AOAC Official Method 2002.04 Amylase-Treated Neutral Detergent Fiber in Feeds Using Refluxing in Beakers or Crucibles First Action 2002

A. Principle

Fiber in feeds is a nutritionally defined entity that represents the indigestible and slowly digesting fraction of feeds. Neutral detergent (ND) solution and heat-stable -amylase are used to dissolve easily digested proteins, lipids, sugars, starches, and pectins in feeds, leaving a fibrous residue that is primarily cell wall components in plant materials (cellulose, hemicellulose, and lignin) and indigestible nitrogenous matter in animal products. aNDF is a gravimetric method that best estimates the total insoluble fiber, and is inversely related to digestibility and intake potential of a feed. ND soluble matter is almost completely digested by most animals; however, the digestibility of aNDF is variable among feeds and is related to lignin and other constituents in fiber.
Sodium lauryl sulfate, an anionic detergent, and sodium sulfite are used to solubilize nitrogenous matter; EDTA is used to chelate calcium and enhance removal of pectins at boiling temperatures; triethylene glycol helps remove some nonfibrous matter from concentrated feeds; disodium phosphate and sodium borate are used as buffers to maintain a neutral pH.
Hot ND solution has limited ability to solubilize starch; therefore, a heat-stable amylase is used to hydrolyze starch to saccharides that can be easily removed from fiber by filtration. Heat-stable amylases are used in hot solutions to inactivate potential contaminating enzymes in crude amylase extracts that might degrade fibrous constituents. To ensure that the amylase activity is sufficient to remove most starch and to reduce filtering difficulties, the amount of any specific amylase source needed to measure aNDF is determined under the conditions of the aNDF method.
Although boiling ND solution dissolves most proteins, lipids, and nonfibrous carbohydrates, these constituents are nonviscous only in water that is near boiling temperature. Therefore, boiling water is needed for washing fibrous residues and removing nonfibrous matter. Because fiber is particulate matter, mass-action equilibration during soaking is needed to migrate ND and contaminating soluble matter from the interior of fiber particles. Fibrous residues must be soaked, instead of being simply rinsed, in near boiling water to remove nonfibrous matter.
Boiling ND solution solubilizes lipids, and acetone soaking of the residue completes the extraction of lipids and pigments in most materials. However, excessive amounts of lipids in materials can complex with the detergent and reduce extraction efficiency. Because extraction of lipids by ND and acetone may be incomplete when feeds contain >10% lipid, these materials are pre-extracted to ensure complete removal of lipid contamination from fiber. Pre-extract materials with 5–10% lipid to minimize filtration difficulties and avoid variable aNDF results.