An improved method for a rapid determination of phytase activity in animal feed

The current direct colorimetric assay for phytase activity in feeds has interference from high P background and other factors. Our objective was to develop a rapid and reliable spin column method to accurately determine phytase activity in feed ingredients or complete diets. After the feed sample was extracted by stirring in 0.2 M citrate buffer, pH 5.5, for 30 min at room temperature, the oily layer of the supernatant fraction was removed by passing through an acrodisc syringe filter (0.45-microm HT Tuffryn membrane, Gelman Laboratory, Ann Arbor, MI). The filtrate was then loaded onto a spin column (MW cutoff 30,000, Millipore, Bedford, MA) to remove free phosphate before the phytase activity assay. Compared with the direct assay, this new procedure improved both accuracy and reproducibility. When diets contained phytase at 0 to 1,500 U/kg (as fed), the CV for multiple assays of the same samples (n = 6) by the new method ranged from 1 to 6% compared with 28 to 39% by the direct method. A linear relationship was found between the added phytase activity in practical diets and the analyzed activity by the new method (r2 = 0.99; P < 0.01). In conclusion, the spin column method is an improved assay for phytase activity in animal feed, and may be used for quality control of phytase supplementation.