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GB 1886.316-2021: National food safety standard - Food additives - Bixin
GB 1886.316-2021
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Food Additives -
BIXIN
ISSUED ON: FEBRUARY 22, 2021
IMPLEMENTED ON: AUGUST 22, 2021
Issued by: National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
1 Scope ... 3
2 Molecular Formula, Structural Formula, and Relative Molecular Mass of Iconic
Components ... 3
3 Classification of Product ... 4
4 Technical Requirements ... 5
Appendix A Inspection Methods ... 7
Appendix B Solvent Residue Chromatogram ... 14
National Food Safety Standard - Food Additives -
BIXIN
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
2.1 Molecular formula
Bixin: C25H30O4
Norbixin: C24H28O4
2.2 Structural formula
Bixin
Norbixin
2.3 Relative molecular mass
Bixin: 394.51 (as per 2018 international relative atomic mass)
Norbixin: 380.48 (by R1=H, R2=H) (as per 2018 international relative atomic mass)
3 Classification of Product
3.1 BIXIN (bixin)
3.1.1 BIXIN (bixin extracted by the solvent)
The product that takes the outer seed coat of annato (Bixa orellana L.) seed as the raw
material, and obtains through solvent extraction and refining (acidification precipitation,
solvent removal, drying and crushing).
3.1.2 Bixin (bixin extracted by the alkaline aqueous solution)
The product that takes the outer seed coat of annato (Bixa orellana L.) seed as the raw
materials, and obtains through being extracted by the cold weak alkaline aqueous
solution, and refining (acidification precipitation, solvent removal, drying and crushing).
3.2 BIXIN (norbixin)
3.2.1 BIXIN (norbixin extracted by solvent)
The product that takes the outer seed coat of annato (Bixa orellana L.) seed as the raw
materials, and obtains through the solvent extraction, refining (solvent removal,
crystallisation and drying), then hydrolysing through heating the alkaline aqueous
solution, and refining (cooling, filtering, drying, and crushing).
3.2.2 BIXIN (norbixin extracted by alkaline aqueous solution-acidification
precipitation-free)
The product that takes the BIXIN (bixin extracted by alkaline aqueous solution) as raw
materials, and obtains through hydrolysing the hot alkaline aqueous solution, and
refining (cooling, filtering, drying, and crushing).
3.2.3 BIXIN (norbixin extracted by alkaline aqueous solution-acidification
precipitation)
The product that takes the BIXIN (bixin extracted by alkaline aqueous solution) as raw
materials, and obtained through hydrolysing the hot alkaline aqueous solution, and
refining (cooling, filtering, acidification precipitation, drying and crushing).
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: 250μm; specification: 5cm×20cm)
at 110°C for 1h. Prepare a specimen solution with a concentration of 5% (solvent is
95% ethanol); spot 10μL; 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 5μm,
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 15μL 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;
50 – conversion factor of mass;
m4 – mass of the internal standard substance in the internal standard solution, in mg;
C - the ratio of the peak area of ...