32.1.18
AOAC Official Method 992.16 (j) Freeze-dryer.—For drying food samples with minimum heat
Total Dietary Fiber
Enzymatic-Gravimetric Method
First Action 1992
Final Action 1997
(Applicable to determination of total dietary fiber in cereals, beans,
vegetables, and fruits.)
Method Performance (dry weight basis):
Turnip, 20.71%:
sr = 1.01; sR = 1.37; RSDr = 4.85%; RSDR = 6.60%
Wheat bran, 46.30%:
sr = 0.69; sR = 1.91; RSDr = 1.48%; RSDR = 4.13%
Bean, 18.19%:
sr = 0.90; sR = 2.06; RSDr = 4.93%; RSDR = 11.30%
Rice, 1.21%:
sr = 0.18; sR = 0.22; RSDr = 14.73%; RSDR = 17.94%
Whole wheat bread, 10.29%:
sr = 0.74; sR = 0.80; RSDr = 7.18%; RSDR = 7.81%
A. Principle
Food samples, dried and ground, are fat extracted if containing
>5% fat. A portion of sample is treated in autoclave with heat stable
amylase, amyloglucosidase, and protease to remove starch and
protein. Enzymatically undigested fiber is precipitated by ethanol
and filtered. Residue is dried, weighed, ashed, and reweighed. A
second portion of sample is refluxed with neutral detergent and
treated with α-amylase from porcine pancreas to remove water
solube carbohydrates and protein. Residue is dried, weighed, ashed,
and reweighed. Total dietary fiber is calculated as sum of the 2
residues.
B. Apparatus
(a) Autoclave or pressure cooker.—Capable of 15 psi.
(b) Tubes.— 50 mL, heavy duty, with screw caps (Pyrex, Fisher
No. 05–558–5B, Fisher Scientific, Pittsburgh, PA 15219, USA or
Corning No. 8422, Corning, Inc., Corning, NY 14831, USA are
suitable).
(c) Ovens.—(1) Forced draft, capable of maintaining 105 ± 1°.
(2) Capable of maintaining 55 ± 0.5°.
(d) Water baths.—(1) Boiling. (2) Capable of maintaining 60 ±
0.5°.
(e) Balance.—Analytical, sensitive to 0.1 mg.
(f) Muffle furnace.—With temperature regulator capable of 525 ± 1°
(Fisher Isotemp, Model 497, or Thermoline equipped with Furnatrol
controller are suitable).
(g) Neutral detergent fiber extraction system.—Extraction appa-
ratus with (1) condenser to fit 600 mL tall-form beaker without
spout, (2) hot plate capable of bringing 100 mL neutral detergent to
boiling in 5–10 min, and (3) filtering device equipped with suitable
holder for crucible (Fibertec system 1, Tecator, Fisher No. TC
1010-001 is suitable).
(h) Filtering system.—Gooch crucible with suitable holder and suc-
tion flask (Fibertec-E, with incubation flasks, Tecator, Fisher No.
TC-1023-002 is suitable).
(i) Fritted (sintered) glass crucibles.—(1) Gooch type, 50 mL,
coarse, ASTM 40–60 µm; or P2 crucibles 40–90 µm (Tecator No. 1000
1172). (2) Gooch type, 50 mL, medium ASTM, 10–15 µm (Fisher No.
08-237-1B) with rubber ring adaptors. Heat 2 h at 525° before use, if
not used regularly.
damage (Virtis freeze mobile, with drying chamber No. 10-MR-TR is
suitable).
(k) Cutting mill.—Low-speed rotating blades in chamber bot-
tomed with interchangeable 20 mesh screen (Wiley, intermediate
model, Fisher No. 08-338 is suitable). (Note: High-speed rotary
mills are not suitable because they produce fine particles which may
pass through fritted glass crucible.)
(l) Desiccator.
C. Reagents
(a) Neutral detergent solution.— Dissolve 148.5 g sodium
lauryl sulfate in 3 L H2O. Dissolve 92.12 g disodium EDTA and
33.70 g sodium tetraborate (Na2B4O7⋅10 H2O) in 1 L H2O by
stirring and heating and add to sodium lauryl sulfate solution.
Dissolve 22.57 g sodium phosphate dibasic anhydrous
(Na2HPO4) in 1 L H2O by stirring and heating and add to other
solution. Mix well. pH must be 6.9–7.1; adjust using NaOH or
HCl.
(b) Phosphate buffer.—0.1M, pH 7.0 ± 0.1. Mix 610 mL 0.1M
Na2HPO4 with 390 mL 0.1M sodium phosphate monobasic monohy-
drate (NaH2PO4⋅H2O).
(c) Sodium acetate solution.—2.0M. Dissolve 164.06 g sodium
acetate in H2O and dilute to 1 L.
(d) Acetate buffer.—2.0M, pH 4.5 ± 0.1. Mix 200 mL 2.0M sodium
acetate, (c), with 300 mL 2.0M acetic acid. Adjust pH, if needed, by
adding sodium acetate or acetic acid.
(e) Ethanol solution.—80%. Dilute 800 mL anhydrous ethanol to 1
L with H2O.
(f) Acetone.—Glass distilled.
(g) Filter aid.—Celite (No. C-211, Fisher Scientific). No known
suitable substitute. (Caution: Celite is a lung, skin, and eye irritant; avoid
inhalation, and contact with skin and eyes.)
(h) Glass wool.—Borosilicate fiber glass, 8 µm diameter, free from
fluorine, alumina, and heavy metals (Pyrex is suitable).
(i) α-Amylase solution.—Stir 5.0 g α-amylase, Type VI-B
(No. A-3176, Sigma Chemical Co., St Louis, MO 63178, USA is
suitable source), with 100 mL phosphate buffer, (b), 15 min.
Centrifuge 10 min at 1500 × g and filter through coarse sintered
glass crucible containing glass wool. Prepare daily and store at
4° when not in use.
(j) Amyloglucosidase solution.—Amyloglucosidase from Asper-
gillus niger (No. A9913, Sigma Chemical Co. is suitable source).
Store at 4°.
(k) Protease solution.—Protease for total dietary fiber assay (No.
3910, Sigma Chemical Co. is suitable source). Store at 4°. Prepare 50
mg/mL H2O just before use.
(l) Heat stable amylase.— Alpha-amylase for total dietary fiber
assay (No. A3306, Sigma Chemical Co. is suitable source). Store at 4°.
(Caution: Dried enzymes may cause allergic reaction. Handle in
fume hood and avoid inhalation.)
D. Enzyme Suitability Test
Every 3 months or each time enzyme lot changes, verify
full enzyme activity and absence of undesirable enzymatic
activities by running the standards listed in Table 992.16 in
this method.
© 1998 AOAC INTERNATIONAL
Table 992.16 Standards for Testing Enzyme Activity Dry crucible and contents overnight at 105° in forced-draft oven. Cool
in desiccator to room temperature and weigh to nearest 0.1 mg (C1r).
Weight of Activity Expected Ash residue 4 h at 525°. Cool in desiccator to room temperature and
Standard standard, g tested recovery, %
weigh to nearest 0.1 mg (C1a).
Cornstarch 0.5 Amylase 0–1 (b) Accurately weigh (S2) second set of duplicate 0.5 g portions
(Sigma S-2388) of sample to nearest 0.1 mg, into 600 mL tall form beaker or P2
Wheat starch 0.5 Amylase 0–1 crucible. Add 100 mL neutral detergent solution to beaker; place
(Sigma S-1514) on hot plate and fit condenser (or fit P2 crucible in hot extractor and
Casein 0.5 Protease 0–1 add 100 mL neutral detergent solution, preheated to 80°, to boiling
(Sigma C-7906) column). Heat to boiling within 5–10 min; then reduce heat and reflux
β-Glucan 0.1 β-Glucanase 90–95 60 min from onset of boiling. Filter through coarse Gooch, or P2
(Sigma G-7391) crucible. Wash residue with ca 100 mL hot H2O. If filtration is
Citrus pectin 0.1 Pectinase 80–85 difficult, apply any of following procedures to facilitate filtration: (1)
(Sigma P-7536) apply back pressure, (2) add 100 µL heat stable amylase, (3) add ca 0.5
Arabinogalactan 0.2 Hemicellulase 95–100 g Celite to crucible, (4) reduce sample weight to 0.3 g and analyze in
(Sigma A-9788) triplicate, or (5) clean crucible or (6) use new crucible.
Add 10 mL cold α-amylase solution and 15 ± 2 mL hot H2O to
E. Sample Preparation crucible and hold 5 min on filtering device or hot extractor without
Freeze-dry wet samples. Grind samples using cutting mill fitted heating. Apply suction to remove enzyme solution and wash residue
with 20 mesh screen at bottom of cutting chamber. If fat content is with ca 20 mL hot H2O. Stopper bottom of crucible (use No. 8 rubber
≥5%, defat dried sample by adding 4 volumes acetone, stirring 1 h stopper for Gooch crucible, or No. 7 for P2 crucible) and add 10 mL cold
at room temperature, and evaporate acetone 2 h at 55°. Record α-amylase solution and 15 ± 2 mL hot H2O. Incubate 60 min in 55°
weight loss due to fat and/or H2O removal and make appropriate oven. Filter on filtering device or cold extractor and wash residue
correction to sample weight in calculation of % dietary fiber. successively with ca 100 mL hot H2O and two 20 mL portions of
acetone. Discard eluates.
F. Fiber Determination Dry crucible and contents overnight at 105° in forced-draft oven. Cool
(a) Accurately weigh (S1) duplicate 0.5 g portions of sample to in desiccator to room temperature and weigh to nearest 0.1 mg (C2r).
nearest 0.1 mg into 50 mL screw cap tubes. Run duplicate reagent Ash residue 4 h at 525°. Cool in desiccator to room temperature and
blanks. Add 20 mL H2O and 2 mL acetate buffer and mix. Autoclave reweigh (C2a).
60 min at 120° and 15 psi (with cap loosened). Decrease autoclave
G. Calculations
pressure slowly before removing tube from autoclave. Add 0.1 mL
heat stable amylase, mix, and incubate in boiling H2O bath 30 min. Total dietary fiber:
Filter through coarse Gooch (or P2 with Fibertec) crucible, contain-
TDF, % = {[(C2r – C2a)/S2] + [(C1r – C1a – B)/S1]} × 100
ing ca 0.5 g Celite, on filtering apparatus equipped with suction flask
(or Fibertec E equipped with incubation flask) to receive filtrate. Blank (B) = Cb – Ca
Rinse tube with 10 mL hot H2O and add rinse to crucible (adding to
filtrate). Remove crucible and rinse the filtering device, or Fibertec
E tubing, with 5 mL hot (95–100°) H2O (adding to filtrate). To where Cb = weight of crucible with blank; Ca = weight of crucible
combined filtrate, add 4 mL sodium acetate solution and mix. Add with blank after ashing; C1r = weight of crucible with residue, F(1);
0.3 mL amyloglucosidase solution, cover, mix, and incubate 30 min C1a = weight of crucible with residue after ashing, F(1); C2r = weight
in 60° H2O bath. Add 0.1 mL protease solution, and continue 60° of crucible with residue, F(2); C2a = weight of crucible with residue
incubation for 30 min. after ashing, F(2); and S1 and S2 = weights of dry samples.
Add 4 volumes (166 mL) of anhydrous ethanol and mix. Let precipi- References: Cereal Foods World 35, 319(1990);
tate form at room temperature ≥60 min. Filter mixture through medium J. AOAC Int. 76, 923(1993).
Gooch crucible containing glass wool. Rinse residue successively with
two 20 mL portions 80% ethanol and two 20 mL portions of acetone. Revised: March 1998
Discard eluates.

© 1998 AOAC INTERNATIONAL