GB 5009.276-2016National food safety standard - Determination of Glucono Delta-Lactone in Foods (English PDF)

3.1 Reagents

3.1.1 Absolute ethanol (C2H6O).

6-gluconate gluconate

Gluconate adenosine triphosphate

6-phosphogluconate ribulose 5-phosphate adenosine diphosphate 6-phosphate gluconate Gluconokinase

4.2 Analytical balance: the sensitivity is 1 mg and 0.1 mg.

4.3 Homogenizer.

5.1 Sample preparation

5.1.1 Solid samples such as meat, meat products and soy products

Take no less than 200 g of representative samples; then use homogenizer to make them into homogenate. Analyze the finished sample as soon as possible, or seal and freeze the sample if it is not analyzed immediately. The stored sample should be remixed when activated.

5.1.2 Liquid samples such as beverage

Shake up and take samples directly.

5.2 Extraction

Meat products: weigh 2 g ~ 10 g of sample (accurate to 0.001g) into a 50 mL beaker;

add 10 mL of petroleum ether to wash; use glass rod to stir evenly; let stand for 15 min; then use dry filter paper to filter; repeat washing three times. Then use 10 mL of absolute ethanol to wash for three times. Then, transfer the residue on the filter paper to the original small beaker; add 30 mL of water to boil. After cooling, use potassium hydroxide solution (2 mol/L) to adjust the solution pH to 10; use water to fix-volume to a 100 mL volumetric flask. Filter; put the filtrate as standby for the machine.

Soy products: weigh 2 g ~ 10 g of sample (accurate to 0.001 g) into a 50 mL beaker;

add 30 mL of water to extract; after boiling and cooling, use potassium hydroxide solution (2 mol/L) to adjust the solution pH to 10.0; use water to fix-volume to a 100 mL volumetric flask. Filter; put the filtrate as standby for the machine.

Beverage: filter, accurately draw 30 mL of filtrate; use potassium hydroxide solution (2 mol/L) to adjust solution pH to 10; use water to fix-volume to a 100 mL volumetric flask;

put as standby for the machine.

5.3 Determination

Take two 1 cm cuvettes; one of them is test cuvette and the other one is blank cuvette.

Add 2.50 mL of buffer (3.2.2), 0.1 mL of NADP solution (10 mg/mL) and 0.1 mL of ATP solution (50 mg/mL) respectively and orderly; cover and mix; add 0.2 mL of extract to the test cuvette and 0.2 mL of water to the blank cuvette respectively; then cover and mix. Then add 0.05 mL of 6-PGDH solution (2.0 mg/mL); cover and mix; then put aside for 5 min; with reference to the blank, measure the absorbance at the wavelength of m -- sample quality; the unit is g.

The calculation result shall keep three significant figures.

10.1 Reagents

10.1.1 Monopotassium phosphate (KH2PO4).

10.1.2 Phosphoric acid (H3PO4).

10.2 Reagent preparation

Monopotassium phosphate-phosphoric acid buffer: weigh 1.36 g of monopotassium phosphate and 0.5 mL of phosphoric acid; use water to dissolve them; then fix-volume

12.3 Apparatus reference conditions

12.3.1 Chromatographic column: organic acid column with a column length of 4.6 mm, an inner diameter of 250 mm and a film thickness of 5 µm, or other silica gel bonding column with equivalent separation effect.

12.3.2 Column temperature: 30°C.

12.3.3 Differential detector detection cell temperature: 35°C.

12.3.4 Mobile phase: monopotassium phosphate-phosphoric acid buffer.

12.3.5 Flow velocity: 0.8 mL/min.

12.3.6 Injection volume: 1 µL.

12.4 Preparation of the standard curve

Respectively inject standard series working solution into the high performance liquid chromatography to test the corresponding peak area of gluconic acid; use the concentration of standard working solution as the vertical ordinate, the peak area as the abscissa; draw the standard curve. See Figure A.1 for the chromatogram of glucono delta-lactone standard.

12.5 Test of sample solution

Inject the sample solution into the high performance liquid chromatography to obtain the peak area of gluconic acid; compare with the peak area from glucono delta-lactone standard-hydrolysis into gluconic acid; obtain the content of glucono delta-lactone in the to-be-test solution according to the standard curve.