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

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March | April 2013Effect of dietary inclusion of seaweeds on

intestinal proteolytic activity of juvenile seabream, Sparus aurata

The International magazine for the aquaculture feed industry

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In thelast years considerable attention

has beenpaidon the useofseaweeds

(SW)asapossibleingredientforaqua-feeds.Red,greenandbrownSWcanbe

takenfromtheirnaturalhabitatandbrought

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 interest-

ing alternative to reduce

the ecological impact of

domestic, industrial or

aquaculture e ff luents .

Generally,high-qualityalgalbiomass is yielded from

algalcultivation,represent-

inganexcellentsourceof

hydrocolloids,carotenoids,

and bioactive substances,

which allows dif ferent

industrial applications. In

addition,thereiscurrently

an increas ing interest

for the potential of SW

in human and animal

nutrition.

Seaweed asingredientin aquafeeds

AlthoughnutritionalpropertiesofSWare

notaswellknownasarethoseoflandplant-

basedingredients,theirchemicalcomposition

maybecharacterisedbylowcontentinlipids,

moderate in protein, but rich in non-starch

polysaccharides, minerals and vitamins. Lipid

contents range from 0.3 to 7.2 percent,

althoughalgallipidsarerichinPUFAsuchasC20:5n3 (eicosapentaenoic acid, EPA) and

C22:6n3 (docosahexaenoicacid,DHA).The

proteincontributionisrangedfrom10to30

g/100 g dry weight, whichmay vary greatly

amongSWspecies,environmentalconditions

(especiallyundernitrogen-enrichedcondition)

andseason.

Thehighbiologicalvalueofalgalproteinsmakes algae suitable for inclusion both in

animalfeeds(especiallymarinespecies)andin

humandiets.Thehighcarbohydratecontent

(30to60%)isaverymarkedcharacteristicin

most SW, comprising mainly soluble carbo-

hydrates,likesugars,andpectins,alginicacid,

agar and carrageenan as well. Besides their

potential nutritional value, from a techno-

logicalpointofview,SWcanalsobeusedas

additivesinthefeedindustry,forinstance,as

excellentfeedagglutinants(improvingtexture

andwaterstabilityofpellets),orasattractants

(increasingfeedintake).

The effects of seaweeds on fish

SeveralstudieshaveprovedthatadditionofsmallamountofSWinaquafeedsresulted

in considerable positive effect on growth

performance and feed utilisation efficiency,

carcass quality, physiological activity, intesti-

nalmicrobiota, disease resistance,and stress

response(Valente et al.,2006).Nonetheless,

ithas been alsonotedin otherpublications

that high SW inclusion reduces fish growthandfeedefficiency.Fromtheliteratureavail-

ableitcanbedeductedthattheresponseof

animalstoSWseemstobedose-dependent

and species-specific. Moreover, certain sub-

stances with antinutritive activity may be

present in SW, like lectins, tannins, phytic

acid, and protease and amylase inhibitors

(Oliveiraet al.,2009).Such

antinutritionalfactorsmight

interferewithbioavailability

and/ordigestibilityofnutri-

ents.Specialemphasisshould

be focused on protease

inhibitors. Binding of pro-

tease inhibi tors to pro-

teolyt ic enzymes causes

the pancreas to secrete

largeramountsofdigestive

enzymestoovercomethe

negative effects of inhibi-

tors on the digestion of

dietary protein. This fact

can lead to decreased

weightgain,andpancreatic

hypertrophy in some fish

species. For this reason,

studies aimed to include

SW in aquafeeds must also bring up their

possible effects on fish digestive physiology.

Todate,thereisscarceliteratureanalysingif

SW inclusion causes negative consequences

ondigestivephysiologyoffish.

Evaluating the effect of seaweedson digestive proteases

Inarecentstudy,weevaluatedtheeffectofinclusionoftwoSWasdietaryingredients

onintestinalproteolyticactivityofjuvenilesea

bream.Gracilaria cornea(GR)andUlva rigida

(UL)werechoseninthepresentstudyowing

to its fast growth, low-cost production and

Effect of dietary inclusion

of seaweeds on intestinal

proteolytic activity of juvenile

sea bream, Sparus aurata

by Mara Isabel Sez, Toms Martnez and Javier Alarcn,Universidad de Almera-CEIA3, Spain

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

38 | IntenAtIonA AquAFeed | March-April 2013

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successfulintegratedcultureinfish-farmefflu-

ents.BiomasswasobtainedfromtheMarine

Biotechnology Centre (ULPGC, Spain). SW

werecultivatedin750Lsemicircularfibreglass

tanks filled with seawater plus the fishpond

effluentsofapilotaquaculturesystem(11m 3

withanoptimaldensityofSparus aurataof20

kgm-3,andawaterrenovationrateof68

volday-1).RedandgreenSWwerewashed

withseawater,sun-driedfor48hours,groundandsievedthrough0.1mmsievebeforebeing

usedasadietaryingredient.

Dry algal biomass was incorporated into

six experimental diets (40% crude protein

and 12% crudelipid) atincreasing levels (5,

15 and 25%). A feed without SW served

as a control diet. Feeds were made at the

UniversityofAlmeria-CEIA 3facilities(Service

of Experimental Diets; http://www.ual.es/

stecnicos_spe).Every experimental feedwas

randomly assigned to triplicate group of

fifteenseabreamjuveniles(15.4ginitialbody

weight). Fish were fed by hand twice per

day (9:00and17:00) ata rateof 3percent

of their body weight for 70 days. At the

endofthetrial,fishwerekilledaccordingto

the requirements of the Directive 2010/63/

UE, and digestive tract was removed, and

thenprocesses toobtainenzymaticextracts.

Intestinal proteases were analysed by two

differentapproaches:a) quantifyingthe level

ofintestinalproteolyticactivity,andb)visual-

izing the profile of intestinal proteases in

zymograms(Alarcnet al.,1998).Inaddition,

thepresenceofproteaseinhibitorsinSWwastestedaccordingtoAlarcnet al.(1999).

Checking the presence ofprotease inhibitors in SW

Resultsrevealedthe presenceofprotease

inhibitors in SW.

Dose-response

curves showed

that UL contained

substances able to

reduce digestive

proteolytic activity

in sea bream (up

to77%),whereasa

negligible inhibition

by GR was found

(4%). Obvious dif-

ferences in the

kinetic of inhibition

of protease activity

werefoundforUL.

Equation defining

such curvemay be

used topredictthe

expected percent-

age of reduction

in protease activ-

ity, once proteaseactivityinthediges-

tive tract and the

amount of feed ingested are known. For

instance,inthecaseof40gseabream,total

proteaseactivityreleasedafteramealisaround

1,300units.Thosefishthatconsumed0.5gof

afeedcontaining15percentofUL,showeda

ratiomgULperunit ofactivityof50,which

determined a

reduction nearly

40percentinthe

activity of diges-

tive proteases.

Fortunately, fish

havemechanisms

to compensate

theeffectof die-

taryantinutrients.

Zymograms

obtained after

electrophoretic.

separation of

proteinsisause-

ful tool toknow

indetailthetype

of inhibition

caused by pro-

tease inhibitors.

From the zymo-

gram, it is clear

that Ulva pro-

duces a general-

ised inhibition in

alkaline proteas-

esofseabream.

On the contrary

Gracilariadidnotaffectanyofthe

activebands.

The same

results were

observed after

incubationofdigestiveproteaseswithextracts

oftheexperimentaldiets.Themeaninhibition

ranged from 11 to 48 percent. In general,

UL-supplemented feeds showed inhibition

values higher than the GR-supplemented

diets,whichdidnotexceed16percent.For

ULdiets,itwasfoundthatpercentageofinhi-

bitionwaspositivelycorrelatedwiththeSW

inclusion level, whichagreeswith theabove

mentioned dose-response curve. Inhibition

producedbyGRfeedscannotbeassociated

totheuseof thisSW.

Effect of seaweed on digestiveproteases of sea bream

Digestiveenzymeswereaffectedbydiets,

asfishhaddifferentenzyme activitylevel of

alkaline proteases after 70 days of feeding

experimental diets. In general, a decrease

in alkaline protease activity was evidenced

whenfeedsincludedULorGR.Inparticular,

the proteolytic activities of fish fed Ulva

supplemented-feeds were significantly lowerthan those of fish fed on control diet. The

presence of protease inhibitors in SWmay

be the reason of the progressive decrease

intheproteolyticactivityinfishfeddietwith

increasing levels of Ulva meal. Supporting

this hypothesis, it has been confirmed that

aqueousextractsofUlvamealinhibitalkaline

proteasesofS. aurata.Moreover,thedropin

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

fixed amount of proteolytic activity (1 U) in the inhibitoryassay. Protease inhibition was expressed as the percentage ofreduction 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

March-April 2013 | IntenAtIonA AquAFeed | 39

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thelevelofalkalineprotease activitywasnot

accompanied by a decrease of fish growth

andfeedutilization,sinceallfishgrewequally

(unpublished data). Santigosa et al. (2008)

reportedasimilarfindingwhentroutwerefed

ondietsincludingplantproteins.

On the other hand, the ana lysi s of

zymograms revealed that the pattern of

intestinal proteases was not modified by

inclusion ofSW. Allsea breamspecimens

showed the same number and distr ibu-

tion of active fractions as in control group

(afterelectrophoreticalseparation,thepat-

tern of intestinal proteases in this speciesis characterized by five groups of active

bands). These results confirmed that the

typeofalkaline proteases secreted into the

intestinal lumen was notmodified by any

of experimental diets. The existence of a

compensation mechanism against dietary

protease inhibitors in juvenile sea bream

hasbeenpreviouslyprovedbySantigosaet

al.(2010),whofoundsimilarresultswhen

fish were fed diets with soybean trypsin

inhibitor.

According tothe results,it isclear that

the amount of the pancreat ic proteases

secretedintotheintestinallumeninjuvenile

S. aurataisaffectedbytheuseofSW,par-ticularlyUlva.Nevertheless,itisalsoevident

that these ingredientsdidnot cause qualita-

tive changes in the composition of alkaline

proteases, given that all fish showed the

samepatternofproteolyticenzymesintheir

intestines, and thatgrowth performance of

fishwasnotaffected,asdeducedfromthein

vivofeedingtrial.

Conclusions

In vitroproteaseinhibitionassaysareause-

fultooltoassessthepresenceofantinutrients

inSWwithpotentialuseinaquafeeds.Based

on the results of this study, SW, especially

Ulva rigida, have antinutritive factors able to

inhibitdigestiveproteasesofS. aurata.Feeding

juvenile S. aurata on seaweed-based diets

decreased the amount of proteolytic activ-

ity secreted intothe intestine. However, the

inclusion of SWdoes not alter the pattern

of proteolytic enzymes in sea bream, which

reveals a compensating mechanism in this

species.Researchisbeingcurrentlyconducted

toassess theeffectofSWonotherdigestive

enzymes, intestinal microbiota, blood andtissue metabolites, and intestine and liver

histologyafter 70daysof feedingSW-based

diets. Further research is needed in order

to known the impact of SW in a long-term

feedingassay.

References

AlarcnFJ,DazM,MoyanoFJandAbellnE.

(1998)Characterizationandfunctionalproperties

ofdigestiveproteasesintwosparids;gilthead

seabream(Sparus aurata)andcommondentex

(Dentex dentex).FishPhysiolBiochem.19:257-267.

Alarcn,FJ,Moyano,FJandDaz,M.(1999).

Effectofinhibitorspresentinproteinsourceson

digestiveproteasesofjuvenileseabream( 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-Catanho

deSena,MM.(2009)Nutritiveandnon-nutritive

attributesofwashed-upseaweedsfromthecoast

ofCear,Brazil.FoodChem.11:254-259.

Santigosa,E,Snchez,J,Mdale,F,Prez-Snchez,J

andGallardo,MA.(2008).Modificationsofdigestive

enzymesintrout(Onchorynchus mykiss)andsea

bream(Sparus aurata)inresponsetodietary

fishmealreplacementbyplantproteinsources.

Aquaculture252:68-74.

Santigosa,E,SezdeRodigraez,MA,Rodiles,A,

GarcaBarroso,FandAlarcn,FJ.(2010).Effectof

dietscontainingapurifiedsoybeantrypsininhibitor

ongrowthperformance,digestiveproteasesand

intestinalhistologyinjuvenileseabream( Sparus

aurataL.).AquacultureRes.41:e187-e198.

Valente,LMP,Gouveia,A,Rema,P,Matos,J,Gomes,

EFandPinto,IS.(2006)Evaluationofthree

seaweedsGracilariabursa-pastoris,Ulva rigida

andGracilaria corneaasdietaryingredientsin

Europeanseabass(Dicentrarchus labrax)juveniles.

Aquaculture252:85-91.

Figure 4: Inhibition of seabream intestinal proteases

after incubation of extractswith 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 ofintestinal proteolytic enzymes

by Gracilaria corneaandUlva rigida meal. Qualitative

analysis: visualization ofinhibition of active fractions in

zymograms

MoreinforMation:

Mara Isabel Sez Casado

Email: [email protected]

40 | IntenAtIonA AquAFeed | March-April 2013

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

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