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Fiber

AACC International Method

32-05.01
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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 α-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 40–60 μm; or Corning No. 36060 Büchner, fritted disk,
Pyrex 60 ml, ASTM 40–60 μm). 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 (NaH2PO4⋅H2O) 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

freeze-dry before milling. Hydrochloric acid solution.1 1. run materials listed below through entire procedure. weigh. Store dry-milled sample in capped jar in desiccator until analysis is run.Fiber AACC International Method 32-05. Cool and dilute to volume with water. 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. If high fat content (>10%) prevents proper milling. If sample cannot be heated.0 1. . Each new lot of enzymes should be tested..3–0. 0.275N. always extract fat before determining TDF. Dissolve 11. Heat-stable α-amylase solution. 8. and record weight loss due to drying.1 0.0N HCl) to 1 liter with water in volumetric flask. 10. and dry overnight in 70° vacuum oven. 6. 7391) Pectinase Hemicellulase Amylase Amylase Protease 0. 9. defat with petroleum ether (three times with 25-ml portions per gram of sample) before milling. Amyloglucosidase. Dilute stock solution of known titer (i. Protease. 0. Cool in desiccator. Method Run blank through entire procedure along with samples to measure any contribution from reagents to residue. Test Sample Activity Tested Sample Weight (g) Expected Recovery (%) Citrus pectin Stractan (larch gum) Wheat starch Corn starch Casein β-Glucan (barley gum) (Sigma. Keep refrigerated. Sodium hydroxide solution.325N. 325 ml of 1.e.0 0. using appropriate handling precautions.01 Page 2 of 4 Total Dietary Fiber (continued) 5. in 1-liter volumetric flask. 7. Drymill portion of dried sample to 0.3 95–100 95–100 0–1 0–2 0–2 β-Glucanase 0. When analyzing mixed diets. Celite.1 95–100 Preparation of sample Total dietary fiber should be determined on as-is basis on dried low-fat or fat-free sample. Correct final percent dietary fiber determination for both moisture and fat removed. Homogenize sample.00 g ACS grade NaOH in about 700 ml distilled water. Record weight loss due to fat. acid-washed. reweigh. as should enzymes that have not been tested for the previous 6 months.5 mm mesh. Keep refrigerated.

5 ± 0. Maintain suction and quantitatively transfer precipitate from enzyme digest to crucible. break surface film with spatula to improve filtration.0 phosphate buffer to each beaker and check pH with pH meter. Analyze residue from one sample of a set of duplicates for protein by Method 46-13. Use thermometer to indicate that 15 min at 100° is attained. 9. and two 10-ml portions of acetone. If so.325N HCl solution to adjust pH to 4. 11. Weigh duplicate 1-g samples. trapping liquid in residue. Adjust if pH does not equal 6. Add 5 mg protease. 14. Note: Increase incubation time when number of beakers in bath makes it difficult for beaker contents to reach internal temperature of 100°. Total of 30 min in boiling water bath should be sufficient. Adjust to pH 7. Wash residue successively with three 20-ml portions of 78% EtOH. Subtract crucible and Celite weights to determine weight of residue. 8. 10. 6. gums may form during filtration. Check pH with pH meter. Long filtration times can be avoided by careful intermittent suction throughout filtration. 15.1 by adding 10 ml 0. two 10-ml portions of 95% EtOH.25 as conversion factor. 13. 12.3 ml amyloglucosidase.2 ml heat-stable α-amylase solution.275N NaOH solution. In some cases.01.2. accurate to 0. cover with aluminum foil. Dry crucible containing residue overnight in 70° vacuum oven or 105° air oven. then wet and distribute bed of Celite in crucible by using stream of 78% EtOH from wash bottle.1. 16. and incubate 20 min at 60° with continuous agitation. Protease sticks to spatula. 7. . Add 0. Shake gently at 5-min intervals. Add 280 ml 95% EtOH preheated to 60° (measure volume before heating). Cool solutions to room temperature. 4. Apply suction to draw Celite onto fritted glass as even mat.1 mg. 5.1 mg.01 Page 3 of 4 Total Dietary Fiber (continued) 1. Cover beaker with aluminum foil and incubate at 60° with continuous agitation for 30 min. Check pH with pH meter. Cover beaker with aluminum foil and place in boiling water bath for 15 min. Weigh crucible containing Celite to nearest 0.5 ± 0. so it may be preferable to prepare enzyme solution (5 mg in 0.0 ± 0. 3. Cool in desiccator and weigh to nearest 0. Let precipitate form at room temperature for 60 min.1 mg. using N × 6.Fiber AACC International Method 32-05. Sample weights should differ <20 mg from each other.1 ml phosphate buffer) just before use and pipet amount required. into 400-ml tall-form beakers. Add 0. 2. Add 50 ml pH 6. Cool and add 10 ml 0.

and Harland. 71:1017. J. Furda. Official Methods of Analysis of AOAC International. 4th rev. The Association. Method 985. 1988..Fiber AACC International Method 32-05. 2.01 Page 4 of 4 Total Dietary Fiber (continued) 17. Determination of insoluble. L. Chem.. and total dietary fiber in foods and food products. MD. F. DeVries.. N.29. G. Assoc. 16th ed. AOAC International.. J. I. Prosky. T. J. Asp. B. 3.. Cool in desiccator and weigh to 0. I. J. DeVries. 1998. Chem. N. T. F.. soluble. Off. 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. Subtract crucible and Celite weights to determine ash.. G. References 1. . Gaithersburg. W. F. Determination of total dietary fiber in foods and food products: Collaborative study. L. Schweizer. Anal.. Asp. Prosky.1 mg. Schweizer. 68:677. and Furda.. Incinerate second residue sample of duplicate for 5 hr at 525°. Assoc. Off. 1985.. W. Anal.