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The effects of different environmental salinities (0, 12, 40, and 55 ppt) on pepsinogen 2 (pga2), trypsinogen 2 (try2), chymotrypsinogen (ctr), and pancreatic alpha-amylase (amy2a) gene expression, and on the total activities of their corresponding enzymes, were assessed in Chelon labrosus juveniles, after their corresponding full-complementary DNA sequences were cloned. Furthermore, the quantitative effect of different salinities on the hydrolysis of feed protein by fish digestive enzymes was evaluated using an in vitro system. Relative pga2 expression levels were significantly higher in animals maintained at 12 ppt, while a significantly higher gene expression level for ctr and try2 was observed at 40 ppt. amy2a gene expression showed its maximum level at 40 ppt and the lowest at 55 ppt. A significant reduction in the activity of amylase with the increase in salinity was observed, whereas the maximum activity for alkaline proteases was observed in individuals maintained at 40 ppt. A negative effect of high salinity on the action of proteases was confirmed by the in vitro assay, indicating a decreased efficiency in the digestive function in C. labrosus when maintained at high environmental salinities. Nevertheless, individuals can live under different environmental salinities, even though gene expression is different and the enzymatic activities are not maintained at the highest studied salinity. Therefore, compensatory mechanisms should be in place. Results are discussed on the light of the importance as a new species for aquaculture, This work was funded by project AGL2010-14876 from MICINN to JMM.

The present work uses a novel approach to develop a mathematical model explaining the effect of three main factors - temperature, total reaction time and enzyme:substrate ratio - in the hydrolysis of protein by the intestinal enzymes of the Senegalese sole. The methodology combines a factorial design based in the Response Surface Methodology and in vitro digestibility assays adapted to the physiology and culture conditions of the species. The model revealed that, within the physiological ranges considered in the study, the efficiency of protein hydrolysis by the intestinal enzymes of the sole was linearly correlated to two of the selected factors; directly to incubation time and inversely to the amount of substrate. The effect of temperature was not linear but quadratic, hence showing a maximum at 23 °C. The information obtained can be used as a tool to maximize the biological response under variable conditions and may orientate some on-farm feeding practices., This research was funded by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by projects EFISHDIGEST (AGL2014-52888-R) with FEDER/ERDF contribution granted to M.Y. and WISEFEED funded by the European Union's H2020 programme (Marie Skłodowska-Curie grant No 691150). Neda Gilannejad was supported by a doctoral fellowship (BES-2015-071662) from MINECO (Spain).

Fil: Márquez, Lorenzo. Universidad Católica de Temuco. Facultad de Recursos Naturales. Departamento de Ciencias Agropecuarias y Acuícolas. Núcleo de Investigación en Producción Alimentaria. Temuco, Chile. Fil: Hernández, Adrián J. Universidad Católica de Temuco. Facultad de Recursos Naturales. Departamento de Ciencias Agropecuarias y Acuícolas. Núcleo de Investigación en Producción Alimentaria. Temuco, Chile. Fil: Carvalho, Edison S. M. Instituto Gulbenkian de Ciência, Telomeres and Genome Stability Group. Oeiras, Portugal. Fil: Morales, Gabriel A. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Animal. Buenos Aires, Argentina. Fil: Dantagnan, Patricio. Universidad Católica de Temuco. Facultad de Recursos Naturales. Departamento de Ciencias Agropecuarias y Acuícolas. Núcleo de Investigación en Producción Alimentaria. Temuco, Chile. Fil: Díaz, Manuel. Universidad de Almería. Departamento de Biología y Geología. Grupo de Nutrición y Alimentación Animal. Almería, España. Fil: Moyano, Francisco J. Universidad de Almería. Departamento de Biología y Geología. Grupo de Nutrición y Alimentación Animal. Almería, España. The activity of fish acid proteases is strongly dependent on the pH, and many basic and applied works had focused on researching the pH-profile of digestive proteases using different buffered media. Nevertheless, enzymatic activity is also known to be affected by the ionic strength (IS) of the medium. The present work is aimed at researching the combined effects of pH (2.5, 3.0, 3.5, 3.75 and 4.0) and IS (50, 100 and 200 mM) on the protease activity of gastric extracts of juvenile Coho salmon Oncorhynchus kisutch. It is concluded that the activity of Coho salmon gastric proteases is maximal at pH 2.5-3.0, drops towards pH 4.0 irrespective of ionic strength, and they showed a lower activity at IS 50 mM in comparison to IS 100 or 200 mM. The present results point out to the convenience of considering the effect of ionic strength when measuring the activity of fish gastric proteases. tbls.

The products resulting from the non-enzymatic browning in processed diets are known to exert negative effects on the digestive enzymes of vertebrates. In addition, browning heavy products (BHP's) are known to co-precipitate with proteins depending on the pH and ionic strength of the medium and on the isoelectric point of the protein. As the manufacture of aquafeeds as well as aquafeed ingredients frequently implies heating processes, the effects of BHP's from a well-known model mixture (glucose + glycine) on the gastric proteases of the rainbow trout Oncorhynchus mykiss were investigated in the present work. The results support that BHP's interact with gastric proteases most probably to form a co-precipitate, thus potentially reducing the protease activity in the fish stomach. The maximal loss of activity found in the experiments was above 20%. The interaction is pH and ionic strength-dependent and relatively stable against mechanical perturbation. In the range of BHP concentration 500-1000 μg/mL, the interaction was dose-dependent at pH 3.5 with a maximum above 20%, and apparently independent of the dose at pH 4.0. Above an ionic strength of 100 mM due to monovalent ions (NaCl), the intensity of the interaction is reduced. On the other hand, divalent cations such as Ca2+ and Mg2+ in the range 30-80 mM disturb the interaction between BHP's and gastric proteases even at ionic strengths below 100 mM, thus alleviating the inactivating effect of BHP's. Fil: Serrano, Xavier. Universidad Católica de Temuco; Chile Fil: Hernández, Adrián J.. Universidad Católica de Temuco; Chile Fil: Larson, Majorie. Universidad Católica de Temuco; Chile Fil: Morales, Gabriel Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Animal; Argentina Fil: Díaz, Manuel. Universidad de Almería; España Fil: Moyano, Francisco J.. Universidad de Almería; España Fil: Márquez, Lorenzo. Universidad Católica de Temuco; Chile

In order to identify daily changes in digestive physiology in developing gilthead seabream larvae, the enzyme activity (trypsin, lipases and α-amylase) and gene expression (trypsinogen-try, chymotrypsinogen-ctrb, bile salt-activated lipase-cel1b, phospholipase A2-pla2 and α-amylase-amy2a) were measured during a 24 h cycle in larvae reared under a 12 h light/12 h dark photoperiod. Larvae were sampled at 10, 18, 30 and 60 days post-hatch. In each sampling day, larvae were sampled every 3 h during a complete 24 h cycle. The enzyme activity and gene expression exhibited a marked dependent behavior to the light/darkness cycle in all tested ages. The patterns of activity and expression of all tested enzymes were compared to the feeding pattern found in the same larvae, which showed a rhythmic feeding pattern with a strong light synchronization. In the four tested ages, the activities of trypsin, and to a lesser extent lipases and amylase, were related to feeding activity. Molecular expression of the pancreatic enzymes tended to increase during the night, probably as an anticipation of the forthcoming ingestion of food that will take place during the next light period. It follows that the enzymatic activities are being regulated at translational and/or post-translational level. The potential variability of enzyme secretion along the whole day is an important factor to take into account in future studies. A particularly striking consequence of the present results is the reliability of studies based in only one daily sample taken at the same hour of the day, as those focused to assess ontogeny of digestive enzymes., This research was funded by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by project RIDIGEST (AGL2011-23722) with FEDER/ERDF contribution. J.A. Mata-Sotres was supported by a doctoral grant (ID 215473) from the Mexican National Council for Science and Technology (CONACYT).

There is a large amount of scientific literature on digestive enzymes activity during fish larval development. Nevertheless, most of this information refers to descriptive aspects of the ontogeny of hydrolyzing capacities, and only few studies looked into functional aspects and its relation with feeds and feeding activity. A comparison of published data normalized by days-degrees reveals at least three groups of species (precocious, medium and late) in relation to the time required for reaching maximum activity of digestive enzymes capacities, irrespective of the taxonomic group. Recent progress has allowed enlarging our knowledge on fish larvae digestive function. Such progress is partially based on new methodologies, but also on extended experimental designs and sampling protocols. On one side, the molecular basis of the onset of the digestive functionality has been examined in several species, cloning the mRNAs codifying the precursors of the main digestive enzymes and determining the gene expression profiles. On the other side, the daily rhythms coupling feeding and digestive enzyme production activities started to be investigated, giving a new dimension to our understanding of the digestive capacity during these early stages. Comparison between molecular expression and actual activity of the different enzymes is providing new insights on the regulation mechanisms. The connection between feeding behavior and digestive response both at genomic and biochemical levels is a required step towards the rationalization of feeding protocols with both live and inert diets, as a way to increase the nutrients utilization and the developmental performance at these early stages.

Trypsin enzymes have been studied in a wide variety of animal taxa due to their central role in protein digestion as well as in other important physiological and biotechnological processes. Crustacean trypsins exhibit a high number of isoforms. However, while differences in properties of isoenzymes are known to play important roles in regulating different physiological processes, there is little information on this aspect for decapod trypsins. The aim of this review is to integrate recent findings at the molecular level on trypsin enzymes of the spiny lobster Panulirus argus, into higher levels of organization (biochemical, organism) and to interpret those findings in relation to the feeding ecology of these crustaceans. Trypsin in lobster is a polymorphic enzyme, showing isoforms that differ in their biochemical features and catalytic efficiencies. Molecular studies suggest that polymorphism in lobster trypsins may be non-neutral. Trypsin isoenzymes are differentially regulated by dietary proteins, and it seems that some isoenzymes have undergone adaptive evolution coupled with a divergence in expression rate to increase fitness. This review highlights important but poorly studied issues in crustaceans in general, such as the relation among trypsin polymorphism, phenotypic (digestive) flexibility, digestion efficiency, and feeding ecology., Most of the work outlined on this topic was supported by the International Foundation for Science (No. A/4306-1 and No. A/4306-2) granted to EP, the Agencia Española de Cooperación Internacional/Asociación Universitaria Iberoamericana de Postgrado (AUIP/AECI) from Spain, and the Program ʻDoctorado Iberoamericano en Ciencias’ at the University of Cadiz, Spain.

The Mugilidae are a group of fish with a great interest for aquaculture due to their omnivorous profile, rapid growth, and resistance to environmental variations. The selection of feed ingredients for these species is currently focused on an extensive use of plant by-products, with this being limited by their content in anti-nutritive factors (mainly phytate and non-starch polysaccharides, NSPs). Nevertheless, specific enzymes can be used to counteract some of those negative effects. In the present study, the effect of pretreating two high-plant feeds with a mixture of enzymes (glucanases + phytase) on the digestive use of protein and phosphorus by juvenile mullets (Mugil cephalus) was assessed using both in vitro and in vivo assays. The enzymatic treatment significantly modified the potential bioavailability of some nutrients, such as a reduction of sugars, pentoses, and phytic phosphorus. Also, it increased the digestibility of protein in one of the feeds but reduced that of phosphorus in both of them. The potential usefulness of enzyme treatment and the information provided by the two types of assays are discussed.

To assess to what extent addition of phytase to a plant-based diet results in spatio-temporal changes of phytate, available P, soluble protein, total amino acids and the activity of the main digestive proteases in gilthead sea bream, fish were fed two plant-based diets with or without phytase. Stomach, proximal intestine and distal intestine contents were monitored for these parameters at 0, 1, 2, 4 and 6 h after feeding. A reduction (P < 0.0001) of the soluble P–IP6 in the stomach when phytase was added to the diet was observed. Within stomach, most of the total P–IP6 was precipitated (86%), possibly due to the low acidification capacity of the sea bream (pH > 4), but 57% of the dietary P–IP6 was dephosphorylated, suggesting that phytase could have the capacity to dephosphorylate insoluble IP6 at such pH. An increment (60%) (P < 0.01) in total gastric protease activity was observed by phytase addition, this being the first demonstration of the in vivo effect of IP6 on the pepsin activity in fish stomach. Gastric pH and residence time of the digesta inside the stomach are critical factors for an efficient phytase action and improve P and N bioavailability in plant-based diets used in fish aquaculture. Fil: Morales, Gabriel Alejandro. Universidad de Almería; España. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Marquez, L.. Universidad de Almería; España Fil: Saenz de Rodrigañez, M.. Universidad de Almería; España Fil: Bermúdez, L.. Fundación Centro Tecnológico de Acuicultura de Andalucía; España Fil: Robles, R.. Fundación Centro Tecnológico de Acuicultura de Andalucía; España Fil: Moyano, F. J.. Universidad de Almería; España

In the present study, the protective effect of nucleotides over damages induced by the consumption of a diet containing a high amount of vegetable ingredients (560 g kg(-1)) in the intestinal epithelia of the meagre (Argyrosomus regius) was assessed by assays performed with an Ussing-type chamber. Two experimental feeds were prepared including or not a commercial mixture of nucleotides (1 g kg(-1)). Nucleotides significantly enhanced fish growth during the experiment. On the other hand, differences in the integrity and functionality of intestinal epithelia were evidenced by a change in the polarity of intestinal trans-epithelial potential. Samples of fish fed on the control diet showed a preferentially secretory short-circuit current, while those of fish receiving the nucleotide-supplemented diet showed a significantly lower and preferentially absorptive negative current. It is concluded that alterations of intestinal physiology juvenile meagre resulting from the intake of high amounts of plant ingredients could be minimized by nucleotide supplementation.

The aim of the present study was to determine the optimum dietary levels of soybean lecithin (SBL) for seabream (Sparus aurata) larvae, and its influence on production performance and digestive enzymes activity. Larvae were fed five formulated microdiets with five levels of SBL. Complete replacement of live preys with the experimental microdiets for seabream larvae at 16 dph produced over 55% survival rates, particularly in fish fed with the highest levels of SBL. Moreover, increase in dietary SBL up to 80 g kg−1 significantly improved larval growth, leading to high final total length and body weight. An increase in alkaline phosphatase activity with the elevation up to 80 g kg−1 SBL was also found denoting a better maturation of the digestive system. Besides, there was a stimulatory effect of dietary SBL on PLA2 activity. Finally, increasing dietary SBL lead to better utilization of dietary highly unsaturated fatty acid, as it was reflected in their higher content in both neutral and polar lipid of the larvae. In summary, elevation of dietary SBL up to 80 g kg−1 in microdiets for seabream significantly improved digestive enzymes activities, enterocyte maturation, utilization and deposition of dietary essential fatty acids and larval growth, as a consequence of a better digestion, absorption, transport and deposition of dietary nutrients.

The objective of the present experiment was to investigate effects of autoclaving and addition of sodium diformate to diets on amino acid (AA) composition, in vivo coefficient of total tract apparent digestibility (CTTAD) in mink (Neovison vison) of protein and AA and in vitro bioavailability of total AA using crude enzyme extracts from Atlantic salmon (Salmo salar). Diets were also evaluated for content of individual AA, reactive and available lysine, formic acid, pH and color analysis following autoclaving. The experimental treatments were organized according to a 4 × 2 factorial arrangement with four autoclave temperatures (100, 110, 120 and 130 °C), without or with (10.6 g/kg) sodium diformate. The CTTAD was determined for all experimental treatments, whereas the in vitro bioavailability was estimated only for the sodium diformate treatments at 100 and 130 °C. Autoclaving at 130 °C reduced (P

Six different commercial feeds used in weaning and postweaning of marine fish were analysed from the perspective of some of their main physico-chemical features and in vitro digestibility. While the range of particle sizes in dry state closely agreed to those claimed by the producing companies, dramatic changes were observed after suspension in water. Sedimentation rates measured in some of the feeds with higher particle sizes may be too high to ensure intake by young fish. The label claims of the products overestimate the amount of total protein but underestimate that of lipids. The analysis of amino acid profiles evidenced important differences in total amino acid composition between feeds of the same company but a great similitude in the profile of free amino acids (FAA). The evaluation of leaching evidenced that the fraction of FAA, which represented

Crustacean serine proteases (Brachyurins, EC 3.4.21.32) exhibit a wide variety of primary specificities and no member of this family has been reported for spiny lobsters. The aim of this work was to study the diversity of trypsins in the digestive gland of Panulirus argus. Several trypsin-like proteases were cloned and the results suggest that at least three gene families encode trypsins in the lobster. Three-dimensional comparative models of each trypsin anticipated differences in the interaction of these enzymes with proteinaceous substrates and inhibitors. Most of the studied enzymes were typical trypsins, but one could not be allocated to any of the brachyurins groups due to amino acid substitutions found in the vicinity of the active site. Among other changes in this form of the enzyme, conserved Gly216 and Gly226 (chymotrypsin numbering) are substituted by Leu and Pro, respectively, while retaining all other key residues for trypsin specificity. These substitutions may impair the access of bulky residues to the S1 site while they make the pocket more hydrophobic. The physiological role of this form of the enzyme could be relevant as it was found to be highly expressed in lobster. Further studies on the specificity and structure of this variant must be performed to locate it within the brachyurins family. It is suggested that specificity within this family of enzymes is broader than is currently believed.

BACKGROUND: Although several types of in vitro digestibility assays have been applied to nutritional evaluation of feeds for aquatic organisms, all of them are based on the use of closed reactors and do not simulate the gastric phase of the digestion. Our objective was to evaluate the suitability of a gastrointestinal model based on the use of a digestion cell provided by a semi-permeable reaction chamber, which allows continuous removal of digestion products as they are produced. We tested the effects of some factors, like the inclusion of a gastric phase, reaction temperature or bile salts on the hydrolysis of feed proteins by fish enzymes. RESULTS: We found that the most suitable operational conditions to simulate the digestion process must include a short acid pre-digestion as well as the use of bile salts in the reaction mixture. Acid pre-digestion resulted in a significant increase in the liberation of amino acids which represented more than twice that measured when using a single phase. The addition of two bile salts (45 µmol L−1 sodium taurocholate + chenodesoxycolate) resulted in almost a threefold increase in the hydrolysis of feed protein. The use of the described open system also allows the evaluation of carbohydrate hydrolysis as well as determination of residual undigested matter, in a similar manner to that carried out in ruminants with the DAISY system. CONCLUSION: Results suggest the system can be a very suitable model for evaluation of bioaccessibility in fish feeds. Copyright © 2009 Society of Chemical Industry

The potential use of fluorescent substrates for measuring digestive enzyme activities has been adapted to decapod crustacean larvae and compared to traditional analytical techniques based on spectrophotometry. The ontogeny of three digestive enzymes (trypsin, amylase, and esterase) from the hepatopancreas of Maja brachydactyla has been selected to illustrate the advantages and disadvantages of both spectrophotometric (SP) and fluorometric (FL) methods. The three enzyme activities showed the same pattern with either of the techniques used. Standardised discriminant function coefficients indicated that the stage of development in spider crab larvae was most strongly discriminated by the activity of amylase, whichever method of analysis was considered. The FL method allows the detection of up to three enzyme activities in a single larva (

The potential use of digestive activities as indicators of the nutritional status in bivalves is discussed in relation to the results obtained in two clam species exposed to starvation and refeeding. Activities of some digestive enzymes (amylase, laminarinase, cellulase, and protease) were measured in juveniles of two commercially interesting species of clams, Ruditapes decussatus and Venerupis pullastra. The specimens were fed normally, being after subjected to a 15-days starvation and a further refeeding period. Samples were obtained at different moments of such feeding schedule to evaluate enzymes as well as weight (live, dry and organic) and length, in order to calculate growth rates and feeding efficiencies. Starvation led to a major decrease in clam growth as measured by dry weight and a negative growth as measured by organic weight, this coinciding with a certain degree of growth of the shell and a consumption of soft tissue. This response occurred more rapidly in R. decussatus but was of a lower magnitude than in V. pullastra. Activity of carbohydrases decreased rapidly in both species with starvation, although protease activity was maintained in R. decussatus. Recovery after the end of starvation was not similar in both species; while R. decussatus attained similar growth rates and enzyme activities to those measured prior to nutritional stress, V. pullastra only recovered 50% of its initial values. For both species of bivalves it can be concluded that digestive enzymes, and more specifically amylase, could be used as indicative of their nutritional condition.

The effect of variations in the amount or quality of food provided on the amino acid profile of larvae, was tested in two marine fish species, the Senegal sole (Solea senegalensis) and the sea bass (Dicentrarchus labrax). The objective was to assess whether such nutritionally suboptimum dietary patterns, which simulate situations that may occur in hatcheries, result in variations in the concentration of one or more amino acids that could be used as indicators of nutritional condition of larvae. Restriction in the normal food ration by 60% had no clear effect on the amino acid profile of sea bass, while the concentration of some amino acids (Arg, Ala and Phe) showed significant variations in sole. Feeding on artificial diets, which have shown their inadequacy a priori, resulted also in no significant effect on the profile of sea bass, but in a dramatic modification of the amino acid profile in sole. In addition, changes in the amino acid profiles considered as a whole were evaluated using the cluster analysis instead of paired comparisons between amino acid concentrations in each treatment. The analysis clearly separated profiles of larvae fed restrictedly or inadequately from their controls, irrespective of the species. This demonstrated the potential value of evaluating whole changes in amino acid profile as nutritional indicator. Besides, it was demonstrated that sensitivity of larvae to nutritional changes that may be reflected in the amino acid profile is greatly conditioned by the species and developmental stage.

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.

The inefficiency of starch utilisation by ruminants fed readily fermentable cereal grains, such as barley, decreases the metabolic potential of such feeds and can cause serious dysfunctions related to acidosis or pre-acidosis status in animals with higher energy requirements. The rate and extent of ruminal disappearance of grain dry matter (DM) are determined largely by the morphological features of the seed endosperm, but the presence of polyphenols may also constitute a limiting factor in ruminal hydrolysis. To assess the impact of tannins on the rate and extent of ruminal fermentation of barley grain, commercial grade tannic acid (TA) was included at 0, 1.0, 2.5 or 5.0% (wt/wt, DM basis) with ground barley grain in incubationin situ, and disappearance of DM and crude protein were monitored over 48h. A dose-dependent effect of TA on in situ degradation of barley meal was observed; significance was attained (p < 0.05) at the 5% treatment level. Scanning electron microscopy revealed limited microbial hydrolysis of endosperm cell walls in TA-treated samples, although TA did not prevent microbial attachment to or hydrolysis of starch granules. Tannins may be effective for slowing ruminal disappearance of barley to improve starch utilisation by ruminants.  2005 Society of Chemical Industry

The inefficiency of protein utilisation by ruminants fed protein concentrates (based on legume meals) causes serious economic loss and environmental damage owing to their rapid hydrolysis and deamination in the rumen. Thus efforts aimed at slowing the ruminal fermentation of such feeds are needed, and recent studies have observed potentially positive effects of tannins on ruminant nutrition under certain circumstances. Tannins are a complex group of naturally occurring plant polyphenols characterised by their ability to bind with proteins. This property of tannins is considered responsible for the decreased ruminal digestibility of forages both in vivo and in vitro. Under that perspective, commercial tannic acid was added at three proportions (10, 25 and 50gkg −1 on a dry matter basis) to four different legume meals (horse bean, kidney bean, soybean and pea), and the effect on in situdry matter and crude protein ruminal disappearance was assessed. The results confirmed the dose-dependent (although not persistent after 48h) slowing of in situ digestibility, this effect being significant at the highest tannin treatment when compared with untreated samples. Scanning electron microscopy revealed that soybean seed endosperm cell walls were protected from digestion by the ruminal microbiota, while the digestion of starch granules was relatively unaffected by tannic acid. Electrophoresis of the protein fractions confirmed the lower digestibility of tannin-treated seeds as well as the relative lack of alteration of the electrophoretic profile of individual proteins. Implications for the digestion of concentrates in ruminants are discussed.  2004 Society of Chemical Industry

Activity of some of the main enzymes involved in protein digestion and absorption (acid and alkaline professes, leucine-aminopeptidase, acid and alkaline phosphatases) as well as those of amylase and lipase, was assessed during larval development of while sea bream Diplodus sargus. All enzyme activity was detected at the moment of mouth opening. The variations observed in the activity profiles of the digestive enzymes were correlated either to developmental events. such as the functional start of the stomach (22 days after hatching), or to changes in the nature of the diet. The early and noticeable development of digestive enzyme activities was linked to a high survival after weaning.

The pH-stat system has been used to assess the effect of tannic acid (TA) on solubility and in vitro enzyme hydrolysis of different proteins. Added TA (from 10 to 50 g kg−1) decreased the extent of hydrolysis of bovine serum albumin. Enzymic hydrolysis of casein, pea meal, soybean meal, and haemoglobin (HB) was increased, as measured by total amino acids released and by the degree of hydrolysis. SDS-PAGE confirmed the results of the in vitro enzymatic hydrolysis. These findings suggest that, under in vitro conditions, when simulating the gastrointestinal environment of domestic mammals, the negative effects of TA described from in vivo experiments are not necessarily due to reduced hydrolysis of proteins. Copyright © 2003 Society of Chemical Industry

This paper presents the results of some experiments oriented to optimise and standardise the measurement of protein hydrolysis by fish 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 fish 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 modifications in protein concentration resulted in significant variations in DH. Protease inhibitors present in various plant protein sources produced a reduction in DH values. Cet article presente les resultats de quelques experimentations menees pour optimiser et normaliser la mesure de l'hydrolyse des proteines par les proteases de poissons en utilisant une technique de pH stat. Differents facteurs affectant la degre d'hydrolyse sont consideres: autohydrolyse des sources de proteines, type d'enzyme utilise, ratio substrat/enzyme et effet des inhibiteurs.

The evolution of digestive proteases during larval development of Rhynchophorus ferrugineus (Olivier, 1790) has been studied. A progressive increase of protease activity has been found. The optimum pH for proteolytic activity against azocasein was determined. Caseinograms revealed an active complex of alkaline proteases from the early stages of the development. From the apparent molecular masses, three groups of proteases have been found - high molecular-mass proteases, medium molecular-mass proteases, and low molecular-mass proteases. Studies using specific protease inhibitors showed the major presence of serine proteases in gut extracts. The results obtained from larvae reared on different substrates have made possible a comparative assessment of the influence of diet on the development of the digestive enzymatic system. Larvae fed on an artificial diet showed a complete pattern of digestive proteases. Data suggest that this diet seems to be suitable for future research with this insect pest.

A sensitive and quick method was developed to determine the presence of α-amylase in the gut of aquatic organisms, as well as its sensitivity to inhibitors. The assay is based on the utilization of Petri dishes filled with starch–agarose gel as a substrate for the enzyme solution, which is placed in small wells punched in the surface. Circular zones produced by the action of amylase remain colourless after staining with lugol. Pure commercial porcine amylase was used to fit the better conditions for developing the assay (1 g L–1 starch in the gels, 4 h of incubation). The diameter of the cleared zones were related to the activity of enzyme and the method detected linearly amylase activity in a range of 2–20 U well–1, so it was used to reveal the presence of amylase in digestive extracts obtained from different sparid fish. The method was also used to evaluate the effect produced by a specific inhibitor on fish amylases, showing a linear response when the ratio inhibitor:enzyme (in units) changed from 20:1 to 2:1. Comparison of the cleared zones produced by amylases of sparid fish in the presence or absence of inhibitor, revealed differences in their sensitivity to inhibition, which ranged from 15 to 50% of total activity. The assay is proposed for a preliminary evaluation of possible inhibitors contained in feedstuffs used in fish feeding.

The presence of digestive carbohydrases was determined in seabream intestines, and the main activity identified was amylase. Optimum activity for this enzyme was found at pH 8.0 at 40 °C. Amylase activity was highly sensitive to extreme pH, and temperatures exceeding 50 °C. The use of SDS–PAGE zymograms allowed identification of amylase in the form of a high molecular mass fraction exceeding 100 kDa. Results confirm the existence of a well developed amylase equipment in this species which supports the possibility of increasing the amount of carbohydrates in the formulation of its commercial feeds.

Inthe field of fish nutrition, the preliminary evaluation of feedstuffs usingin vitro techniques may be an alternative to invivo assays. Degradation of the protein fraction in feedstuffs byseabream (Sparus aurata) digestive enzymes was studiedunder different conditions simulating either acid, alkaline, or acid + alkalinedigestion using a modified pH-stat technique. In addition, a sequential sodiumdodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the releasedproducts was used both to visualise and to quantify the level of proteinhydrolysis. Gels were analysed by optical densitometry and results wereexpressed as a Coefficient of Protein Degradation (CPD). Values of CPD showedclear differences related to the type of protein digested or the steps followedin the digestion. CDP index provides information based on the enzymaticbreakdown of the proteins visualised in SDS-PAGE gels. The pH-stat evaluation(Degree of Hydrolysis) supplies measurement of the total number of peptidebondsbroken down in a given protein. CDP index mainly focuses on the action ofendoproteases, whereas degree of hydrolysis (DH) includes both endo- andexoproteases. CPD and DH are two complementary indexes capable of measuring theprotein hydrolysis by fish enzymes of a given feedstuffs or diet. Both methodswere in agreement with respect to assessing the hydrolysis of protein. Thesignificance of the SDS-PAGE techniques in the assessment of aquafeeds by fishdigestive enzymes is discussed.

The presence of protease inhibitors in different ingredients used in fish feeds may negatively affect their digestive utilisation. The effect of inhibitors present in several protein sources on the activity of digestive proteases of sea bream was assessed using `in vitro' assays. Inhibition produced by extracts of plant proteins ranged from 25 to 50 % of total activity, whereas that obtained using animal protein sources ranged from 1 to 20 %. The exception was ovoalbumin, which showed the highest measured value (62 %). A plot of the inhibition values obtained by changing the relative concentrations enzyme/inhibitor resulted in different curves for different protein sources. The use of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) zymograms allowed visualisation of the aforementioned differences in inhibition. The importance of measuring protease inhibition in the preliminary evaluation of fish feed ingredients is discussed.

In a previous study, several histological alterations were found in the liver of gilthead seabream fed with a diet containing 30% soybean meal (SBM). In the current study, SBM containing diets were supplemented with either potassium phosphate, zinc sulfate or phytase ( Aspergillus ficuum ), or increasing the dietary n −3/ n −6 fatty acids ratio to meet that of a fish meal based diet. Diet composition did not affect fish growth, feed efficiency (FE) or protein productive value (PER). Phosphorous supplementation significantly reduced hepatosomatic indexes (HSI), although it did not alter liver lipid content. Both phosphorous supplementation and correcting the dietary n −3/ n −6 fatty acid ratio significantly altered the lipid and protein content in fish muscle. Only these two treatments and principally the corrected dietary n −3/ n −6 fatty acid ratio, improved the liver histological alterations observed in fish fed with SBM based diets.

Two experiments were conducted to compare the effect of the partial substitution of fish meal by two different protein sources, corn gluten (CGM) and meat and bone meals (MBM) in diets for juveniles gilthead seabream. Growth, feed efficiency, protein efficiency ratio and protein productive values were not significantly affected by the source of dietary protein, although higher values were observed with diets containing increasing levels of meat and bone meal. No differences were found in fish proximate composition at the end of the experiment. Histological studies of liver tissue showed no liver alteration in fish fed diets including increasing levels of corn gluten meal. On the contrary, an increased deposition of lipids, nuclei polarization and isolated necrotic focus were found in hepatocites of fish fed diets exceeding 20% of meat and bone protein. Apparent protein digestibility (ADC) in diets containing CGM showed similar results to those obtained with the control diet. However, a significant reduction in digestibility was observed when MBM was used as partial substitute of fish meal protein. A negative correlation between dietary ash content and protein digestibility was observed. Higher amounts of nitrogen were excreted as levels of CGM and MBM increased in the diets, being significantly higher in the case of 40% substitution either with CGM or MBM protein.

Identification of alkaline proteases produced during larval stages of gilthead sea bream, Sparus aurata was achieved using SDS-PAGE and specific inhibitors. Such techniques were also applied to determine proteases existing in rotifers, Brachionus plicatilis, and Artemia nauplii, which are used as live food for these larvae, as well as proteases of adult fish. The results show a great prominence of trypsin-like proteases during the 4 weeks after hatching, but the number of enzyme species was reduced in adult fish. Alkaline proteases present in the rotifers and Artemia showed clear differences when compared with those of the larvae and were not detected in extracts obtained from fed larvae. The results obtained provide information about the role of exogenous enzymes in larval feeding of sea bream.

The effect of different factors (protein composition, thermal treatment, presence of and acidsalt) on the potential bioavailability of amino acids (AA) in rainbow trout diets was evaluated with a gastro-intestinal model (GIM). The in vitro assay consisted in a detailed evaluation of the rates of release of individual AA, as well as of the whole and AA profile of the dyalisates produced after hydrolysis performed using enzyme extracts obtained from stomach and intestine of fish. Results were compared to values of apparent amino acid digestibility measured in vivo. In addition, two important parameters conditioning potential bioavailability of dietary protein during digestion, solubility and buffering capacity (BC) were evaluated. Protein solubility and BC were significantly affected by the presence of a vegetable protein in the diet. As a result of enzyme hydrolysis, clear differences in the rates of release of different AA's, mainly for those being target for digestive proteases (Arg, Lys, Phe) were detected. A significant correlation between total masses of AA's dialysed in vitro and their digestibility estimated from digestibility coefficients calculated in vivo was obtained although this was not possible when using values of individual AA's. The concept and framework for future assays involving in vitro-in vivo correlations is discussed.

The present study was aimed to establish the relationship between the functionality of the digestive gland and physiological rates including SFG (scope for growth) in wild mussels, Mytilus galloprovincilis. The experimental set-up consisted in the evaluation of changes in the morphology of the gland, as well as in the activity of some key digestive enzymes (amylase, laminarinase, cellulase and protease) within a broad range of SFG obtained through manipulation of food ration. The higher SFG values were correlated to an increase in both the size of the digestive gland and the activities of enzymes when expressed in relation to individual. In contrast, no clear relations were observed when the activity of enzymes was expressed in relation to soluble protein, with the exception to amylase. The higher protease activities measured in mussels showing lower SFG may reflect an initial stage of catabolic processes intended to compensate the energy deficit produced by food restriction. The potential use of parameters measured in digestive glands in studies of marine pollution was discussed.

Hydrolysis of three different proteins by either crude fish digestive extracts or purified mammal proteases was assayed using two different in vitro systems. The closed system was a modification of the pH-stat method including a previous acid digestion. The open system used a digestion cell containing a semi-permeable membrane which allowed continuous separation of the final products of hydrolysis with a molecular cut-off of 1000 Da. Assays in both systems resulted a similar arrangement of the tested proteins in relation to their ability to be hydrolyzed, with casein>fish meal> or =soybean meal. With the exception of casein, no significant differences were found between results produced by any of the enzyme sources using the closed system. In constrast, significantly higher hydrolysis of all proteins was produced by mammal enzymes under conditions operating in the open system. Differences in the rate of release of amino acids measured in this latter system were related both to the type of protein and the origin of the enzymes. When using purified mammal enzymes, release of lysine or phenylalanine from casein and soybean was high, but low from fishmeal. Isoleucine and valine present in fishmeal were preferentially hydrolyzed by commercial enzymes, but glycine and proline by fish enzymes.

The evolution of the digestive enzyme equipment in seabream from hatching to 30 days old larvae was studied; there was a progressive increase in the activity of protease, amylase and acid and alkaline phosphatase from day 15 onwards. The use of specific inhibitors, and SDS-PAGE provided evidence to suggest that most of the proteases belonged to the serine group. A high α-amylase activity was also denoted. Zymograms of larval extracts indicated that exogenous food has more a qualitative than a quantitative role in the secretion of digestive enzymes in this species.