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GB 5009.276-2016: Meat and meat products -- Determination of glucono-delta-lactone content
GB 5009.276-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Glucono Delta-Lactone in Foods
ISSUED ON: DECEMBER 23, 2016
IMPLEMENTED ON: JUNE 23, 2017
Issued by: National Health and Family Planning Commission of the PRC;
China Food and Drug Administration.
Table of Contents
Foreword ... 3
1 Application Scope ... 4
2 Principle ... 4
3 Reagents and Materials ... 4
4 Instruments and Apparatuses ... 5
5 Analysis Steps ... 6
6 Description of the Analysis Result ... 7
7 Precision ... 8
8 Others ... 8
9 Principle ... 8
10 Reagents and Materials ... 8
11 Instruments and Apparatuses ... 9
12 Analysis Steps ... 9
13 Description of the Analysis Result ... 10
14 Precision ... 11
15 Others ... 11
Appendix A Chromatogram of Glucono Delta-Lactone Standard Solution ... 12
National Food Safety Standard -
Determination of Glucono Delta-Lactone in Foods
1 Application Scope
This Standard specifies methods for the determination of glucono delta-lactone in
foods.
Method 1 of this standard is applicable to the determination of glucono delta-lactone
content in meat, meat products, soy products and beverages; method 2 of this
standard is applicable to the determination of glucono delta-lactone content in foods.
Method 1 -- Spectrophotometry
2 Principle
Use boiling water to extract glucono delta-lactone in the sample; filter the supernatant;
under the action of potassium hydroxide, glucono delta-lactone is converted to
gluconate. Under the action of gluconokinase and 6-phosphogluconate
dehydrogenase, gluconate produces ribulose 5-phosphate and reduces nicotinamide
adenine dinucleotide phosphate (NADP). Use a spectrometer to measure the
absorbance of the reduced nicotinamide adenine dinucleotide phosphate; then
calculate the content of glucono delta-lactone.
3 Reagents and Materials
Unless otherwise specified, all the reagents in this method are analytical reagents; the
water is grade-3 water specified by GB/T 6682.
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 Analysis Steps
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.
7 Precision
The absolute difference of two independent test results under repeatability cannot
exceed 10% of the arithmetic mean value.
8 Others
When the sample weight is 10 g, the method detection-limit is 0.007 5%; the
quantitation-limit is 0.025%.
Method 2 -- High Performance Liquid Chromatography
9 Principle
Glucono delta-lactone slowly hydrolyzes in water to form gluconic acid and a small
amount of glucono-γ-lactone and achieves hydrolysis equilibrium. After the sample is
boiled, extracted, fix-volume and filtered, use high performance liquid chromatography
to separate it. By comparing the peak area of gluconic acid in the test solution with the
peak area of glucono delta-lactone standard-hydrolysis into gluconic acid, use external
standard method to fix-volume and calculate the content of glucono delta-lactone in
the sample.
10 Reagents and Materials
Unless otherwise specified, all the reagents in this method are analytical reagents; the
water is grade-1 water specified by GB/T 6682.
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.
13 Description of the Analysis Result
Calculate content of glucono delta-lactone in the sample according to Equation (2):
Where:
X -- content of glucono delta-lactone in the sample, %
ρ -- concentration of glucono delta-lactone in the sample solution; the unit is mg/mL.
V -- fix-volume of the extract; the unit is mL;...