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Fiber

AACC International Method

32-05.01
Page 1 of 4

Total Dietary Fiber


Final approval October 16, 1991; Reapproval November 3, 1999

Objective
Total dietary fiber (TDF) is determined by gelatinizing duplicate samples
(previously fat-extracted if fat content is >10%) with heat-stable -amylase,
digesting with protease and amyloglucosidase to remove protein and starch, and
diluting the aqueous digest with four volumes ethanol to precipitate soluble
dietary fiber. The residue is filtered; washed with 78% ethanol, 95% ethanol,
and acetone; dried; and weighed. One duplicate is analyzed for protein, the other
incinerated at 525 to determine ash. The TDF collected is corrected for method
blanks, which include protein and ash determinations. This method is applicable
to cereal grains and cereal-based products for nutritional labeling purposes.
Apparatus
1. Water baths. One for boiling water or steam and the other set at 60 and
equipped with either multistation shaker or multistation magnetic stirrer to
provide constant agitation of digestion beakers during enzymatic hydrolysis.
2. Fritted crucibles. Two per sample assayed. Porosity No. 2 (Pyrex No.
32940, coarse ASTM 4060 m; or Corning No. 36060 Bchner, fritted disk,
Pyrex 60 ml, ASTM 4060 m). Clean thoroughly. Heat 8 hr at 525, cool and
soak, then rinse in distilled water. Add about 0.5 g Celite to air-dried crucibles
and dry at 130 to constant weight (usually 1 hr). Cool and store in desiccator
until used.
3. Vacuum source. Vacuum pump or water aspirator equipped with in-line
double vacuum flask to prevent contamination of residue in case of water
backup.
4. Vacuum oven, set at 70. Alternatively, 105 air oven can be used.
5. Desiccator.
6. Muffle furnace.
7. Beakers, tall-form, 400-ml.
8. Balance, analytical, capable of weighing to 0.1 mg.
9. pH meter, calibrated to accuracy of 0.1 pH unit, using pH 4.0 and 7.0
buffers.
Reagents
1. Ethanol, 95% v/v, technical grade.
2. Ethanol, 78%. Place 207 ml water into 1-liter volumetric flask. Dilute to
volume with 95% ethanol. Mix well. Prepare more, if necessary.
3. Acetone, reagent grade.
4. Phosphate buffer, 0.08M, pH 6.0. Dissolve 1.400 g Na phosphate
anhydrous (Na2HPO4) or 1.753 g dihydrate and 9.68 g Na phosphate monobasic
monohydrate (NaH2PO4H2O) or 10.94 g dihydrate in about 700 ml distilled
water. Dilute to 1 liter with water. Check pH with pH meter.
doi: 10.1094/AACCIntMethod-32-05.01

Fiber

AACC International Method

32-05.01
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Total Dietary Fiber (continued)


5. Heat-stable -amylase solution.
6. Protease. Keep refrigerated.
7. Amyloglucosidase. Keep refrigerated.
8. Sodium hydroxide solution, 0.275N. Dissolve 11.00 g ACS grade NaOH in
about 700 ml distilled water, using appropriate handling precautions, in 1-liter
volumetric flask. Cool and dilute to volume with water.
9. Hydrochloric acid solution, 0.325N. Dilute stock solution of known titer
(i.e., 325 ml of 1.0N HCl) to 1 liter with water in volumetric flask.
10. Celite, acid-washed.
Procedure
Enzyme purity
To ensure presence of appropriate enzyme activity and absence of undesirable
enzyme activity when this procedure is used for cereal grains and products, run
materials listed below through entire procedure. Each new lot of enzymes
should be tested, as should enzymes that have not been tested for the previous 6
months.
Test Sample

Activity Tested

Sample Weight (g)

Expected Recovery (%)

Citrus pectin
Stractan (larch gum)
Wheat starch
Corn starch
Casein
-Glucan (barley gum)
(Sigma, 7391)

Pectinase
Hemicellulase
Amylase
Amylase
Protease

0.1
0.1
1.0
1.0
0.3

95100
95100
01
02
02

-Glucanase

0.1

95100

Preparation of sample
Total dietary fiber should be determined on as-is basis on dried low-fat or
fat-free sample. Homogenize sample, weigh, and dry overnight in 70 vacuum
oven. Cool in desiccator, reweigh, and record weight loss due to drying. Drymill portion of dried sample to 0.30.5 mm mesh. If sample cannot be heated,
freeze-dry before milling. If high fat content (>10%) prevents proper milling,
defat with petroleum ether (three times with 25-ml portions per gram of
sample) before milling. When analyzing mixed diets, always extract fat
before determining TDF. Record weight loss due to fat. Correct final percent
dietary fiber determination for both moisture and fat removed. Store dry-milled
sample in capped jar in desiccator until analysis is run.
Method
Run blank through entire procedure along with samples to measure any
contribution from reagents to residue.

Fiber

AACC International Method

32-05.01
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Total Dietary Fiber (continued)


1. Weigh duplicate 1-g samples, accurate to 0.1 mg, into 400-ml tall-form
beakers. Sample weights should differ <20 mg from each other. Add 50 ml pH
6.0 phosphate buffer to each beaker and check pH with pH meter. Adjust if pH
does not equal 6.0 0.1.
2. Add 0.2 ml heat-stable -amylase solution.
3. Cover beaker with aluminum foil and place in boiling water bath for 15
min. Shake gently at 5-min intervals. Note: Increase incubation time when
number of beakers in bath makes it difficult for beaker contents to reach internal
temperature of 100. Use thermometer to indicate that 15 min at 100 is
attained. Total of 30 min in boiling water bath should be sufficient.
4. Cool solutions to room temperature.
5. Adjust to pH 7.5 0.1 by adding 10 ml 0.275N NaOH solution. Check pH
with pH meter.
6. Add 5 mg protease. Protease sticks to spatula, so it may be preferable to
prepare enzyme solution (5 mg in 0.1 ml phosphate buffer) just before use and
pipet amount required.
7. Cover beaker with aluminum foil and incubate at 60 with continuous
agitation for 30 min.
8. Cool and add 10 ml 0.325N HCl solution to adjust pH to 4.5 0.2. Check
pH with pH meter.
9. Add 0.3 ml amyloglucosidase, cover with aluminum foil, and incubate 20
min at 60 with continuous agitation.
10. Add 280 ml 95% EtOH preheated to 60 (measure volume before
heating). Let precipitate form at room temperature for 60 min.
11. Weigh crucible containing Celite to nearest 0.1 mg, then wet and
distribute bed of Celite in crucible by using stream of 78% EtOH from wash
bottle.
12. Apply suction to draw Celite onto fritted glass as even mat. Maintain
suction and quantitatively transfer precipitate from enzyme digest to crucible.
13. Wash residue successively with three 20-ml portions of 78% EtOH, two
10-ml portions of 95% EtOH, and two 10-ml portions of acetone. In some cases,
gums may form during filtration, trapping liquid in residue. If so, break surface
film with spatula to improve filtration. Long filtration times can be avoided by
careful intermittent suction throughout filtration.
14. Dry crucible containing residue overnight in 70 vacuum oven or 105 air
oven.
15. Cool in desiccator and weigh to nearest 0.1 mg. Subtract crucible and
Celite weights to determine weight of residue.
16. Analyze residue from one sample of a set of duplicates for protein by
Method 46-13.01, using N 6.25 as conversion factor.

Fiber

AACC International Method

32-05.01
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Total Dietary Fiber (continued)


17. Incinerate second residue sample of duplicate for 5 hr at 525. Cool in
desiccator and weigh to 0.1 mg. Subtract crucible and Celite weights to determine ash.
Calculations
Uncorrected average blank residue (UABR) = Average blank residue of duplicate blanks
(from step 15) in mg
Blank protein residue (BPR) = UABR % protein in blank (step 16)/100
Blank ash residue (BAR) = UABR % ash in blank (step 17)/100
Corrected blank (CB) = UABR BPR BAR
Uncorrected average sample residue (UASR) = Average sample residue of duplicate samples
(from step 15) in mg
Sample protein residue (SPR) = UASR % protein in sample (step 16)/100
Sample ash residue (SAR) = UASR % ash in sample (step 17)/100
Corrected sample residue (CSR) = UASR SPR SAR CB
Percent TDF = 100 CSR/mg sample

Correct final percent TDF for fat and/or water if defatting and/or drying of
sample was done during sample preparation step.
References
1. AOAC International. 1998. Official Methods of Analysis of AOAC International, 16th ed., 4th
rev. Method 985.29. The Association, Gaithersburg, MD.
2. Prosky, L., Asp, N. G., Furda, I., DeVries, J. W., Schweizer, T. F., and Harland, B. F. 1985.
Determination of total dietary fiber in foods and food products: Collaborative study. J. Assoc. Off.
Anal. Chem. 68:677.
3. Prosky, L., Asp, N. G., Schweizer, T. F., DeVries, J. W., and Furda, I. 1988. Determination of
insoluble, soluble, and total dietary fiber in foods and food products. J. Assoc. Off. Anal. Chem.
71:1017.