A. Reagents

(a) Neutral Detergent Solution (ND) -- Add 30.0 g sodium lauryl sulfate, USP; 18.61 g Ethylenediaminetetraacetic Disodium Salt, Dihydrate; 6.81 g sodium tetraborate decahydrate; 4.56 g sodium phosphate dibasic, anhydrous; and 10.0 ml triethylene glycol, in 1 L distilled H2O (ANKOM Technology, premixed chemical solution - FND20 or FND20C). Agitate and heat to facilitate solubility. Check pH range to 6.9 to 7.1.

(b) Alpha-amylase -- Heat-stable bacterial alpha-amylase: activity = 340,000 Modified Wohlgemuth Units / ml (ANKOM Technology - FAA). One Modified Wohlgemuth Unit is that activity which will dextrinize 1.0 mg of soluble starch to a defined size dextrin in 30 minutes.

(c) Sodium sulfite -- Na2SO3, anhydrous (ANKOM Technology - FSS).

(d) Acetone -- Use grade that is free from color and leaves no residue upon evaporation (ANKOM Technology - FACE).

B. Safety Precautions -- see MSDSs in ringed binder located near eye wash station

(a) Acetone is highly flammable. Use fume hood when handling acetone and avoid inhaling or contact with skin. Make sure bags are completely dry and that all the acetone has evaporated before placing in oven.

(b) Sodium lauryl sulfate will irritate the mucous membranes. A dust mask and gloves should be worn when handling this chemical.

C. Apparatus

(a) Digestion apparatus - ANKOM200/220 Fiber Analyzer

(b) Filtration device - ANKOM Technology - F57 Filter Bags

(c) Impulse bag sealer - Requires high enough temperature to melt and seal polymer in filter bags (ANKOM Technology - 1915/1920).

(d) Desiccator -- ANKOM Technology - MoistureStop weigh pouch - F39

D. Procedure

(a) Prepare Sample

1) Weigh Filter Bag (W1) record weight and tare balance.

2) Weigh 0.5g (±0.05 g) of air-dried sample (W2), ground to pass through a 1mm screen, directly into filter bag. Weigh one blank bag and include in digestion to determine blank bag correction (C1).

3) Seal the bags closed within 0.5cm from the open edge using the heat sealer.

4) Spread sample uniformly inside the filter bag by shaking and lightly flicking the bag to eliminate clumping.

5) 1.A maximum of 24 bags may be placed in the bag suspender. All nine trays are used regardless of the number of bags being processed. Place three bags per tray and then stack trays on center post with each level rotated 120 degrees. The weight is placed on top of the empty 9th tray to keep the bag suspender submerged.
2. NOTE: SAMPLES CONTAINING SOY PRODUCT OR >5% FAT - Extract fat from samples by placing 24 bags with samples into a 500 ml bottle with a top. Pour enough acetone into bottle to cover bags and secure top. Shake the container 10 times and allow bags to soak for 10 minutes. Repeat with fresh acetone. Pour out acetone and place bags on a wire screen to air-dry (approximately 5 minutes). EXCEPTION: Roasted Soy - Due to special properties of Roasted Soy a modification to the fat extraction is required. Place Roasted Soy samples into a 500ml bottle with a top. Pour enough acetone into bottle to cover bags and secure top. Shake the container 10 times and pour off acetone. Add fresh acetone and allow samples to soak for twelve hours. After soak time, drain off acetone as stated above and allow to air-dry before next step.

(b) When processing 24 sample bags add 1900 - 2000ml of ambient Neutral Detergent solution into ANKOM Fiber Analyzer vessel. If processing less than 20 bags add 100 ml/bag of detergent solution (minimum of 1500 ml (ensure Bag Suspender is covered)). Add 20 g (0.5 g/50 ml of ND solution) of sodium sulfite to the solution in the vessel and 4.0 ml of heat stable alpha-amylase.

(c) Place bag suspender with samples into the solution in vessel. Turn Agitate and Heat ON and confirm that Bag Suspender is agitating properly. Set timer for 75 minutes and push Start. Close and seal lid of vessel.

(d) After 75 minutes (timer will beep) turn Agitate and Heat OFF, open the drain valve and exhaust hot solution before opening lid. WARNING: The solution in vessel is under pressure. The valve should be opened first to remove pressure before lid can be opened. Ensure exhaust hose is securely positioned for safe disposal of effluent.

(e) After the solution has been exhausted close valve and open the lid. Add approximately 2000 ml of hot (90-100° C) H2O and 4.0 ml of alpha-amylase to the first and second rinses. Lower lid but do not tighten. Turn Agitate ON and leave Heat OFF. Each rinse should last 3-5 minutes. Exhaust water and repeat rinse two more times (total of three rinses).

(f) Remove filter bags from bag suspender and gently press out excess water. Place in beaker and soak in acetone. Allow bags to soak 3 minutes then remove and lightly press out excess acetone.

(g) Spread bags out and allow acetone to evaporate. Complete drying in oven at 105° C for at least 2 hours. WARNING: Do not place bags in the oven until acetone has completely evaporated. Longer drying period may be required depending on oven and frequency of sample introduction into the oven. Remove bags from oven, place directly into MoistureStop weigh pouch and flatten to remove air. Cool to ambient temperature and weigh bags (W3).

E. Calculate percent: