July | August 2012

Unlocking the hidden potential of plant proteins using solid state fermentation

enzymes

The International magazine for the aquaculture feed industry

International Aquafeed is published five 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 2012 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

In the terrestrial animal feed industrythe use of exogenous enzymes is arelatively common practice and todaytheglobalfeedenzymemarketisworth

more than US$550 million.This saves theglobal feed market an estimated US$3-5billionperyear.Todatetheuseofenzymesin aquaculture feeds has been limited, butinterestisgrowingduetotheincreasinguseofplantbasedprotein ingredientsandtheirby-products.

The incorporationofSynergen™(AlltechInc., USA), a natural solid state fermenta-tion complex that improves profitability bymaximizing nutrient release, has enabled thefishmeal content of diets for several com-mercialcarnivorousfishspeciestobereducedby up to 65 percent. These diets have alsomaintainedthesameorachievedevenbettergrowthperformancewhencompared to thehighfishmealdiets.

This article highlights the recent workcarriedoutwithGilthead seabream (Sparus aurata),redseabream(Pagrus major),amber-jack (Serioli dumerili), pangasius catfish andNiletilapia.

Modern aquafeed challenges: maximising nutrient availability

Theeconomicsuccessandsustainabilityofaquaculture depends on minimising produc-tion cost to maintain profitability, and feedgenerallycomprisessome50to60percentofthetotalcostinintensiveculture.Traditionallyfishmeal has been the preferred ingredientdue to its high protein content, favourableaminoacidandmineralprofiles,oilandotherbenefits.Itsreplacementwithplantbasedpro-tein ingredients has been brought about bytheincreasingcostoffishmeal,itslimitedavail-abilityandtherequirementtoensurethatthisexpanding industry remains both financiallyprofitableandenvironmentallysustainable.

Plant derived feed ingredients howeverhave several limitations. Non starch polysac-charides (NSPs) are a complex group ofpolysaccharides which act as energy storagecarbohydratesingrainsandseed.Whiletheymayprovideacheapsourceofdietaryenergy

NSPscannotbebrokendownbythedigestiveenzymes of many fish species especially thecarnivorousones.

A wide range of anti-nutritional factors(ANFs) such as protease inhibitors, non-digestiblecarbohydrates,lectins,saponinsandphytatesmayalsobepresentinanumberofplantderivedmaterialssuchaslegumeseeds,soybeanmeal, rapeseedmealetc.Thesecanimpede digestion in fish often decreasingintestinal viscosity and bacterial loads whichinturnaffecttheanimals’performance.TheseANFshave therefore tobe removedbeforetheycanbeincorporatedincommercialfeeds.

TheuseofhighlydigestibleandprocessedsoybeanmealsuchaslowantigenSPC(soypro-tein concentrates) or proc-essed corn gluten to replacefishmealispossiblebutthesehighly processed ingredientsare expensive and the eco-nomic savings can thereforeberelativelylow.

Anothersolutionforcoun-terbalancing the digestibilityproblemsofsuchingredientsistouseenzymesthat improvesubstratedigestibilityresultingin higher nutrient availabilitythus improving growth, foodconversion rates and there-foreeconomicefficiency.

Today a better under-standing of how enzymeswork inanimal feedsandtheincreasing evidence of theirabilitytoimprovefeedquality,shown in the last 10 years,has allowed a more flexibleapproachbyfeedformulators.

Solid State Fermentation Technology

Alltech has pioneeredthe production of SSF tech-nologies for the animal feedindustry. Eight years of col-laborative research between

Alltech’s Bioscience Centers in Dunboyne,Ireland; Bangkok, Thailand; and Kentucky,USA,haveresulted ina$40millionstate-of-the-artproductionfacilityinSerdan,Mexico.

The SSF process involves the carefulselectionofspecificstrainsofnaturallyoccur-ring fungiwhichhave the ability to fermentawiderangeofagriculturalproductssuchasDDGS, corncob, palm kernel, wheat bran,rapeseedoilcakeandsoybean.Theselectedfungiarefirstpropagatedinaliquidmediatoproduce a large volume of inoculum whichis mixed with pre-sterilised selected solidsubstratemediatoproduceamixtureknown

Unlocking the hidden potential of plant proteins using solid state fermentation technologyby John Sweetman1, Ioannis Nengas2 and Serge Corneillie3

table 1: Diet composition for Gilthead sea bream

Diet 1 2 3

Control natural complex

Pre-treated with natural

complex

Fish meal 25 25 25

Soybean meal 40 40 40

Wheat meal 16.7 16.65 16.65

Corn gluten 5 5 5

Fish oil 13 13 13

Vit-Min 0.3 0.3 0.3

Synergen 0.05 0.05

table 2: effect of diet on the performance of Gilthead sea bream after 12 weeks.

Control natural complex

Pre-treated with natural

complex

Initial weight (g) 31,11±0,65 30,77±1,37 31,65±0,80

final weight (g) 84,04±5,76 95,57±3,57 100,75±1,90

FCr 1,45±0,13a 1,18±0,01b 1,13±0,02b

SGr 0,83±0,05a 0,94±0,07b 0,97±0,04b

Values are means of three replicates expressed with the standard deviation between tanks. Values with common superscripts demonstrate no significant differences among groups (p<0.05)

Specific growth rate (SGR) = (ln final weight - ln initial weight)*100/days

Feed conversion ratio (FCR) = feed consumed g / weight increase g

32 | InternAtIonAl AquAFeed | July-August 2012

FEATURE

July-August 2012 | InternAtIonAl AquAFeed | 33

as ‘Koji’.Under strict aseptic conditions theKojiisthenevenlydistributedontotraysandintroduced into environmentally controlledSSF culture chambers for up to five days.During this time the fungus grows rapidly,breaking down the fibrous and non-fibrousportions of the chosen substrate. Doing sodramatically changes the nutritional profileofthematerialandresultsinthegenerationofproductsthatcanbeusedtoreformulatediets.Onday five, theKoji isextractedandtheby-productisdried.

The product from the Alltech SSF proc-ess, Synergen™, allows for a more flexibleapproach to feed formulation through theinclusionofby-productsorbyreducingnutri-ent constraints in the diet. It has also beenshown,throughanimalperformance,toremain

effective over awide range of feedprocessing condi-tions.

Carnivorous fish: Gilthead sea bream

At the Instituteof Aquaculture ofthe Hellenic Centrefor Marine researchan experiment wasperformed in whichapproximately 31 gjuvenileGiltheadseabream were fed 3differentdiets(Table

table 3: experimental diet composition for red sea bream.

1 2 3 4 5 6

Ingredients FM50 FM20 Ft FtP Fte0.05 Fte 0.1

anchovy meal 50 20 20 20 20 20

Soybean meal 0 18 18 18 18 18

Corn gluten meal 5 23 23 23 23 23

Fish oil 5 5 5 5 5 5

Soybean oil 5 7.4 7.4 7.4 7.4 7.4

Ca(H2Po4)2 1 1 1 1 1 1

taurine - - 0.2 0.2 0.2 0.2

Phytase - - - - - -

(IU/g) - - - 1000 - -

natural complex - - - - 0.05 0.1

others* 34 25.6 25.4 25.2 25.35 25.3

others: Starch, vitamin premix and wheat flour

Figure 1. Feed conversion ratio (a) and Specific growth rate (b) of the Gilthead sea bream fed different diets

For more information: contactlfa@lesaffre.fr

Innovative and proven yeast products in aquaculture

Wellbeing, the source of performance

Consistent products supported bythe Lesaffre group experience andits unique know-how in biotechnologyand nutrition;

Selected strains and controlled production;

Designed to solve nutritional and sanitary issues;

Dedicated range of products:live yeast, yeast cell wall, yeast extractand enriched yeast.

32 | InternAtIonAl AquAFeed | July-August 2012 July-August 2012 | InternAtIonAl AquAFeed | 33

FEATURE

1)intriplicateatawatertemperatureof18±2°C.Theseabreamwerefedwithacontroldiet(1)withamoderate25percentfishmealinclusion and two other diets in which aportion of the wheat meal was replaced bySynergen, incorporated at 0.05%. In diet 2,‘Natural complex’, Synergen was incorpo-rated into thewhole ingredientmixprior toextrusion while in diet 3, ‘Pre-treated with

natural complex’, the plant ingredients werehydrolyzedwith theSynergen for fourhoursat40°Cpriortoextrusion.

The diet whose ingredients were pre-treated by hydrolysing them with Synergenbefore the extrusion gave slightly better butnot significantly different results than thediet which incorporated Synergen as a sup-plemented ingredient, mixed directly withthe rest of the ingredients. Both these dietsperformedsignificantlybetterthanthecontroldiet with improved growth, SGR and FCR(Table2andFigure1).

Pre-treatment of raw materials in com-mercial feedproductionunits isaprocedure

that requiresadditionalfacilities andtherefore itwould need achange in theproductionline. Sincethe resultsobtainedfromthe ‘naturalcomplex’diet have nosignificant dif-

ference from the ‘pre-treated with naturalcomplex’ diet, this indicates that the naturalcomplexcanbeeffectivelyaddeddirectly totheingredientmix.The‘naturalcomplex’dietgave an improvement of 18 percent in FCRanda13percentimprovementinSGRwhencomparedtothecontroldiet.

Currently industrial trials and commercialapplication are underway. Based solely on

the improvement in FCR, areturn on investment of 1:43 can be expected whenSynergen is added in thismannerforthisspecieswithadditional increased profitbenefits to be added fromthe growth rate improve-ments.

Carnivorous fish: Red sea bream

Improved growth rateshave also been achievedbySatohetal.(2011)withjuvenile red sea bream. Inthis case red sea bream,of approximately 13.5 gwere fedsixdifferentdiets(Table 3). A high fish-meal diet with 50 percentanchovymealwas fedasacontrolandfiveotherdietsallwith low fishmeal inclu-sion (20%) but to whichdifferent components wereadded (taurine, taurine and

phytase enzyme, taurine and Synergen). Inthesediets the fishmealwas replacedwithordinary corn gluten and soybean meal,whicharecheaperand industriallyavailableingredients.

Diet2waslowfishmeal,diet3waslowfishmealandtaurine,diet4lowfishmealandtaurineandphytaseenzyme,diet5waslowfishmeal and taurine and500 g Synergen/tonne feed and diet 6 low fishmeal andtaurineand1kgofSynergen/tonnefeed.

Thebestperformancewasobtainedwiththepositivehighfishmealdiet(bestgrowthand lowest FCR).Comparable growth andperformance to the high fishmeal dietwas

onlyobtainedbythelowfishmealdietscon-taining Synergen. The lowest growth andhighestFCRwereobtainedwithdiet2(lowfishmeal).Addingtaurinealoneortaurine/phytase to the low fishmeal diet improvedtheperformancebut thisperformancewasstillmuchlowerthanthehighfishmealdietortheSynergentreatedgroups.

Adding higher amounts of the naturalcomplex (1 kgof Synergen/tonne) did notfurther improve the results. These resultsshow clearly that highly carnivorous fish(RSB) can be fed with low fishmeal levelsandthatordinaryplantproteinscanbeusedifappropriateingredientsareadded.

Field trials in Japan with red sea breamhaveconfirmedtheseresults.Incommercialcages, red sea bream performed betterwhenSynergenwasincorporatedinthedietwith an increased SGR (0.70%when com-paredto0.55%inthecontrolgroup)andafinalweightgainof175gwhichwasgreaterthan the 138 g achieved by the controlgroups. The FCRwas 21 percent lower inthefishfedthedietincorporatingSynergenand the feed efficiency of the Synergenincorporateddietwasalsoimproved.(86.6versus 68%). Amberjack (Serioli dumerili)alsoshowedsimilargrowthresultsgrowingfrom2.2kgto2.6kg(withoutSynergen)or3.1kg(withSynergen).

Pangasius and tilapiaIt is strongly believed that omnivo-

rous and herbivorous fish can digest plantproteins better than carnivorous fish andtherefore do not need additional ingredi-entsintheirdiets.HowevertheinclusionofSynergenindietsforfishspeciessuchastraand basa catfish and tilapia has resulted insignificantly improved growth and loweredsignificantly the FCR in both low and highfishmealdiets.

Inrecenttrials,byHungandKim(2007),a comparison was made between a 15percent fishmeal diet and a five percentfishmeal diet (65% reduction)with addedSynergen (200 or 500 g per tonne feed)to both diets. They demonstrated thatirrespective of the diet used (low or highfishmeal) theadditionof thenaturalcom-plex resulted in dramatic improvements inperformance parameters. Growth reachedapproximatelydoublethatofthedietwith-out the enzyme present. Tra catfish grewfrom14gto36g(withoutSynergen)or70g (with Synergen) while Basa catfish grewfrom6gto57g(withoutSynergen)or82g(withSynergen).

SimilarresultshavebeenrecordedforNileTilapia inwhicha zeropercent fishmealdietwas used as the control diet where growthfrom3gto21goccurredwithoutSynergenandto35g(withSynergen).

This indicates thatsupplementing the low

table 4: effect of dietary treatment on the performance of red sea bream after 12 weeks.

treatments

Parameter FM50 FM20 Ft FtP Fte0.05 Fte 0.1

Final body weight (g) 71.5c 49.8a 55.2ab 59.7ab 74.5c 67. 8bc

Weight gain(g) 57.7b 35.9a 40.4ab 59.7ab 59.9b 53.7b

SGr (%/day) 1.96c 1.52a 1.57ab 1.70ab 1.94c 1.87c

FCr 1.12a 1.30b 1.27b 1.25b 1.16ac 1.21ac

Feed intake (g/day) 64.7a 46.6b 51.2ab 56.6ab 69.6a 64.9a

a,b Means differ P<0.05.

Figure 3: Growth of Amberjack with and without Synergen

Figure 2: Growth of red sea bream fed different diets

34 | InternAtIonAl AquAFeed | July-August 2012

FEATURE

July-August 2012 | InternAtIonAl AquAFeed | 35

52 | InternAtIonAl AquAFeed | July-August 2012

andhighfishmealdietswiththenaturalcom-plex has enhanced the availability of dietarynutrients and compensated for the poorerquality of the raw ingredient characteristicsin the low fishmealdiet so improvingoveralldietaryperformance.

ConclusionTheapplicationofthisSSFtechnologyin

this manner opens the door more flexiblefeed formulation and allows the incorpo-ration of lower cost vegetable proteinsubstitutessuchassimplesoybeanmealandcorn gluten. Improved nutrient availabilityimpactsdirectlygrowthandperformancesoincreasingdietaryefficiencybothintermsofcostandenvironmental impact.Thesavingsin fishmealusageaddressesconsumercon-cernsandsustainabilityissueintheindustry.There is still much to learn about thesecomplex interactions but the indicatorsshowthepotentialofthistechnology. ■

References

HungLTandTNHKim.2007.ReducingfishmealutilizationinPangasiusCatfishfeedsthroughapplicationofenzymes.PresentedatAsianPacificAquaculture2007,Hanoi,Vietnam,5-8August2007.

SatohS,HaniniI,SarkerMSA,HagaY,OhkumaTandHNakayama2011.EffectofTaurine,PhytaseandenzymecomplexsupplementationtolowfishmealdietongrowthofjuvenileredseabreamPagrus major.PresentedatWorldAquaculture2011,Natal,Brazil,6-10June2011.

1Alltech Aqua, 28200 Lixouri, Kefalonia, Greece

2Institute of Aquaculture, Hellenic Centre of Marine Research, Agios Kosmas, Elliniko, 16610 Athens, Greece

3Alltech Japan, Shiba-Koen 2-3-27, Minato, Tokyo 105-0011, Japan

34 | InternAtIonAl AquAFeed | July-August 2012 July-August 2012 | InternAtIonAl AquAFeed | 35

FEATURE

www.evonik.com/feed-additives | feed-additives@evonik.com

Your challenge is our passion.MetAMINO® is the best choice for your aquafeed.

projectincludedfourtilapiafarmsinChina.Thesefarms represented both small- and commercial-scale production facilities utilising two differentproductionsystems(pondandcages).Asidefromidentifyingsimilaritiesanddifferencesamongcriteriaand requirements used by the three standards,thisprojectalsoidentifiedoutstandingissuesinthefarms,whichmostproducerswereabletoaddressasaresultofthetrialaudit.Todate,allfourfarmsarenowcertifiedunderoneormoreofthecom-mercialaquaculturestandards.

SFP is widely acknowledged for its expertiseby stakeholders in Chinese tilapia, including keyUSandEuropeanbuyersandretailers,aswellasproducers and processors in China, aquacultureinstitutes, industry associations, and localChinesegovernments.GiventhehighleveloftrustthatSFPenjoyswiththetilapiasupplychainitwasappropri-atethatatilapiaAquacultureImprovementProject(AIP)wasofficiallylaunchedin2011.

SFPhasnowinitiatedtworesearchprojectstoassess the impactof tilapia farmingon theexternalenvironment.Thefirstproject,startedinApril2011, involvesmonitoringwaterqual-ityonselected farms inHainanprovince,andwas undertaken by the Hainan Institute ofAquaculture.Dozens ofwater quality param-eterssuchaschemicaloxygendemand(COD),nitrogen and phosphorus content, and heavymetalswereanalysed for five farmsover twocroppings (10 months). The study helped

identifythekeyproblemsandcausesrelatedtowatermanagement.

The second project is an assessment of theregionalenvironmentalimpactsoffishfarmclusters,whichwillbejointlyconductedbySFPandHainanResearchAcademyofEnvironmentalSciences,theleadingenvironmentalresearchinstituteinHainan.Thestudywillexaminethepotential forregionalscaleimprovementbylookingatcarryingcapacityandthepotentialforzoninginaspecificarea.

As more first-hand data becomes available(along with a more in-depth understanding ofexistingpoliciesandmanagementmeasures), theAIPwillestablishaworkinggroupthatconvenesthekeybuyers,suppliersandproducersalongtheChinesetilapiasupply-chaintosharethescientificfindings.TheAIPwillthenformamulti-stakeholderpolicyroundtabletofurtherdiscusstheproblemsandsolutions.TheAIPparticipantswilleventuallyagreeontheactionsandtimetablesnecessarytoachieve the sustainability objectives defined bythegroup.SFPwillplayaleadingroleinengagingstakeholders,providingscientificadviceandfacilitat-ingcommunication.

Up-to-date progressSFPhasworkedcloselywithlocaltilapiaassocia-

tions to assessdifferent tilapia standards that areavailable in the market. A workshop introducingthreeinternationalstandardsfortilapiafarming,i.e.BAP,GlobalGAP, andASC,was held inHaikou

inApril,2011.Over40farmers,processors,tech-nicians and government officers attended theworkshop.Participants found theworkshopveryinformativeandhelpful.Thisenhancedtheproduc-ers’awarenessofincreasingdemandsforcertifiedsustainable seafood from overseas markets, thusfurther facilitating the engagement of Chinesestakeholders into a supply-chaindialogue aroundsustainability.

SFPiscurrentlyworkingwithlocalinstitutesofaquacultureandenvironmentalsciencestoidentifyand evaluate both qualitatively and quantitativelythe environmental impacts of tilapia farming inHainan.Thisincludesanecologicalstudyaswellassocio-politicalanalysistoadviselocalgovernmentsandindustrialassociationsabouthowtoefficientlyaddress theenvironmental issuesassociatedwithtilapiafarminginHainan.ThepreliminaryresultswillbesharedwithkeystakeholdersattheAquaculturePolicyRoundtablethisfallinChina.

SFP is also developing partnerships withChineseuniversitiesandlargefeedmanufacturersto improve feed sourcing for tilapia farming inChina. This work is to be undertaken throughresearch projects on improving feeding efficien-cy and developing alternative feeds with fewerimpactsonwildfisheries.

More InforMatIon:Sustainable Fisheries PartnershipWebsite: www.sustainablefish.org

26 | InternAtIonAl AquAFeed | July-August 2012 July-August 2012 | InternAtIonAl AquAFeed | 27

EXPERTT●PIC

www.aquafeed.co.uk

LINKS

• Seethefullissue• VisittheInternationalAquafeedwebsite

• ContacttheInternationalAquafeedTeam

• SubscribetoInternationalAquafeed

VOLUME 15 I S SUE 4 2 012

THE INTERNATIONAL MAGAZINE FOR THE AQUACULTURE FEED INDUSTRY

Tough environment produces world’s best Barramundi

EXPERT TOPIC - Tilapia– a collection of articles creating a worldwide

perspective

Noise– a source of stress for farmed fish

Enzymes– Unlocking the hidden potential of plant

proteins using solid state fermentation technology

Enzymes to improve water and soil quality in

aquaculture ponds

IAF12.04.indd 1 19/07/2012 17:15

Thisdigitalre-printispartoftheJuly|August2012editionofInternationalAquafeedmagazine.Contentfromthemagazineisavailabletoviewfree-of-charge,bothasafullonlinemagazineonourwebsite,andasanarchiveofindividualfeaturesonthedocstocwebsite.Pleaseclickheretoviewourotherpublicationsonwww.docstoc.com.

Topurchaseapapercopyofthemagazine,ortosubscribetothepapereditionpleasecontactourCirculationandSubscriptionsManageronthelinkabove.

INFORMATIONFORADVERTISERS-CLICKHERE