Phosphate buffer— Prepare a pH 6.0 phosphate buffer (see Buffer Solutions under Solutions in the section Reagents, Indicators, and Solutions).

Protease solution— Dissolve 5 mg of protease in 0.1 mL of Phosphate buffer.

Sample preparation— Prepare two samples in parallel. In order to correct for any contribution from reagents, also perform examinations of blanks, which are treated similarly to the samples. Proceed as directed for Limit of fat. Mill to a coarse powder, and store in a desiccator.

Procedure— Transfer about 1.0 g of each Sample preparation, accurately weighed, into separate 400-mL, tall-form beakers. Add 50 mL of Phosphate buffer, and adjust the pH, if necessary, to 6.0 ± 0.1. Add 0.2 mL of heat-stable -amylase solution. Cover the beaker with aluminum foil, place in a boiling water bath for 15 minutes at 100, shaking gently every 5 minutes, and cool to room temperature. Adjust with about 10 mL of 0.275 N sodium hydroxide solution to a pH of 7.5 ± 0.1. Add freshly prepared Protease solution, cover the beaker with aluminum foil, and incubate for 30 minutes at 60 with continuous agitation. Cool, and add about 10 mL of 0.325 N hydrochloric acid and adjust to a pH of 4.5 ± 0.2. Add 0.3 mL of amyloglucosidase, cover with aluminum foil, and incubate for 20 minutes at 60 with continuous agitation. Heat 280 mL of alcohol to 60, add to the digest, and allow the precipitate to form at room temperature for 60 minutes. Place 0.5 g of chromatographic siliceous earth in a crucible with fritted disk, dry at 130 to constant weight, and weigh. Wet the chromatographic siliceous earth in the crucible by using a stream of 78% alcohol from a washing bottle, and apply suction to evenly distribute the chromatographic siliceous earth over the fritted disk. Maintain suction, and quantitatively transfer the enzyme digest precipitate to the crucible. Wash the residue successively with three 20-mL portions of 78% alcohol, two 10-mL portions of alcohol, and two 10-mL portions of acetone. In some cases, gums may form during filtration, trapping liquid in residue. If so, break the surface film with a spatula to improve filtration. Dry the crucible containing the residue at 105 in an air oven for 16 hours, cool in a desiccator, and determine the weight of the residue. Determine the percentage of protein in the first Sample preparation as directed for Limit of protein. Incinerate the residue from the second Sample preparation as directed for Total Ash under Articles of Botanical Origin 561. Calculate the corrected weight, W, of the sample residue by the formula: in which WU and WB are the average weights of the sample residues and the blank residues, respectively; PU and PB are percentages of protein present in the sample and in the blank, respectively; and AU and AB are percentages of ash found in the sample and in the blank, respectively. Then calculate the percentage of the total dietary fiber in the portion of Wheat Bran taken by the formula: in which WI is the weight of the Sample preparation taken. Correct the final percentage of the total dietary fiber for fat and for water: not less than 36.0% is found.