Effect of dietary inclusion of seaweeds on intestinal proteolytic activity of juvenile sea bream, Sparus aurata


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In the last years considerable attention has been paid on the use of seaweeds (SW) as a possible ingredient for aquafeeds. Red, green and brown SW can be taken from their natural habitat and brought to the shore by the action of winds and tides. Otherwise, biomass can be obtained from secondary and tertiary treatment of effluents. Wastewater treatment utilising photosynthetic organisms is an interesting alternative to reduce the ecological impact of domestic, industrial or aquaculture effluents. Generally, high-quality algal biomass is yielded from algal cultivation, representing an excellent source of hydrocolloids, carotenoids, and bioactive substances, which allows different industrial applications. In addition, there is currently an increasing interest for the potential of SW in human and animal nutrition.

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Effect of dietary inclusion of seaweeds on intestinal proteolytic activity of juvenile sea bream, Sparus aurata

  1. 1. I N C O R P O R AT I N G f i s h far m ing t e c h no l og y March | April 2013 Effect of dietary inclusion of seaweeds onintestinal proteolytic activity of juvenile sea bream, Sparus aurata International Aquafeed is published six times a year by Perendale Publishers Ltd of the United Kingdom. All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies, the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis of information published. ©Copyright 2013 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any form or by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058The International magazine for the aquaculture feed industry
  2. 2. FEATURE Effect of dietary inclusion of seaweeds on intestinal proteolytic activity of juvenile sea bream, Sparus aurata by María Isabel Sáez, Tomás Martínez and Javier Alarcón, Universidad de Almería-CEIA3, SpainI n the last years considerable attention (especially under nitrogen-enriched condition) response (Valente et al., 2006). Nonetheless, has been paid on the use of seaweeds and season. it has been also noted in other publications (SW) as a possible ingredient for aqua- The high biological value of algal proteins that high SW inclusion reduces fish growth feeds. Red, green and brown SW can be makes algae suitable for inclusion both in and feed efficiency. From the literature avail-taken from their natural habitat and brought animal feeds (especially marine species) and in able it can be deducted that the response of to the shore by the action of winds and human diets. The high carbohydrate content animals to SW seems to be dose-dependent tides. Otherwise, biomass can be obtained (30 to 60%) is a very marked characteristic in and species-specific. Moreover, certain sub-from secondary and tertiary treatment of most SW, comprising mainly soluble carbo- stances with antinutritive activity may be effluents. Wastewater treatment utilising hydrates, like sugars, and pectins, alginic acid, present in SW, like lectins, tannins, phytic photosynthetic organisms is an interest- agar and carrageenan as well. Besides their acid, and protease and amylase inhibitors ing alternative to reduce (Oliveira et al., 2009). Such the ecological impact of antinutritional factors might domestic, industrial or interfere with bioavailability aquaculture effluents. and/or digestibility of nutri-Generally, high-quality algal ents. biomass is yielded from Special emphasis should algal cultivation, represent- be focused on protease ing an excellent source of inhibitors. Binding of pro-hydrocolloids, carotenoids, tease inhibitors to pro-and bioactive substances, teolytic enzymes causes which allows different the pancreas to secrete industrial applications. In larger amounts of digestive addition, there is currently enzymes to overcome the an increasing interest negative effects of inhibi-for the potential of SW tors on the digestion of in human and animal dietary protein. This fact nutrition. can lead to decreased Figure 1: Detail of experimental feeds. UL-25 weight gain, and pancreatic percent (above) and control (below) hypertrophy in some fish Seaweed as species. For this reason, ingredient studies aimed to include in aquafeeds potential nutritional value, from a techno- SW in aquafeeds must also bring up their Although nutritional properties of SW are logical point of view, SW can also be used as possible effects on fish digestive physiology. not as well known as are those of land plant- additives in the feed industry, for instance, as To date, there is scarce literature analysing if based ingredients, their chemical composition excellent feed agglutinants (improving texture SW inclusion causes negative consequences may be characterised by low content in lipids, and water stability of pellets), or as attractants on digestive physiology of fish.moderate in protein, but rich in non-starch (increasing feed intake).polysaccharides, minerals and vitamins. Lipid Evaluating the effect of seaweedscontents range from 0.3 to 7.2 percent, The effects of seaweeds on fish on digestive proteasesalthough algal lipids are rich in PUFA such as Several studies have proved that addition In a recent study, we evaluated the effect C20:5n3 (eicosapentaenoic acid, EPA) and of small amount of SW in aquafeeds resulted of inclusion of two SW as dietary ingredients C22:6n3 (docosahexaenoic acid, DHA). The in considerable positive effect on growth on intestinal proteolytic activity of juvenile sea protein contribution is ranged from 10 to 30 performance and feed utilisation efficiency, bream. Gracilaria cornea (GR) and Ulva rigida g/100 g dry weight, which may vary greatly carcass quality, physiological activity, intesti- (UL) were chosen in the present study owing among SW species, environmental conditions nal microbiota, disease resistance, and stress to its fast growth, low-cost production and 38 | InternAtIonAl AquAFeed | March-April 2013
  3. 3. FEATURE FEATURE Need for novel a partial dietary substitute for fish oil within ies have been recently reviewed (Nasopoulou sources inhibitors in SW. incubation of digestive proteases with extracts compound feeds. The same is true of linseed and Zabetakis, 2012). Dose-response In order to reduce oil and rapeseed oil, although to a lesser of the experimental diets. The mean inhibition New, alternative and in a way ‘non- dependence on fish oil, sig- extent. curves showed orthodox’, from 11 to 48 percent. In general, ranged sources of lipids need to be nificant breakthroughs have that UL contained UL-supplemented feeds showed inhibition Furthermore, the use of palm oil in diets of identified and valorised in order to achieve occurred over the past few Atlantic salmon and rainbow trout has given to sustainable production of fish GR-supplemented substances able values higher than the feeds and thus years in replacing it with plant reduce digestive diets, which did not exceed 16 percent. For oils. By substituting feeds with proteolytic activity UL diets, it was found that percentage of inhi- plant oils, it also serves to reduce in sea bream (up B) bition was positively correlated with the SW costs due to the fact that vegeta- to 77%), whereas a inclusion level, which agrees with the above ble oils have steadily increasing produc- negligible inhibition mentioned dose-response curve. Inhibition tion, high availability and better economic by GR was found produced by GR feeds cannot be associated value. Several studies have been carried out (4%). Obvious dif- to the use of this SW. to investigate certain vegetable oils as pos- ferences in the sible sustainable partial substitutes for fish kinetic of inhibition Effect of seaweed on digestive oils in compounded fish feeds. The most of protease activity proteases of sea breamcommon vegetable oils used for fish feed pro- were found for UL. Digestive enzymes were affected by diets, duction have been soybean, linseed, rapeseed, Equation defining as fish had different enzyme activity level of Figure 2: Dose-response curves obtained when2: Representative optic micrographs xalkaline proteases sections stained with Figure different such curve may be 100 of aortic wallsunflower, palm oil and olive oil. (0 to 300 µg) were incubated with a and eosin from the two experimental groups,after 70 days of feeding amounts of SW meal haematoxylin where atherosclerotic used to predict the experimental diets. In general, a decrease Soybean and amount of proteolytic activity (1 U) in the inhibitory appear as foam cells (↑). (A) Group A (atherogenic diet); (B) Group B fixed rapeseed oil are considered lesions assay. Protease inhibition was salmo- (atherogenic diet expected percent- in alkaline protease activity was evidenced possible alternative lipid sources for expressed as the percentage of enriched with sea bream polar lipids) (adopted from Nasopoulou etnids, freshwater and marine fish since Such curves are a simple way reduction in proteolytic activity. they age of reduction when feeds included UL or GR. In particular, al., 2010). Copyright, “Food Chemistry” Elsevier to evaluate how hypothetical variations in the inclusion of in protease activ- the proteolytic activities of fish fed Ulva are rich in PUFAs, especially linoleic (18:2 SW might affect sea bream digestive proteases ity, once protease supplemented-feeds were significantly lower ω−6) and oleic acid (18:1 ω−9), but devoid growth and feed utilization efficiency compa- enabling the further development of aquacul- activity in the diges- ture applications. Such promising lipid sources of n-3 PUFA. However, in some cases, fish rable to fish fed with equivalent levels of fish than those of fish fed on control diet. The tive tract partial are vegetable oils (VO). The use of VO based oil substitution by 60 percent rapeseed oil oil. Olive oil could also be used as a and the presence of protease inhibitors in SW may has been found to decrease European sea substitute for dietary fish oil are European aquafeeds has some strong advantages. Olive successful integrated culture in fish-farm efflu- amount of feed ingested in known. For be the reason of the progressive decrease instance, in the case of 40 g sea bream, total in the proteolytic activity in fish fed diet with ents. Biomass was obtained from the Marine sea bass culture, during growth out phase, pomace (OP) and olive pomace oil (OPO) bass (Dicentrarchus labrax) growth. Soybean protease activity released after a meal is around increasing levels of of olive oil produc- Biotechnology Centre (ULPGC, Spain). SW Atlantic salmon (salmo salar) and rainbow are natural by-products Ulva meal. Supporting oil appears to be a better plant lipid source 1,300 units. Those fish that consumed 0.5 g of this hypothesis, it has been confirmed that were cultivated in 750 L semicircular fibreglass trout (Oncorhynchus mykiss) with data showing tion, which contain micro constituents with regarding gilthead sea bream (Sparus aurata) a feed containing 15 percent of UL, showed a aqueous extracts of Ulva meal inhibit alkaline growth while with seawater savings in fishpond similar growth rates to the ones when fish was atheroprotective (substances) activity such as tanks filled considerable plus the feed effluents of a pilot aquaculture system (11 m3 fed on 100 percent fish oil diet. All these stud- PAF-inhibitors and phenolic/polyphenolic mol-costs could be achieved if it could be used as ratio mg UL per unit of activity of 50, which proteases of S. aurata. Moreover, the drop in with an optimal density of Sparus aurata of 20 determined a 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 diges- and sieved through 0.1 mm sieve before being tive proteases. used as a dietary ingredient. Fortunately, fish 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 die- VIV Russia 2013 15 and 25%). A feed without SW served tary antinutrients. as a control diet. Feeds were made at the Zymograms University of Almeria-CEIA3 facilities (Service obtained after 4 BioMarine Business Convention th of Experimental Diets; World Trade and Convention Centre Halifax http://www.ual.es/ electrophoretic. May 21-23, 2013 | Moscow, Russia stecnicos_spe). Every experimental feed was separation of Halifax, Nova Scotia, Canada randomly assigned to triplicate group of proteins is a use- fifteen sea bream juveniles (15.4 g initial body ful tool to know Are you a decision-maker looking for business opportunities in weight). Fish were fed by hand twice per biomarine one of the many biomarine industries? Are you a in detail the type day (9:00 looking for a research partner percent of company and 17:00) at a rate of 3 or financing? inhibition of their body weight for 70 days. At the caused by pro- Join us at BioMarine 2013 in Halifax, Nova Scotia, Canada on end of the trial, fish were killed according to tease inhibitors. September 9-12, 2013. Meet CEOs, R&D partners and discuss the the requirements of the Directive 2010/63/ From the zymo- latest advances in: UE, and digestive tract was removed, and gram, it is clear  Aquaculture and Aquafeed REGISTER NOW then processes to obtain enzymatic extracts. that Ulva pro-  Marine BioTechs for Health & Environment Intestinal proteases were analysed by two duces a general- for FREE entrance at  Algae and Seaweeds different approaches: a) quantifying & Nutraceuticals inhibition in  Marine Ingredients the level ised www.viv.net  Bioprocessing of intestinal proteolytic activity, and b) visual- alkaline proteas- izing the profile of intestinal Biofuels  Biorefinery & proteases in es of sea bream. zymograms (Alarcón et al., 1998). In addition, On register: For more information about the program and to the contrary Special themes Opening the gates to the the presence of protease inhibitors in SW was Gracilaria did not Visit www.biomarine.org Russian Feed to Meat trade. tested according to Alarcón et al. (1999). affect any of the or contact us at Biomarine2013@nrc-cnrc.gc.ca active bands. Checking the BioMarine International Business Convention is co-organized same The fourth edition of the presence of The protease inhibitors in SW with the National Research Council of Canada. results were Results revealed the presence of protease observed after March-April 2013 | InternAtIonAl AquAFeed | 23 March-April 2013 | InternAtIonAl AquAFeed | 39
  4. 4. FEATURE Figure 3: Inhibition of intestinal proteolytic enzymes by Gracilaria cornea and Ulva rigida meal. Qualitative analysis: visualization of inhibition of active fractions in zymograms Figure 5: Total alkaline protease activity measured in extracts of sea bream fed different experimental diets containing graded levels of SW compensation mechanism against dietary References protease inhibitors in juvenile sea bream Alarcón FJ, Díaz M, Moyano FJ and Abellán E. has been previously proved by Santigosa et (1998) Characterization and functional properties al. (2010), who found similar results when of digestive proteases in two sparids; gilthead fish were fed diets with soybean trypsin sea bream (Sparus aurata) and common dentex inhibitor. (Dentex dentex). Fish Physiol Biochem. 19: 257-267. According to the results, it is clear that Alarcón, FJ, Moyano, FJ and Díaz, M. (1999). the amount of the pancreatic proteases Effect of inhibitors present in protein sources on secreted into the intestinal lumen in juvenile digestive proteases of juvenile sea bream (Sparus S. aurata is affected by the use of SW, par- aurata). Aquatic Living Res. 12: 233-238. ticularly Ulva. Nevertheless, it is also evident Oliveira, MN, Ponte-Freitas, AL, Urano-Carvalho, AF, that these ingredients did not cause qualita- Taveres-Sampaio, TM, Farias, DF, Alves-Teixera, DI, tive changes in the composition of alkaline Figure 4: Inhibition of sea Gouveia, ST, Gomes-Pereira, J and Castro-Catanho proteases, given that all fish showed the de Sena, MM. (2009) Nutritive and non-nutritive bream intestinal proteases after incubation of extracts same pattern of proteolytic enzymes in their attributes of washed-up seaweeds from the coast with solutions prepared using intestines, and that growth performance of of Ceará, Brazil. Food Chem. 11: 254-259. experimental diets containing 5, fish was not affected, as deduced from the in 15 and 25 percent of Ulva (UL) Santigosa, E, Sánchez, J, Médale, F, Pérez-Sánchez, J vivo feeding trial. and Gracilaria (GR) meal and Gallardo, MA. (2008). Modifications of digestive enzymes in trout (Onchorynchus mykiss) and sea Conclusions bream (Sparus aurata) in response to dietary In vitro protease inhibition assays are a use- fish meal replacement by plant protein sources. the level of alkaline protease activity was not ful tool to assess the presence of antinutrients Aquaculture 252: 68-74.accompanied by a decrease of fish growth in SW with potential use in aquafeeds. Based Santigosa, E, Sáez de Rodigrañez, MA, Rodiles, A, and feed utilization, since all fish grew equally on the results of this study, SW, especially García Barroso, F and Alarcón, FJ. (2010). Effect of (unpublished data). Santigosa et al. (2008) Ulva rigida, have antinutritive factors able to diets containing a purified soybean trypsin inhibitor reported a similar finding when trout were fed inhibit digestive proteases of S. aurata. Feeding on growth performance, digestive proteases and on diets including plant proteins. juvenile S. aurata on seaweed-based diets intestinal histology in juvenile sea bream (Sparus On the other hand, the analysis of decreased the amount of proteolytic activ- aurata L.). Aquaculture Res. 41: e187-e198.zymograms revealed that the pattern of ity secreted into the intestine. However, the Valente, LMP, Gouveia, A, Rema, P, Matos, J, Gomes, intestinal proteases was not modified by inclusion of SW does not alter the pattern EF and Pinto, IS. (2006) Evaluation of three inclusion of SW. All sea bream specimens of proteolytic enzymes in sea bream, which seaweeds Gracilaria bursa-pastoris, Ulva rigida showed the same number and distribu- reveals a compensating mechanism in this and Gracilaria cornea as dietary ingredients in tion of active fractions as in control group species. Research is being currently conducted European sea bass (Dicentrarchus labrax) juveniles. (after electrophoretical separation, the pat- to assess the effect of SW on other digestive Aquaculture 252: 85-91.tern of intestinal proteases in this species enzymes, intestinal microbiota, blood and is characterized by five groups of active tissue metabolites, and intestine and liver bands). These results confirmed that the histology after 70 days of feeding SW-based More inforMation:type of alkaline proteases secreted into the diets. Further research is needed in order María Isabel Sáez Casadointestinal lumen was not modified by any to known the impact of SW in a long-term Email: msc880@ual.esof experimental diets. The existence of a feeding assay. ■ 40 | InternAtIonAl AquAFeed | March-April 2013
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