Commission Regulation (EU) 2025/652 of 2 April 2025 amending Regulation (EC) No 1333/2008 of the European Parliament and the Council and Commission Regulation (EU) No 231/2012 as regards the use of Steviol glycosides produced by fermentation using Yarrowia lipolytica

• CELEX: Regulation (EU) 2025/652
• Articles: 5

Article 1

Annex II to Regulation (EC) No 1333/2008 is amended in accordance with Annex I to this Regulation.

Article 2

The Annex to Regulation (EU) No 231/2012 is amended in accordance with Annex II to this Regulation.

Article 3

This Regulation shall enter into force on the twentieth day following that of its publication in the Official Journal of the European Union .

Schedules & Appendices

ANNEX I

ANNEX I

Annex II to Regulation (EC) No 1333/2008 is amended as follows:

(a)

in point 2 (Sweeteners) of Part B, the following entry is inserted after the entry for E 960a:

‘E 960b

Steviol glycosides from fermentation’

(b)

in point (5) (Other additives that may be regulated combined) of Part C, point (v) is replaced by the following:

‘(v)

E 960a – 960d: Steviol glycosides

E-number

Name

E 960a

Steviol glycosides from stevia

E 960b

Steviol glycosides from fermentation

E 960c

Enzymatically produced steviol glycosides

E 960d

Glucosylated steviol glycosides’

ANNEX II

ANNEX II

In the Annex to Regulation (EU) No 231/2012 the following entry is inserted after the entry for food additive E 960a:

‘ E 960B(I)     REBAUDIOSIDE M FROM FERMENTATION PRODUCED BY YARROWIA LIPOLYTICA

Synonyms

Definition

Rebaudioside M from fermentation produced by Yarrowia lipolytica consist of a mixture of steviol glycosides composed of rebaudioside M as the main componant, 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 simple sugar source by a non-toxigenic non-pathogenic strain of Yarrowia lipolytica that has been genetically modified with heterologous genes to overexpress genes which are involved in the synthesis of steviol glycosides to result in the strain VRM (CBS 147477). 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 cristallisation of the steviol glycosides from ethanol, resulting in a final product containing not less than 95 % of rebaudiosides M, D, A and B. Viable cells and DNA of Yarrowia lipolytica VRM shall 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

C 44 H 70 O 23

0,33

Rebaudioside B

C 38 H 60 O 18

0,40

Rebaudioside D

C 50 H 80 O 28

0,29

Rebaudioside M

C 56 H 90 O 33

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

1 129,15

Rebaudioside M

1220616-44-3

1 291,30

Assay

Not less than 95 % of rebaudioside M, rebaudioside D, rebaudioside A and rebaudioside B, on the dried basis.

Particle size

Not more than 0,5 % of particles of less than 300 nm (by STEM method)

Description

White to light yellow powder, approximately between 200 and 350 times sweeter than sucrose (at 5 % sucrose equivalency).

Identification

Solubility

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 5 000  mg/kg ethanol

Kaurenoic acid

Not more than 0,3 mg/kg (measured by liquid chromatography-mass spectrometry detection)

Arsenic

Not more than 0,01 mg/kg

Lead

Not more than 0,01 mg/kg

Cadmium

Not more than 0,01 mg/kg

Mercury

Not more than 0,05 mg/kg

Microbiological criteria

Total (aerobic) plate count

Not more than 1 000 CFU/g

Yeast and moulds

Not more than 100 CFU/g

Escherichia coli

Negative in 1 g

Salmonella spp.

Negative in 25 g

Residual protein

Not more than 20 mg/kg’