Gum benzoin

Description and assay

Specification history

Prepared at the 79 th JECFA (2014), published in FAO JECFA Monographs 16 (2014) replacing the tentative specifications prepared at the 74 th JECFA (2011), published in FAO JECFA Monographs 11 (2011). No safety concern at current estimated dietary exposures.

C.A.S. number

9000-72-0

DESCRIPTION

White-yellow to reddish splits of flattened almond-like grains with a strong vanilla smell.

FUNCTIONAL USES

Flavouring agent

Identification

Solubility (Vol. 4)

Insoluble in water; soluble in ethanol

Benzoic acid, vanillin and benzyl benzoate

The sample contains benzoic acid, vanillin and benzyl benzoate identified by their characteristic peaks in the gas-chromatogram. See description under TESTS

Coniferyl benzoate

The sample contains coniferyl benzoate identified by its characteristic peak in the HPLC-chromatogram. See description under TESTS

Cinnamic acid

Not present See description under TESTS

Purity criteria

Loss on drying (Vol. 4)

Not more than 5.0% (105°, 4 h). Test 2 g of sample.

Total ash

Not more than 2.0% Evenly distribute 1.0 g of the powdered sample in a crucible. Dry at 100-105° for 1 h and ignite to constant mass in a muffle furnace at 600 ± 25°.

Alcohol-insoluble matter

Not more than 5%. To 2.0 g of the powdered sample add 25 ml of ethanol 90% v/v. Boil until almost completely dissolved. Filter through a previously tared sintered-glass filter and wash with three 5 ml portions of boiling ethanol 90% v/v. Dry the residue at 100-105° for 2 h and weigh after cooling.

Acid value (Vol. 4)

Between 160 and 206

Benzoic acid

Between 15 and 45% See description under TESTS

Coniferyl benzoate

Between 15 and 60% See description under TESTS

Lead (Vol. 4)

Not more than 2 mg/kg Triturate the sample in a centrifugal grinder to a particle size < 200 μ m. Determine using an AAS (Electrothermal atomization 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 in Volume 4 (under “General Methods, Metallic Impurities”).

Microbiological criteria (Vol. 4)

Salmonella spp.: absent in 25 g Escherichia coli: absent in 1 g Yeast and moulds: less than 20 CFU/g

Benzoic acid, vanillin, benzyl benzoate and cinnamic acid

Benzoic acid, vanillin and benzyl benzoate are identified and benzoic acid is quantified by gas chromatography. The absence of cinnamic acid is confirmed. Reagents Ethanol 96% Reference standard of benzoic acid (purity >99%) Reference standard of vanillin (purity > 99%) Reference standard of benzyl benzoate (purity > 99%) Reference standard of cinnamic acid (purity > 99%) Gas Chromatographic system Detector: Flame ionization detector Column: HP-1 (30 m x 0.2 mm I.D., 0.33 μm-film) or equivalent Carrier gas: helium Flow rate: 1 ml/min Temperatures: -Injector: 250° -Oven: 5 min at 80°; to 300° at 8°/min; then hold for 40 min -Detector: 300° Injection mode: split 1/10 Injection volume: 1 μ L Benzoic acid elutes at 11.7 min. Peaks at 16.1 min, 14.8 min and 20.4 min corresponds to cinnamic acid, vanillin and benzyl benzoate, respectively. Benzoic acid standard stock solution (20 mg/ml): Weigh accurately 0.4 g (±0.1 mg) of benzoic acid reference standard, transfer to a 20 ml volumetric flask and bring to volume with ethanol. Benzoic acid standard solutions: Prepare five solutions from the standard stock solutions within the concentration range of 0.25 to 20 mg/ml. Cinnamic acid, vanillin and benzyl benzoate solution: Prepare a 10 mg/ml solution containing cinnamic acid, vanillin and benzyl benzoate in ethanol. Sample solution Accurately weigh about 5.0 g (±0.1 mg) (ws) of the previously crushed Benzoe tonkinensis and solubilize in 20 ml of ethanol (96%). Sonicate the mixture and filter. Re-extract the residue with a second portion of 20 ml ethanol (96%), sonicate and filter. Combine the ethanol extracts and evaporate the solvent under vacuum. Weigh accurately the extracted resin (wex). Dissolve the resin with ethanol to a final concentration of 30 mg/ml. Procedure Inject 1 μl of each benzoic acid standard solutions and record the peak areas. Inject 1 μl of the 10 mg/ml solution containing cinnamic acid, vanillin and benzyl benzoate. Plot a standard curve (concentration of benzoic acid (mg/ml) (X-axis) vs. peak area of benzoic acid (Y-axis)) and determine the slope (m) and the linear coefficient (a). Inject 1 μl of the sample solution and record the peak area. For identification of cinnamic acid, vanillin and benzyl benzoate, compare the retention times of the corresponding peaks of cinnamic acid, vanillin and benzyl benzoate in the chromatograms obtained with the standard solution and sample solution. Calculation Calculate the content of benzoic acid as follows: ABzA − a 1 ws Benzoic acid ( w w ⁄ , % ) = � � × × × 100 m 30 wex where ABzA is the peak area of benzoic acid in the sample; a is the linear coefficient of the standard curve; m is the slope of the standard curve; wex is the weight of resin extracted with ethanol (g); and ws is the weight of sample Benzoe tonkinensis (g).

Coniferyl benzoate

Coniferyl benzoate is identified and quantified by high performance liquid chromatography. Reagents Acetonitrile, HPLC grade Formic acid Reference standard of coniferyl benzoate (>95%) Chromatographic system HPLC system with a diode array detector (DAD), auto sampler or injector. Detector wavelength for quantitation: 300 nm Column: Luna C18 Phenomenex (150 mm x 4.6 mm, 5 μ m) or equivalent. Mobile phase: solvent A: water added of 0.1% formic acid and solvent B: acetonitrile added of 0.1% formic acid Gradient elution: A:B 65:35 v/v (0 to 5 min) to A:B 0:100 v/v (5 to 25 min) Column temperature: 25° Flow rate: 1 ml/min Injection volume: 10 μ l Coniferyl benzoate elutes at 14.9 min. Standard stock solution (coniferyl benzoate 8 mg/ml): Weigh accurately 0.04 g (±0.1 mg) of coniferyl benzoate reference standard and transfer to a 5 ml volumetric flask and bring to volume with ethanol. Standard solutions: Prepare five solutions, by the dilution of the standard stock solution of coniferyl benzoate with ethanol, in the concentration range of 0.05 to 0.8 mg/ml. Sample preparation Accurately weigh about 5 g (±0.1 mg) (ws) of the previously crushed Benzoe tonkinensis sample and solubilize in 20 ml of ethanol (96%). Sonicate the mixture and filter. Re-extract the residue with a second portion of 20 ml ethanol (96%), sonicate and filter. Combine the ethanol extracts and evaporate the solvent under vacuum. Weigh accurately the extracted resin (wex). Dilute the resin with ethanol to a final concentration (Cresin) of 1 mg/ml. Procedure Inject 10 μl of each standard solution and record the peak areas. Plot a standard curve (concentration of coniferyl benzoate (mg/ml) (X-axis) vs. peak area of coniferyl benzoate (Y-axis)) and determine the slope (m) and the linear coefficient (a). Inject 10 μl of the sample and record the peak area. Calculation Calculate the content of coniferyl benzoate as follows: AConBz − a 1 ws Coniferyl benzoate ( w w ⁄ , % ) = � � × × × 100 m Cresin wex where AConBz is the peak area of coniferyl benzoate in the sample; a is the linear coefficient of the standard curve; m is the slope of the standard curve; Cresin is the final concentration of the extracted resin diluted in ethanol; wex is the weight of extracted resin with ethanol (g); and ws is the weight of sample of Benzoe tonkinensis (g).