ANNEX IISupplementary provisions
ANNEX II The Annex to Regulation (EU) No 231/2012 is amended as follows: (1) In the entry for E 960 Steviol glycosides, the heading is replaced by the following: ‘ E 960a STEVIOL GLYCOSIDES FROM STEVIA ’ (2) The following new entry is inserted after the entry for E 960: ‘ E 960c(i) REBAUDIOSIDE M PRODUCED VIA ENZYME MODIFICATION OF STEVIOL GLYCOSIDES FROM STEVIA Synonyms Definition Rebaudioside M is a steviol glycoside composed predominantly of rebaudioside M with minor amounts of other steviol glycosides such as rebaudioside A, rebaudioside B, rebaudioside D, rebaudioside I, and stevioside. Rebaudioside M is obtained via enzymatic bioconversion of purified steviol glycoside leaf extracts (95% steviol glycosides) of the Stevia rebaudiana Bertoni plant using UDP-glucosyltransferase and sucrose synthase enzymes produced by the genetically modified yeasts K. phaffi (formerly known as Pichia pastoris ) UGT-a and K. phaffi UGT-b 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 rebaudioside M resulting in a final product containing not less than 95 % of rebaudioside M. Viable cells of the yeasts K. phaffii UGT-a and K. phaffii UGT-b or 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 C 56 H 90 O 33 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 200 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] ’