http://www.legislation.gov.uk/id/ssi/2024/156/schedule/2

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
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
C₄₄H₇₀O₂₃
0.33
Rebaudioside B
C₃₈H₆₀O₁₈
0.40
Rebaudioside D
C₅₀H₈₀O₂₈
0.29
Rebaudioside M
C₅₆H₉₀O₃₃
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

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	C₄₄H₇₀O₂₃	0.33
	Rebaudioside B	C₃₈H₆₀O₁₈	0.40
	Rebaudioside D	C₅₀H₈₀O₂₈	0.29
	Rebaudioside M	C₅₆H₉₀O₃₃	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