STUDIES ONTH E CONTROL OF FUNGAL CONTAMINATION AND AFLATOXIN PRODUCTION BY ASPERGILLUS FLAWS LINK INA CEREAL GRAIN BY THE COMBINATION TREATMENT OF HEAT AND IRRADIATION G.T.Odamtte n CENTRALE LANDBOUWCATALOGUS OOOO 0174 6094 Promotoren: dr. E.H. Kampe!mâcher, emeritus hoogleraar in de voedingsmiddelenmicrobiologie en -hygiene dr.ir. F.M. Rombouts, hoogleraar in de levensmiddelenhygiëne en -microbiologie LANû30UV.iiu!,,:.. (>0L WAGJKNJï'iG^V ^/UOÏZD',/'00 G.T. ODAMTTEN STUDIES ON THE CONTROL OF FUNGAL CONTAMINATION AND AFLATOXIN PRODUCTION BY ASPERGILLUS FLAWS LINK IN A CEREAL GRAIN BY THE COMBINATION TREATMENT OF HEAT AND IRRADIATION Proefschrift ter verkrijging van de graad van doctor in de landbouwwetenschappen, op gezag van de rector magnificus, dr. C.C. Oosterlee, in het openbaar te verdedigen op dinsdag 4 november 1986 des namiddags te vier uur in de aula van de Landbouwuniversiteit te Wageningen iÜ 3<i4lbW ACKNOWLEDGEMENTS I am indebted toProf .E.H . Kampelmacher and Prof.F.M . Rombouts,m y promoters,fo r their guidance andassistanc e during thepreparatio n of this thesis. My thanks are also due toProf .F.K . Alloteywhos e good foresight and influence spurredm e on topursu e this study. Iexpres s my sincere appreciation to the InternationalAtomi cEnerg yAgenc y IAEAfo r the award ofApplie d ResearchFellowshi p at the International Facility forFoo d Irradiation Technology, IFFIT,Wageningen . Ia mals o grateful to theDutc h and Ghana Government for financial support. Many thanks toal lm y colleagues at ITAL,Wageningen ,Ghan aAtomi c Energy Commission,th e BotanyDepartment ,Universit yo f Ghana,Legon ,especiall y Prof.E .Lain g for inspiring discussions and criticisms.Specia l thanks go to my dearwife ,Catherine , forhe rprayerfu l andphysica l support. Finally, Ia mgratefu l to theman y other friends,to onumerou s tomentio n whohelpe d me inon ewa y or theother . jUrJoS^o^WOO STELLINGEN (THEOREMS) 1. Moulds contaminatingstore d cerealgrain san dgrai nproduct sbelon gpre ­ dominantly toth egener a Aspergillus and Pénicillium. Aspergillus spp.an d Pénicillium spp.impar tpoten tmycotoxin sint ostore dproduce . Thecontaminatin gmycoflor aexhibit s fourpattern so finfectio nan dalthoug h somecanno tb e isolatedafte r4- 5 monthsstorag e (e.g. Pénicillium digitatum, P. expansum, Fusarium moniliforme, Paecilomyces varioti); theywoul dhav eal ­ readyrelease d thetoxi nint oth efood . 2. The conventionalaeria lprophylacti c sprayingo fstore d grains andcereal s with fumigantsan dinsecticide s temporarilyreduce s aerialspore so ffungi .O n eachoccasio no fspraying ,th eaeroso l isinhale db y theworkers ;th echemica l burdeno ngrain sbecome s additivea smor esprayin g iscarrie dou ta ta late r date. 3. Insecticidesd ono tkil l fungi.Th econtaminatin gmould sreappea rafte ra lapseo fa fe wday si nlarg enumbers . 4. Hightropica lenvironmenta l conditions formos tpar to fth eyea r (>85°sR.H . and28± 3 C)ar econduciv e forrapi dproliferatio no ffung io nstore dcerea l grainsan dgrai nproduct sa sth emoistur e contents (m.c.)increase s aboveth e stipulated safem.c . forstorage .Insec tactivit y (producingmetaboli cwater ) combineswit hhig hambien tR.H . toinduc emoul dgrowth .I naddition ,insect s ingestan dcarr ymoul dspore so nthei rbodie s anddropping s andspore sma y spread todistance sa sfa ra sa ninsec tca ng owithi na storag esack . 5.A combinationo fmois thea t (60° Cfo r3 0min. )an dirradiatio n (4.0KGy ) candecontaminat emaiz eo ffungi .Thi sresul tcoul db eextende d toothe r cerealgrain san d grainproducts . 6.Unlik e dryheat ,mois thea tha shig hpenetratin gpower ,increasin gtarge t sizean dcausin grapi dcoagulatio no fprotein . Thegenera lmutationa leffec to firradiatio n ist ocaus edamag e toth egeneti c andbiologica lmechanisms ,creatin g impairmento ffunction . V LAIS'L'.-;' .,i?Oi W.i,, -; . ,t,, \ Dedicated to my parents, and to my beloved wife Catherine, who through the years, through thick and thin, has lovingly and patiently encouraged me to pursue God's best for my life and profession. ABSTRACT Traditional storageo fmaiz ei ntropica l countries sucha sGhan a results inth erapi ddevelopmen to fnumerou s fungi,includin gpotentia lmycotoxi npro ­ ducers sucha sAspergillus flavus (aflatoxins) , A. oohraaeus (ochratoxins, pe- nicillicacid) , Fusarium moniliforme (moniliformin), Paeailomyaes varioti and Pénicillium expansion (patulin).Treatmen to fmaiz ewit ha combinatio no fmois t heat (30min .a t6 0° C andrelativ ehumidit y >85l)an dgamm airradiatio n(4. 0 kGy)prove dt ob eeffectiv ei ninactivatin g the residentpopulatio no ffunga l spores.Thi s resultwa s confirmedb y in vitro studieswit h sporeso f Aspergil­ lus flavus NRRL 5906.I na comparativ e studyo fpackagin gmaterial si twa s found thatfoo dcommoditie s storedi nwove npolypropylen e bags forsi xmonth s at85 1R.H .ha dmoul d andyeas tcount swhic hwer e2- 3lo gcycle s lowertha n thoseo fproduct skep ti njut ebags .Also ,th eviabilit yo fth eseed swa sbet ­ terpreserve di npolypropylen e sacks. Iti srecommende d thatth ecombinatio n treatmentb ecarrie dou to ngoo d quality grains,an dtha twove npolypropylen e sacks areuse di nbaggin gprio r toirradiation , formaximu mextensio no fshelf-life . CONTENTS Introduction 1 1. Asurve yo fth emycoflor ao fmaiz egrain sstore da t26+ 3 C 18 and75+5 4R.H . In: Proceedings 12thBiennia l Conference andNationa l Science Festival of the Ghana Science Association. University ofGhana , April 5-11, 1981, 14pp . 2. Studieso nth etechnologica lfeasibilit yo fth eapplicatio no f 31 dryo rmois thea tt ograin san dgrai nproduct sprio rt ogamm a irradiation. IFFITRepor tNo . 10, 1980:1-15 . 3. In vitro studieso nth eeffec to fth ecombinatio ntreatmen t 46 ofhea tan dirradiatio no nspore so f Aspergillus flavus LinkNRR L5906 . ActaAlimentaria ,Vol . 14 (2),1985 : 139-150. 4. Preliminarystudie so nth eeffect so fhea tan dgamm airridiatio n 58 onth eproductio no faflatoxi nB -i nstati cliqui dcultur eb y Aspergillus flavus LinkNRR L5906 . Int. J. Food Microbiol., 1986.I nPress . 5. Influenceo finoculu msiz eo f Aspergillus flavus Linko nth e 69 productiono faflatoxi nB- ,i nmaiz emediu mbefor ean dafte r exposuret ocombinatio ntreatmen to fhea tan dgamm aradiation . Int. J. Food Microbiol., 1986.I nPress . 6. Studieso nth ecomparativ eefficienc yo fwove npolypropylen e 85 andjut esac sa spossibl epackagin gmateria lfo rpre -an d post-irradiationstorag eo fsom ecerea lgrains . In: Proceedings Post-harvest losses and their prevention. African Biological Network, ICSU Workshop,Dept .o f Zoology, University of Ghana, 1985 7. Studieso nth eselectio no fa suitabl epackagin gmateria lfo r 118 pre-an dpost-irradiatio nstorag eo fcerea lgrain si nGhana . Inc.J .Foo dMicrobiol. ,1985 a(2) :151-158 . 8. Mycologicalan dphysica lparameter sfo rselectin gsuitabl e 126 packagingmateria lfo rpre -an dpost-irradiatio nstorag eo f cerealgrain si nGhana . Int. J. Food Microbiol., 1985b (2):227-237 . 9. Influenceo fpackagin gmateria lo nmoistur esorptio nan dth e 137 multiplicationo fsom etoxigeni can dnon-toxigeni e Aspergillus spp.infectin gstore dcerea lgrains ,cowpe aan dgroundnut . Int. J. FoodMicrobiol. , 1986 (3):57-70 . 10. Sensoryevaluatio nan dsom equalit yparameter so fmaiz e 1S1 combined-treatedwit hhea tan dgamm airradiation . ReportIAEA-SM-271 . 11. Microbiologicalqualit yan dproductio no faflatoxi nB 1b y 155 Aspergillus flavus LinkNRR L590 6durin gstorag eo f artificiallyinoculate dmaiz egrain streate db ya combinatio n ofhea tan dradiation . In:Proc . Int.Symp . Food Irrad.Processing ,Washington , 1985. 12. Discussion 159 13. Sumnary 182 14. Samenvatting 186 INTRODUCTION Maize [Zea mays L.)ha sbee nunde r cultivation forsevera l thousands of years inth eAmericas ,bu t itwa s Columbuswh o introduced iti nth e early 1500's intoWes tAfric awher e itsuse shav e increased. Maize,a nexampl e ofcerea l grain is aver y important staple food crop in Africa and isa s arul e stored for longperiods ,a sbuffe r stock,fo rbot h human consumption and lately as ingredient forpoultr y and livestock feed. Maize production inGhan a isprimaril y forhuma n consumption and a largepro ­ portion of thepopulatio n (>70%)depen d onmaiz e as theprincipa l source of food. Indeed,a sman y as twenty differentkind s offoo d canb eprepare d from maize (Eyeson andAnkrah , 1975). The crop is cultivated throughout the countrywit hvaryin g degrees of success,dependin g on the edaphic andclimati c factors. InGhana ,th e areas of maize cultivation include thewhol e ofSouther nGhana ,Ashanti ,Bron gAhafo , theNorther nRegio n andpart s ofth eUppe rRegion .Th emos t suitable soils which enhancemaiz eproductio n and theirdistributio n are theKumasi ,Asuansi , Kroso, Swedru,Nsaba ,Bekwa i andKokof userie s found inth efores t areas of Ashanti,Central ,Eastern ,Wester n andBron gAhaf oRegions . Inth e transitional zone,Bedeis i and Sutuwaserie s inth eBron gAhafo ,Central ,Ashant i and EasternRegion s are alsover y suitable formaiz e cultivation.Th emos t suitable soils inth e SavannahVegetatio n Zonear e theDamongo ,Ejura ,an dValempel e series found inth eUppe r andNorther n Regions andpart s ofAshanti . Suitable soils ofCoasta l Savannah thicket are theOyarifa ,Toje ,Abouk u and Bajiase series among several others. In theVolta ,Easter n andCentra lRegions ,th e Afeyi,Kpand u andAsokw a series form the suitable soils formaiz e cultivation. Intensive commercial production ofmaiz e inGhan a ishoweve r found in four main centres: (a)th e SomanyaDistric t ofEaster nRegion ; (b)th eMidlan