Determination of dietary starch in animal feeds and pet food by an enzymatic-colorimetric method: collaborative study

Starch, glycogen, maltooligosaccharides, and other α-1,4- and α-1,6-linked glucose carbohydrates exclusive of resistant starch are collectively termed "dietary starch". This nutritionally important fraction is increasingly measured for use in diet formulation for animals as it can have positive or negative effects on animal performance and health as it affects energy supply, glycemic index, and formation of fermentation products by gut microbes. AOAC method 920.40 that was used to measure dietary starch in animal feeds, was invalidated due to discontinued production of a required enzyme. As a replacement, enzymatic-colorimetric starch assay developed in 1997 that had advantages in ease of sample handling and accuracy compared to other methods being considered. The assay was further modified to improve utilization of lab resources and reduce time required for the assay. The assay was quasi-empirical: glucose is the analyte detected, but its release is determined by run conditions and specification of enzymes. The modified assay was tested in an AOAC collaborative study to evaluate its accuracy and reliability for determination of dietary starch in animal feedstuffs and pet foods. In the assay, samples are incubated in screw cap tubes with thermostable α-amylase in pH 5.0 sodium acetate buffer for 1 h at 100°C with periodic mixing to gelatinize and partially hydrolyze α-glucan. Amyloglucosidase is then added and the reaction mixture is incubated at 50°C for 2 h and mixed once. After subsequent addition of water, mixing, dilution as needed, and clarification, free + enzymatically released glucose are measured. Values from a separate determination of free glucose are subtracted to give values for enzymatically released glucose. Dietary starch equals enzymatically released glucose multiplied by 162/180 (or 0.9) divided by the weight of the as received sample. Fifteen laboratories representing feed company, regulatory, research, and commercial feed testing laboratories analyzed 10 homogenous animal feedstuffs in duplicate using the dietary starch assay. The test samples ranged from 1% to 70% in dietary starch content and included moist canned dog food, alfalfa pellets, distillers grains, ground corn grain, poultry feed, low starch horse feed, dog food kibbles, complete dairy cattle feed, soybean meal, and corn silage. The average within-laboratory repeatability SD(S(r)) for percentage dietary starch in the test samples was 0.49 with a range of 0.03 to 1.56, and