Animal Feed Science and Technology 170 (2011) 209–221 Contents lists available at SciVerse ScienceDirect Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci In vitro assessment of the effects of phytate and phytase on nitrogen and phosphorus bioaccessibility within fish digestive tract Gabriel Alejandro Morales ∗ , Francisco Javier Moyano, Lorenzo Marquez Departamento Biología Aplicada, Escuela Politécnica Superior, Universidad de Almería, La Ca˜ nada S Urbano s/n, 04120 Almería, Spain a r t i c l e i n f o Article history: Received 31 March 2011 Received in revised form 18 August 2011 Accepted 19 August 2011 Keywords: Aquaculture In vitro digestion Phytases Phytate–protein complexing Digestive proteases a b s t r a c t The aim of the present work was to assess different possible interactions between phytate (IP6), dietary protein and digestive proteases, as well as the preventive effect of phytase in the formation of protein-IP6 complexes, through the use of a gastrointestinal model sim- ulating conditions existing in the digestive tract of rainbow trout (Oncorhynchus mykiss). Two commercial phytases, expressed in P. lycii and E. coli, were evaluated. Fungal phy- tase showed an optimum at pH 5.5 and bacterial phytase showed two peaks at pH 2.5 and 4.5. These phytases evidenced different resistance against gastric proteolytic degradation; E. coli phytase retained 90% of its initial activity after 4 h incubation in the presence of either porcine pepsin or fish stomach proteases, while the dephosphorylation activity of P. lycii phytase was reduced by 90% (P<0.001). The presence of IP6 determined a reduction of 80% in protein solubility (P<0.001) when casein was used as substrate of fish acid pro- teases. The formation of IP6-protein complexes resulted in lower amino acids liberation from casein (P<0.05). Addition of phytase to soybean meal prevented the formation of such IP6-protein complexes and improved amino acid release by 60% (P<0.01). In contrast, IP6 had not effect on protein solubilisation or amino acid liberation during the intestinal diges- tion. Under conditions simulating gastric digestion a great amount of IP6 was combined to either porcine pepsin or soluble protein in fish stomach crude extract, precipitating 68 and 100% of such proteins, respectively. A similar, but lower reduction in soluble protein from either pancreatin or fish intestinal extract was observed when simulating intestinal diges- tion (47 and 33%, respectively). The formation of IP6-protein complexes reduced the activity of all gastric and intestinal proteases assayed (P<0.01) with the exception of chymotrypsin. A positive effect (P<0.05) of low pH on IP6-dephosphorylation was measured when the effect of bacterial phytase (2500 FTU/kg) on the release of soluble P from native IP6 present in soybean meal was tested under different digestion temperatures (6 and 16 ◦ C) and pH (2.0, 3.0 and 4.0). A positive effect of temperature was also observed when the reaction was carried out at pH 2.0 and 3.0 (P<0.001), but not at 4.0, suggesting the existence of a limit in the solubility of IP6 and therefore in its availability for phytase action at this last pH value. © 2011 Elsevier B.V. All rights reserved. Abbreviations: BAPNA, N˛-benzoyl-dl-arginine-p-nitroanilide substrate; FTU, phytase units; GD, gastric digestion; ID, intestinal digestion; IP6, myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate; N, nitrogen; P, phosphorus; SAPNA, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide substrate; TCA, trichloroacetic acid. ∗ Corresponding author. Tel.: +34 950 014135; fax: +34 950 015476. E-mail address: moralesg@ual.es (G.A. Morales). 0377-8401/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2011.08.011