose synthase enzymes produced by the genetically modified yeasts K. phaffii UGT-a and K. phaffii UGT-b (EFSA FAF Panel, 2019). Regulation (EU) No 231/2012 was also amended accordingly, with the inclusion of a new entry for 'E 960c(i) Rebaudioside M produced via enzyme modification of steviol glycosides from Stevia'. Assessment of a new application requesting an amendment of the specifications of steviol glycosides in order to include the 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, was completed by the FAF Panel (EFSA FAF Panel, 2021). The Joint FAO/WHO Expert Committee on Food Additives (JECFA) established an ADI for steviol glycosides of 0–4 mg/kg bw per day, expressed as steviol (JECFA, 2009). In 2016, JECFA confirmed that rebaudioside A from multiple gene donors66 According to JECFA specifications: 'Rebaudioside A is obtained from the fermentation of a non-toxigenic non-pathogenic strain of Yarrowia lipolytica that is genetically modified with heterologous genes from multiple donor organisms to [over]express steviol glycosides'. expressed in Yarrowia lipolytica is included in the ADI of 0–4 mg/kg bw, expressed as steviol. 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 issued new 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 (FAO and WHO, 2020). The framework adopted in 2019 has been subsequently amended by JECFA at its 91st meeting in February 2021. Specifications for steviol glycosides manufacturing using four different methods have been established, including specifications for 'Enzyme modified glucosylated Steviol Glycosides' (FAO and WHO, 2021). 2 Data and methodologies 2.1 Data The present evaluation is based on the data submitted in the application dossier ('Documentation provided to EFSA' No 1) and additional information submitted by the applicant during the assessment process either spontaneously (Documentation provided to EFSA No 2) or following request by EFSA ('Documentation provided to EFSA' No 3-5). A clarification meeting during risk assessment was held on 06 December 2019. 2.2 Methodologies This opinion was formulated following the principles described in the EFSA Guidance 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 Guidance for submission for food additive evaluations (EFSA ANS Panel, 2012) was followed for the assessment. 3 Assessment 3.1 Technical data 3.1.1 Identity of the proposed food additive Glucosylated steviol glycoside preparations were described by the applicant as being comprised of a mixture of glucosylated steviol glycosides, containing 1–20 additional glucose units bound to the parent steviol glycoside. The purified steviol glycosides (≥ 95%), extracted from the leaves of the Stevia rebaudiana Bertoni plant, are enzymatically modified such that additional glucose moieties are conjugated to the parent steviol glycosides structure via α-(1–4) linkages. Glucosylated steviol glycoside preparations consist of not less than 95% total steviol glycosides, made up of any combination of glucosylated steviol glycosides of different molecular weights as well as any remaining unreacted steviol glycosides (parent steviol glycosides). According to the applicant, the percentage of steviol glycosides containing additional molecules of glucose is between 80% and 92% w/w and the parent glycosides between 5% and 15% w/w (Documentation provided to EFSA n. 1, 4). According to the applicant, all constituents of glucosylated steviol glycosides share the same steviol backbone structure. As an example, the representative chemical structures of a glucosylated steviol glycoside are depicted in Figure 1. Figure 1Open in figure viewerPowerPoint Representative structures of α-monoglucosylated rebaudioside A According to the applicant, glucosylated steviol glycoside preparations are white to off-white powders that have a clean taste with a mild odour. According to the applicant, glucosylated steviol glycosides are soluble in water as determined by the standard JECFA method (JECFA, 2006). Information on the composition of three preparations depending on the composition of the starting steviol glycosides was included in the application dossier: from steviol glycoside starting material of ≥ 95% rebaudioside A (giving product GSG-RA); from steviol glycoside starting material of ≥ 95% rebaudioside D (giving product GSG-RD); and from a mixed steviol glycoside starting material of ≥ 95% total steviol glycosides (giving product GSG-SG95). The applicant stated that the parent steviol glycoside and glucosylated steviol glycoside content was characterised in accordance with the Japanese Ministry of Health, Labour and Welfare (MHLW) assay for α-glucosyltransferase-treated stevia (MHLW, 2009) and the JECFA method for measuring steviol glycosides (JECFA, 2008). Description of an adsorption chromatography and HPLC method to determine the concentration of steviol glycosides, residual dextrin and unreacted steviol glycosides was provided (Documentation provided to EFSA n. 1). Adsorption chromatography is used for separating and quantifying the dextrin and steviol glycoside fractions. The adsorption chromatographic method also helps in estimating the total amount of steviol glycosides (as the percentage of glucosylated steviol glycoside components and residual parent steviol glycosides). HPLC analysis is used for the determination of total parent steviol glycosides (by summation of the different steviol glucoside components). The total amount of glucosylated steviol glycosides is calculated by the difference between the total amount of steviol glycosides, estimated by adsorption chromatography and the total parent steviol glycosides calculated by HPLC. The Panel noted that this is the method indicated in the current JECFA specifications (FAO and WHO, 2021) to calculate total glucosylated steviol glycosides and unreacted parent steviol glycosides. The total glucosylated steviol glycosides amount may also be quantified using a method that employs a two-stage process involving the analysis of parent steviol glycosides and glucose content separately (MHLW, 2009). This is achieved by treating the glucosylated steviol glycoside sample with glucoamylase to cleave the added α-glucosyl residues from the parent steviol glycosides. The α-glucosyl residues liberated are quantified through the determination of glucose using d-glucose as standard and the steviol glycosides content is also measured. The total content of glucosylated and unreacted steviol glycosides is obtained as the sum of the content of steviol glucosides and α-glucosyl residues. According to the applicant, the approach including adsorption chromatography is preferred because it estimates the total steviol equivalency of a glucosylated steviol glycoside preparation which is needed to estimate the use of glucosylated steviol glycosides in different food and beverage applications, where the additive level is defined/expressed as steviol equivalents. The applicant provided a description of how steviol equivalence is calculated based on the compositional analysis of the glucosylated mixture (by the absorption chromatography/HPLC method) along with a correction for the molecular weight of the glucosylated steviol glycoside components in comparison with the MW of steviol itself (mw = 318). An example was provided for this compositional analysis and calculation, for a glucosylated mixture named 'GSG-RA'. This was made from a starting material of ≥ 95% rebaudioside A, showing that the average degree of glucosylation of this GSG-RA was 2.95 additional glucose units. The steviol equivalency for this GSG-RA mixture was reported as 0.222, meaning that 4.50 g of the GSG-RA mixture was
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