Journal of the Science of Food and Agriculture J Sci Food Agric 87:120–126 (2007) The effect of proteinase inhibitors in food protein hydrolysis by digestive proteinases of white shrimp (Penaeus vannamei ) larvae Francisco J Alarc ´ on, 1∗ Carmen de O ˜ na, 1 Manuel D´ ıaz, 1 Fernando L Garc´ ıa-Carre ˜ no, 2 Francisco J Moyano 1 and Mar´ ıa A Navarrete del Toro 2 1 Dpto. Biolog´ ıa Aplicada, Escuela Polit ´ ecnica Superior, CITE II-B. Universidad de Almer´ ıa, La Ca ˜ nada S. Urbano, 04120-Almer´ ıa, Spain 2 Centro de Investigaciones Biol ´ ogicas del Noroeste (CIBNOR), PO Box 128, La Paz, BCS, 23000 M ´ exico Abstract: This study describes the digestive capacities of 10-day-old white shrimp postlarvae (PL10) and how some inhibitors of proteinases affect the digestion of protein in aquafeeds by using in vitro hydrolysis techniques. Biochemical data showed eight active proteinases in the PL10 hepatopancreas extract. Enzymes belong to the metallo- and serine-proteinase classes. The effect of inhibitors present in protein ingredients and aquafeeds on PL10 proteinases showed that ovalbumin alone and in commercial microcapsules yielded a significant inhibition in proteolytic activity of PL10 hepatopancreas enzymes. The capacity of PL10 proteinases to hydrolyse the protein fraction within different sources and microcapsules was demonstrated by two in vitro approaches, the pH-stat (degree of hydrolysis, DH) and electrophoresis (coefficient of protein degradation, CPD). It was shown that PL10 proteinases hydrolyse, in different extent protein sources and microcapsules. Casein, cuttlefish meal and feeds containing these ingredients are quickly hydrolysed. By contrast, ovalbumin and microcapsules containing ovalbumin are not hydrolysed.  2006 Society of Chemical Industry Keywords: digestive enzymes; Penaeus vannamei ; postlarvae; proteinase inhibitor; pH-stat; SDS-PAGE; white shrimp INTRODUCTION The Pacific white shrimp, Penaeus vannamei, is the second most important aquafarmed penaeid species. With the development and expansion of production, processing and international marketing, farmed shrimp quality is becoming increasingly important. Aquaculture production of penaeid shrimp larvae is constrained by costly and unreliable supplies of live food, especially Artemia nauplii. Despite Japanese and European companies attempts to produce high quality larvae feed commercially, a complete replacement for Artemia is not yet possible. For this reason, larvae production constitutes a bottleneck for shrimp production. Microcapsulated feeds have been developed to replace live food. One of the most important questions in the formulation of inert feeds is the quality of the raw materials, mainly the quality of protein. 1 In addition to the amino acid composition, quality involves digestibility, which is dependent on the presence and amount of antinutritional factors such as proteinase inhibitors. So, one limitation in choosing a feedstuff for aquafeeds is the presence of enzyme inhibitors. Inhibitors can affect several proteinase classes, such as serine-, cysteine-, metallo- and acid proteinases. 2 The effect of inhibitors on the shrimp digestive system needs to be studied in order to determine possible adverse side effects of the feedstuff. Likewise, a detection procedure for proteinase inhibitors in raw materials should be included in the quality control of shrimp- feed production. Since biological experiments are both expensive and time-consuming, and yield results that are only approximate, many authors have sought for appropriate laboratory methods to evaluate protein quality. 3,4 Dimes et al . 5 developed an in vitro method using enzyme fractions from trout pyloric caeca to estimate the degradation of protein using a pH- stat assay. The use of in vitro assays in aquaculture nutrition provides advantages in both economy and animal well-being because fewer animals and facilities are needed. The crustacean hepatopancreas produces and releases several enzymes into the digestive tract, including proteinases and peptidases. 6,7 Proteinases from shrimp hepatopancreas have been used to evaluate the in vitro protein degradation of ingredients for aquafeeds. 4,8 ∗ Correspondence to: Francisco J Alarc ´ on, Dpto. Biolog´ ıa Aplicada, Escuela Polit ´ ecnica Superior, CITE II-B, Universidad de Almer´ ıa, La Ca ˜ nada S. Urbano, 04120-Almer´ ıa, Spain E-mail: falarcon@ual.es Contract/grant sponsor: Ministerio de Educaci ´ on y Cultura, Spain (Received 21 February 2005; revised version received 8 November 2005; accepted 15 August 2006) Published online 11 October 2006; DOI: 10.1002/jsfa.2686  2006 Society of Chemical Industry. J Sci Food Agric 0022–5142/2006/$30.00