Evaluation of different protein sources for aquafeeds by an optimised pH‐stat system

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.

The effect of proteinase inhibitors in food protein hydrolysis by digestive proteinases of white shrimp (Penaeus vannamei) larvae

“…Kinetics of protein hydrolysis Because we know from previous experience 28 that, in spite of the presence of proteinase inhibitors in some feeds for aquaculture, there is some digestion of the protein evaluated as digestibility, we conducted some assays to evaluate the in vitro digestibility of protein in the ingredients and microcapsules. The highest DH values were obtained for MC 4, MC 3, casein and cuttlefish meal (Table 5).…”

Digestão in vitro de proteína e fósforo utilizando enzimas digestivas espécie-específicas: potencial de desenvolvimento e aplicação para a aquicultura de espécies de peixes

“…The consistency of in vitro techniques employing species-specific digestive enzymes depends on the standardization of the hydrolytic capacity of the recovered extract. Most commonly, extracts have been standardized to the activities of trypsin and or chymotrypsin (BASSOMPIERRRE, et al, 1997b;CHONG et al, 2002;HAARD, 1994;GRABNER, 1985;RUNGRUANGSAK-TORRISSEN, et al, 2002;TIBBETTS et al, 2011a), or total alkaline proteinase (ALARCÓN, et al, 2002;EL-SAYED, et al, 2000;EZQUERRA et al, 1997;HAMDAN, et al, 2009;LEMOS et al, 2009;MÁRQUEZ et al, 2013). The present study introduces the degree of protein hydrolysis (DH) as a standardization method using analytical grade proteinaceous substrates under the same conditions (pH-stat) as of the in vitro digestion assays.…”

“…1.3, 1.4 and 1.5). In vivo, the role of the stomach in protein digestion is to initiate breaking down the food, partially hydrolyzing proteins into peptides and/or mechanical disruption via muscular contraction in preparation for further hydrolysis in the intestine (ALARCÓN et al, 2002;BASSOMPIERRE et al, 1997b;CLEMENTS;RAUBENHEIMER, 2006;HAMDAN et al, 2009). Gastric digestion could also increase the speed of intestinal hydrolysis, leading to a significant shift from soluble polypeptides to oligoand dipeptides (GRABNER;HOFER, 1985HOFER, , 1989 and potentially increasing availability of soluble protein by inactivation of protease inhibitors by low pH and/or pepsin activity (HAMDAN et al, 2009;EGGUM, 1983).…”

“…For aquatic animals, the focus has been on protein hydrolysis, as this nutrient may be more relevant in the metabolism and requirement than carbohydrates and lipids. The in vitro methods may include open or closed vessels, measure different products, e.g., released amino acids, degree of peptide bond hydrolysis, use commercially available enzyme sources or from the target species (ALARCÓN et al, 2002;BASSOMPIERRE, et al, 1997a;HAARD 1994;EZQUERRA et al, 1998;HAMDAN, et al, 2009;LAZO et al, 1998;MOYANO, 2010;SHIPTON;BRITZ, 2002;TIBBETTS et al, 2011a;TONHEIM et al, 2007). Substrate and enzymes may be incubated in aqueous solution under controlled conditions and final products measured after a given time (BASSOMPIERRE et al, 1998;DIVAKARAN et al, 2004;LAN;PAN, 1993); membrane reactors in which an inner reaction chamber is separated from the outer chamber by a dialysis membrane of variable molecular weight cut off (1000 -3500 Da) through which passes amino acids released during hydrolysis and are continuously removed from the outer chamber and quantified (HAMDAN et al, 2009;SAVOIE, 2001;MOYANO, 2010;GAUTHIER, 1986); substrate and enzymes may be incubated in aqueous solution under controlled conditions and the course of protein hydrolysis be evaluated by changes in pH, either by recording the change in pH (pH-drop or pH-shift) (LAZO et al, 1998) or by keeping the pH constant with continuous addition of titrant and measuring consumption (pH-stat) (CÓRDOVA-MURUETA; GARCÍA- CARREÑO, 2002;DIMES et al, 1994a;EL-MOWAFI et al, 2000;EZQUERRA et al, 1998;LEMOS et al, 2009;YASUMARU, 2010;TIBBETTS et al, 2011b).…”

“…This motivated the development of in vitro methods based on the digestion of small amounts of feedstuff samples. Reported in vitro protein digestion methods include the use of digestive enzymes that may be available from commercial sources (LAZO et al, 1998;SHIPTON;BRITZ, 2002;TONHEIM et al, 2007) or recovered from the target species (ALARCÓN et al, 2002;HAARD, 1994;EZQUERRA et al, 1998;TIBBETTS et al, 2011a). Nevertheless, different results in the in vitro digestion have been found depending on the enzyme origin, suggesting a speciesspecific feature of in vitro protein digestion (ALARCÓN et al, 2002;EZQUERRA et al, 1998;LEMOS et al, 2004;NUNES, 2008;SAVOIE, 2001).…”