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OMA 2014.10 C: Collaborative Study Manuscript Expert Review Panel Use Only September, 2017

2013 Dietary Starch in Animal Feeds Collab Study Protocol 072513

TITLE PAGE: Determination of Dietary Starch in Animal Feeds and Pet Food by an Enzymatic-Colorimetric Method. Collaborative Study Protocol Background: There has been increased interest in the analysis of starch in animal feeds for its positive effects on animal performance, and potential undesirable effects on glycemic response and animal health (1). The AOAC Method 14.075 for starch in animal feeds (2) is no longer valid because of discontinued production of the enzyme “Rhozyme-S” (Rohm and Haas, Philadelphia, PA) specified in the procedure. Accordingly, another approved method for starch in animal feeds is needed. Additionally, new terminology is needed to define “starch” to more accurately describe the nutritionally relevant fraction of interest and what is actually being measured. The definition for the nutritionally relevant fraction can be used to specify the needed analysis. Starch has long been defined as a natural vegetable polymer consisting of long linear unbranched chains of 1,4- α -D-glucose units (amylose) and/or long α -1,6-branched chains of α -1,4- linked glucose units (amylopectin) (3). However, the amylases and amyloglucosidases that specifically hydrolyze the linkages in plant starch, also hydrolyze those same linkages in glycogen from animal (4) or microbial (5) sources and in maltooligosaccharides that are breakdown products of starch, but are not polysaccharides. Accordingly, enzymatic starch methods do not measure plant starch alone (6), unless animal and microbial ingredients and the feedstuffs that contain them are excluded from analysis. From a nutritional stand point, inclusion of glycogen, starch, and maltooligosaccharides more completely describes the pool of carbohydrate that is potentially available to digestion by salivary or small intestinal amylases or amyloglucosidases (7), but the pool can’t be called “starch” because that term is well established as referring to a plant polysaccharide. Recognizing the aim of nutritional characterization, the AAFCO Laboratory Methods & Services Committee with involvement of researchers and industry arrived at a definition for “Dietary Starch”: An alpha-linked-glucose carbohydrate of or derived from plants, animals, or microbes from which glucose is released through the hydrolytic actions of purified alpha-amylases and amyloglucosidases that are specifically active only on alpha-(1-4) and alpha-(1-6) linkages in feed materials that have been gelatinized in heated, mildly acidic buffer. Its concentration in feed is determined by enzymatically converting the alpha-linked-glucose carbohydrate to glucose and then measuring the liberated glucose. This definition encompasses plant starch, glycogen, maltooligosaccharides and maltose/isomaltose. The use of mildly acidic buffer for the gelatinization excludes the use of alkali or dimethyl sulfoxide and thus excludes resistant starch from inclusion in the dietary starch fraction. The next candidate method for starch analysis in animal feeds should be accurate, repeatable, robust, and avoid known analytical defects. Bach Knudsen published such an assay (7) that was then compared to other starch assays, and slightly modified to improve use of lab resources, reduce run time, and maintain starch recovery (8). It is similar in chemistry to AOAC Method 996.11 (9), but yielded a greater recovery of starch. The candidate method also differs in buffer used and in sample handling procedures. Specific to this assay, all enzymatic reactions are carried out in an acidic buffer which improves recovery by limiting the production of maltulose, an isomerization product produced at more neutral pH (10). Maltulose is resistant to enzymatic hydrolysis and reduces starch recovery. The use of a screw cap tube as a reaction vessel allows for more vigorous mixing which is useful for all types of feed materials, but may be essential for those that clump and do not behave like dry, ground powders. Although enzymes used in AOAC Research Institut ERP Use Only

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