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 960c(iv): ‘E 960d GLUCOSYLATED STEVIOL GLYCOSIDES Synonyms Definition Mixture of larger glycosides of steviol derived by glucosylation of steviol glycosides extracted from leaves of Stevia rebaudiana Bertoni plant. The mixture is composed of glucosylated steviol glycosides and residual parent steviol glycosides from Stevia leaf. Glucosylated steviol glycosides are produced by treating the steviol glycosides, extracted from Stevia leaf, and starch suitable for human consumption with Cyclomaltodextrin glucanotransferase (EC 2.4.1.19) derived from a non-GMO strain of Anoxybacillus caldiproteolyticus St-88 . The enzyme transfers glucose units from the starch to the steviol glycosides. The resulting material is heated and treated with activated carbon to remove the enzyme, then passed through adsorption/desorption resin to remove residual hydrolysed starch (dextrin), followed by purification and preparation of the final product using processes that may include decolourisation, concentration and spray drying. Chemical name Steviolbioside: 13-[(2- O -β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]kaur-16-en-18-oic acid Rubusoside: 13-β-D-glucopyranosyloxykaur-16-en-18-oic acid, β-D-glucopyranosyl ester Dulcoside A: 13-[(2- O -α–L-rhamnopyranosyl-β–D-glucopyranosyl)oxy]kaur-16-en-18-oic acid, β-D-glucopyranosyl ester Stevioside: 13-[(2- O -β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]kaur-16-en-18-oic acid, 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 Rebaudioside B: 13-[(2- O -β–D-glucopyranosyl-3- O -β–D-glucopyranosyl-β-D-glucopyranosyl)oxy]kaur-16-en-18-oic acid Rebaudioside C: 13-[(2- O -α–L-rhamnopyranosyl-3- O -β–D-glucopyranosyl-β-D-glucopyranosyl)oxy]kaur-16-en-18-oic acid, β-D-glucopyranosyl ester 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 E: 13-[(2- O -β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]kaur-16-en-18-oic acid, 2- O -β-D-glucopyranosyl-β-D-glucopyranosyl ester Rebaudioside F: 13-[(2- O -β-D-xylofurananosyl-3- O -β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]kaur-16-en-18-oic acid, 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 And their glucosylated derivatives (1-20 added glucose units) Molecular formula Trivial name Formula Conversion factor n-Glucosylated Steviolbioside C (32+n*6) H (50+n*10) O (13+n*5) n-Glucosylated Rubusoside C (32+n*6) H (50+n*10) O (13+n*5) n-Glucosylated Dulcoside A C (38+n*6) H (60+n*10) O (17+n*5) n-Glucosylated Stevioside C (38+n*6) H (60+n*10) O (18+n*5) n-Glucosylated Rebaudioside A C (44+n*6) H (70+n*10) O (23+n*5) n-Glucosylated Rebaudioside B C (38+n*6) H (60+n*10) O (18+n*5) n-Glucosylated Rebaudioside C C (44+n*6) H (70+n*10) O (22+n*5) n-Glucosylated Rebaudioside D C (50+n*6) H (80+n*10) O (28+n*5) n-Glucosylated Rebaudioside E C (44+n*6) H (70+n*10) O (23+n*5) n-Glucosylated Rebaudioside F C (43+n*6) H (68+n*10) O (22+n*5) n-Glucosylated Rebaudioside M C (56+n*6) H (90+n*10) O (33+n*5) n: number of glucose units enzymatically added to the parent steviol glycoside (n = 1-20) Typical conversion factor for glucosylated steviol glycosides mixtures = 0,20 (on the dried, dextrin-free, basis) Steviol C 20 H 30 O 3 1,00 Steviolbioside C 32 H 50 O 13 0,50 Rubusoside C 32 H 50 O 13 0,50 Dulcoside A C 38 H 60 O 17 0,40 Stevioside C 38 H 60 O 18 0,40 Rebaudioside A C 44 H 70 O 23 0,33 Rebaudioside B C 38 H 60 O 18 0,40 Rebaudioside C C 44 H 70 O 22 0,34 Rebaudioside D C 50 H 80 O 28 0,29 Rebaudioside E C 44 H 70 O 23 0,33 Rebaudioside F C 43 H 68 O 22 0,34 Rebaudioside M C 56 H 90 O 33 0,25 Molecular weight and CAS No Trivial name CAS Number Molecular weight (g/mol) n-Glucosylated Steviolbioside Not available 642,73+n*162,15 n-Glucosylated Rubusoside Not available 642,73+n*162,15 n-Glucosylated Dulcoside A Not available 788,87+n*162,15 n-Glucosylated Stevioside Not available 804,88+n*162,15 n-Glucosylated Rebaudioside A Not available 967,01+n*162,15 n-Glucosylated Rebaudioside B Not available 804,88+n*162,15 n-Glucosylated Rebaudioside C Not available 951,02+n*162,15 n-Glucosylated Rebaudioside D Not available 1129,15+n*162,15 n-Glucosylated Rebaudioside E Not available 967,01+n*162,15 n-Glucosylated Rebaudioside F Not available 936,99+n*162,15 n-Glucosylated Rebaudioside M Not available 1291,30+n*162,15 Steviol 318,46 Steviolbioside 41093-60-1 642,73 Rubusoside 64849-39-4 642,73 Dulcoside A 64432-06-0 788,87 Stevioside 57817-89-7 804,88 Rebaudioside A 58543-16-1 967,01 Rebaudioside B 58543-17-2 804,88 Rebaudioside C 63550-99-2 951,02 Rebaudioside D 63279-13-0 1 129,15 Rebaudioside E 63279-14-1 967,01 Rebaudioside F 438045-89-7 936,99 Rebaudioside M 1220616-44-3 1 291,30 Assay Not less than 95 % of total steviol glycosides, comprised of above mentioned steviol glycosides along with their glucosylated derivatives (1-20 added glucose units), on the dried, dextrin-free, basis. Description White to light yellow powder, approximately between 100 and 200 times sweeter than sucrose (at 5 % sucrose equivalency). Identification Solubility 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, 2 h) Residual solvent Not more than 200 mg/kg methanol Not more than 3 000 mg/kg ethanol Arsenic Not more than 0,015 mg/kg Lead Not more than 0,1 mg/kg Cadmium Not more than 0,1 mg/kg Mercury Not more than 0,1 mg/kg Microbiological criteria Total (aerobic) plate count Not more than 1 000 CFU/g Yeast and moulds Not more than 200 CFU/g E. coli Negative in 1 g Salmonella Negative in 25 g’.