http://www.legislation.gov.uk/id/wsi/2023/343/schedule/2

SCHEDULE 2Amendments to Annex to Regulation (EU) No 231/2012 concerning the specification for E 960a steviol glycosides from Stevia (formerly E 960 steviol glycosides) and for the addition of a specification for E 960c rebaudioside M produced via enzyme modification of steviol glycosides from Stevia

3

In the appropriate place, insert the following entry—

E 960c REBAUDIOSIDE M PRODUCED VIA ENZYME MODIFICATION OF STEVIOL GLYCOSIDES FROM STEVIA
Synonyms
Definition
Rebaudioside M is a steviol glycoside composed predominantly of rebaudioside M with minor amounts of other steviol glycosides such as rebaudioside A, rebaudioside B, rebaudioside D, rebaudioside I, and stevioside.
Rebaudioside M is obtained via enzymatic bioconversion of purified steviol glycoside leaf extracts (95% steviol glycosides) of the Stevia rebaudiana Bertoni plant using UDP-glucosyltransferase and sucrose synthase enzymes produced by the genetically modified yeasts K. phaffi (formerly known as Pichia pastoris) UGT-a and K. phaffi UGT-b 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 rebaudioside M by resin adsorption, followed by recrystallisation of rebaudioside M resulting in a final product containing not less than 95% of rebaudioside M. Viable cells or the DNA of the yeasts K. phaffi UGT-a or K. phaffi UGT-b 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
Molecular formula
Trivial name
Formula
Conversion factor
Rebaudioside M
C₅₆ H₉₀ O₃₃
0.25
Molecular weight and CAS number
Trivial name
CAS Number
Molecular weight (g/mol)
Rebaudioside M
1220616-44-3
1291.29
Assay
Not less than 95% 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)
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
Particle size
Not less than 74 μm (using a mesh #200 sieve with a particle size limit of 74 μm)

Synonyms
Definition	Rebaudioside M is a steviol glycoside composed predominantly of rebaudioside M with minor amounts of other steviol glycosides such as rebaudioside A, rebaudioside B, rebaudioside D, rebaudioside I, and stevioside. Rebaudioside M is obtained via enzymatic bioconversion of purified steviol glycoside leaf extracts (95% steviol glycosides) of the Stevia rebaudiana Bertoni plant using UDP-glucosyltransferase and sucrose synthase enzymes produced by the genetically modified yeasts K. phaffi (formerly known as Pichia pastoris) UGT-a and K. phaffi UGT-b 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 rebaudioside M by resin adsorption, followed by recrystallisation of rebaudioside M resulting in a final product containing not less than 95% of rebaudioside M. Viable cells or the DNA of the yeasts K. phaffi UGT-a or K. phaffi UGT-b 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
Molecular formula	Trivial name	Formula	Conversion factor
	Rebaudioside M	C₅₆ H₉₀ O₃₃	0.25
Molecular weight and CAS number	Trivial name	CAS Number	Molecular weight (g/mol)
	Rebaudioside M	1220616-44-3	1291.29
Assay	Not less than 95% 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)
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
Particle size	Not less than 74 μm (using a mesh #200 sieve with a particle size limit of 74 μm)