GB/T 22224-2008 English PDF (GBT22224-2008)

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GB/T 22224-2008: Determination of dietary fiber in foods -- Enzymatic gravimetric method and enzymatic gravimetric method -- Liquid chromatography
GB/T 22224-2008
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 67.050
X 04
Determination of dietary fiber in foods - Enzymatic
gravimetric method and enzymatic gravimetric
method-liquid chromatography
ISSUED ON: MAY 16, 2008
IMPLEMENTED ON: OCTOBER 01, 2008
Issued by: General Administration of Quality Supervision, Inspection and
Quarantine;
Standardization Administration of the People's Republic of
China.
Table of Contents
Foreword ... 4 
1 Method One - Enzymatic-gravimetric method ... 5 
1.1 Scope ... 5 
1.2 Normative references ... 5 
1.3 Terms and definitions ... 5 
1.4 Method summary ... 6 
1.5 Reagents and solutions ... 6 
1.6 Apparatus and equipment ... 8 
1.7 Sample preparation ... 9 
1.8 Analysis steps ... 9 
1.9 Result calculation ... 11 
1.10 Tolerance ... 12 
2 Method Two - Enzymatic-gravimetric method and liquid chromatography
method ... 12 
2.1 Scope ... 12 
2.2 Normative references ... 13 
2.3 Terms and definitions ... 13 
2.4 Method summary ... 13 
2.5 Reagents and solutions ... 14 
2.6 Apparatus and equipment ... 14 
2.7 Sample preparation ... 15 
2.8 Analysis steps ... 15 
2.9 Result calculation ... 18 
2.10 Tolerance ... 20 
Determination of dietary fiber in foods - Enzymatic
gravimetric method and enzymatic gravimetric
method-liquid chromatography
1 Method One - Enzymatic-gravimetric method
1.1 Scope
Method One of this Standard specifies the conditions and detailed analysis
steps for the determination of total, soluble and insoluble dietary fiber in foods
by enzymatic-gravimetric method.
Method One of this Standard is applicable to the determination of total, soluble
and insoluble dietary fiber in cereals, vegetables and fruits as well as their
products. But it is not applicable to the determination of dietary fiber in foods
containing low-molecular-mass resistant maltodextrin, oligofructose, galacto-
oligosaccharides, polyglucose and resistant starch.
1.2 Normative references
The provisions in following documents become the provisions of this Standard
through reference in this Standard. For dated references, the subsequent
amendments (excluding corrigendum) or revisions do not apply to this Standard,
however, parties who reach an agreement based on this Standard are
encouraged to study if the latest versions of these documents are applicable.
For undated references, the latest edition of the referenced document applies.
GB/T 5009.3-2003, Determination of moisture in foods
GB/T 5009.4-2003, Determination of ash in foods
GB/T 5009.5-2003, Determination of protein in foods
1.3 Terms and definitions
For the purposes of Method One of this Standard, the following terms and
definitions apply.
1.3.1 dietary fiber
μmol of p-nitrophenyl per minute).
1.5.3 Protease: CAS 9014-01-1, IUS 3.4.21.14, cannot contain glycerol
stabilizer. Use MES-TRIS buffer to prepare protease solution of which the
concentration is 50 mg/mL. Prepare when it is needed. Store at 0°C ~ 5°C.
1.5.3.1 Enzyme activity representation 1: Casein test, 300 units/mL ~ 400
units/mL, or 7 units/mg ~ 15 units/mg.
NOTE: 1 enzyme activity unit is defined as: at 40°C, when pH8.0, the amount of enzyme
required to hydrolyze 1 μmol of tyrosine (and is soluble in trichloroacetic acid) from soluble
casein;
Or it is defined as: at 37°C, when pH7.5, the amount of enzyme required to hydrolyze a
certain amount of tyrosine from casein per minute (equivalent to the color change caused
by 1.0 μmol of tyrosine in coloration reaction; use Folin-Ciocalteau for coloration).
1.5.3.2 Enzyme activity representation 2: Azo-casein test, 300 units/mL ~ 400
units/mL.
NOTE: 1 endopeptidase activity unit is defined as: at 40°C, when pH8.0, the amount of enzyme required
to hydrolyze 1 μmol of tyrosine from soluble casein (and is soluble in trichloroacetic acid).
1.5.4 Amyloglucosidase solution: cannot contain glycerol as stabilizer, CAS
9032-08-0, IUS 3.2.1.3. Store at 0°C ~ 5°C.
1.5.4.1 Enzyme activity representation 1: Starch/glucose oxidase-peroxidase
method, 2,000 units/mL ~ 3,300 units/mL.
NOTE: 1 endopeptidase activity unit is defined as: at 40°C, when pH4.5, the amount of enzyme required
to release 1 μmol of glucose per minute.
1.5.4.2 Enzyme activity representation 2: p-Nitrophenyl-β-maltoside (PNPBM)
method, 130 units/mL ~ 200 unit /mL.
NOTE: 1 enzyme activity unit is defined as (1 PNP unit): at 40°C, in the presence of excessive β-
glucosidase, the amount of enzyme required to release 1 μmol of p-nitrophenyl from p-nitrophenyl-β-
maltoside per minute.
1.5.5 Pickled diatomite: CAS 68855-54-9. Take 200 g of diatomite in 600 mL of
hydrochloric acid (HCl : H2O = 1:4, volume ratio). Soak overnight. Filter. Use
distilled water to wash until the filtrate is neutral. Place in 525°C ± 5°C muffle
furnace to burn the ash. Leave it for use.
1.5.6 2-(N-morpholino)-sulfonate ethane (MES): CAS 4432-31-9, purity >99.5%.
1.5.7 Trihydroxymethyl aminomethane (TRIS): CAS 77-86-1, purity >99%.
1.6.13 Pipette: 100 µL, 5 mL; disposable pipette tip.
1.7 Sample preparation
1.7.1 Food with fat content less than 10%
Take well-mixed sample at 70°C vacuum to dry overnight. Place in the dryer to
cool. After the dry sample is smashed, sieve through 0.3mm ~ 0.5mm sieve. If
the sample cannot be heated, then lyophilize it first. Smash and sieve. Store
the smashed and sieved dry sample in the dryer for use.
1.7.2 Food with fat content greater than 10%
Take an appropriate amount of high-temperature-dried or lyophilized sample.
Use petroleum ether to respectively degrease three times, 25 mL for each. Mix
well. dry overnight at 70°C vacuum. Place in the dryer to cool. Dry and record
the sample loss caused by petroleum ether. At last, calibrate while calculating
the dietary fiber content. Store the smashed-sieved dry sample in the dryer for
use.
1.7.3 Food with high sugar content
Take an appropriate amount of sample. Add 10 mL of 85% ethanol per gram of
sample. Process the sample 2 ~ 3 times to de-sugar. Dry overnight at 40°C.
Store the smashed-sieved dry sample in the dryer for use.
1.8 Analysis steps
1.8.1 Determination of moisture content
Determine the moisture content in sample according to GB/T 5009.3-2003. Use
it for result calculation.
1.8.2 Enzymatic hydrolysis
1.8.2.1 Accurately weigh double samples (ms1 and ms2), 1 g for each. The mass
difference between two samples is ≤0.005 g, to the nearest of 0.1 mg. Place in
400mL or 600mL high-type beaker (1.6.1). Prepare double blank samples at the
same time. Add 40 mL of pH8.2 MES-TRIS buffer into each beaker.
Magnetically stir until the sample is completely dispersed in the buffer.
1.8.2.2 Enzymatic hydrolysis of thermostable α-amylase: Add 50 µL of
thermostable α-amylase solution (1.5.2). Cover aluminum foil. Place in 95°C
constant-temperature oscillating water bath to shake continuously. Start timing
when the temperature inside the beaker rises to 95°C. React for 30 min.
residue mass.
1.8.3.1.4 Determination of protein and ash: For the mixture of residue and
diatomite of which their masses have been weighed, one is used to determine
nitrogen (N) content according to GB/T 5009.5-2003: use N × 6.25 as
conversion factor to calculate protein mass. The other sample is used to
determine ash according to GB/T 5009.4-2003. That is: Ash at 525°C for 5 h.
Cool in dryer. Accurately weigh the total weight of cru...