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GB 5009.9-2023 English PDF (GB5009.9-2023)

GB 5009.9-2023 English PDF (GB5009.9-2023)

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GB 5009.9-2023: National food safety standard - Determination of starch in foods
This Standard specifies the methods for the determination of starch in food. Method I and method II of this Standard apply to the determination of starch in food (except meat products); method III applies to the determination of starch in meat products. This Standard does not apply to the determination of starch in foods with added substances that produce reducing sugars upon hydrolysis (except maltodextrin and soluble sugars). Method I: Enzymatic hydrolysis method
GB 5009.9-2023
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
GB 5009.9-2023
National food safety standard - Determination of starch in
foods
ISSUED ON: SEPTEMBER 06, 2023
IMPLEMENTED ON: MARCH 06, 2024
Issued by: National Health Commission of the People’s Republic of China; State Administration for Market Regulation.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Principle ... 4
3 Reagents and materials ... 4
4 Instruments and equipment ... 6
5 Analysis procedure ... 6
6 Expression of analysis results ... 9
7 Precision ... 11
8 Principle ... 12
9 Reagents and materials ... 12
10 Instruments and equipment ... 13
11 Analysis procedure ... 13
12 Expression of analysis results ... 14
13 Precision ... 15
14 Principle ... 15
15 Reagents and materials ... 15
16 Instruments and equipment ... 17
17 Analysis procedure ... 18
18 Expression of analysis results ... 19
19 Precision ... 20
Annex A Conversion relationship between millimoles of sodium thiosulfate and glucose content (m3) ... 21
National food safety standard - Determination of starch in
foods
1 Scope
This Standard specifies the methods for the determination of starch in food. Method I and method II of this Standard apply to the determination of starch in food (except meat products); method III applies to the determination of starch in meat products. This Standard does not apply to the determination of starch in foods with added substances that produce reducing sugars upon hydrolysis (except maltodextrin and soluble sugars).
Method I: Enzymatic hydrolysis method
2 Principle
After the fat and soluble sugars are removed from the sample, the starch is hydrolyzed into glucose by amylase and hydrochloric acid in sequence. Determine the glucose content and convert it into the starch content in the sample.
3 Reagents and materials
Unless otherwise stated, the reagents used in this method are of analytical regent, and the water is Grade 3 water specified in GB/T 6682.
3.1 Reagents
3.1.1 Iodine (I2).
3.1.2 Potassium iodide (KI).
3.1.3 α-amylase: EC 3.2.1.1, enzyme activity ≥ 1.6 U/mg.
3.1.4 Absolute ethanol (C2H5OH) or 95 % ethanol.
3.1.5 Petroleum ether: the boiling range is 60 ℃ ~ 90 ℃.
3.1.6 Diethyl ether (C4H10O).
3.1.7 Toluene (C7H8).
containing maltodextrin, first wash it with 100 mL of ethanol solution (85 %, volume fraction), and then wash it with ethanol solution (40 %, volume fraction) until the microsugar test result is negative. Drain the ethanol solution, transfer the residue into a 250 mL beaker, wash the filter paper with 50 mL of water, put the washing liquid into the beaker, place the beaker in a boiling water bath and heat until complete gelatinization, which usually takes 15 minutes, let it cool to below 60 ℃, add 20 mL of amylase solution, incubate at 55 ℃ ~ 60 ℃ for 1 hour, and stir frequently. Then take 1 drop of this solution and add 1 drop of iodine solution, there shall be no blue color. If it appears blue, reheat to gelatinize and add 20 mL of amylase solution, and continue to incubate until it no longer appears blue after adding the iodine solution. Heat to boiling, cool down and transfer to a 250 mL volumetric flask, wash the beaker with an appropriate amount of water, transfer to the volumetric flask, add water to the mark, mix well, filter, and discard the primary filtrate.
Take 50.00 mL of the filtrate, place it in a 250 mL Erlenmeyer flask, add 5 mL of hydrochloric acid (1 + 1); install a reflux condenser, reflux in a boiling water bath for 1 hour; cool down and add 2 drops of methyl red indicator solution, use sodium hydroxide solution (200 g/L) to neutralize to neutrality; transfer the solution into a 100 mL volumetric flask, wash the Erlenmeyer flask, merge the washing liquid into the 100 mL volumetric flask, add water to the mark, mix well for later use.
5.2 Microsugar test method
Take 2 mL of the washing liquid in a small test tube, add 4 drops of α-naphthol ethanol solution (10 g/L), and slowly add 1 mL of concentrated sulfuric acid along the tube wall. If a purple ring appears at the interface between water and acid, it is judged as positive; if a yellow-green ring appears at the interface of water and acid, it is judged as negative. NOTE: Since the washing liquid contains ethanol and water, when adding concentrated sulfuric acid, it needs to add it slowly along the wall of the test tube and ensure that the mouth of the test tube does not face anyone.
5.3 Determination
5.3.1 Calibration of alkaline copper tartrate solution
Pipette 5.00 mL of alkaline copper tartrate solution A and 5.00 mL of alkaline copper tartrate solution B, place them in a 150 mL Erlenmeyer flask; add 10 mL of water, add 2 glass beads, drop about 9 mL of glucose standard solution from the burette; control it to heat to boil within 2 minutes, keep the solution in a boiling state, continue to add glucose at a rate of 1 drop every 2 seconds, until the blue color of the solution just fades, which it taken as the end point; record the total volume of glucose standard solution consumed. Operate 3 copies in parallel at the same time, and take their average value. Calculate the mass m1 (mg) of glucose, which is equivalent to the alkaline copper tartrate solution (half of solution A and half of solution B) used for calibration, according to formula (1).
NOTE: It can also calibrate 4 mL ~ 20 mL of alkaline copper tartrate solution (half of solution A and half of solution B) according to the above method to adapt to the concentration changes of reducing sugars in the sample. The volume of alkaline copper tartrate solution used for the determination of the sample solution is the same as the volume of alkaline copper tartrate solution used for calibration.
5.3.2 Prediction of sample solution
Pipette 5.00 mL of alkaline copper tartrate solution A and 5.00 mL of alkaline copper tartrate solution B, place them in a 150 mL Erlenmeyer flask; add 10 mL of water, add 2 glass beads; control it to heat to boil within 2 minutes, keep boiling and add the sample solution dropwise from the burette at a fast speed first and then a slow speed, and maintain the solution in a boiling state. When the color of the solution becomes lighter, titrate at a rate of 1 drop per 2 seconds until the blue color of the solution just fades, which it taken as the end point. Record the consumed volume of the sample solution. When the glucose concentration in the sample solution is too high, it shall be appropriately diluted before formal determination, so that the volume of the sample solution consumed in each titration is controlled to be similar to the volume of the glucose standard solution consumed when calibrating the alkaline copper tartrate solution, about 10 mL.
5.3.3 Determination of sample solution
Pipette 5.00 mL of alkaline copper tartrate solution A and 5.00 mL of alkaline copper tartrate solution B, place them in a 150 mL Erlenmeyer flask; add 10 mL of water, add 2 glass beads, and drop the sample solution that is 1 mL less than the predicted volume from the burette into the Erlenmeyer flask; heat it to boil within 2 minutes, maintain the boiling state and continue to titrate at a rate of 1 drop per 2 seconds until the blue color just fades, which is taken as the end point. Record the consumed volume of the sample solution. Operate 3 copies in parallel with the same method, and get the average consumed volume. Calculate the results according to formula (2).
When the concentration of the sample solution is too low and it cannot be titrated to the end point with 25.00 mL, directly add 10.00 mL of sample solution without adding 10 mL of water, and then use the glucose standard solution to titrate to the end point. Calculate the mass of glucose contained in 10 mL of the sample solution, which is equivalent to the difference between the volume consumed and the volume of the glucose standard solution consumed during calibration. Calculate the results according to formula (3) and formula (4).
5.3.4 Determination of reagent blank
At the same time, measure 20.00 mL of water and the same amount of amylase solution as when processing the sample solution, and carry out a reagent blank test according to the back titration method. That is, use the glucose standard solution to titrate the reagent blank solution to the end point, and record the mass of glucose contained in 10 mL of Method II: Acid hydrolysis method
8 Principle
After the fat and soluble sugar are removed from the sample, the starch is hydrolyzed into glucose by hydrochloric acid. The glucose content is measured, and converted into the starch content in the sample.
9 Reagents and materials
Unless otherwise stated, the reagents used in this method are of analytical regent, and the water is Grade 3 water specified in GB/T 6682.
9.1 Reagents
9.1.1 Hydrochloric acid (HCl).
9.1.2 Sodium hydroxide (NaOH).
9.1.3 Lead acetate (PbC4H6O4 · 3H2O).
9.1.4 Sodium sulfate (Na2SO4).
9.1.5 Petroleum ether: the boiling range is 60 ℃ ~ 90 ℃.
9.1.6 Diethyl ether (C4H10O).
9.1.7 Absolute ethanol (C2H5OH) or 95 % ethanol.
9.1.8 Methyl red (C15H15N3O2): indicator.
9.1.9 Precision pH test paper: 6.8 ~ 7.2.
9.1.10 α-naphthol (C10H8O).
9.1.11 Concentrated sulfuric acid (H2SO4).
9.2 Preparation of reagents
9.2.1 Methyl red indicator solution (2 g/L): Weigh 0.20 g of methyl red, dissolve in 95 % ethanol and dilute to 100 mL.
9.2.2 Sodium hydroxide solution (400 g/L): Weigh 40 g of sodium hydroxide, add water to dissolve and dilute to 100 mL.
9.2.3 Lead acetate solution (200 g/L): Weigh 20 g of lead acetate, add water to dissolve and dilute to 100 mL.
9.2.4 Sodium sulfate solution (100 g/L): Weigh 10 g of sodium sulfate, add water to dissolve and dilute to 100 mL.
9.2.5 Hydrochloric acid solution (1 + 1): Measure 50 mL of hydrochloric acid and 50 mL of water and mix them well.
9.2.6 Ethanol solution (85 %, volume fraction): Take 85 ml of absolute ethanol, add water to dilute to 100 mL and mix well. It can also be prepared with 95 % ethanol. 9.2.7 Ethanol solution (40 %, volume fraction): Take 40 ml of absolute ethanol, add water to dilute to 100 mL and mix well. It can also be prepared with 95 % ethanol. 9.2.8 α-naphthol ethanol solution (10 g/L): Weigh 1 g of α-naphthol, dissolve in 95 % ethanol and dilute to 100 mL.
9.3 Reference material
Same as 3.3.
9.4 Preparation of standard solution
Same as 3.4.
10 Instruments and equipment
10.1 40 mesh sieve: the aperture is 0.425 mm.
10.2 Balances: the minimum divisions are 1 mg and 0.1 mg.
10.3 Constant temperature water bath: it can be heated to 100 ℃.
10.4 Reflux device, accompanied with a 250 mL Erlenmeyer flask.
10.5 Tissue masher.
10.6 Electric stove.
10.7 Burette: 25 mL.
11 Analysis procedure
11.1 Preparation of samples
A1 - the mass of glucose in the sample hydrolyzate for determination, in milligrams (mg);
A2 - the mass of glucose in the reagent blank, in milligrams (mg);
0.9 - the conversion coefficient for converting glucose into starch;
m - the mass of the sample, in grams (g);
V - the volume of the sample hydrolyzate for determination, in milliliters (mL); 500 - the total volume of the sample liquid, in milliliters (mL).
If the result is < 1 g/100 g, keep 2 significant figures. If the result is ≥ 1 g/100 g, keep 3 significant figures.
13 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10 % of the arithmetic mean.
Method III: Saponification-acid hydrolysis method
14 Principle
After the sample is saponified with potassium hydroxide-ethanol to remove fat, and then soluble sugar is removed, starch is hydrolyzed into glucose by hydrochloric acid, and the glucose content is measured and converted into starch content in the sample. 15 Reagents and materials
Unless otherwise stated, the reagents used in this method are of analytical regent, and the water is Grade 3 water specified in GB/T 6682.
15.1 Reagents
15.1.1 Potassium hydroxide (KOH).
15.1.2 Absolute ethanol (C2H5OH) or 95 % ethanol.
15.1.3 Hydrochloric acid (HCl).
15.1.4 Sodium hydroxide (NaOH).
15.1.5 Potassium ferricyanide (C6FeK3N6).
15.1.6 Zinc acetate (C4H6O4Zn).
15.1.7 Glacial acetic acid (CH3COOH).
15.1.8 Copper sulfate (CuSO4 · 5H2O).
15.1.9 Anhydrous sodium carbonate (Na2CO3).
15.1.10 Citric acid (C6H8O7 · H2O).
15.1.11 Potassium iodide (KI).
15.1.12 Sodium thiosulfate (Na2S2O3 · 5H2O).
15.1.13 Bromothymol blue (C27H28Br2O5S): indicator.
15.1.14 Soluble starch: indicator.
15.1.15 α-Naphthol (C10H8O).
15.1.16 Concentrated sulfuric acid (H2SO4).
15.2 Preparation of reagents
15.2.1 Potassium hydroxide-ethanol solution: Weigh 50 g of potassium hydroxide, dissolve in 95 % ethanol and dilute to 1000 mL.
15.2.2 Ethanol solution (80 %, volume fraction): Take 80 ml of absolute ethanol, add water to dilute to 100 mL and mix well. It can also be prepared with 95 % ethanol. 15.2.3 Ethanol solution (40 %, volume fraction): Take 40 mL of absolute ethanol, add water to dilute to 100 mL and mix well. It can also be prepared with 95 % ethanol. 15.2.4 Hydrochloric acid solution (1.0 mol/L): Measure 83 mL of hydrochloric acid, and dilute with water to 1000 mL.
15.2.5 Sodium hydroxide solution (300 g/L): Weigh 30 g of sodium hydroxide, dissolve in water and dilute to 100 mL.
15.2.6 Protein precipitating agents, which include protein precipitating solution A and protein precipitating solution B:
Protein precipitation solution A: Weigh 106 g of potassium ferricyanide, dissolve in water and dilute to 1000 mL.
Protein precipitation solution B: Weigh 220 g of zinc acetate, add 30 ml of glacial acetic acid, and dilute to 1000 mL with water.
15.2.7 Alkaline copper reagents:
17 Analysis procedure
17.1 Preparation of samples
Take a representative sample of no less than 200 g, grind it twice with a meat grinder and mix well.
The ground samples shall be analyzed as soon as possible. If not analyzed immediately, they shall be sealed and refrigerated to prevent deterioration and composition changes. The stored samples shall be remixed before use.
17.2 Starch separation
Weigh 25 g of the sample (accurate to 0.01g, starch content is about 1 g) in a 500 mL beaker, add 300 mL of hot potassium hydroxide-ethanol solution, stir evenly with a glass rod, cover with a watch glass, and heat on a boiling water bath for 1 hour, stirring occasionally. Then, completely transfer the precipitate to the funnel and filter, and wash the precipitate several times with hot ethanol solution (80 %, volume fraction) until the microsugar test result is negative. Samples containing maltodextrin are first washed with 100 mL of hot ethanol solution (80 %, volume fraction), and then washed with ethanol solution (40 %, volume fraction) until the microsugar test result is negative. 17.3 Microsugar test method
Same as 5.2.
17.4 Hydrolysis
Drill holes in the filter paper, use 100 mL of 1.0 mol/L hydrochloric acid solution to wash the precipitate completely into a 250 mL beaker, cover with a watch glass, and hydrolyze in a boiling water bath for 2.5 h, stirring occasionally.
The solution is cooled to room temperature and neutralized with sodium hydroxide solution to a pH of approximately 6 (not exceeding 6.5). Transfer the solution into a 200 mL volumetric flask, add 3 mL of protein precipitation solution A, mix well and then add 3 mL of protein precipitation solution B, and dilute to the mark with water. Shake well and filter through filter paper. Add 1 to 2 drops of sodium hydroxide solution to the filtrate to make it alkaline to the bromothymol blue indicator.
17.5 Determination
Accurately take a certain amount of filtrate (V4) and dilute it to a certain volume (V5), then take 25.00 mL (preferably containing 40 mg ~ 50 mg of glucose) and transfer it into an iodine volume bottle, add 25.00 mL of alkaline copper reagent, install a condenser tube, and boil on an electric stove within 2 min. Then switch to warm fire and continue boiling for 10 min, quickly cool to room temperature, remove the condenser tube, add 30 mL of potassium iodide solution, carefully add 25.00 mL of 7.5 mol/L hydrochloric acid solution, cover it, and wait for titration.
Titrate the iodine released in the above solution with a sodium thiosulfate standard solution. When the solution turns light yellow, add 1 mL of starch indicator, continue titration until the blue color disappears, and record the consumed volume of sodium thiosulfate standard solution (V3).
Carry out two determinations on the same sample and carry out a blank test. 18 Expression of analysis results
18.1 Calculation of glucose amount
The millimoles of sodium thiosulfate consumed, X3, is calculated according to formula (9).
where:
X3 - the millimoles of the sodium thiosulfate consumed;
Vempty - the volume of the sodium thiosulfate standard solution consumed by the blank test, in milliliters (mL);
V3 - the volume of sodium thiosulfate standard solution consumed by the sample solution, in milliliters (mL);
c - the concentration of sodium thiosulfate standard solution, in moles per liter (mol/L).
According to X3, find the corresponding glucose amount (m3) from Table A.1 of Annex A.
18.2 Calculation of starch content
The starch content is calculated according to formula (10).
where:
X - the starch content, in grams per hundred grams (g/100 g);
m3 - the glucose content, in milligram (mg);
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