ANNEX IISupplementary provisions
ANNEX II In the Annex to Regulation (EU) No 231/2012 the following entry is inserted after the entry for food additive E 960a: ‘ E 960B(I) REBAUDIOSIDE M FROM FERMENTATION PRODUCED BY YARROWIA LIPOLYTICA Synonyms Definition Rebaudioside M from fermentation produced by Yarrowia lipolytica consist of a mixture of steviol glycosides composed of rebaudioside M as the main componant, with some rebaudioside D, and smaller amounts of rebaudioside A and rebaudioside B. The manufacturing process comprises two main phases. The first phase involves fermentation of a simple sugar source by a non-toxigenic non-pathogenic strain of Yarrowia lipolytica that has been genetically modified with heterologous genes to overexpress genes which are involved in the synthesis of steviol glycosides to result in the strain VRM (CBS 147477). Removal of biomass by solid-liquid separation and heat treatment is followed by concentration of the steviol glycosides. The second phase involves purification by employing ion exchange chromatography, followed by cristallisation of the steviol glycosides from ethanol, resulting in a final product containing not less than 95 % of rebaudiosides M, D, A and B. Viable cells and DNA of Yarrowia lipolytica VRM shall not be detected in the food additive. Chemical name 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 Rebaudioside B: 13-[(2- O -β–D-glucopyranosyl-3- O -β–D-glucopyranosyl-β-D-glucopyranosyl)oxy]kaur-16-en-18-oic acid 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 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 A C 44 H 70 O 23 0,33 Rebaudioside B C 38 H 60 O 18 0,40 Rebaudioside D C 50 H 80 O 28 0,29 Rebaudioside M C 56 H 90 O 33 0,25 Molecular weight and CAS No Trivial name CAS Number Molecular weight (g/mol) Rebaudioside A 58543-16-1 967,01 Rebaudioside B 58543-17-2 804,88 Rebaudioside D 63279-13-0 1 129,15 Rebaudioside M 1220616-44-3 1 291,30 Assay Not less than 95 % of rebaudioside M, rebaudioside D, rebaudioside A and rebaudioside B, on the dried basis. Particle size Not more than 0,5 % of particles of less than 300 nm (by STEM method) Description White to light yellow powder, approximately between 200 and 350 times sweeter than sucrose (at 5 % sucrose equivalency). Identification Solubility 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 Kaurenoic acid Not more than 0,3 mg/kg (measured by liquid chromatography-mass spectrometry detection) Arsenic Not more than 0,01 mg/kg Lead Not more than 0,01 mg/kg Cadmium Not more than 0,01 mg/kg Mercury Not more than 0,05 mg/kg Microbiological criteria Total (aerobic) plate count Not more than 1 000 CFU/g Yeast and moulds Not more than 100 CFU/g Escherichia coli Negative in 1 g Salmonella spp. Negative in 25 g Residual protein Not more than 20 mg/kg’