The Food Additives and Novel Foods (Authorisations and Miscellaneous Amendments) and Food Flavourings (Removal of Authorisations) (England) Regulations 2024

Regulation 3(6)

SCHEDULE 2 Insertion of entry in the Annex to Commission Regulation (EU) 231/2012 for E 960c(ii) for Rebaudioside M, AM and D Produced via Enzymatic Conversion of Highly Purified Steviol Glycosides from Stevia Leaf Extracts

“E 960c(ii) REBAUDIOSIDE M, AM AND D PRODUCED VIA ENZYMATIC CONVERSION OF HIGHLY PURIFIED STEVIOL GLYCOSIDES FROM STEVIA LEAF EXTRACTS

Synonyms
Definition	Steviol glycosides produced via enzymatic conversion of highly purified steviol glycosides (rebaudioside A or stevioside) stevia leaf extracts are composed predominantly of rebaudioside M, rebaudioside D, and rebaudioside AM. Rebaudiosides D, M and AM are produced via enzymatic conversion of highly purified steviol glycoside (rebaudioside A or stevioside) extracts (95% steviol glycosides) obtained from Stevia rebaudiana Bertoni plant using UDP-glucosyltransferase and sucrose synthase enzymes produced by genetically modified strains of Escherichia coli (pPM294, pFAH170, and pSK041) 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 steviol glycosides by resin adsorption, followed by recrystallisation of the steviol glycosides resulting in a final product containing not less than 95 % of total steviol glycosides, including one or more of rebaudiosides D, M and AM. Viable cells or DNA of Escherichia coli (pPM294, pFAH170, and pSK041) must 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 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 AM: 13-[(2-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	C56 H90O33	0.25
	Rebaudioside D	C50H80O28	0.29
	Rebaudioside AM	C50H80O28	0.29
Molecular weight and CAS No	Trivial name	CAS Number	Molecular weight (g/mol)
	Rebaudioside M	1220616-44-3	1291.29
	Rebaudioside D	63279-13-0	1129.15
	Rebaudioside AM	2222580-26-7	1129.15
Assay	Not less than 95 % of steviol glycosides on the dried basis, including one or more of rebaudiosides D, M and AM.
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 5000 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”