FEATURE inhibitors in SW. Dose-response curves showed that UL contained substances able to reduce digestive proteolytic activity in sea bream (up to 77%), whereas a negligible inhibition by GR was found (4%). Obvious differences in the kinetic of inhibition of protease activity were found for UL. Equation defining Figure 2: Dose-response curves obtained when different such curve may be amounts of SW meal (0 to 300 µg) were incubated with a used to predict the fixed amount of proteolytic activity (1 U) in the inhibitory expected percentassay. Protease inhibition was expressed as the percentage of age of reduction reduction in proteolytic activity. Such curves are a simple way to evaluate how hypothetical variations in the inclusion of in protease activSW might affect sea bream digestive proteases ity, once protease activity in the digestive tract and the successful integrated culture in fish-farm efflu- amount of feed ingested are known. For ents. Biomass was obtained from the Marine instance, in the case of 40 g sea bream, total Biotechnology Centre (ULPGC, Spain). SW protease activity released after a meal is around were cultivated in 750 L semicircular fibreglass 1,300 units. Those fish that consumed 0.5 g of tanks filled with seawater plus the fishpond a feed containing 15 percent of UL, showed a effluents of a pilot aquaculture system (11 m3 ratio mg UL per unit of activity of 50, which a with an optimal density of Sparus aurata of 20 determined kg m-3, and a water renovation rate of 6–8 reduction nearly vol day-1). Red and green SW were washed 40 percent in the with sea water, sun-dried for 48 hours, ground activity of digesand sieved through 0.1 mm sieve before being tive proteases. Fortunately, fish used as a dietary ingredient. Dry algal biomass was incorporated into have mechanisms six experimental diets (40% crude protein to compensate and 12% crude lipid) at increasing levels (5, the effect of die15 and 25%). A feed without SW served tary antinutrients. Zymograms as a control diet. Feeds were made at the University of Almeria-CEIA3 facilities (Service obtained after of Experimental Diets; http://www.ual.es/ electrophoretic. of stecnicos_spe). Every experimental feed was separation randomly assigned to triplicate group of proteins is a usefifteen sea bream juveniles (15.4 g initial body ful tool to know weight). Fish were fed by hand twice per in detail the type inhibition day (9:00 and 17:00) at a rate of 3 percent of of their body weight for 70 days. At the caused by proend of the trial, fish were killed according to tease inhibitors. the requirements of the Directive 2010/63/ From the zymoUE, and digestive tract was removed, and gram, it is clear then processes to obtain enzymatic extracts. that Ulva proIntestinal proteases were analysed by two duces a generaldifferent approaches: a) quantifying the level ised inhibition in of intestinal proteolytic activity, and b) visual- alkaline proteasizing the profile of intestinal proteases in es of sea bream. Special themes zymograms (Alarcón et al., 1998). In addition, On the contrary
incubation of digestive proteases with extracts of the experimental diets. The mean inhibition ranged from 11 to 48 percent. In general, UL-supplemented feeds showed inhibition values higher than the GR-supplemented diets, which did not exceed 16 percent. For UL diets, it was found that percentage of inhibition was positively correlated with the SW inclusion level, which agrees with the above mentioned dose-response curve. Inhibition produced by GR feeds cannot be associated to the use of this SW.
Effect of seaweed on digestive proteases of sea bream Digestive enzymes were affected by diets, as fish had different enzyme activity level of alkaline proteases after 70 days of feeding experimental diets. In general, a decrease in alkaline protease activity was evidenced when feeds included UL or GR. In particular, the proteolytic activities of fish fed Ulva supplemented-feeds were significantly lower than those of fish fed on control diet. The presence of protease inhibitors in SW may be the reason of the progressive decrease in the proteolytic activity in fish fed diet with increasing levels of Ulva meal. Supporting this hypothesis, it has been confirmed that aqueous extracts of Ulva meal inhibit alkaline proteases of S. aurata. Moreover, the drop in
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the presence of protease inhibitors in SW was tested according to Alarcón et al. (1999).
Checking the presence of protease inhibitors in SW Results revealed the presence of protease
Gracilaria did not affect any of the active bands. The same results were observed after March-April 2013 | International AquaFeed | 39
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