Revised: March 1996 A.

Principle
Duplicate samples of dried foods, fat-extracted if containing >10%
32.1.17 fat, undergo sequential enzymatic digestion by heat stable α-amylase,
AOAC Official Method 991.43 protease, and amyloglycosidase to remove starch and protein. For total
Total, Soluble, and Insoluble Dietary Fiber in Foods dietary fiber (TDF), enzyme digestate is treated with alcohol to precipi-
Enzymatic-Gravimetric Method, MES–TRIS Buffer tate soluble dietary fiber before filtering, and TDF residue is washed
First Action 1991 with alcohol and acetone, dried, and weighed. For insoluble and
Final Action 1994 soluble dietary fiber (IDF and SDF), enzyme digestate is filtered, and
Codex-Adopted—AOAC Method* residue (IDF) is washed with warm water, dried and weighed. For SDF,
combined filtrate and washes are precipitated with alcohol, filtered,
(Applicable to processed foods, grain and cereal products, fruits, and
dried, and weighed. TDF, IDF, and SDF residue values are corrected for
vegetables.)
protein, ash, and blank.
Method Performance: B. Apparatus
See Table 991.43A for method performance data. (a) Beakers.—400 or 600 mL tall-form.

Table 991.43A Method Performance for Total, Soluble, and Insoluble Dietary Fiber in Foods (Fresh Weight Basis),
Enzymatic-Gravimetric Method, MES-TRIS Buffer
Food Mean, g/100 g sr sR RSDr % RSDR %
Total dietary fiber (TDF)
Barley 12.25 0.36 0.85 2.88 6.89
High-fiber cereal 33.73 0.70 0.94 2.08 2.79
Oat bran 16.92 1.06 2.06 6.26 12.17
Soy bran 67.14 1.01 1.06 1.50 1.58
Apricots 1.12 0.01 0.01 0.89 0.89
Prunes 9.29 0.13 0.40 1.40 4.31
Raisins 3.13 0.09 0.15 2.88 4.79
Carrots 3.93 0.13 0.13 3.31 3.31
Green beans 2.89 0.07 0.07 2.42 2.42
Parsley 2.66 0.07 0.14 2.63 5.26
Soluble dietary fiber (SDF)
Barley 5.02 0.40 0.62 8.01 12.29
High-fiber cereal 2.78 0.44 0.56 15.83 20.14
Oat bran 7.17 0.72 1.14 10.04 15.90
Soy bran 6.90 0.30 0.60 4.35 8.70
Apricots 0.53 0.02 0.02 3.77 3.77
Prunes 5.07 0.11 0.31 2.17 6.11
Raisins 0.73 0.05 0.16 6.85 21.92
Carrots 1.10 0.07 0.18 6.36 16.36
Green beans 1.02 0.08 0.11 7.84 10.78
Parsley 0.64 0.03 0.10 4.69 15.63
Insoluble dietary fiber (IDF)
Barley 7.05 0.61 0.61 8.62 8.62
High-fiber cereal 30.52 0.44 0.71 1.44 2.33
Oat bran 9.73 0.85 1.17 8.74 12.02
Soy bran 60.53 0.70 0.70 1.16 1.16
Apricots 0.59 0.02 0.02 3.39 3.39
Prunes 4.17 0.07 0.09 1.68 2.16
Raisins 2.37 0.04 0.07 1.69 2.95
Carrots 2.81 0.09 0.16 3.20 5.69
Green beans 2.01 0.08 0.08 3.98 3.98
Parsley 2.37 0.12 0.24 5.06 10.13
Total dietary fiber (SDF + IDF)
Barley 12.14 0.39 0.70 3.21 5.77
High-fiber cereal 33.30 0.63 0.90 1.89 2.70
Oat bran 16.90 0.99 1.49 5.86 8.82
Soy bran 67.56 0.56 0.94 0.83 1.39
Apricots 1.12 0.02 0.02 1.79 1.79
Prunes 9.37 0.12 0.30 1.28 3.20
Raisins 3.10 0.05 0.18 1.61 5.81
Carrots 3.92 0.11 0.13 2.81 3.32
Green beans 3.03 0.09 0.12 2.97 3.96
Parsley 3.01 0.12 0.23 3.99 7.64

Copyright 1998 AOAC INTERNATIONAL

Table 991.43B Standards for Testing Enzyme Activity
Standard Activity Tested Weight of Standard, g Expected Recovery, (%)
Citrus pectin Pectinase 0.1–0.2 95–100
Arabinogalactan Hemicellulase 0.1–0.2 95–100
β-Glucan β-Glucanase 0.1–0.2 95–100
Wheat starch α-Amylase + AMG 1.0 0–1
Corn starch α-Amylase + AMG 1.0 0–1
Casein Protease 0.3 0–1

(b) Filtering crucible.—With fritted disk, coarse, ASTM 40–60 (h) TRIS.—Tris(hydroxymethyl)aminomethane (No. T-1503,
µm pore size, Pyrex 60 mL (Corning No. 36060 Bchner, Corning, Sigma Chemical Co., or equivalent).
Inc., Science Products, Corning, NY 14831, USA, or equivalent). (i) MES–TRIS buffer solution.—0.05M MES, 0.05M TRIS, pH
Prepare as follows. Ash overnight at 525° in muffle furnace. Let 8.2 at 24°. Dissolve 19.52 g MES and 12.2 g TRIS in 1.7 L H2O.
furnace temperature fall below 130° before removing crucibles. Adjust pH to 8.2 with 6N NaOH, and dilute to 2 L with H2O.
Soak crucibles 1 h in 2% cleaning solution at room temperature. (Note: It is important to adjust pH to 8.2 at 24°. However, if
Rinse crucibles with H2O and then deionized H2O; for final rinse, buffer temperature is 20°, adjust pH to 8.3; if temperature is 28°,
use 15 mL acetone and then air-dry. Add ca 1.0 g Celite to dry adjust pH to 8.1. For deviations between 20 and 28°, adjust by
crucibles, and dry at 130° to constant weight. Cool crucible ca 1 interpolation.)
h in desiccator, and record weight, to nearest 0.1 mg, of crucible (j) Hydrochloric acid solution.—0.561N. Add 93.5 mL 6N HCl
plus Celite. to ca 700 mL H2O in 1 L volumetric flask. Dilute to 1 L with H2O.
(c) Vacuum system.—Vacuum pump or aspirator with regulating D. Enzyme Purity
device. Heavy walled filtering flask, 1 L, with side arm. Rubber To ensure absence of undesirable enzymatic activities and pres-
ring adaptors, for use with filtering flasks. ence of desirable enzymatic activities, run standards listed in Table
(d) Shaking water baths.—(1) Capable of maintaining 98 2°, 991.43B each time enzyme lot changes or at maximum interval of 6
with automatic on-and-off timer. (2) Constant temperature, adjust- months.
able to 60°.
(e) Balance.—Analytical, sensitivity 0.1 mg. E. Sample Preparation and Digestion
(f) Muffle furnace.—Capable of maintaining 525 5°. Prepare samples as in 985.29E (see 45.4.07) (if fat content of
(g) Oven.—Capable of maintaining 105 and 130 3°. sample is unknown, defat before determining dietary fiber). For high
(h) Desiccator.—With SiO2 or equivalent desiccant. Biweekly, sugar samples, desugar before determining dietary fiber by extract-
dry desiccant overnight at 130°. ing 2–3 times with 85% ethanol, 10 mL/g, decanting, and then
(i) pH meter.—Temperature compensated, standardized with pH drying overnight at 40°.
4.0, 7.0, and 10.0 buffer solutions. Run 2 blanks/assay with samples to measure any contribution
(j) Pipetters.—With disposable tips, 100–300 µL and 5 mL capacity. from reagents to residue.
(k) Dispensers.—Capable of dispensing 15 0.5 mL for 78% Weigh duplicate 1.000 0.005 g samples (M1 and M2), accurate to
ethanol, 95% ethanol, and acetone; 40 0.5 mL for buffer. 0.1 mg, into 400 mL (or 600 mL) tall-form beakers. Add 40 mL
(l) Magnetic stirrers and stir bars. MES–TRIS buffer solution, pH 8.2, to each. Stir on magnetic stirrer
until sample is completely dispersed (to prevent lump formation,
C. Reagents
which would make test material inaccessible to enzymes).
Use deionized water throughout. Add 50 µL heat-stable α-amylase solution, stirring at low speed.
(a) Ethanol solutions.—(1) 85%. Place 895 mL 95% ethanol into Cover beakers with Al foil, and incubate in 95–100° H2O bath 15
1 L volumetric flask, dilute to volume with H2O. (2) 78%. Place
min with continuous agitation. Start timing once bath temperature
821 mL 95% ethanol into 1 L volumetric flask, dilute to volume with reaches 95° (total of 35 min is normally sufficient).
H2O. Remove all beakers from bath, and cool to 60°. Remove foil.
(b) Heat-stable α-amylase solution.—Catalog Number A 3306, Scrape any ring from inside of beaker and disperse any gels in bottom
Sigma Chemical Co., St. Louis, MO 63178, USA, or Termamyl of beaker with spatula. Rinse beaker walls and spatula with 10 mL
300L, Catalog Number 361-6282, Novo-Nordisk, Bagsvaerd, Den- H2O.
mark, or equivalent. Add 100 µL protease solution to each beaker. Cover with Al foil,
(c) Protease.—Catalog Number P 3910, Sigma Chemical Co, or and incubate 30 min at 60 1° with continuous agitation. Start timing
equivalent. Prepare 50 mg/mL enzyme solution in MES/TRIS buff- when bath temperature reaches 60°.
er fresh daily. Remove foil. Dispense 5 mL 0.561N HCl into beakers while
(d) Amyloglucosidase solution.—Catalog Number AMG A9913, stirring. Adjust pH to 4.0–4.7 at 60°, by adding 1N NaOH solution
Sigma Chemical Co, or equivalent. Store at 0–5°. or 1N HCl solution. (Note: It is important to check and adjust pH
(e) Diatomaceous earth.—Acid washed (Celite 545 AW, No. while solutions are 60° because pH will increase at lower tempera-
C8656, Sigma Chemical Co. or equivalent). tures.) (Most cereal, grain, and vegetable products do not require
(f) Cleaning solution.—Liquid surfactant-type laboratory pH adjustment. Once verified for each laboratory, pH checking
cleaner, designed for critical cleaning (Micro®, International Prod- procedure can be omitted. As a precaution, check pH of blank
ucts Corp., Burlington, NJ 08601, USA, or equivalent). Prepare 2%
routinely; if outside desirable range, check samples also.)
solution in H2O.
Add 300 µL amyloglucosidase solution while stirring. Cover with
(g) MES.—2-(N-Morpholino)ethanesulfonic acid (No. M-8250,
Al foil, and incubate 30 min at 60 1° with constant agitation. Start
Sigma Chemical Co., or equivalent.)

Copyright 1998 AOAC INTERNATIONAL

timing once bath reaches 60°. H. Determination of Soluble Dietary Fiber
F. Determination of Total Dietary Fiber Proceed as for insoluble dietary fiber determination through in-
To each digested sample, add 225 mL (measured after heating) struction to combine the filtrate and water washings in pretared 600
95% ethanol at 60°. Ratio of ethanol to sample volume should be mL tall-form beakers. Weigh beakers with combined solution of
4:1. Remove from bath, and cover beakers with large sheets of Al filtrate and water washings, and estimate volumes.
foil. Let precipitate form 1 h at room temperature. Add 4 volumes of 95% ethanol preheated to 60°. Use portion of
Wet and redistribute Celite bed in previously tared crucible B(b), 60° ethanol to rinse filtering flask from IDF determination. Alterna-
using 15 mL 78% ethanol from wash bottle. Apply suction to tively, adjust weight of combined solution of filtrate and water
crucible to draw Celite onto fritted glass as even mat. washings to 80 g by addition of H2O, and add 320 mL 60° 95%
Filter alcohol-treated enzyme digestate through crucible. Using ethanol. Let precipitate form at room temperature 1 h.
wash bottle with 78% ethanol and rubber spatula, quantitatively Follow TDF determination, F, from “Wet and redistribute Celite
transfer all remaining particles to crucible. (Note: If some samples bed . . . .”
form a gum, trapping the liquid, break film with spatula.) I. Calculations
Using vacuum, wash residue 2 times each with 15 mL portions of Blank (B, mg) determination:
78% ethanol, 95% ethanol, and acetone. Dry crucible containing
residue overnight in 105° oven. Cool crucible in desiccator ca 1 h. B = [(BR1 + BR2)/2] – PB – AB
Weigh crucible, containing dietary fiber residue and Celite, to near-
where BR1 and BR2 = residue weights (mg) for duplicate blank
est 0.1 mg, and calculate residue weight by subtracting weight of dry
determinations; and PB and AB = weights (mg) of protein and ash,
crucible with Celite, B(b).
respectively, determined on first and second blank residues.
Use one duplicate from each sample to determine protein, by
Dietary fiber (DF, g/100 g) determination:
method 960.52 (see 12.1.07), using N × 6.25 as conversion factor.
For ash analysis, incinerate second duplicate 5 h at 525°. Cool in DF = {[(R1 + R2)/2] – P – A – B}/[(M1 + M2)/2] × 100
desiccator, and weigh to nearest 0.1 mg. Subtract weight of crucible
and Celite, B(b), to determine ash weight. where R1 and R2 = residue weights (mg) for duplicate samples; P
and A = weights (mg) of protein and ash, respectively, determined
G. Determination of Insoluble Dietary Fiber on first and second residues; B = blank weight (mg); and M1 and M2
Wet and redistribute Celite bed in previously tared crucible, B(b), using = weights (mg) for samples.
ca 3 mL H2O. Apply suction to crucible to draw Celite into even mat. Total dietary fiber determination: Determine either by independent
Filter enzyme digestate, from E, through crucible into filtration flask. analysis, as in F, or by summing IDF and SDF, as in G and H.
Rinse beaker, and then wash residue 2 times with 10 mL 70° H2O.
Reference: J. AOAC Int. 75, 395(1992).
Combine filtrate and water washings, transfer to pretared 600 mL tall-
form beaker, and reserve for determination of soluble dietary fiber, H. *Adopted as a Codex Defining Method for gravimetry/enzymatic di-
Using vacuum, wash residue 2 times each with 15 mL portions of gestion of total dietary fiber in infant formula and follow-up for-
78% ethanol, 95% ethanol, and acetone. (Note: Delay in washing mula.
IDF residues with 78% ethanol, 95% ethanol, and acetone may cause
inflated IDF values.) © AOAC INTERNATIONAL
Use duplicates to determine protein and ash as in F.

Copyright 1998 AOAC INTERNATIONAL