32.1.18 not used regularly. AOAC Official Method 992.16 (j) Freeze-dryer.—For drying food samples with minimum heat Total Dietary Fiber damage (Virtis freeze mobile, with drying chamber No. 10-MR-TR is Enzymatic-Gravimetric Method suitable). First Action 1992 (k) Cutting mill.—Low-speed rotating blades in chamber bot- Final Action 1997 tomed with interchangeable 20 mesh screen (Wiley, intermediate model, Fisher No. 08-338 is suitable). (Note: High-speed rotary (Applicable to determination of total dietary fiber in cereals, beans, mills are not suitable because they produce fine particles which may vegetables, and fruits.) pass through fritted glass .) (l) Desiccator. Method Performance (dry weight basis): Turnip, 20.71%: C. Reagents sr = 1.01; sR = 1.37; RSDr = 4.85%; RSDR = 6.60% (a) Neutral detergent solution.—Dissolve 148.5 g sodium

Wheat bran, 46.30%: lauryl sulfate in 3 L H2O. Dissolve 92.12 g disodium EDTA and ⋅ sr = 0.69; sR = 1.91; RSDr = 1.48%; RSDR = 4.13% 33.70 g sodium tetraborate (Na2B4O7 10 H2O) in 1 L H2O by Bean, 18.19%: stirring and heating and add to sodium lauryl sulfate solution. sr = 0.90; sR = 2.06; RSDr = 4.93%; RSDR = 11.30% Dissolve 22.57 g sodium phosphate dibasic anhydrous

Rice, 1.21%: (Na2HPO4) in 1 L H2O by stirring and heating and add to other sr = 0.18; sR = 0.22; RSDr = 14.73%; RSDR = 17.94% solution. Mix well. pH must be 6.9–7.1; adjust using NaOH or Whole wheat bread, 10.29%: HCl. sr = 0.74; sR = 0.80; RSDr = 7.18%; RSDR = 7.81% (b) Phosphate buffer.—0.1M, pH 7.0 ± 0.1. Mix 610 mL 0.1M Na HPO with 390 mL 0.1M sodium phosphate monobasic monohy- A. Principle 2 4 drate (NaH PO ⋅H O). Food samples, dried and ground, are fat extracted if containing 2 4 2 (c) Sodium acetate solution.—2.0M. Dissolve 164.06 g sodium >5% fat. A portion of sample is treated in with heat stable acetate in H O and dilute to 1 L. amylase, amyloglucosidase, and protease to remove starch and 2 (d) Acetate buffer.—2.0M, pH 4.5 ± 0.1. Mix 200 mL 2.0M sodium protein. Enzymatically undigested fiber is precipitated by ethanol acetate, (c), with 300 mL 2.0M acetic acid. Adjust pH, if needed, by and filtered. Residue is dried, weighed, ashed, and reweighed. A adding sodium acetate or acetic acid. second portion of sample is refluxed with neutral detergent and (e) Ethanol solution.—80%. Dilute 800 mL anhydrous ethanol to 1 treated with α-amylase from porcine pancreas to remove water L with H O. solube carbohydrates and protein. Residue is dried, weighed, ashed, 2 (f) Acetone.—Glass distilled. and reweighed. Total dietary fiber is calculated as sum of the 2 (g) Filter aid.—Celite (No. C-211, Fisher Scientific). No known residues. suitable substitute. (Caution: Celite is a lung, skin, and eye irritant; avoid B. Apparatus inhalation, and contact with skin and eyes.) µ (a) Autoclave or pressure cooker.—Capable of 15 psi. (h) Glass wool.—Borosilicate fiber glass, 8 m diameter, free from (b) Tubes.—50 mL, heavy duty, with screw caps (Pyrex, Fisher fluorine, alumina, and heavy metals (Pyrex is suitable). α α No. 05–558–5B, Fisher Scientific, Pittsburgh, PA 15219, USA or (i) -Amylase solution.—Stir 5.0 g -amylase, Type VI-B Corning No. 8422, Corning, Inc., Corning, NY 14831, USA are (No. A-3176, Sigma Chemical Co., St Louis, MO 63178, USA is suitable). suitable source), with 100 mL phosphate buffer, (b), 15 min. × (c) Ovens.—(1) Forced draft, capable of maintaining 105 ± 1°. Centrifuge 10 min at 1500 g and filter through coarse sintered (2) Capable of maintaining 55 ± 0.5°. glass crucible containing glass wool. Prepare daily and store at (d) Water baths.—(1) Boiling. (2) Capable of maintaining 60 ± 4° when not in use. 0.5°. (j) Amyloglucosidase solution.—Amyloglucosidase from Asper- (e) Balance.—Analytical, sensitive to 0.1 mg. gillus niger (No. A9913, Sigma Chemical Co. is suitable source). (f) Muffle furnace.—With temperature regulator capable of 525 ± 1° Store at 4°. (Fisher Isotemp, Model 497, or Thermoline equipped with Furnatrol (k) Protease solution.—Protease for total dietary fiber assay (No. controller are suitable). 3910, Sigma Chemical Co. is suitable source). Store at 4°. Prepare 50 (g) Neutral detergent fiber extraction system.—Extraction appa- mg/mL H2O just before use. ratus with (1) to fit 600 mL tall-form beaker without (l) Heat stable amylase.—Alpha-amylase for total dietary fiber spout, (2) capable of bringing 100 mL neutral detergent to assay (No. A3306, Sigma Chemical Co. is suitable source). Store at 4°. boiling in 5–10 min, and (3) filtering device equipped with suitable (Caution: Dried enzymes may cause allergic reaction. Handle in holder for crucible (Fibertec system 1, Tecator, Fisher No. TC and avoid inhalation.) 1010-001 is suitable). (h) Filtering system.—Gooch crucible with suitable holder and suc- D. Enzyme Suitability Test tion flask (Fibertec-E, with incubation flasks, Tecator, Fisher No. Every 3 months or each time enzyme lot changes, verify TC-1023-002 is suitable). full enzyme activity and absence of undesirable enzymatic (i) Fritted (sintered) glass .—(1) Gooch type, 50 mL, activities by running the standards listed in Table 992.16 in µ µ coarse, ASTM 40–60 m; or P2 crucibles 40–90 m (Tecator No. 1000 this method. 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

© 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. α ± Add 10 mL cold -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 α ± at room temperature, and evaporate acetone 2 h at 55°. Record -amylase solution and 15 2 mL hot H2O. Incubate 60 min in 55° oven. Filter on filtering device or cold extractor and wash residue weight loss due to fat and/or H2O removal and make appropriate 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 pressure slowly before removing tube from autoclave. Add 0.1 mL G. Calculations Total dietary fiber: heat stable amylase, mix, and incubate in boiling H2O bath 30 min. Filter through coarse Gooch (or P with Fibertec) crucible, contain- 2 TDF, % = {[(C – C )/S ] + [(C – C – B)/S ]} × 100 ing ca 0.5 g Celite, on filtering apparatus equipped with suction flask 2r 2a 2 1r 1a 1

(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 where C = weight of crucible with blank; C = weight of crucible E tubing, with 5 mL hot (95–100°) H2O (adding to filtrate). To b a 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 of crucible with residue, F(2); C = weight of crucible with residue in 60° H2O bath. Add 0.1 mL protease solution, and continue 60° 2a 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