Effects of temperature, pH, incubation time and pepsin concentration on the in vitro stability of intrinsic phytase of wheat, barley and rye

Three experiments were conducted to evaluate the effects of temperature (T), pH, time of incubation and pepsin on the stability of intrinsic phytase of different cereals. Experiment one was conducted as a 3x4x3x3 factorial arrangement with three feed ingredients (rye, wheat and barley). four T (20, 38, 55 and 80 degrees C), three pH levels (3.0, 5.5 and 8.0) and three incubation times (30,60 and 120 min), with two replicates per treatment. Stability was calculated as the ratio of the residual phytase activity of a treated sample to the non-treated (reference sample) times 100. Phytase activity of the reference samples of rye, wheat and barley was 3.14, 1.77 and 0.66 FTU/g, respectively. Overall, rye showed the highest and barley the lowest stability (0.58 vs. 0.47, P<0.001). Stability decreased with increasing T to 0.07 at 80 degrees C across ingredients, pH levels and time of incubation (P<0.001). Stability of intrinsic phytase decreased with increasing incubation time (P<0.001). Stability was highest at pH 3.0 and lowest at pH 8.0, respectively (0.65 vs. 0.34 across ingredients, T and time of incubation, P<0.001). Combinations of low pH and low temperatures (20 and 38 degrees C) resulted in the highest stability of intrinsic phytase (>0.90). Wheat and rye phytases were stable at pH 3.0 and 5.5(20 and 38 degrees C). In experiment two, the effect of shorter period of time (10 and 30 min) was assessed on the stability of cereal phytases. Barley phytase showed the highest residual phytase activity among cereals at 65 degrees C. In experiment three, stability of intrinsic phytase of these feed ingredients was determined after incubation in different concentrations of pepsin (0,5 and 10 mg/mL) at pH 2.0. Intrinsic phytases of wheat and rye were resistant to pepsin, but barley phytase was susceptible to pepsin and its stability decreased to 0.57 after pre-incubation for 60 min in 5 mg pepsin/mL. This knowledge regarding the effect of T, incubation time and pH on stability of cereal intrinsic phytases may contribute to optimize available P contents of diets, thereby reducing phosphorus excretion of monogastrics. (C) 2012 Elsevier B.V. All rights reserved.