National Food Safety Standard
Determination of Fructan in foods
食品安全国家标准
食品中果聚糖的测定
1 Scope
This standard specifies the determination of fructan content in foods by ion chromatography method.
This standard is applicable to the determination for the content of fructooligosaccharide, polyfructan or inulin separately added in milk and dairy products, formula foods and cereal-based complementary foods for infants and young children, solid beverages, blended wines.
2 Principle
Extract specimen with hot water, the sucrose in sample solution is hydrolyzed into glucose and fructose by sucrase, then restore the glucose and fructose to corresponding sugar alcohol by sodium borohydride and neutralize extra sodium borohydride with acetic acid. Hydrolyze the fructan in sample solution into fructose and glucose by inulinase, determine the fructose content by ion chromatography pulsed amperometric detector, and then obtain the fructan content through conversion coefficient.
3 Reagents and Materials
3.1 Reagents
Unless otherwise specified, analytically-pure reagents and first-grade water (defined in GB/T 6682) are adopted for the purposes of this method.
3.1.1 Sodium hydroxide (NaOH).
3.1.2 Maleic acid (C4H4O4).
3.1.3 Sucrase: derived from yeast, enzyme activity ≥300U.
3.1.4 Sodium borohydride (NaBH4).
3.1.5 Glacial acetic acid (CH3COOH).
3.1.6 Sodium acetate trihydrate (CH3COONa·3H2O).
3.1.7 Inulinase: derived from Aspergillus niger, enzyme activity ≥10000U.
3.1.8 50% sodium hydroxide solution (NaOH): chromatographically pure.
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 (1mol/L): weigh 40g sodium hydroxide (with an accuracy of 0.01g), dissolve it into water and dilute to 1000mL, and place it under room temperature for 2 months.
3.2.2 Sodium maleate buffer solution (100mmol/L, pH 6.5): weigh 1.16g maleic acid (with an accuracy of 0.01g), place it in a 150mL-scaled beaker, add about 70mL water for dissolution, regulate pH to 6.5 with 1mol/L sodium hydroxide solution and dilute it to 100mL with water, then place it at 4℃ for 3 months.
3.2.3 Sucrase solution (4.5U/mL): dissolve sucrase (with activity of 300U) into 66mL sodium maleate buffer solution, sub-pack it to 2mL-scaled centrifugal tubes and place it at -20℃ for 6 months. Determine the enzyme activity prior to use.
3.2.4 Sodium hydroxide solution (50mmol/L): weigh 2g sodium hydroxide (with an accuracy of 0.01g), dissolve it into water and dilute to 1000mL, and place it under room temperature for 2 months.
3.2.5 Sodium borohydride solution (10mg/mL): accurately weigh appropriate sodium borohydride (with an accuracy of 0.001g) into centrifugal polypropene tube, dissolve it with 50mmol/L sodium hydroxide solution, the final mass concentration is 10mg/mL and prepare immediately before use.
3.2.6 Acetic acid solution (200mmol/L): pipet 0.6mL glacial acetic acid, and dilute to 50mL with water, and place it at 4℃ for 2 months.
3.2.7 Sodium acetate solution (200mmol/L): weigh 1.36g sodium acetate trihydrate (with an accuracy of 0.01g), dissolve it into water and dilute to 50mL, and place it at 4℃ for 2 months.
3.2.8 Sodium acetate buffer solution (pH 4.5): pipet 14mL acetic acid solution and 11mL sodium acetate solution, dilute them to 50mL with water and prepare immediately before use.
3.2.9 Inulinase solution (455U/mL): dissolve inulinase (with activity of 10000U) into 22mL sodium acetate buffer solution, sub-pack it to 2mL centrifugal tubes and place it at -20℃ for 6 months. Determine the enzyme activity prior to use.
3.2.10 Sodium hydroxide solution (200mmol/L): take 10.4mL of 50% sodium hydroxide solution, dilute it to 1000mL with water, inlet nitrogen for protection and shake slowly and well, and place it under room temperature for 7d.
3.2.11 Mixed solution of sodium hydroxide (150mmol/L) and sodium acetate (500mmol/L): weigh 41g anhydrous sodium acetate (with an accuracy of 0.01g), dissolve it with about 500mL water, filter it with 0.22μm filter membrane, deaerate for 10min and add 7.8mL of 50% sodium hydroxide solution, and dilute to 1000mL with water, inlet nitrogen for protection and shake slowly and well, and place it under room temperature for 7d.
3.3 Standard substance
Fructose standard substance (C6H12O6): purity ≥99.0%.
3.4 Preparation of standard solution
3.4.1 Fructose stock solution (2000mg/L): accurately weigh 0.05g (with an accuracy of 0.1mg) fructose standard substance dried to constant weight at 80℃ into a 50mL-scaled beaker, add about 10mL hot water, cool it to room temperature after the fructose dissolved, dilute it with water into a 25mL-scaled volumetric flask and shake well, place it at 4℃ for 1 month.
3.4.2 Fructose intermediate solution (80.0mg/L): pipet 1mL fructose stock solution, dilute it with water into a 25mL-scaled volumetric flask and prepare immediately before use.
3.4.3 Fructose working solution of standard curve: take appropriate fructose intermediate solution, prepare it into the working solution of standard curve with mass concentration of 0.800mg/L, 1.60mg/L, 4.00mg/L, 8.00mg/L and 16.0mg/L with water.
3.5 Material
3.5.1 Purification column: reversed phase solid-phase extraction column, the filler is 2.5mL styrene divinyl benzene.
3.5.2 Microfiltration membrane: water phase, 0.22μm.
4 Instruments and Equipment
4.1 Ion chromatograph: it is equipped with ternary or above gradient pump, pulsed amperometric detector and Au working electrode.
4.2 Balance: with sensibility of 0.1mg, 0.001g and 0.01g.
4.3 pH meter: with accuracy of 0.01pH.
4.4 Vortex oscillator.
4.5 Shaking bath: with temperature control accuracy of ±1℃.
4.6 Centrifuge: with rotation speed ≥3000r/min.
5 Analysis Procedures
5.1 Specimen preparation
5.1.1 Specimen pretreatment
5.1.1.1 Solid sample: divide into about 200g sample with "quartering method" and pulverized through grinder, then mix uniformly for future use.
5.1.1.2 Liquid sample: put the specimen into a covered container which is able to contain 2 times of specimen, and shake it well for future use.
5.1.2 Extraction
Accurately weigh 1~5g specimen (with an accuracy of 0.001g, at least containing 5mg fructan), put it into a 150mL-scaled conical flask, add about 50mL hot water (80±1℃), place it in a 80±1℃ shaking bath, shake it for 15min at 150r/min, then take it out and cool to room temperature, transfer it to a 100mL-scaled volumetric flask, wash the conical flask with water for three times, scale the volume and shake well, filter or centrifuge the solution with filter paper, determine the dilution ratio of filtrate or supernatant based on the linear range of standard curve, and reserve it for future use.
Take 200μL of the above-mentioned stock sample solution into a 10mL-scaled glass tube with plug, estimate the possible sucrose content of sample solution, add 300μL sucrase solution into sucrose per mg, mix uniformly by means of vortex shaking and place it in a 40±1℃ shaking bath, add 300μL sodium borohydride solution after shaking for 60min at 150r/min, mix uniformly by means of vortex shaking and place it in a 40±1℃ shaking bath, then take it out after shaking for 30min at 150r/min and cool to room temperature. Add 750μL acetic acid solution and keep it still for 10min. Estimate the possible fructan content of sample solution, add 1.2mL inulinase solution per mg, mix uniformly by means of vortex shaking and place it in a 40±1℃ shaking bath, take it out after shaking for 30min at 150r/min and cool to room temperature. Transfer it to a 10mL-scaled volumetric flask, wash the glass tube with water for three times, scale the volume and shake well.
Activate the purification column, the specimen solution passes through 0.22μm water-phase filter membrane and purification column in sequence, discard the former eluent equivalent to 3 times of the column volume, collect the remained eluent to be tested, and conduct reagent blank test at the same time.
5.2 Reference conditions of instruments
5.2.1 Reference condition I of instruments
5.2.1.1 Chromatographic column: high-capacity anion exchange chromatographic column, compatible with gradient elution.
5.2.1.2 Eluate: water for A, sodium hydroxide solution (200mmol/L) for B, mixed solution of sodium hydroxide (150mmol/L) and sodium acetate (500mmol/L) for C. See Table 1 for the gradient elution conditions.
Table 1 Gradient Elution Conditions
Time
min Flow velocity
mL/min Eluate
% Curve
A B C
0~20 1.0 80 20 0 Linearity
20.1~30 1.0 0 0 100 Linearity
30.1~40 1.0 0 100 0 Linearity
40.1~50 1.0 80 20 0 Linearity
5.2.1.3 Detector: pulsed amperometric detector, Au working electrode, Ag/AgCl reference electrode, temperature of detection cell is 30℃ and see Table 2 for the detection waveform of fructose.
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Equipment
5 Analysis Procedures
6 Expression of Analysis Result
7 Accuracy
8 Other
Appendix A Chromatogram of Fructose Standard Solution and Milk Powder Sample
Appendix B Method for Determination of Enzyme Activity
National Food Safety Standard
Determination of Fructan in foods
食品安全国家标准
食品中果聚糖的测定
1 Scope
This standard specifies the determination of fructan content in foods by ion chromatography method.
This standard is applicable to the determination for the content of fructooligosaccharide, polyfructan or inulin separately added in milk and dairy products, formula foods and cereal-based complementary foods for infants and young children, solid beverages, blended wines.
2 Principle
Extract specimen with hot water, the sucrose in sample solution is hydrolyzed into glucose and fructose by sucrase, then restore the glucose and fructose to corresponding sugar alcohol by sodium borohydride and neutralize extra sodium borohydride with acetic acid. Hydrolyze the fructan in sample solution into fructose and glucose by inulinase, determine the fructose content by ion chromatography pulsed amperometric detector, and then obtain the fructan content through conversion coefficient.
3 Reagents and Materials
3.1 Reagents
Unless otherwise specified, analytically-pure reagents and first-grade water (defined in GB/T 6682) are adopted for the purposes of this method.
3.1.1 Sodium hydroxide (NaOH).
3.1.2 Maleic acid (C4H4O4).
3.1.3 Sucrase: derived from yeast, enzyme activity ≥300U.
3.1.4 Sodium borohydride (NaBH4).
3.1.5 Glacial acetic acid (CH3COOH).
3.1.6 Sodium acetate trihydrate (CH3COONa·3H2O).
3.1.7 Inulinase: derived from Aspergillus niger, enzyme activity ≥10000U.
3.1.8 50% sodium hydroxide solution (NaOH): chromatographically pure.
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 (1mol/L): weigh 40g sodium hydroxide (with an accuracy of 0.01g), dissolve it into water and dilute to 1000mL, and place it under room temperature for 2 months.
3.2.2 Sodium maleate buffer solution (100mmol/L, pH 6.5): weigh 1.16g maleic acid (with an accuracy of 0.01g), place it in a 150mL-scaled beaker, add about 70mL water for dissolution, regulate pH to 6.5 with 1mol/L sodium hydroxide solution and dilute it to 100mL with water, then place it at 4℃ for 3 months.
3.2.3 Sucrase solution (4.5U/mL): dissolve sucrase (with activity of 300U) into 66mL sodium maleate buffer solution, sub-pack it to 2mL-scaled centrifugal tubes and place it at -20℃ for 6 months. Determine the enzyme activity prior to use.
3.2.4 Sodium hydroxide solution (50mmol/L): weigh 2g sodium hydroxide (with an accuracy of 0.01g), dissolve it into water and dilute to 1000mL, and place it under room temperature for 2 months.
3.2.5 Sodium borohydride solution (10mg/mL): accurately weigh appropriate sodium borohydride (with an accuracy of 0.001g) into centrifugal polypropene tube, dissolve it with 50mmol/L sodium hydroxide solution, the final mass concentration is 10mg/mL and prepare immediately before use.
3.2.6 Acetic acid solution (200mmol/L): pipet 0.6mL glacial acetic acid, and dilute to 50mL with water, and place it at 4℃ for 2 months.
3.2.7 Sodium acetate solution (200mmol/L): weigh 1.36g sodium acetate trihydrate (with an accuracy of 0.01g), dissolve it into water and dilute to 50mL, and place it at 4℃ for 2 months.
3.2.8 Sodium acetate buffer solution (pH 4.5): pipet 14mL acetic acid solution and 11mL sodium acetate solution, dilute them to 50mL with water and prepare immediately before use.
3.2.9 Inulinase solution (455U/mL): dissolve inulinase (with activity of 10000U) into 22mL sodium acetate buffer solution, sub-pack it to 2mL centrifugal tubes and place it at -20℃ for 6 months. Determine the enzyme activity prior to use.
3.2.10 Sodium hydroxide solution (200mmol/L): take 10.4mL of 50% sodium hydroxide solution, dilute it to 1000mL with water, inlet nitrogen for protection and shake slowly and well, and place it under room temperature for 7d.
3.2.11 Mixed solution of sodium hydroxide (150mmol/L) and sodium acetate (500mmol/L): weigh 41g anhydrous sodium acetate (with an accuracy of 0.01g), dissolve it with about 500mL water, filter it with 0.22μm filter membrane, deaerate for 10min and add 7.8mL of 50% sodium hydroxide solution, and dilute to 1000mL with water, inlet nitrogen for protection and shake slowly and well, and place it under room temperature for 7d.
3.3 Standard substance
Fructose standard substance (C6H12O6): purity ≥99.0%.
3.4 Preparation of standard solution
3.4.1 Fructose stock solution (2000mg/L): accurately weigh 0.05g (with an accuracy of 0.1mg) fructose standard substance dried to constant weight at 80℃ into a 50mL-scaled beaker, add about 10mL hot water, cool it to room temperature after the fructose dissolved, dilute it with water into a 25mL-scaled volumetric flask and shake well, place it at 4℃ for 1 month.
3.4.2 Fructose intermediate solution (80.0mg/L): pipet 1mL fructose stock solution, dilute it with water into a 25mL-scaled volumetric flask and prepare immediately before use.
3.4.3 Fructose working solution of standard curve: take appropriate fructose intermediate solution, prepare it into the working solution of standard curve with mass concentration of 0.800mg/L, 1.60mg/L, 4.00mg/L, 8.00mg/L and 16.0mg/L with water.
3.5 Material
3.5.1 Purification column: reversed phase solid-phase extraction column, the filler is 2.5mL styrene divinyl benzene.
3.5.2 Microfiltration membrane: water phase, 0.22μm.
4 Instruments and Equipment
4.1 Ion chromatograph: it is equipped with ternary or above gradient pump, pulsed amperometric detector and Au working electrode.
4.2 Balance: with sensibility of 0.1mg, 0.001g and 0.01g.
4.3 pH meter: with accuracy of 0.01pH.
4.4 Vortex oscillator.
4.5 Shaking bath: with temperature control accuracy of ±1℃.
4.6 Centrifuge: with rotation speed ≥3000r/min.
5 Analysis Procedures
5.1 Specimen preparation
5.1.1 Specimen pretreatment
5.1.1.1 Solid sample: divide into about 200g sample with "quartering method" and pulverized through grinder, then mix uniformly for future use.
5.1.1.2 Liquid sample: put the specimen into a covered container which is able to contain 2 times of specimen, and shake it well for future use.
5.1.2 Extraction
Accurately weigh 1~5g specimen (with an accuracy of 0.001g, at least containing 5mg fructan), put it into a 150mL-scaled conical flask, add about 50mL hot water (80±1℃), place it in a 80±1℃ shaking bath, shake it for 15min at 150r/min, then take it out and cool to room temperature, transfer it to a 100mL-scaled volumetric flask, wash the conical flask with water for three times, scale the volume and shake well, filter or centrifuge the solution with filter paper, determine the dilution ratio of filtrate or supernatant based on the linear range of standard curve, and reserve it for future use.
Take 200μL of the above-mentioned stock sample solution into a 10mL-scaled glass tube with plug, estimate the possible sucrose content of sample solution, add 300μL sucrase solution into sucrose per mg, mix uniformly by means of vortex shaking and place it in a 40±1℃ shaking bath, add 300μL sodium borohydride solution after shaking for 60min at 150r/min, mix uniformly by means of vortex shaking and place it in a 40±1℃ shaking bath, then take it out after shaking for 30min at 150r/min and cool to room temperature. Add 750μL acetic acid solution and keep it still for 10min. Estimate the possible fructan content of sample solution, add 1.2mL inulinase solution per mg, mix uniformly by means of vortex shaking and place it in a 40±1℃ shaking bath, take it out after shaking for 30min at 150r/min and cool to room temperature. Transfer it to a 10mL-scaled volumetric flask, wash the glass tube with water for three times, scale the volume and shake well.
Activate the purification column, the specimen solution passes through 0.22μm water-phase filter membrane and purification column in sequence, discard the former eluent equivalent to 3 times of the column volume, collect the remained eluent to be tested, and conduct reagent blank test at the same time.
5.2 Reference conditions of instruments
5.2.1 Reference condition I of instruments
5.2.1.1 Chromatographic column: high-capacity anion exchange chromatographic column, compatible with gradient elution.
5.2.1.2 Eluate: water for A, sodium hydroxide solution (200mmol/L) for B, mixed solution of sodium hydroxide (150mmol/L) and sodium acetate (500mmol/L) for C. See Table 1 for the gradient elution conditions.
Table 1 Gradient Elution Conditions
Time
min Flow velocity
mL/min Eluate
% Curve
A B C
0~20 1.0 80 20 0 Linearity
20.1~30 1.0 0 0 100 Linearity
30.1~40 1.0 0 100 0 Linearity
40.1~50 1.0 80 20 0 Linearity
5.2.1.3 Detector: pulsed amperometric detector, Au working electrode, Ag/AgCl reference electrode, temperature of detection cell is 30℃ and see Table 2 for the detection waveform of fructose.
Contents of GB 5009.255-2016
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Equipment
5 Analysis Procedures
6 Expression of Analysis Result
7 Accuracy
8 Other
Appendix A Chromatogram of Fructose Standard Solution and Milk Powder Sample
Appendix B Method for Determination of Enzyme Activity
