Regulation 4(4)
SCHEDULE 2Amendment to the Annex to Commission Regulation (EU) No 231/2012 for the authorisation of steviol glycosides from fermentation (Yarrowia lipolytica) (E 960b)
1. In the appropriate place, insert the following entry—
“E 960b STEVIOL GLYCOSIDES FROM FERMENTATION (YARROWIA LIPOLYTICA)
Synonyms | |||
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Definition | Steviol glycosides from Yarrowia lipolytica consist of a mixture predominantly composed of rebaudioside M, 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 non-toxigenic non-pathogenic strain of Yarrowia lipolytica VRM that has been genetically modified with heterologous genes to overexpress steviol glycosides. 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 recrystallisation of the steviol glycosides resulting in a final product containing not less than 95% of rebaudiosides M, D, A, and B. Viable cells or the DNA of Yarrowia lipolytica VRM must 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 | C44 H70 O23 | 0.33 | |
Rebaudioside B | C38 H60 O18 | 0.40 | |
Rebaudioside D | C50 H80 O28 | 0.29 | |
Rebaudioside M | C56 H90 O33 | 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 | 1129.15 | |
Rebaudioside M | 1220616-44-3 | 1291.29 | |
Assay | Not less than 95 % of rebaudioside A, rebaudioside B, rebaudioside D and 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) | ||
hPurity | |||
Total ash | Not more than 1 % | ||
Loss on drying | Not more than 6 % (105°C, 2h) | ||
Residual solvent | Not more than 5000 mg/kg ethanol | ||
Arsenic | Not more than 0.1 mg/kg | ||
Lead | Not more than 0.1 mg/kg | ||
Cadmium | Not more than 0.01 mg/kg | ||
Mercury | Not more than 0.05 mg/kg | ||
Residual protein | Not more than 20 mg/kg | ||
Microbiological criteria | |||
Total (aerobic) plate count | Not more than 1000 CFU/g | ||
Yeast | Not more than 100 CFU/g | ||
Moulds | Not more than 100 CFU/g | ||
Escherichia coli | Negative in 1g | ||
Salmonella spp. | Negative in 25g”. |