beta-CAROTENE FROM BLAKESLEA TRISPORA

New specifications prepared at the 57th JECFA (2001) and published in FNP 52 Add 9 (2001). A group ADI with synthetic ß-carotene of was established at the 57^th JECFA (2001).

Information required on method of analysis for residual solvents (ethyl acetate and isobutyl acetate).

SYNONYMS

CI Food Orange 5; INS No. 160a

DEFINITION

Obtained by a fermentation process using the two sexual mating types (+) and (-) of the fungus Blakeslea trispora. The colour is isolated from the biomass by solvent extraction and crystallised. The colouring principle consists predominantly of trans β-carotene together with variable amounts of cis isomers of β-carotene. Minor amounts of other carotenoids of which γ-carotene accounts for the major part may also be present. The only organic solvents used in the extraction and purification are ethanol, isopropanol, ethyl acetate and isobutyl acetate.

The main articles of commerce are suspensions in food grade vegetable/plant oil and water dispersible powders. This is for ease of the use and to improve stability as carotenes easily oxidise.

Carotenoid

Chemical names

β-carotene, β,β-carotene

C.A.S. number

7235-40-7

Chemical formula

C₄₀H₅₆

Structural formula

Formula weight

536.88

Assay

Not less than 96.0% of total colouring matter (expressed as β-carotene)

DESCRIPTION

Red to brownish-red cyrstals or crystalline powder

FUNCTIONAL USES

Colour

CHARACTERISTICS

IDENTIFICATION

Solubility (FNP 5)

Insoluble in water; practically insoluble in ethanol, slightly soluble in vegetable oil.

Spectrometry

Determine the absorbance of the diluted sample solution used in the Method of Assay at 455 nm and 483 nm. The ratio is between 1.14 and 1.19.

Spectrometry

Determine the absorbance of the diluted sample solution used in the Method of Assay at 455 nm and 340 nm. The ratio is not lower than 0.75.

Test for carotenoid

The colour of a solution of the sample in acetone disappears after successive addition of a 5% solution of sodium nitrite and 0.5 M of sulfuric acid.

PURITY

Sulfated ash (FNP 5)

Not more than 0.2%

Carotenoids other than β-carotene

Not more than 3.0% of total colouring matters

See description under TESTS

Residual solvent (FNP 5)

Ethanol:

}

Not more than 0.8% singly or in combination

Ethyl acetate:

Isopropanol:

Not more than 0.1%

Isobutyl acetate:

Not more than 1.0%

Lead

Not more than 2 mg/kg

Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described on FNP 5, "Instrumental methods".

TESTS

PURITY TESTS

Carotenoids other than

β-carotene

Determine by liquid chromatography (FNP 5) using the following the following procedure:

Apparatus:

HPLC system equipped with

- UV detector (445 nm)

- Column heater (30°)

- Refrigerated autosampler (0-10°)

- Column: 250 mm x 4.6 mm, Vydac 218 TP54, 5 μm, or equivalent

- Solvent system: 99% methanol and 1% tetrahydrofuran containing 50 mg/l of L-ascorbic acid.

Working conditions:

- Flow rate: 0.6 ml/min

- Injection 10 μl

- Run time: approximately 25 min

Sample preparation:

Results:

γ-carotene as a % of total β-carotene equals:

where

A₁ is the area of the ã-carotene peak

A₂ is the area of the all-trans β-carotene peak

A₃ is the combined area of the peaks from the isomers of all trans β-carotene

METHOD OF ASSAY

Proceed as directed in Colouring matters, Total Content by Spectrophotometry (FNP 5), procedure 2 using the following conditions:

W = 0.08 g

V₁ = V₂ = V₃ = 100 ml

v₁ = v₂ = 5 ml

A^1%_{1 cm} = 2500

A_max = about 455 nm