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

Authors: Hall, Mary Beth

Submitted to: Journal of Association of Official Analytical Chemists International
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/13/2015
Publication Date: 3/1/2015
Publication URL: http://handle.nal.usda.gov/10113/62060
Citation: Hall, M. 2015. Determination of dietary starch in animal feeds and pet food by an enzymatic-colorimetric method: collaborative study. Journal of Association of Official Analytical Chemists International. 98:397-409.

Interpretive Summary: Animal nutritionists have increasingly measured dietary starch in feeds for use in diet formulation for animals. Dietary starch 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. Presently, there is no official method to measure dietary starch in animal feeds because an older starch assay can no longer be used. Without an official assay, there is no method with which companies can provide nutritionists and consumers with information on the dietary starch content of animal feeds. A new assay for measurement of dietary starch in animal feeds was tested. Fifteen laboratories that represented feed company, regulatory, research, and commercial feed testing laboratories analyzed 10 diverse feedstuffs that ranged from 1% to 73% in dietary starch content. The precision of the assay within and among laboratories indicated that the assay is suitable for use as an official regulatory method. This is an important step in providing companies with a reliable way to describe the nutritional value of animal feeds for consumers and nutritionists.

Technical Abstract: Starch, glycogen, maltooligosaccharides, and other alpha-1,4- and alpha-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 14.075, used to measure dietary starch in animal feeds, was invalidated due to discontinued production of a required enzyme. An enzymatic-colorimetric starch assay developed in 1997 was considered as a replacement assay, and 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 action of specific 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. In the assay, samples are incubated in screw cap tubes with thermostable alpha-amylase in pH 5.0 sodium acetate buffer for 1 h at 100C with periodic mixing to gelatinize and partially hydrolyze alpha-glucan. Amyloglucosidase is then added and the reaction mixture is incubated at 50C for 2 h and mixed once. After subsequent addition of water, mixing, dilution as needed, and clarification, free and 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 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 73% 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 standard deviation for percentage dietary starch in feeds was 0.48 with a range of 0.03 to 1.52, and among-laboratory repeatability standard deviations averaged 1.00 with a range of 0.11 to 2.69. The Horwitz Ratio (HORRAT) averaged 2.0 for test samples with greater than 2% dietary starch, which is comparable to results found for AOAC method 996.11. For all test samples, HORRAT averaged 2.2. It is recommended that the dietary starch method be accepted for Official First Action status.