The enzyme preparation is suitable if more than 90% of the original weight of pectin, arabinogalactan, and -glucan is recovered; NMT 2% of the original weight of casein and corn starch is recovered; and NMT 1% of the original weight of wheat starch is recovered. [Note—Test the enzyme purity of every new lot of enzyme and at 6-month intervals thereafter. ]

Add 125 µL of heat-stable -amylase solution, and stir to ensure uniform mixing. Cover the beaker with aluminum foil, and incubate over a water bath maintained at 95–100 for 15 min, with continuous agitation. [Note—Start timing once the water bath temperature reaches 95. A total time of 35 min is usually sufficient. ]

Remove the beaker from the water bath, and cool to 60. Remove the aluminum foil, scrape any ring from inside the beaker, and disperse any gels in the bottom of the beaker with a spatula. Rinse the walls of the beaker and the spatula with 10 mL of water, collecting the rinsings in the beaker. Add 500 µL of Protease solution. Cover with aluminum foil, and incubate over a water bath maintained at 60 ± 3 for 30 min with continuous agitation. [Note—Start timing when the bath temperature reaches 60. ]

Remove the foil, and transfer 5 mL of Acid solution while stirring. Adjust, if necessary, with 1 N sodium hydroxide or 1 N hydrochloric acid to a pH of 4.28 ± 0.07 at 60. [Note—It is important to adjust the pH to 4.28 while the solution in the beaker is maintained at 60; otherwise, the pH will increase at lower temperatures. ]

Add 150 µL of amyloglucosidase solution while stirring. Cover with aluminum foil, and incubate over a water bath maintained at 60 ± 3 for 30 min with constant agitation. [Note—Start timing once the water bath reaches 60. ]

Transfer 40 mL of the beaker contents to a 50-mL centrifuge tube, and sonicate the tube contents for 3 min.1 Centrifuge at 10,000–14,000 rpm for 10 min. Carefully pour the supernatant into a labeled 600-mL tared beaker. Do not disturb any pellet in the bottom of the centrifuge tube. Add the remaining sample from the original 400-mL beaker into the centrifuge tube still containing the pellet. Rinse the 400-mL beaker with 15–20 mL of water, and add the rinsing to the 50-mL centrifuge tube. Centrifuge the sample at 10,000–14,000 rpm for 10 min. Carefully pour the supernatant into the 600-mL beaker containing the first supernatant. Add 390 mL (measured before heating) of alcohol at 60 to the 600-mL beaker. Cover the beaker, and allow to stand for at least 1 h to form a precipitate.

Place 3 g of chromatographic siliceous earth into a clean air-dried crucible with a fritted disk. Heat the crucible containing chromatographic siliceous earth at 525 in a muffle furnace for at least 4 h. Cool. Pass deionized water through the crucible while applying constant suction. Rinse with acetone, and allow to air-dry. Store the crucible in a convection oven at 130 for at least 2 h before use. Weigh the prepared crucible to 0.1 mg before use. Wet the chromatographic siliceous earth in the crucible using a stream of Alcohol solution from a washing bottle, and apply suction to evenly distribute the chromatographic siliceous earth over the fritted disk. Maintaining the suction, transfer the supernatant and precipitate from the beaker to the crucible, and filter. Transfer any solid residue in the beaker with the aid of Alcohol solution. [Note—In some cases, gums may form during filtration, trapping liquid in the residue. If so, break the surface film with a spatula to improve filtration. ]

Wash the residue in the crucible sequentially with 30 mL of Alcohol solution, 20 mL of alcohol, and 20 mL of acetone. Dry the crucible containing the residue at 100 in a convection oven for at least 4 h, and cool to room temperature in a desiccator.

Determine the weight of the residue (R).

Use one of the duplicate residues from the Sample solution and one of the blank residues from the Blank solution to determine the protein content, in mg, by placing the residue in a 500-mL Kjeldahl flask, and proceeding as directed for Nitrogen Determination 461, Method I. The protein content is determined by multiplying the content of nitrogen found by 6.25. Incinerate the residue from the remaining duplicate of the Sample solution and the Blank solution as directed for Articles of Botanical Origin 561, Total Ash at a reduced temperature of 525, and determine the ash content as directed.

Calculate the corrected average weight of the blank (B), in mg:

Calculate the percentage of soluble dietary fiber:

(See Chromatography 621, System Suitability.)

Calculate the percentage of alcohol in the portion of Psyllium Hemicellulose taken: