GB 5009.303-2025 PDF English
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National food safety standard - Determination of Yeast β -glucan in Food
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GB 5009.303-2025: National food safety standard - Determination of Yeast β -glucan in Food---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GB5009.303-2025
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard - Determination of yeast β-
glucan in food
Issued on: MARCH 16, 2025
Implemented on: SEPTEMBER 16, 2025
Issued by. National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
1 Scope... 3
2 Principle... 3
3 Reagents and materials... 3
4 Instruments and equipment... 6
5 Preparation of samples... 6
6 Analysis steps... 7
7 Expression of analysis results... 9
8 Precision... 11
9 Others... 11
National food safety standard - Determination of yeast β-
glucan in food
1 Scope
This Standard specifies the method for the determination of insoluble yeast β-glucan in
food.
This Standard applies to the determination of insoluble yeast β-glucan in milk and dairy
products, protein powder, candy and beverages.
2 Principle
After the sample is freed of fat and protein by protease and/or lipase, yeast β-glucan is
precipitated by centrifugation. The precipitated yeast β-glucan is hydrolyzed into
glucose under the action of multiple enzymes. Glucose oxidase (GOD) is used to
catalyze the oxidation of glucose under aerobic conditions to generate D-glucuronic
acid-d-lactone and hydrogen peroxide, hydrogen peroxide is catalyzed by peroxidase
(POD) to react with 4-aminoantipyrine and p-hydroxybenzoic acid to generate red
quinone imine (GODPOD method). The absorbance of quinone imine is measured at a
wavelength of 510 nm using a spectrophotometer. Finally, the content of yeast β-glucan
in the sample is calculated by the conversion coefficient of yeast β-glucan and glucose
(0.9).
3 Reagents and materials
Unless otherwise specified, all reagents used in this method are analytically pure, and
the water is grade 3 water specified in GB/T 6682.
3.1 Reagents
3.1.1 Alkaline protease, liquid, enzyme activity ≥ 240000 U/mL.
3.1.2 Neutral protease, liquid, enzyme activity ≥ 80000 U/mL.
3.1.3 Acidic protease, liquid, enzyme activity ≥ 50000 U/mL.
3.1.4 Lipase, liquid, enzyme activity ≥ 20000 U/mL.
3.1.5 Tetrasodium ethylenediaminetetraacetic acid dihydrate (C10H12N2Na4O8 • 2H2O).
3.1.6 Tris(hydroxymethyl)aminomethane (tris) (C4H11NO3).
3.2.5 Sodium acetate buffer solution A (0.2 mol/L). respectively weigh 5.25 g of
anhydrous sodium acetate and 2.16 g of glacial acetic acid into 400 mL of water, adjust
the pH to 5.0 ± 0.05 with sodium hydroxide solution or glacial acetic acid, add water to
make up to 500 mL, and mix well.
3.2.6 Sodium acetate buffer solution B (1.2 mol/L). respectively weigh 4.96 g of
anhydrous sodium acetate and 32.32 g of glacial acetic acid into 400 mL of water, adjust
the pH to 3.8 ± 0.05 with sodium hydroxide solution or glacial acetic acid, add water to
make up to 500 mL, and mix well.
3.2.7 Buffer solution C (pH 7.5). dissolve 1.212 g of tris(hydroxymethyl)aminomethane,
1.169 g of sodium chloride, and 0.416 g of tetrasodium ethylenediaminetetraacetic acid
dihydrate in 90 mL of water. Adjust the pH to 7.5 ± 0.05 with hydrochloric acid solution
or sodium hydroxide solution, add water to make up to 100 mL, and mix well. Before
use, transfer an appropriate amount and dilute 10 times with water. Prepare before use.
3.2.8 Lytic enzyme solution (5 U/μL). weigh an appropriate amount of lytic enzyme
into buffer solution C to make the final concentration of lytic enzyme 5 U/μL. Prepare
before use.
3.2.9 β-(1,6)-glucanase solution. dissolve an appropriate amount of β-(1,6)-glucanase
in sodium acetate buffer solution A to make a suspension with the final concentration
of 1 U/300 μL. Prepare before use.
3.2.10 Mixed enzyme solution. pipette an appropriate amount of β-(1,3)-glucanase and
β-glucosidase mixed enzyme, add an appropriate amount of sodium acetate buffer
solution A to dilute and mix well, so that the final concentrations are 20 U/mL and 4
U/mL respectively. Prepare before use. It shall be stored in an ice water bath during use.
3.2.11 Glucose oxidase-peroxidase (GODPOD) buffer. add about 160 mL of water to a
200 mL volumetric flask, then add 27.2 g of potassium dihydrogen phosphate, 8.4 g of
sodium hydroxide, and 6.0 g of p-hydroxybenzoic acid, stir to dissolve completely, and
adjust the pH to 7.4 ± 0.05.Finally, add 0.8 g of sodium azide, add water to make up to
the mark after dissolution, and prepare before use. It shall be diluted before use.
Measure 48 mL of GODPOD buffer into a 1000 mL volumetric flask, add water to make
up to the mark, and mix thoroughly. Prepare before use.
3.2.12 GODPOD working solution. weigh 400 U of glucose oxidase, 1000 U
peroxidase, and 31.3 mg of 4-aminoantipyrine into a 1000 mL volumetric flask, add 20
mL diluted GODPOD buffer, shake gently to fully dissolve, then make up to the mark
with GODPOD buffer, mix well and store at 4 °C in the dark. Prepare before use.
NOTE. 3.2.8 ~ 3.2.12 can also use commercial reagents or kits.
3.3 Standards
NOTE. Samples that cannot be crushed, such as soft candy, shall be cut into pieces as much as
possible.
6 Analysis steps
6.1 Preparation of matrix control sample
Weigh 3 mg ~ 10 mg of yeast β-glucan reference substance (accurate to 0.0001 g, and
the deviation from the yeast β-glucan content in the sample to be tested is not more than
20 %) in a conical centrifuge tube, add 10.0 g (accurate to 0.01 g) of the corresponding
blank sample, and mix thoroughly.
6.2 Precipitation
6.2.1 Liquid sample
Respectively weigh 10.0 g (accurate to 0.01 g, or a sample mass equivalent to 3 mg ~
10 mg yeast β-glucan) of the mixed liquid sample and the corresponding matrix control
sample in 15 mL conical centrifuge tubes, each add 200 μL of protease mixed solution
(add 200 μL of acidic protease for acidic samples such as yogurt), and incubate at 40 ℃
for 2 h and cool to room temperature. After standing for 10 min, centrifuge at 8000
r/min for 5 min, slowly remove the upper fat and intermediate enzymatic hydrolysate
with a dropper, and retain the precipitate.
6.2.2 Solid sample
Respectively weigh 10.0 g (accurate to 0.01 g, or a sample mass equivalent to 3 mg ~
10 mg of yeast β-glucan) of the solid sample treated in 5.1 and the corresponding matrix
control sample in 50 mL centrifuge tubes, and each add 20 mL of water to fully dissolve.
Add 500 μL of protease mixed solution and mix well. Incubate at 40 ℃ for 2 h and then
cool to room temperature. After standing for 10 min, centrifuge at 8000 r/min for 5 min,
slowly remove the upper fat and the intermediate enzymatic hydrolysate with a dropper,
and retain the precipitate.
NOTE. For samples with no yeast β-glucan precipitation after centrifugation or samples with a fat
content of ≥ 10 %, add 500 μL of lipase after protease incubation for 2 h, and incubate at 40 ℃ for
2 h. Or weigh 10.0 g of sample, refer to 6.1.3 of GB 5009.88-2023 to remove excess fat in the
sample, and then use protease to hydrolyze the protein.
6.3 Precipitation cleaning
Add 5 mL of water to the precipitate treated in 6.2, fully disperse it, let stand for 10 min,
centrifuge at 8000 r/min for 5 min, and slowly remove the supernatant with a dropper.
The precipitation cleaning needs to be repeated more than 3 times until no glucose is
detected in the supernatant according to the method in 6.4.The bottom precipitate can
be subjected to the next step of enzymatic hydrolysis.
6.4 Determination of supernatant
Take an appropriate amount of the supernatant treated in step 6.3 and filter it with a
0.45 μm filter membrane, transfer 100 μL to a 5 mL centrifuge tube, each add 3 mL of
GODPOD working solution, and place in a 40 ℃ water bath for 20 min. Remove from
the water bath, cool to room temperature, transfer to a 1 cm cuvette, use the GODPOD
working solution as blank, measure the absorbance at 510 nm with a spectrophotometer,
and record. Calculate the concentration of glucose in the sample solution using the
standard curve.
6.5 Enzymatic hydrolysis
6.5.1 Sample and matrix control sample
Add 400 μL of cold potassium hydroxide solution to the sample and matrix control
sample treated in 6.3, place in an ice-water bath for 20 min. During the ice-water bath,
vortex briefly several times until all the precipitates are dispersed and no lumps are
visible, add 1.6 mL of sodium acetate buffer solution B, then add 500 μL of lytic enzyme
solution, and record the total volumes as V1 and V'1 at this moment. Incubate the reaction
solutions in a 50 ℃ constant temperature water bath for 12 h ~ 18 h, and then cool to
room temperature. Respectively transfer 150 μL (V2 and V'2) of the enzymatic
hydrolysate to 2 mL centrifuge tubes, add 300 μL of β-(1,6)-glucanase solution,
incubate at 80 ℃ for 15 min, and cool to room temperature. Add 300 μL of mixed
enzyme solution, record the total volumes as V3 and V'3, incubate at 40 ℃ for 1 h, and
then cool to room temperature. Pipette an appropriate amount of solution, filter through
a 0.45 μm filter membrane, and then test.
6.5.2 Enzyme blank solution
Pipette 200 μL of potassium hydroxide solution into a 5 mL centrifuge tube, add 0.8 mL
of sodium acetate buffer solution B, mix well and then pipette 120 μL into a 2 mL
centrifuge tube, add 30 μL of lytic enzyme solution, and mix well. Incubate at 50 ℃ for
12 h ~ 18 h, cool to room temperature. Add 300 μL of β-(1,6)-glucanase solution,
incubate at 80 ℃ for 15 min, and cool to room temperature. Finally, add 300 μL of
mixed enzyme solution, incubate at 40 ℃ for 1 h, and cool to room temperature. Pipette
an appropriate amount of solution, filter through a 0.45 μm filter membrane, and then
test.
6.6 Determination
6.6.1 Plotting of standard curve
Respectively pipette 100 μL of glucose series standard solutions into 5 mL centrifuge
tubes, each add 3 mL of GODPOD working solution, and incubate in a 40 ℃ water bath
for 20 min. Remove from the water bath, cool to room temperature, pipette an
appropriate amount of solution, filter through a 0.45 μm filter membrane, and transfer
to 1 cm cuvettes, use GODPOD working solution as blank, measure the absorbance at
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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