Evaluation of different protein sources for aquafeeds by an optimised pH-stat system Francisco J Alarco ´n,* Francisco J Moyano and Manuel Dı ´az Dpto Biologı´a Aplicada, Escuela Polite ´cnica Superior,CITE II-B, Universidad de Almerı´a, La Can ˜ada de San Urbano, E-04120 Almerı´a, Spain Abstract: This paper presents the results of some experiments oriented to optimise and standardise the measurement of protein hydrolysis by ®sh proteases using the pH-stat technique. Various factors affecting the degree of hydrolysis (DH) were considered (autohydrolysis of the protein sources, type of enzymes utilised, substrate/enzyme ratio and effect of inhibitors). The crude extracts obtained from ®sh digestive tissues showed their suitability for the determination of protein hydrolysis, rendering better results than those obtained using mixtures of commercial enzymes. DH values for a given protein were greatly affected by the substrate/enzyme ratio, since small modi®cations in protein concentration resulted in signi®cant variations in DH. Protease inhibitors present in various plant protein sources produced a reduction in DH values. # 2002 Society of Chemical Industry Keywords: aquafeed; digestibility; enzyme; ®sh; hydrolysis; inhibitor; pH-stat; protease; protein; Sparus aurata INTRODUCTION The determination of the digestibility of the major nutrients is one of the main steps in the evaluation of their bioavailability for a given species. This is the reason why studies aimed at the measurement of digestibility of protein and energy are key to the formulation of adequate feeds for any species, being routinely performed in a great number of research centres all over the world. In the last few years, interest in the feasibility of developing invitro digestibility tests has been renewed for a number of reasons, most of them related to both the high cost of analyses and the social actions concerned with animal welfare. Some of the advantages supporting the use of such methodol- ogies are cost-effectiveness, easy performance and ready availabilityofresults. 1,2 In this context the evaluation of a great number of ingredients employed in aquafeed formulation may bene®t from the recent advances in various methodologies applied to the invitro measure- ment of digestibility in feeds for aquatic animals. 3 One the most interesting in vitro techniques is the measurement of protein hydrolysis using the pH-stat system, developed by Pedersen and Eggum, 4 who adapted the method previously assayed in Carlsberg laboratories. This method has proved its accuracy in predicting digestibility in both human foods and feeds for various terrestrial animals. 5±9 Dimes and Haard 10 suggested its utilisation in the evaluation of feeds for salmonids, providing the good correlation the authors found with digestibility measurements in those ®sh. More recently, the technique has been used for the optimisation of the protein fraction of microcapsules employed in feeding of marine ®sh larvae. 11 However, the use of the pH-stat technique in the measurement of protein hydrolysis by ®sh digestive enzymes requires a previous standardisation of the method. Several experiments were designed to determine the best conditions for future application of such methodology in ®sh nutritional studies. Studies were performed using digestive extracts of seabream (Sparusaurata L), an omnivorous marine ®sh, whose culture is rapidly expanding along the Mediterranean coasts. EXPERIMENTAL Preparation of active extracts Live specimens of juvenile seabream (S aurata) ranging from 35 to 45g in weight were provided by a local ®sh farmer (Predomar, Almerõ Âa, Spain). Each ®sh had been fed a commercial diet (45% protein) three times a day to reach a total amount of feed representing 3.5% of its body weight. After killing specimens, previously starved for 6 h, by submersion in cold water (4 °C), the digestive tract was removed and cleaned. A portion of the anterior intestine containing pyloric caeca was homogenised (100g l 1 ) in cold distilled water. Supernatants obtained after centrifu- gation (16 000  g for 30 min at 4 °C) were adjusted to pH 8.0 and stored at 20 °C until enzyme analysis. The concentration of soluble protein in enzyme extracts was determined by the Bradford method (Received 6 November 2000; revised version received 15 November 2001; accepted 21 January 2002) * Correspondence to: Francisco J Alarco ´n, Dpto Biologı ´a Aplicada, Escuela Polite ´cnica Superior, CITE II-B, Universidad de Almerı ´a, La Can ˜ada de San Urbano, E-04120 Almerı ´a, Spain Contract/grant sponsor: CICYT (Spain); contract/grant number: MAR97-0924-C02-02 # 2002 Society of Chemical Industry. J Sci Food Agric 0022±5142/2002/$30.00 697 Journal of the Science of Food and Agriculture J Sci Food Agric 82:697±704 (online: 2002) DOI: 10.1002/jsfa.1100