1. In the appropriate place, insert the following entry—
“E 960b STEVIOL GLYCOSIDES FROM FERMENTATION (YARROWIA LIPOLYTICA)
| Synonyms | |||
|---|---|---|---|
| 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”. | ||