GB 5009.255-2016 English PDF

GB 5009.255-2016: National food safety standard - Determination of fructan in foods
Delivery: 9 seconds. Download (& Email) true-PDF + Invoice.
Get Quotation: Click GB 5009.255-2016 (Self-service in 1-minute)
Historical versions (Master-website): GB 5009.255-2016
Preview True-PDF (Reload/Scroll-down if blank)

GB 5009.255-2016
GB
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
National food safety standard
Determination of fructan in foods
ISSUED ON. AUGUST 31, 2016
IMPLEMENTED ON. MARCH 01, 2017
Issued by. National Health and Family Planning Commission of the PRC
Table of contents
National food safety standard ... 3 
1 Scope ... 3 
2 Principles ... 3 
3 Reagents and materials ... 3 
4 Instruments and equipment ... 6 
5 Analytical procedures ... 6 
6 Expression of analysis results ... 9 
7 Precision... 10 
8 Others ... 10 
Appendix A Fructose standard solution and milk powder sample
chromatogram ... 11 
Appendix B Enzyme activity determination method ... 13 
National food safety standard
Determination of fructan in foods
1 Scope
This standard specifies the determination of fructan content in food by ion
chromatography.
This standard applies to the determination of oligofructose, polyfructose or
inulin content separately added in milk and dairy products, infants and young
children's formula, infants and young children cereals, solid beverages and
formulated wines.
2 Principles
The sample is subjected to digestion by hot water, AND the sucrose in the
sample solution is hydrolyzed to glucose and fructose by sucrase. Glucose
and fructose are reduced to corresponding sugar alcohol by sodium
borohydride, AND the excess sodium borohydride is neutralized by acetic acid.
The fructan in the sample solution are hydrolyzed into fructose and glucose by
the fructanase. The fructose content is determined by ion
chromatography-pulsed amperometric detector, AND converted to the fructan
content by the use of conversion factor.
3 Reagents and materials
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).
3.1.3 Sucrase. derived from yeast, enzyme activity ≥ 300 U.
3.1.4 Sodium borohydride (NaBH4).
3.1.5 Glacial acetic acid (CH3COOH).
3.1.6 Sodium trihydrate (CH3COONa•3H2O).
3.1.7 Fructanase. from Aspergillus niger, enzyme activity ≥ 10000 U.
3.1.8 50% sodium hydroxide solution (NaOH). chromatographic 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 (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.
3.2.3 Sucrose solution (4.5 U/mL). DISSOLVE the sucrosease (activity 300 U)
into 66 mL of sodium maleate buffer solution; DISPENSE it into 2 mL
centrifuge tube; PREERVE it at -20 °C, AND it can be stored for 6 months.
Before use, it shall determine the enzyme activity.
3.2.4 Sodium hydroxide solution (50 mmol/L). WEIGH 2 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.
3.2.5 Sodium borohydride solution (10 mg/mL). Accurately WEIGH appropriate
amount of sodium borohydride (accurate to 0.001g) in a polypropylene
centrifuge tube; USE 50 mmol/L sodium hydroxide solution to dissolve it, to
make the final mass concentration of 10 mg/mL; PREPARE it before use.
3.2.6 Acetic acid solution (200 mmol/L). PIPETTE 0.6 mL of glacial acetic acid;
USE water to dilute it to 50 mL; PRESERVE it at 4 °C; AND it can be stored for
2 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.
3.5.1 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 Instruments and equipment
4.1 Ion chromatograph. equipped with ternary and above gradient pump, pulse
amperometric detector, and Au working electrode.
4.2 Balance. sensibility of 0.1 mg, 0.001 g and 0.01 g.
4.3 pH meter. accuracy of 0.01 pH.
4.4 Vortex oscillator.
4.5 Constant temperature water bath shaker. temperature control accuracy ±
1 °C.
4.6 Centrifuge. speed ≥ 3000 r/min.
5 Analytical procedures
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.
5.1.1.2 Liquid sample. PLACE the sample in a capped container capable of
holding 2 times the sample volume; 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; COOL it to room temperature;
TRANSFER it into a 100 mL volumetric flask; USE water to rinse the conical
flask for 3 times; MAKE the volume reach to the mark; SHAKE it uniformly;
MAKE the filtrate subjected to filter paper filtration or centrifuging;
DETERMINE the dilution factor of the filtrate or the supernatant based on the
liner range of the standard curve; PREPARE for use.
0.2 - The volume of sample solution for enzymolysis, in milliliters (mL);
1000 - Unit conversion factor;
n - Average degree of polymerization, it is taken as n = 4 for oligofructose, n
= 23 for polyfructose, AND n = 10 for inulin.
The calculated results are expressed as the arithmetic mean of the two
independent determinations obtained under repeatability conditions, with the
result retaining three significant digits.
7 Precision
The absolute difference between the two independent determinations obtained
under repeatability conditions shall not exceed 10% of the arithmetic mean.
8 Others
When the sample amount is 5 g, the detection limit is 0.4 g/kg AND the
quantification limit is 2.0 g/kg.
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. The 3,5-dinitrosalicylic acid and reducing
sugar will form a brown red amino compound after being heated together, AND
it may conduct determination at the maximum absorption wavelength 540 nm;
within a certain concentration range, the absorbance is in linear relation with
the reducing sugar content, so it can be used for the determination of the
reducing sugar content, thus determining the sucrase activity.
The sucrase activity is defined as below. under the conditions of 37 °C and pH
6.5, the amount of enzyme (activity) required to hydrolyze 1 μmol of glucose
per minute is defined as one enzyme activity unit.
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.1.2.7 Phenol (C6H5OH).
B.2.2.2 Inulin. purity ≥ 90.0%.
B.2.2.3 Glacial acetic acid (CH3COOH).
B.2.2.4 Sodium acetate trihydrate (CH3COONa•3H2O).
B.2.2.5 Dinitrosalicylic acid (C7H4N2O7).
B.2.2.6 Potassium tartrate (C4H4KNaO6).
B.2.2.7 Phenol (C6H5OH).
B.2.2.8 Sodium sulfite (Na2SO3).
B.2.3 Reagent preparation
B.2.3.1 2.5 µmol/mL fructose standard solution. Accurately WEIGH 0.045 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;
after the fructose 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.
B.2.3.2 Acetic acid solution (200 mmol/L). PIPETTE 0.6 mL of glacial acetic
acid; USE water to dilute it to 50 mL; PRESERVE it at 4 °C, AND it can be
stored for 2 months.
B.2.3.3 Sodium ace...
GB 5009.255-2016
GB
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
National food safety standard
Determination of fructan in foods
ISSUED ON. AUGUST 31, 2016
IMPLEMENTED ON. MARCH 01, 2017
Issued by. National Health and Family Planning Commission of the PRC
Table of contents
National food safety standard ... 3 
1 Scope ... 3 
2 Principles ... 3 
3 Reagents and materials ... 3 
4 Instruments and equipment ... 6 
5 Analytical procedures ... 6 
6 Expression of analysis results ... 9 
7 Precision... 10 
8 Others ... 10 
Appendix A Fructose standard solution and milk powder sample
chromatogram ... 11 
Appendix B Enzyme activity determination method ... 13 
National food safety standard
Determination of fructan in foods
1 Scope
This standard specifies the determination of fructan content in food by ion
chromatography.
This standard applies to the determination of oligofructose, polyfructose or
inulin content separately added in milk and dairy products, infants and young
children's formula, infants and young children cereals, solid beverages and
formulated wines.
2 Principles
The sample is subjected to digestion by hot water, AND the sucrose in the
sample solution is hydrolyzed to glucose and fructose by sucrase. Glucose
and fructose are reduced to corresponding sugar alcohol by sodium
borohydride, AND the excess sodium borohydride is neutralized by acetic acid.
The fructan in the sample solution are hydrolyzed into fructose and glucose by
the fructanase. The fructose content is determined by ion
chromatography-pulsed amperometric detector, AND converted to the fructan
content by the use of conversion factor.
3 Reagents and materials
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).
3.1.3 Sucrase. derived from yeast, enzyme activity ≥ 300 U.
3.1.4 Sodium borohydride (NaBH4).
3.1.5 Glacial acetic acid (CH3COOH).
3.1.6 Sodium trihydrate (CH3COONa•3H2O).
3.1.7 Fructanase. from Aspergillus niger, enzyme activity ≥ 10000 U.
3.1.8 50% sodium hydroxide solution (NaOH). chromatographic 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 (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.
3.2.3 Sucrose solution (4.5 U/mL). DISSOLVE the sucrosease (activity 300 U)
into 66 mL of sodium maleate buffer solution; DISPENSE it into 2 mL
centrifuge tube; PREERVE it at -20 °C, AND it can be stored for 6 months.
Before use, it shall determine the enzyme activity.
3.2.4 Sodium hydroxide solution (50 mmol/L). WEIGH 2 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.
3.2.5 Sodium borohydride solution (10 mg/mL). Accurately WEIGH appropriate
amount of sodium borohydride (accurate to 0.001g) in a polypropylene
centrifuge tube; USE 50 mmol/L sodium hydroxide solution to dissolve it, to
make the final mass concentration of 10 mg/mL; PREPARE it before use.
3.2.6 Acetic acid solution (200 mmol/L). PIPETTE 0.6 mL of glacial acetic acid;
USE water to dilute it to 50 mL; PRESERVE it at 4 °C; AND it can be stored for
2 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.
3.5.1 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 Instruments and equipment
4.1 Ion chromatograph. equipped with ternary and above gradient pump, pulse
amperometric detector, and Au working electrode.
4.2 Balance. sensibility of 0.1 mg, 0.001 g and 0.01 g.
4.3 pH meter. accuracy of 0.01 pH.
4.4 Vortex oscillator.
4.5 Constant temperature water bath shaker. temperature control accuracy ±
1 °C.
4.6 Centrifuge. speed ≥ 3000 r/min.
5 Analytical procedures
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.
5.1.1.2 Liquid sample. PLACE the sample in a capped container capable of
holding 2 times the sample volume; MIX it uniformly; PREPARE for use.
5.1.2 Extraction
Accura...