GB 1886.316-2021National food safety standard - Food additives - Bixin (English PDF)

1 Scope

This Standard is applicable to the food additive BIXIN (bixin) obtained by taking the outer seed coat of annato (Bixa orellana L.) seeds as raw materials, and through extracting a solvent or cold weak alkaline aqueous solution and refining; as well as the food additive BIXIN (norbixin) obtained through hydrolysis of the hot alkaline aqueous solution and refining. The used solvents include acetone, methanol, ethanol, n-hexane, isopropanol, ethyl acetate, basic ethanol and/or carbon dioxide.

2 Molecular Formula, Structural Formula, and Relative

Molecular Mass of Iconic Components

3 Classification of Product

Appendix A Inspection Methods

A.1 General provisions If no other requirements are specified, all the reagents and water used in this Standard refer to the analytical reagents and Class-III water specified in GB/T 6682. If no other requirements are specified, all the standard solution used in the test, standard solution for determining the impurity, preparation and product shall be prepared according to the provisions of GB/T 601, GB/T 602, GB/T 603.The solution used in the test refers to the aqueous solution when the solvent is not specified.

A.2 Identification test A.2.1 Solubility BIXIN (bixin) is insoluble in water and slightly soluble in ethanol. Annatto (norbixin) is soluble in alkaline water and slightly soluble in ethanol.

A.2.2 Maximum absorption wavelength A.2.2.1 BIXIN (bixin)

After BIXIN (bixin) was diluted by acetone, the solution showed three maximum absorption peaks at wavelengths of about 425nm, 457nm and 487nm.

A.2.2.2 BIXIN (Norbixin)

After BIXIN (norbixin) was diluted by potassium hydroxide solution (5g/L), the solution showed two maximum absorption peaks at wavelengths of about 453nm and 482nm.

A.2.3 Thin layer chromatography Activate the TLC plate (thickness of the thin layer: 250um; specification: 5cmx20cm)

at 110°C for 1h. Prepare a specimen solution with a concentration of 5% (solvent is 95% ethanol); spot 10uL; after drying it, expand by a mixture of n-butanol, methyl ethyl ketone, and 10% ammonia (volume ratio 3: 2: 2) until the front of the solution raises about 10cm and dry it. BIXIN (bixin) and BIXIN (Norbixin) respectively showed yellow spots at the Rf value of about 0.45 ~ 0.50. After spraying by 5% sodium nitrite solution and 0.5mol/L sulfuric acid successively, the yellow spots faded immediately.

A.3 Determination of bixin content A.3.1 Reagents and materials A.5.1.3 Internal standard substance: 3-methyl-2-pentanone.

A.5.1.4 Methanol: chromatographically pure.

A.5.1.5 Water: Class-I water specified in GB/T 6682-2008.

A.5.2 Apparatus Gas chromatograph equipped with hydrogen flame ionization detector (FID) and headspace sampler.

A.5.3 Reference chromatographic conditions A.5.3.1 Chromatographic column: Quartz capillary column taking 6% cyanopropyl phenyl and 94% dimethyl polysiloxane (or 100% dimethyl polysiloxane) as a stationary phase, with length 60m, inner diameter of 0.53mm, and the coating thickness of 5um, or a chromatographic column with equivalent performance.

A.5.3.2 Carrier gas: helium.

A.5.3.3 Flow rate: 208kPa, 5mL/min.

A.5.3.4 Column temperature: at 35°C for 5min; then raise to 90°C at a rate of 5°C/min;

keep for 19min.

A.5.3.5 Injection port temperature: 140°C.

A.5.3.6 Detector temperature: 300°C.

A.5.4 Reference headspace conditions A.5.4.1 Sample heating temperature: 60°C.

A.5.4.2 Sample heating time: 10min.

A.5.4.3 Injector temperature: 70°C.

A.5.4.4 Transmission temperature: 80°C.

A.5.4.5 Specimen gas injection: split mode, 1.0mL.

A.5.5 Analysis procedures A.5.5.1 Method-I (determined in water)

A.5.5.1.1 Preparation of internal standard solution Pipette 50mL of water into a 50mL injection bottle; seal it; accurately weigh, accurate to 0.0001g. Pipette 15uL of 3-methyl-2-pentanone; inject it into the injection bottle Heat at 60°C for 10min; shake vigorously for 10s.

A.5.5.2.4 Preparation of specimen solution Accurately weigh 0.20g of specimen into the injection bottle; then add 5.0mL of methanol and 1.0mL of internal standard solution; heat it at 60°C for 10min; and shake vigorously for 10s.

A.5.6 Determination Under reference operating conditions in A.5.3 and A.5.4, chromatographically analyse the sample solution, blank solution, and calibration solution. Refer to Figure B.1 in

Appendix B for the solvent residue chromatogram.

A.5.7 Calculation of results A.5.7.1 Calibration factor fi The calibration factor fi of Method-I is calculated according to Formula (A.3).

Where:

m3 - mass of standard product of component to be tested in the standard solution, in mg;

m4 - mass of the internal standard substance in the internal standard solution, in mg;

C - the ratio of the peak area of the component to be tested to the peak area of the internal standard substance in the standard solution chromatogram;

D - the ratio of the peak area of the component to be tested to the peak area of the internal standard substance in the blank solution chromatogram.

The calibration factor fi in Method-II is calculated according to Formula (A.4).

Where:

m3 - mass of standard product of component to be tested in the standard solution, in mg;