March | April 2013

Effect of dietary inclusion of seaweeds on intestinal proteolytic activity of juvenile sea

bream, Sparus aurata

The International magazine for the aquaculture feed industry

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: 1464-0058

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In the last years considerable attentionhas been paid on the use of seaweeds(SW)asapossibleingredientforaqua-feeds.Red,greenandbrownSWcanbe

takenfromtheirnaturalhabitatandbroughtto the shore by the action of winds andtides. Otherwise, biomass can be obtainedfrom secondary and tertiary treatment ofeffluents. Wastewater treatment utilisingphotosynthetic organisms is an interest-ing alternative to reducethe ecological impact ofdomestic, industrial oraquaculture effluents.Generally,high-qualityalgalbiomass is yielded fromalgalcultivation,represent-inganexcellentsourceofhydrocolloids,carotenoids,and bioactive substances,which allows differentindustrial applications. Inaddition,thereiscurrentlyan increasing interestfor the potential of SWin human and animalnutrition.

Seaweed as ingredient in aquafeeds

AlthoughnutritionalpropertiesofSWarenotaswellknownasarethoseoflandplant-basedingredients,theirchemicalcompositionmaybecharacterisedbylowcontentinlipids,moderate in protein, but rich in non-starchpolysaccharides, minerals and vitamins. Lipidcontents range from 0.3 to 7.2 percent,althoughalgal lipidsarerichinPUFAsuchasC20:5n3 (eicosapentaenoic acid, EPA) andC22:6n3 (docosahexaenoic acid,DHA). Theproteincontributionisrangedfrom10to30g/100 g dry weight, which may vary greatlyamongSWspecies,environmentalconditions

(especiallyundernitrogen-enrichedcondition)andseason.

Thehighbiological valueof algalproteinsmakes algae suitable for inclusion both inanimalfeeds(especiallymarinespecies)andinhumandiets.Thehigh carbohydrate content(30to60%)isaverymarkedcharacteristicinmost SW, comprising mainly soluble carbo-hydrates, likesugars,andpectins,alginicacid,agar and carrageenan as well. Besides their

potential nutritional value, from a techno-logicalpointofview,SWcanalsobeusedasadditivesinthefeedindustry,for instance,asexcellentfeedagglutinants(improvingtextureandwaterstabilityofpellets),orasattractants(increasingfeedintake).

The effects of seaweeds on fishSeveral studieshaveproved that addition

ofsmallamountofSWinaquafeedsresultedin considerable positive effect on growthperformance and feed utilisation efficiency,carcass quality, physiological activity, intesti-nal microbiota, disease resistance, and stress

response(Valenteet al.,2006).Nonetheless,it has been also noted in other publicationsthat high SW inclusion reduces fish growthand feedefficiency.Fromthe literatureavail-ableitcanbedeductedthattheresponseofanimalstoSWseemstobedose-dependentand species-specific. Moreover, certain sub-stances with antinutritive activity may bepresent in SW, like lectins, tannins, phyticacid, and protease and amylase inhibitors

(Oliveiraet al.,2009).Suchantinutritionalfactorsmightinterferewithbioavailabilityand/ordigestibilityofnutri-ents.

Specialemphasisshouldbe focused on proteaseinhibitors. Binding of pro-tease inhibitors to pro-teolytic enzymes causesthe pancreas to secretelargeramountsofdigestiveenzymes toovercome thenegative effects of inhibi-tors on the digestion ofdietary protein. This factcan lead to decreasedweightgain,andpancreatichypertrophy in some fishspecies. For this reason,studies aimed to include

SW in aquafeeds must also bring up theirpossible effects on fish digestive physiology.Todate, there isscarce literatureanalysing ifSW inclusion causes negative consequencesondigestivephysiologyoffish.

Evaluating the effect of seaweeds on digestive proteases

Inarecentstudy,weevaluatedtheeffectofinclusionoftwoSWasdietaryingredientsonintestinalproteolyticactivityofjuvenileseabream.Gracilaria cornea (GR)andUlva rigida(UL)werechoseninthepresentstudyowingto its fast growth, low-cost production and

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, Spain

Figure 1: Detail of experimental feeds. UL-25 percent (above) and control (below)

38 | InternAtIonAl AquAFeed | March-April 2013

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March-April 2013 | InternAtIonAl AquAFeed | 39

successfulintegratedcultureinfish-farmefflu-ents.Biomasswasobtained fromtheMarineBiotechnology Centre (ULPGC, Spain). SWwerecultivatedin750Lsemicircularfibreglasstanks filled with seawater plus the fishpondeffluentsofapilotaquaculturesystem(11m3withanoptimaldensityofSparus aurataof20kgm-3, and awater renovation rateof 6–8volday-1).RedandgreenSWwerewashedwithseawater,sun-driedfor48hours,groundandsievedthrough0.1mmsievebeforebeingusedasadietaryingredient.

Dry algal biomass was incorporated intosix experimental diets (40% crude proteinand 12% crude lipid) at increasing levels (5,15 and 25%). A feed without SW servedas a control diet. Feeds were made at theUniversityofAlmeria-CEIA3facilities(Serviceof Experimental Diets; http://www.ual.es/stecnicos_spe). Every experimental feedwasrandomly assigned to triplicate group offifteenseabreamjuveniles(15.4ginitialbodyweight). Fish were fed by hand twice perday (9:00 and 17:00) at a rate of 3 percentof their body weight for 70 days. At theendof thetrial, fishwerekilledaccording tothe requirements of the Directive 2010/63/UE, and digestive tract was removed, andthenprocesses toobtainenzymaticextracts.Intestinal proteases were analysed by twodifferent approaches: a) quantifying the levelofintestinalproteolyticactivity,andb)visual-izing the profile of intestinal proteases inzymograms(Alarcónet al.,1998).Inaddition,thepresenceofproteaseinhibitorsinSWwastestedaccordingtoAlarcónet al.(1999).

Checking the presence of protease inhibitors in SW

Results revealed thepresenceof protease

inhibitors in SW.Dose-responsecurves showedthat UL containedsubstances able toreduce digestiveproteolytic activityin sea bream (upto77%),whereasanegligible inhibitionby GR was found(4%). Obvious dif-ferences in thekinetic of inhibitionof protease activitywere found forUL.Equation definingsuch curve may beused topredict theexpected percent-age of reductionin protease activ-ity, once proteaseactivityinthediges-tive tract and the

amount of feed ingested are known. Forinstance, in thecaseof40g seabream, totalproteaseactivityreleasedafteramealisaround1,300units.Thosefishthatconsumed0.5gofafeedcontaining15percentofUL,showedaratiomgULper unit of activity of 50,whichdetermined areduction nearly40percentintheactivity of diges-tive proteases.Fortunately, fishhavemechanismsto compensatetheeffectof die-taryantinutrients.

Zymogramsobtained afterelectrophoretic.separation ofproteinsisause-ful tool to knowindetailthetypeof inhibitioncaused by pro-tease inhibitors.From the zymo-gram, it is clearthat Ulva pro-duces a general-ised inhibition inalkaline proteas-esof seabream.On the contraryGracilariadidnotaffect any of theactivebands.

The sameresults wereobserved after

incubationofdigestiveproteaseswithextractsoftheexperimentaldiets.Themeaninhibitionranged from 11 to 48 percent. In general,UL-supplemented feeds showed inhibitionvalues higher than the GR-supplementeddiets,whichdidnot exceed16percent. ForULdiets,itwasfoundthatpercentageofinhi-bitionwaspositivelycorrelatedwiththeSWinclusion level, which agrees with the abovementioned dose-response curve. InhibitionproducedbyGR feedscannotbeassociatedtotheuseofthisSW.

Effect of seaweed on digestive proteases of sea bream

Digestiveenzymeswereaffectedbydiets,as fish had different enzyme activity level ofalkaline proteases after 70 days of feedingexperimental diets. In general, a decreasein alkaline protease activity was evidencedwhenfeedsincludedULorGR. Inparticular,the proteolytic activities of fish fed Ulvasupplemented-feeds were significantly lowerthan those of fish fed on control diet. Thepresence of protease inhibitors in SW maybe the reason of the progressive decreaseintheproteolyticactivityinfishfeddietwithincreasing levels of Ulva meal. Supportingthis hypothesis, it has been confirmed thataqueousextractsofUlvameal inhibitalkalineproteasesofS. aurata.Moreover,thedropin

Figure 2: Dose-response curves obtained when different amounts of SW meal (0 to 300 µg) were incubated with a

fixed amount of proteolytic activity (1 U) in the inhibitory assay. Protease inhibition was expressed as the percentage of reduction in proteolytic activity. Such curves are a simple way

to evaluate how hypothetical variations in the inclusion of SW might affect sea bream digestive proteases

38 | InternAtIonAl AquAFeed | March-April 2013 March-April 2013 | InternAtIonAl AquAFeed | 39

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Need for novel sources

In order to reducedependence on fish oil, sig-nificant breakthroughs have

occurred over the past fewyearsinreplacingitwithplantoils.Bysubstitutingfeedswith

plantoils,italsoservestoreducecostsduetothefactthatvegeta-

bleoilshavesteadilyincreasingproduc-tion, high availability and better economicvalue.Several studieshavebeencarriedout

toinvestigatecertainvegetableoilsaspos-siblesustainablepartialsubstitutesforfishoils incompoundedfish feeds.Themost

commonvegetableoilsusedforfishfeedpro-ductionhavebeensoybean,linseed,rapeseed,sunflower,palmoilandoliveoil.

Soybeanand rapeseedoil areconsideredpossible alternative lipid sources for salmo-nids, freshwater and marine fish since theyare rich in PUFAs, especially linoleic (18:2ω−6)andoleicacid(18:1ω−9),butdevoidof n-3 PUFA. However, in some cases, fishoil substitution by 60 percent rapeseed oilhas been found to decrease European seabass (Dicentrarchus labrax) growth. Soybeanoil appears to be a better plant lipid sourceregarding gilthead seabream (Sparus aurata)growth while considerable savings in feedcostscouldbeachievedifitcouldbeusedas

a partial dietary substitute for fish oil withincompoundfeeds.Thesameistrueoflinseedoil and rapeseed oil, although to a lesserextent.

Furthermore,theuseofpalmoilindietsofAtlantic salmonand rainbow trouthas given

growthandfeedutilizationefficiencycompa-rabletofishfedwithequivalent levelsof fishoil. Olive oil could also be used as a partialsubstitute for dietary fish oil in Europeansea bass culture, during growth out phase,Atlantic salmon (salmo salar) and rainbowtrout(Oncorhynchus mykiss)withdatashowingsimilargrowthratestotheoneswhenfishwasfedon100percentfishoildiet.Allthesestud-

ieshavebeenrecentlyreviewed(NasopoulouandZabetakis,2012).

New, alternative and in a way ‘non-orthodox’, sources of lipids need to beidentified and valorised in order to achievesustainableproductionof fish feedsand thus

enablingthefurtherdevelopmentofaquacul-tureapplications.Suchpromisinglipidsourcesarevegetableoils(VO).TheuseofVObasedaquafeedshassomestrongadvantages.Olivepomace (OP) and olive pomace oil (OPO)are natural by-products of olive oil produc-tion, which contain micro constituents withatheroprotective(substances)activitysuchasPAF-inhibitorsandphenolic/polyphenolicmol-

Figure 2: Representative optic micrographs x 100 of aortic wall sections stained with haematoxylin and eosin from the two experimental groups, where atherosclerotic

lesions appear as foam cells (↑). (A) Group A (atherogenic diet); (B) Group B (atherogenic diet enriched with sea bream polar lipids) (adopted from Nasopoulou et

al., 2010). Copyright, “Food Chemistry” Elsevier

B)

22 | InternAtIonAl AquAFeed | March-April 2013 March-April 2013 | InternAtIonAl AquAFeed | 23

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thelevelofalkalineproteaseactivitywasnotaccompanied by a decrease of fish growthandfeedutilization,sinceallfishgrewequally(unpublished data). Santigosa et al. (2008)reportedasimilarfindingwhentroutwerefedondietsincludingplantproteins.

On the other hand, the analysis ofzymograms revealed that the pattern ofintestinal proteases was not modified byinclusion of SW. All sea bream specimensshowed the same number and distribu-tionof active fractions as in control group(afterelectrophoreticalseparation,thepat-tern of intestinal proteases in this speciesis characterized by five groups of activebands). These results confirmed that thetypeofalkalineproteasessecretedintotheintestinal lumen was not modified by anyof experimental diets. The existence of a

compensation mechanism against dietaryprotease inhibitors in juvenile sea breamhasbeenpreviouslyprovedbySantigosaet al. (2010), who found similar results whenfish were fed diets with soybean trypsininhibitor.

According to the results, it is clear thatthe amount of the pancreatic proteasessecretedintotheintestinallumeninjuvenileS. aurata isaffectedby theuseofSW,par-ticularlyUlva.Nevertheless, it isalsoevidentthatthese ingredientsdidnotcausequalita-tive changes in the composition of alkalineproteases, given that all fish showed thesamepatternofproteolyticenzymesintheirintestines, and that growth performance offishwasnotaffected,asdeducedfromthein vivofeedingtrial.

ConclusionsIn vitroproteaseinhibitionassaysareause-

fultooltoassessthepresenceofantinutrientsinSWwithpotentialuseinaquafeeds.Basedon the results of this study, SW, especiallyUlva rigida, have antinutritive factors able toinhibitdigestiveproteasesofS. aurata.Feedingjuvenile S. aurata on seaweed-based dietsdecreased the amount of proteolytic activ-ity secreted into the intestine. However, theinclusion of SW does not alter the patternof proteolytic enzymes in sea bream, whichreveals a compensating mechanism in thisspecies.ResearchisbeingcurrentlyconductedtoassesstheeffectofSWonotherdigestiveenzymes, intestinal microbiota, blood andtissue metabolites, and intestine and liverhistology after 70 days of feeding SW-baseddiets. Further research is needed in orderto known the impact of SW in a long-termfeedingassay. ■

References

AlarcónFJ,DíazM,MoyanoFJandAbellánE.(1998)Characterizationandfunctionalpropertiesofdigestiveproteasesintwosparids;giltheadseabream(Sparus aurata)andcommondentex(Dentex dentex).FishPhysiolBiochem.19:257-267.

Alarcón,FJ,Moyano,FJandDíaz,M.(1999).Effectofinhibitorspresentinproteinsourcesondigestiveproteasesofjuvenileseabream(Sparus aurata).AquaticLivingRes.12:233-238.

Oliveira,MN,Ponte-Freitas,AL,Urano-Carvalho,AF,Taveres-Sampaio,TM,Farias,DF,Alves-Teixera,DI,Gouveia,ST,Gomes-Pereira,JandCastro-CatanhodeSena,MM.(2009)Nutritiveandnon-nutritiveattributesofwashed-upseaweedsfromthecoastofCeará,Brazil.FoodChem.11:254-259.

Santigosa,E,Sánchez,J,Médale,F,Pérez-Sánchez,JandGallardo,MA.(2008).Modificationsofdigestiveenzymesintrout(Onchorynchus mykiss)andseabream(Sparus aurata)inresponsetodietaryfishmealreplacementbyplantproteinsources.Aquaculture252:68-74.

Santigosa,E,SáezdeRodigrañez,MA,Rodiles,A,GarcíaBarroso,FandAlarcón,FJ.(2010).Effectofdietscontainingapurifiedsoybeantrypsininhibitorongrowthperformance,digestiveproteasesandintestinalhistologyinjuvenileseabream(Sparus aurataL.).AquacultureRes.41:e187-e198.

Valente,LMP,Gouveia,A,Rema,P,Matos,J,Gomes,EFandPinto,IS.(2006)EvaluationofthreeseaweedsGracilariabursa-pastoris,Ulva rigidaandGracilaria corneaasdietaryingredientsinEuropeanseabass(Dicentrarchus labrax)juveniles.Aquaculture252:85-91.

Figure 4: Inhibition of sea bream intestinal proteases

after incubation of extracts with solutions prepared using

experimental diets containing 5, 15 and 25 percent of Ulva (UL)

and Gracilaria (GR) meal

Figure 5: Total alkaline protease activity measured in extracts of sea bream fed different experimental diets containing graded levels of SW

Figure 3: Inhibition of intestinal proteolytic enzymes

by Gracilaria cornea and Ulva rigida meal. Qualitative

analysis: visualization of inhibition of active fractions in

zymograms

More inforMation:María Isabel Sáez Casado Email: msc880@ual.es

40 | InternAtIonAl AquAFeed | March-April 2013

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March-April 2013 | InternAtIonAl AquAFeed | 41

40 | InternAtIonAl AquAFeed | March-April 2013 March-April 2013 | InternAtIonAl AquAFeed | 41

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Whilesomemicro-organismsproliferateina narrow range of environmental conditions(pH,oxygen,availability,etc.),certainenzymesareabletoactinmultipleenvironments.

Nevertheless, some products combiningthepositiveeffectsofbeneficialbacteria andenzymesarealreadybeingusedasbioreme-diationagentsinaquaculture.

Efficacy of enzymes in bioremediation

Enzymes have the capacity to stabilizethe soil organic matter and can be usedeffectively tomanage soil quality and rear-ing conditions for aquatic species.There isnot one specific enzyme that works bestinallcases.Ablendcontainingavarietyofenzymesmaybe themosteffectivemeansforbioremediationinaquaculture.Enzymesgreatly reduce sludge accumulation andanaerobic conditions in pond bottoms.They promote a faster degradation ofthe accumulated organic matter especiallyunder intensive production conditions.This organic matter comprises uneatenfeed, dead plankton, mineral soils, faecesandpathogenicmicro-organisms in thesoilwhere the conditions are often anaerobic.However,forallthesebioremediationproc-esses catalyzed by enzymes, the presenceof beneficial bacteria is important as well.Enzymes acceleratemicrobial processes by

breaking apart large sludge particles, thuscreatingwidersurfaceareaswhichcanthenbe fermented by microbes. This reductionof sludge and dead organic matter can beseenvisuallynotonlythroughbetterwaterquality,butalsothroughbettersoilquality.

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Four earth shrimpponds (0.7 – 0.8 ha/pond) with a depth of 1 – 1.2 m werestockedwithjuvenileshrimp(approximately1.4 g/shrimp)with a density of 50 shrimp/m². The trial was carried out for a periodof 57 days with a dosage of 500 g/ha ofproduct applied once a month to thetreatment group (twoponds).The controlpondsconsistedoftwopondswithnormalproductionoperations.

The soil of the AquaStar® ponds inPicture 1 was of yellow colour which isregarded as the best bottom type, while

the soil of the control ponds in Picture 2exhibited a dark black colour, an indica-tion of the accumulation of dead organicmatter.

Resultssuggestedthatwiththecombineduseofbeneficialbacteriaandenzymes,pondsoils containing black and glutinous organicsludgeturnedintoamoreyellowsoil.

Intermsofperformance,theaveragedailyweightgainofshrimpintheAquaStar®groupincreasedby36percentandfeedconversionratio improvedby9percentcomparedwiththe control (no probiotic inclusion). TheresultsareshowninFigure4and5.

Basedon theseresults, itwasconcludedthat in the search for more effective andenvironmentally-friendly treatments, benefi-cialbacteriahaveemergedasaviablealter-native. The application of bioremediationsolutions in aquaculture can also benefitfrom the inclusionof enzymes, especially inintensive productions. AquaStar® positivelyaffects the performance of shrimp whilemaintaining a stable environment in thepond, proving to be an effective manage-menttoolinaquaculture.

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