(2)

the following entries are inserted after the entry for E 960c(i): ‘E 960c(ii) REBAUDIOSIDE M PRODUCED VIA ENZYMATIC CONVERSION OF HIGHLY PURIFIED REBAUDIOSIDE A STEVIA LEAF EXTRACTS Synonyms Definition Rebaudioside M produced via enzymatic conversion of highly purified rebaudioside A stevia leaf extracts is a steviol glycoside composed predominantly of rebaudioside M with minor amounts of other steviol glycosides such as rebaudioside A and rebaudioside D. Rebaudioside M is produced via enzymatic conversion of highly purified steviol glycoside rebaudioside A extracts (95 % steviol glycosides) obtained from Stevia rebaudiana Bertoni plant using UDP-glucosyltransferase and sucrose synthase enzymes produced by the genetically modified strains of E. coli (pPM294, pFAF170 and pSK401) that facilitate the transfer of glucose from sucrose and UDP-glucose to steviol glycosides via glycosidic bonds. After removal of the enzymes by solid-liquid separation and heat treatment, the purification involves concentration of the rebaudioside M by resin adsorption, followed by recrystallisation of the steviol glycosides resulting in a final product containing not less than 95 % of rebaudioside M. Viable cells of E. coli (pPM294, pFAF170 and pSK401) and their DNA shall not be detected in the food additive. Chemical name Rebaudioside M: 13-[(2-O-β-D-glucopyranosyl-3-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 Molecular formula Trivial name Formula Conversion factor Rebaudioside M C56H90O33 0,25 Molecular weight and CAS No Trivial name CAS Number Molecular weight (g/mol) Rebaudioside M 1220616-44-3 1 291,29 Assay Not less than 95 % rebaudioside M on the dried basis. Description White to light yellow powder, approximately between 150 and 350 times sweeter than sucrose (at 5 % sucrose equivalency). Identification Solubility Freely soluble to slightly soluble in water pH Between 4,5 and 7,0 (1 in 100 solution) Purity Total ash Not more than 1 % Loss on drying Not more than 6 % (105 °C, 2h) Residual solvent Not more than 5 000 mg/kg ethanol Arsenic Not more than 0,015 mg/kg Lead Not more than 0,2 mg/kg Cadmium Not more than 0,015 mg/kg Mercury Not more than 0,07 mg/kg Residual protein Not more than 5 mg/kg Particle size Not less than 74 μm [using a mesh #200 sieve with a particle size limit of 74 μm] E 960c(iii) REBAUDIOSIDE D PRODUCED VIA ENZYMATIC CONVERSION OF HIGHLY PURIFIED REBAUDIOSIDE A STEVIA LEAF EXTRACTS Synonyms Definition Rebaudioside D produced via enzymatic conversion of highly purified rebaudioside A stevia leaf extracts is a steviol glycoside composed predominantly of rebaudioside D with minor amounts of other steviol glycosides such as rebaudioside A and rebaudioside M. Rebaudioside D is produced via enzymatic conversion of highly purified steviol glycoside rebaudioside A extracts (95 % steviol glycosides) obtained from Stevia rebaudiana Bertoni plant using UDP-glucosyltransferase and sucrose synthase enzymes produced by the genetically modified strains of E. coli (pPM294, pFAF170 and pSK401) that facilitate the transfer of glucose from sucrose and UDP-glucose to steviol glycosides via glycosidic bonds. After removal of the enzymes by solid-liquid separation and heat treatment, the purification involves concentration of the rebaudioside D by resin adsorption, followed by recrystallisation of the steviol glycosides resulting in a final product containing not less than 95 % of rebaudioside D and rebaudioside A. Viable cells of E. coli (pPM294, pFAF170 and pSK401) and their DNA shall not be detected in the food additive. Chemical name Rebaudioside D: 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]kaur-16-en-18-oic acid, 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester. Rebaudioside A: 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]kaur-16-en-18-oic acid, β-D-glucopyranosyl ester Molecular formula Trivial name Formula Conversion factor Rebaudioside D C50H80O28 0,29 Rebaudioside A C44H70O23 0,33 Molecular weight and CAS No Trivial name CAS Number Molecular weight (g/mol) Rebaudioside D 63279-13-0 1 291,15 Rebaudioside A 58543-16-1 967,01 Assay Not less than 95 % rebaudiosides D and A on the dried basis. Description White to light yellow powder, approximately between 150 and 350 times sweeter than sucrose (at 5 % sucrose equivalency). Identification Solubility Freely soluble to slightly soluble in water pH Between 4,5 and 7,0 (1 in 100 solution) Purity Total ash Not more than 1 % Loss on drying Not more than 6 % (105 °C, 2h) Residual solvent Not more than 5 000 mg/kg ethanol Arsenic Not more than 0,015 mg/kg Lead Not more than 0,2 mg/kg Cadmium Not more than 0,015 mg/kg Mercury Not more than 0,07 mg/kg Residual protein Not more than 5 mg/kg Particle size Not less than 74 μm [using a mesh #200 sieve with a particle size limit of 74 μm] E 960c(iv) REBAUDIOSIDE AM PRODUCED VIA ENZYMATIC CONVERSION OF HIGHLY PURIFIED STEVIOSIDE STEVIA LEAF EXTRACTS Synonyms Definition Rebaudioside AM produced via enzymatic convertion of highly purified stevioside stevia leaf extracts is a steviol glycoside composed predominantly of rebaudioside AM with minor amounts of other steviol glycosides such as stevioside and rebaudioside E. Rebaudioside AM is produced via enzymatic conversion of highly purified steviol glycoside stevioside extracts (95 % steviol glycosides) obtained from Stevia rebaudiana Bertoni plant using UDP-glucosyltransferase and sucrose synthase enzymes produced by the genetically modified strains of E. coli (pPM294, pFAF170 and pSK401) that facilitate the transfer of glucose from sucrose and UDP-glucose to steviol glycosides via glycosidic bonds. After removal of the enzymes by solid-liquid separation and heat treatment, the purification involves concentration of the rebaudioside AM by resin adsorption, followed by recrystallisation of the steviol glycosides resulting in a final product containing not less than 95 % of rebaudioside AM. Viable cells of E. coli (pPM294, pFAF170 and pSK401) and their DNA shall not be detected in the food additive. Chemical name Rebaudioside AM: 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. Molecular formula Trivial name Formula Conversion factor Rebaudioside AM C50 H80 O28 0,29 Molecular weight and CAS No Trivial name CAS Number Molecular weight (g/mol) Rebaudioside AM 2222580-26-7 1 291,15 Assay Not less than 95 % rebaudioside AM on the dried basis. Description White to light yellow powder, approximately between 150 and 350 times sweeter than sucrose (at 5 % sucrose equivalency). Identification Solubility Freely soluble to slightly soluble in water pH Between 4,5 and 7,0 (1 in 100 solution) Purity Total ash Not more than 1 % Loss on drying Not more than 6 % (105 °C, 2h) Residual solvent Not more than 5 000 mg/kg ethanol Arsenic Not more than 0,015 mg/kg Lead Not more than 0,2 mg/kg Cadmium Not more than 0,015 mg/kg Mercury Not more than 0,07 mg/kg Residual protein Not more than 5 mg/kg Particle size Not less than 74 μm [using a mesh #200 sieve with a particle size limit of 74 μm]’