GB 5009.255-2016National food safety standard - Determination of fructan in foods (English PDF)

3.1 Reagents

Unless otherwise stated, the reagents used in this method are of analytical grade AND water is level I water as specified in GB/T 6682.

3.1.1 Sodium hydroxide (NaOH).

3.1.2 Maleic acid (C4H4O4).

3.1.9 Anhydrous sodium acetate (CH3COONa). purity >= 99.0%.

3.1.10 Nitrogen (N2). purity >= 99.9%.

3.2 Reagent preparation

3.2.1 Sodium hydroxide solution (1 mol/L). WEIGH 40 g of sodium hydroxide

(accurate to 0.01 g); DISSOLVE it in water and DILUTE it to 1000 mL; AND it can be preserved for 2 months at room temperature.

3.2.2 Sodium maleate buffer solution (100 mmol/L, pH 6.5). WEIGH 1.16 g of

maleic acid (accurate to 0.01 g); PLACE it in a 150 mL beaker; ADD about 70 mL of water to dissolve it; USE 1 mol/L sodium hydroxide solution to adjust the pH to 6.5; USE water to dilute it to 100 mL; PRESERVE it at 4 °C, AND it can be stored for 3 months.

3.2.7 Sodium acetate solution (200 mmol/L). WEIGH 1.36 g of sodium

trihydrate (accurate to 0.01 g); USE water to dissolve and dilute it to 50 mL;

PRESERVE it at 4 °C; AND it can be stored for 2 months.

3.2.8 Sodium acetate buffer solution (pH 4.5). PIPETTE 14 mL of acetic acid

solution and 11 mL of sodium acetate solution; MIX it; USE water to dilute it to

50 mL; PREPARE it before use.

3.2.9 Fructanase solution (455 U/mL). DISSOLVE the fructanase (activity

10000 U) in 22 mL of sodium acetate buffer solution; DISPENSE it into 2 mL centrifuge tube; PRESERVE it at -20 °C and it can be stored for 6 months. It shall determine the enzyme activity before use.

3.3 Standard substance

Fructose standard substance (C6H12O6). purity >= 99.0%.

3.4 Preparation of standard solution

3.4.1 Fructose stock solution (2000 mg/L). accurately WEIGH 0.05 g (accurate

to 0.1 mg) of fructose standard substance which had been dried to constant weight at 80 °C into a 50 mL beaker; ADD about 10 mL of hot water; until the fructose is dissolved, COOL it to room temperature; USE water to dilute it into a 25 mL volumetric flask; SHAKE it uniformly; PRESERVE it at 4 °C and it can be stored for 1 month.

3.4.2 Fructose intermediate solution (80.0 mg/L). PIPETTE 1 mL of fructose

stock solution; USE water to dilute it into a 25 mL volumetric flask; PREPARE it before use.

3.5 Material

3.5.1 Purification column. reverse phase solid phase extraction column, the

filler is styrene divinyl benzene, 2.5 mL.

3.5.2 Microporous membrane. water phase, 0.22 µm.

4.1 Ion chromatograph. equipped with ternary and above gradient pump, pulse

amperometric detector, and Au working electrode.

4.5 Constant temperature water bath shaker. temperature control accuracy ±

1 °C.

4.6 Centrifuge. speed >= 3000 r/min.

5.1 Sample preparation

5.1.1 Sample pretreatment

5.1.1.1 Solid sample. SPLIT it to about 200 g by “quartering” method; USE

crusher to crush it; MIX it uniformly; PREPARE for use.

5.1.2 Extraction

Accurately WEIGH 1 g ~ 5 g (accurate to 0.001 g, containing at least 5 mg of fructan) of sample in a 150 mL conical flask; ADD about 50 mL of 80 °C ± 1 °C hot water; PLACE it in a 80 °C ± 1 °C constant temperature water bath shaker;

SHAKE it at 150 r/min for 15 min; TAKE it out;

5.2 Instrument reference conditions

5.2.1 Instrument reference conditions 1

5.2.1.1 Columns. High-capacity anion exchange columns that are compatible with gradient elution.

5.2.2.2 Eluent. A. sodium hydroxide solution (90 mmol/L); B. sodium hydroxide

(150 mmol/L) and sodium acetate (500 mmol/L) mixed solution. The gradient elution conditions are as shown in Table 3.

5.2.2.4 Column temperature. 32 °C.

5.2.2.5 Injection volume. 20 µL.

5.3 Production of standard curve

INJECT the standard curve working solution (from high to low) in turn into the ion chromatograph; DETERMINE the corresponding response value (peak area); USE the mass concentration of fructose in the standard working solution as the abscissa AND the response value (peak area) as the ordinate, DRAW the standard curve. The standard solution chromatogram is as shown in

Appendix A.

5.4 Determination of sample solution

INJECT the sample solution into the ion chromatograph; RECORD the chromatogram. MAKE qualitative based on the retention time; RECORD the peak area;

A.1 Fructose standard solution chromatogram (reference condition 1)

The fructose standard solution chromatogram (reference condition 1) is as shown in Figure A.1.

A.2 Milk powder sample chromatogram (reference condition 1)

The milk powder sample chromatogram (reference condition 1) is as shown in

Figure A.2.

Appendix B

Enzyme activity determination method

B.1 Determination of sucrase activity

B.1.1 Principle

Sucrase (Sucrase, EC 3.2.1.26), also known as invertase, it may cut the fructose glycosidic of the sucrose from the fructose ends, to make the sucrose hydrolysis to produce glucose and fructose; the glucose and fructose are reducing sugar, and their content can be determined through the

3,5-dinitrosalicylic acid colorimetric method, in order to measure the enzyme activity. Since the sucrase is easily inactivated under alkaline conditions, it may use alkali to terminate the reaction.

B.1.2 Reagent

Unless otherwise stated, the reagents used in this method are of analytical grade AND water is level I water as specified in GB/T 6682.

B.1.2.1 Glucose standard substance (C6H12O6). purity >= 99.0%.

B.1.2.2 Sucrose (C12H22O11). purity >= 99.0%.

B.1.2.3 Sodium hydroxide (NaOH).

B.1.2.4 Maleic acid (C4H4O4).

B.1.2.5 Dinitrosalicylic acid (C7H4N2O7).

B.1.2.6 Potassium tartrate (C4H4KNaO6).

B.1.3 Regent preparation

B.1.3.1 2.5 µmol/mL glucose standard solution. Accurately WEIGH 0.045 g (accurate to 0.1 mg) of glucose standard substance which had been dried to constant weight at 80 °C into a 50 mL beaker; ADD about 10 mL of hot water;

after the glucose is dissolved; COOL it to room temperature; USE water to dilute it into a 100 mL volumetric flask; SHAKE it uniformly; PRESERVE it at

4 °C, AND it can be stored for 1 month.

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