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
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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 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 ,fo rhe 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 econtent s (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 dMaiz e belt ofAshant i and BrongAhaf o Region; (c)th eHo-Kpand udistric t ofVolt a Region,an d (d)th e Swedru-AdjumakeDistric t ofth eCentra lRegion . Elsewhere substantial amountsma yb eproduce dbu t these areuse d mainly forhom e consumption ona subsistenc e economy and dono t feature in commercial estimates. Being a seasonal crop,especiall y inWes tAfrica ,maiz e isstore d as dry grains and forms anenormou s reserve of food,However ,substantia l amounti n storage are subject to attackb y avariet y of insects,fung i andothe rbio - 1 deterioagents.Losse si nstorag edu et oinsect san dfung ii sestimate da t between30-50 1o fannua lharves t (Rawnsley,1970 ;Adams ,1977) .I nth erecen t 1983estimate sb yth eMinistr yo fAgricultur ei nGhana ,the ypu tlosse sowin g toinsec tan dfung iactivit ya t6 0millio ni nloca lcurrency .Productio nan d Importfigure sfo rmaiz e (1970-80)i spresente di nTabl e1 . Therol eo ffung ii nqualit ydeterioratio no fgrain si swel ldocumente di n developedcountrie s (Bothaste tal. ,1975 ;Christense nan dKaufmann ,1965 ,1969 ; Lagrandeuran dPoisson ,1968 ;Semenuik ,1954 ;Tuite ,1978 ;Tuit ee tal. ,1967 , etc.)bu tther ei spaucit yo finformatio nregardin gthes efung io nmaiz egrain s inWes tAfric aan dth erol esuc hfung iplay . Broadbente tal . (1969)provide da nextensiv elis to ffung iassociate d withmaiz eisolate di nNigeria .Later ,Oyenira n(1973a,b )adde deleve ndifferen t fungibelongin gt oth egener a Aspergillus and Pénicillium toth elist .Othe r workersi nEgyp t(Moubashe re tal. ,1972 )isolate dan didentifie dthirty-fou r specieso ffung ibelongin gt othirtee ngener ao nstore dmaize .Member so fth e genus Aspergillus wereth emos tpredominan tfollowe db y Pénicillium. Danquah (1973)isolate dtwenty-tw oseedborn efung io nmaiz ei nGhan aincludin gonl yon e Aspergillus sp.an don e Pénicillium sp.an dh eshowe dth epathogenicit yo fsom e ofthe mt omaiz eseedlings .H edi dnot ,pe rse ,repor to fan ytoxigeni cspecie s ofpotentia lhuma nhazard . Fungiinfec tgrain si nth efiel dbefor eharvestin go rdurin gharvestin g throughpur emechanica ldamag eo ri nstorag eb yaeria lspore si nth estorag e siloso rbins . Lossesinduce db yfung ian dinsec tactivit y Themajo rtyp eo flosse scause db yfung igrowin gi nstore dgrain sar e (i)decreas ei nviabilit y (ii)discolouratio no fpar t (usuallyth eger mo r embryo)o ral lkerne lo rsee d(iii )heatin go rmustines s(iv )variou sbiochem icalchange s (v)los si nweigh t (vi)productio no ftoxin stha ti fconsume dma y beinjuriou st oma nan ddomesticate danimals .Thes ear eexhaustivel ydiscusse d byChristense ne nKaufman n (1969). Therol eo fstorag efung ia sa sourc eo finsec tfoo dan dthei reffec to n thebreedin go fsom estore dproduct sinsect se.g . Sitophilus oryzae hasbee n reported (Agrawale tal. ,1957 ;Misr ae tal. ,1961 ;Sikirowski ,1964 ;Widstrom , 1979).Result sindicat etha tlarvae ,pupa ean dadult so fvariou sstore dproduct s insectscarr ytoxigeni can dnon-toxigeni cspecie so ffungi ,internall y (Table2) . Themajo reconomi clos scause db ygrain-infestin ginsec ti sno talway sth e actualmateria l they consume,bu t also the amountcontaminate db ythe man d their excretawhic h make foodunfi t forhuma n consumption. Sufficet osa y thatth e cumulative effecto finsec t feeding,breeding , transmissiono ftoxigeni can d saprophytic fungi and associated changesi nth emicroecologica l conditionsi n thebul k grain,haste n themicrobia ldeteriorativ eprocess . Table 1. Production and imported supplement ofmaiz e inGhan a for the period 1970-1980. Production figure Import supplement Year (thousand metric tonnes) (thousand metric tonnes) 1970 483.0 a 529.0 b 1971 465.4 338.0 1972 402.4 2.0 1973 427.0 242.2 1974 486.0 26.0 1975 343.0 44.0 1976 295.0 105.7 1977 299.0 1978 386.0 1979 370.0 1980 424.0 Source: a)Ministr y ofAgriculture , Ghana,Plannin g and Research Division,Accra ; b)Centra l Bureau of Statistic,Accra . The activityo finfestin g insectsmak e metabolicwate r availablewhic h goest ocaus emarke d increasesi nth emoistur e content (m.c.)o fgrains . For eacho fth e common specieso ffung i infecting grains,ther ei sa minimu mm.c . ingrain sbelo wwhic h the fungus cannot grow.Thes eminimu mm.c . havebee n determined formos t commonstorag e fungi growingo nstarch y grains (Christensen and Kaufmann, 1969). Davey andElcoat e (1965)pu tth e safem.c .fo rstorag eo f maizea tabou t 13.01whils t Lutey and Christensen (1963)previousl y suggested thatviabl e field and storage fungima yb egenerall y reducedo reliminate d from grain lota tm.c . 12-141 anda temperatur eo f20-3 0 °C,withou t serious deteri orationo fgrains .I ti sth eintersee dEquilibriu m Relative Humidity (E.R.H.) expresseda sa fractio n thatdetermine s fungal activity (Christensen,1974 , 1978; Koehler, 1938;Semenuik , 1954). This fraction orwate r activitya wi sth e Table2 .Funga lspecie sisolate dfro mstorag einsects . Insectspecie sscreene d Toxigenicspecie s Otherspecie s Sitophilus aryzae, Linn. A. flavus, A. oandidus, A. sydowi, A. ruber, A. oahraoeus, A. niger, A. ohevalieri, A. fumigatus P. rugulosvm, Amblyosporium sp., Cladosporium sp. Tribolium aastaneum A. flavus, A. oandidus, A. versiaolor, A. niger, Hebrst. P. islandiaum A. ruber, A. ohevalieri, Trogoderma granarium A. flavus, A. oandidus, A. ruber, A. niger, Everts P. islandioum A. glauous gr., A. sydowi, A. versiao lor, P. restriction Callosobruahus ohinensis A. flavus, A. oandidus A. ruber, A. sydowi, Linn. A. glauous gr. Orysaephilus surinamensis A. flavus, A. oahraoeus A. restriotus, Linn. A. terreus, A. glauous gr.,P . deoumbens, Cladosporium sp. Stegobium paneaewn Linn. A. oandidus A. glauous gr. Shizopertha dominioa A. oandidus, A. niger, A. glauous gr. Fab. A. oahraoeus Araeoerus fascioulatus A. flavus, A. oandidus A. niger, A. glauous gr. deGee r Coroyra oephalonioa A. flavus, A. oandidus A. sydowi, A. ruber, Staint. A. niger, A. restriotus, A. versioo lor, P. spinulosum, P. aorylophilum, Nigrospora sp. Ephestia oautella Walker A. flavus, A. oandidus A. niger, A. glauous gr. A. terreus, A. ruber, A. versiaolor. ratio ofth ewate r vapourpressur e ofsubstrat e toth ewate rvapou r ofpur e water atsam e temperature andpressur e (Scott, 1957). Water activityha sno w taken theplac e ofmoistur e as themos tusefu l expression ofth e availability ofwate r for growtho fmicroorganism s (Scott, 1957). The a of foodproduc t is low (i.e.lo wm.c. )whe n the solutemolecule s bindwate rmolecules .Then ,th e watermolecule s are less free toescap e fromth e surface ofth e food product into thevapou rphase ,an d the vapourpressur e is low.Whe nbound ,wate rmole cules areno t available for fungal growth.Funga l growth is therefore related toth e a . Indeed, it isbelieve d thatstorag e fungi dono t readily growbelo w anE.R.H . of 751 (aw 0.75) (Christensen, 1978). Unfortunately, theenvironmenta l conditions inth e tropicswit hhig h average humidity >80%R.H . andhig h temperatures (28±3°C )favou r allkind s ofdeterio rative processes of.store dgrains . Odamttenan dLangera k (1980)showe d that theequilibratio n period oftw o varieties ofmaiz e (Ghanaian local variety andDutc hyello wmaize )hel d under R.H. 60-801wa s 12day s afterwhic h therewa sn o further increase inm.c .Th e m.c. of grainskep t at 85-95%R.H . continued rising above the stipulated safe m.c. forstorag e ofgrain s (11-131). Gain inweigh t owing towate r sorption is atth e expense of thequalit y ofgrains ,whos e shelflife is shortenedb y the growth of fungi on suchgrains .Th e equilibriummoistur e content (EMC)o fgrain s is influencedb y genotype (Hubbard et al., 1957),previou s drying temperature (Tuite andFoster , 1963), storage temperature (Pixton andWarburton , 1971), hysteresis (Hubbarde t al.,1957 )o rwhethe r grain is ground orungroun d (Tuite andFoster , 1963). However,Odamtte n andLangera k (1980)foun dtha tmoistur e sorption isotherms (atvaryin ghumidities )obtaine d forGhanaia n localmaiz e varietywer e akin to thatobtaine dwit hDutc h varietymaiz e except that the Dutchvariet ywa s lesshygroscopic ,absorbin g comparatively lowerwate r vapour moisture athig h storagehumiditie s and containing 14.01m.c .a t 801R.H . (a^ 0.8) as compared to 15-171m.c . for theGhanaia n variety under thesam e humidity (80°iR.H. ) condition. They concluded that control and storage conditions which wouldb e ideal for storage ofGhanaia n localmaiz ewoul d alsob e suitable for theDutc h variety and viceversa . Mycotoxin contamination of foodstuffs Foodstuff are contaminatedwit hmycotoxin s indifferen tways . Toxigenic moulds may grow andproduc e mycotoxins inagricultura l produce inth e field as well as during transport andstorag ewhe n conditions are favourable i.e. athig h a >_0.70.Berma n (1958)an d Higginson (1963)showe d that the areas showing high incidenceo fprimar y cancero fth elive r include thesub-Sahar a regiono f Africa (whereGhan ai s located)an dSoutheas tAsia . Interestingly, thesear e all areaso fcontinuou s highhumidit y andhig h tomoderat e temperatures in general.Thes e climatic conditions favourmoul d growtho nsuc hstore d staple foods asrice ,maize ,ry ean dgroundnuts .Th e mycotoxinproble m inthes earea s isbecomin g serious; Aspergillus flavus andmos tothe rtoxigeni cmould s areex tremely commonan dhav ebee n found to growo na variet yo f foodstuffs under a wide range ofconditions . Extensive field investigations concerning contamination ofhuma n foodstuffs withmycotoxin shav ebee nconducte d inman yAfrican ,an dAsia n countries inclu ding SouthAfric a andSwazilan d (1966-1969)a s reportedb yGilma n (1971);Ugand a (1965-1967)reporte db yCrawfor d (1971)an dAlper te t al. (1971); Thailand (1967-1970)reporte db y Shank (1971);Mozambiqu e (1969)reporte db yPurchas e andGonsalve s (1971);Philippine s (1967-1969)reporte db yCampbel l and Salamate (1971);Keny a (Peersan dLindsell ,;1973 ); Ghan a (Lokko, 1978).Ne wGuinea ,Kore a andJapan .Result s ofth e field investigations on isolationo ftoxi c fungi from foodstuffs showed thatmos to fth e fungalspecie sbelon g to the twomajo r genera ofstorag efungi , Aspergillus and Pénicillium. These foodstuffs included rice grains (unhulled,roug h andpolishe d rice)othe rcerea l grains (wheat,barley ) maize,soybean ,re dbean s andothe r legumes,cassav a flour,fermente d foods such asmis o (soybeanpaste) ,shoy a (soyasauce )drie d fishan dpickle d vegetables (mukamiso).Aspergill i andPenicill i aremostl y confined tostore dproduct sbe causeo fthei radaptatio n todr yconditions .Fusari a thrive inwette rconditions . Interestingly,n o significant differencewa s observed inth ekind s and dis tributiono f fungiamon g the investigated areas of theworl d (Purchase, 1971), though there aresevera l characteristic patterns ofmycoflor adistributio n in each localare aan di neac h typeo f foodstuff. Somewor kha sbee ndon e inGhan a withregar d toth e fungi thatinfec t foodstuffs.Lokk o (1978)provide s thefirs t information on aflatoxin incassav a flour.Ther e are also sporadic reports of deatho fpoultr y andhuma nbeing sa sa resul to fingestin gmaize ,cassav aflour , animal feedetc .contamine db y fungi andFoo dResearc h Instituteha s recordo f aflatoxinpresenc e inmaiz e (unpublished data).Thi s isbecaus e the recommended practices for'preventio n ofmycotoxi n formation in food,fee dan d theirpro ductsb y theFA D isno t strictly adhered tob y some farmersan d secondly our ambientenvironmen tnaturall ypromote sfunga ldeterioration .Dryin go fth epro duce isa neffectiv e means topreven t further growth.However ,i fmycotoxin s havebee n formed,th eproces s ofcontaminatio n canb e stoppedbu tno t reversed by drying. Because ofthei rstability ,mycotoxin sma ypas s industrialprocessin g ofth eproduc ewithou t largereductio n andente rint o consumerproduct s (Harwig et al., 1974;Mart h andDoyle ,1979 ;Va nEgmon de t al., 1977).Aflatoxin s them selves,o rderivative s ofthe mhav ebee n detectedi nth eurine ,faeces ,an d milk ofanimals ,an deve n inhuma nurin e (Campbellan d Salamat, 1971). Smithe t al. (1976)showe dtha tincidenc e severity ofaflatoxicosi s isgrea twhe n there ispoo ron-far mstorag eo fmaiz ean d thataflotoxi n formationcontinue s during andafte rmillin gprocess . Aflatoxins formedb y A. flavus and A. parasitions are themos tstudie d mycotoxinsbecaus e ofthei rwel lknow n carcinogenic andetiologica l effecto f human and animalmalignancie s (Rodrick and Stoloff, 1977).Aflatoxin s showed carcinogenic targeteffec to norgan s othertha nth e liver,suc ha sth estomach , kidney,trache aan d intestines (Butleran dBarnes ,1966 ;Dicken se t al., 1966). Zuber andLilleho j (1979)recentl yreviewe dth e statuso fth e aflatoxin problem inmaiz e andstate dtha tcondition s ofstres ssuc ha sdrough thav ebee n shownt oenhanc e aflotoxincontaminatio nproblem . Ifthi s istrue ,the nth e WestAfrica n regionstan d agrea tris ko fqualit ydeterioratio n ofthei r little stock ofmaiz e atthi smateria lDûmen to facut e drought conditions.Prio rt o 1971,aflatoxi n contamination inmaiz ewa s associatedwith stored grain (Christensen andKaufmann , 1968).Afterwards ,i twa s demonstrated inpre-harves t maize (Andersone t al., 1975). Sincemos to fth emaiz e grainso rthei r flour are frequentlyuse di nfoo do r feed,i tbecam enecessar y todevelo pmethod s to prevent toxinformation . Application ofconventiona l technology for preservation Sundryin g isa chea pfor mo fnatura lenerg yuse d inreducin g them.c . of maize-on-the-cob andhulle dmaiz e tosaf e (12-141m.c. )i.e . ana of0.70-0.7 5 forstorage . InGhana ,th e traditionalplatforms ,an dmat suse d forsu ndryin g ofshelle dmaiz e arevariabl e fromon e locality toth eother . Insom e instances (alsoi nth eNigeria ,Keny a andZambia )grain sar e leftt omatur e anddr yo n the cob inth e field.Th emaiz e grains are collected fromth e fieldwhe nthe y are completely dry forthreshing .Anothe rcommo npractic e ist ocu tth estalk s atth ebas e and arrange themi nbundle s orlaye ri nth e field ando nbam s at home,a proces s referred toa sstooking .The y are then allowed todr y inth e sun. Themaiz e cobsma y alsob e tiedtogethe rb yusin gth ehusk s andca nthe n behun g onbundle s fromstave s ina traditiona l fire-place ofa kitche n (t o promote smoke infiltration into thebundle s supposedly tokil l insect and fungi) or froma treea s inth ecas eo fZambi a (Chalwe, 1980).Admittedly ,moul d growthi sstil lpossibl ebecaus eo f the increased graintemperatur e and the attendant insectactivit ymakin gavailabl emetaboli cwate r forfunga lgrowth . Moulds sucha s Aspergillus flavua can invariablyb e detected (Conversee t al., 1978b). High temperature drying isth emetho do fchoic e inconditionin gmaiz e for storage andmarketing .Grai ndrie d athig htemperatur e isdecrease d inhygros - copicity anda sa resul ti si nequilibriu mwit h ahighe rR.H . than lowtempera turedr ygrain s (Tuite, 1979):Dryers ,particularl y thecros s flowan dbatc h driers,operatin ga tto ohig ha temperatur e andhig hdryin grat egiv e brittle kernelscause db yth e formationo fstres s crack inth eendosper m (Peplinskie t al., 1975;Thompso nan dFoster , 1963).A s a result,broke nmaiz eforeig nmatte r isincrease d and thisconstitute s astorag ehazar dbecaus e theyar ehygroscopi c and the surface isexposed ;actin ga sa foodbai t forfunga lspores .Suc hbroke n grainscontai n6- 7 timesa sman yspore sa sthei runbroke nkernel s (Tuite, 1975). Storage situation inGhan a Hermetic storageo fhig hmoistur emaiz epractice d indevelope d countries was the innovative ventureo fVayssièr e (1948)i nFrance .Th e grainundergoe s fermentation,oxyge n isdeplete d andcarbo ndioxid e isincrease db y therespira tiono fgrai nyeas tan dbacteri a (Burmeistera t al., 1966;Isaac se t al., 1959). Areaswit hwarme rclimate s andmuc h rainfall in the tropics,lik eGhan aar emor e subject toinsec tan dmoul d contaminationo fstore d grains.Variabl e traditional storagebin suse d indifferen t sectorso fGhan ahav ebee ncollate d and reviewed byNyanten g (1972). Inadditio n to these traditional structures,concrete ,ply wood,aluminiu man dbutyl silo sar emostl yuse dan dsom ebul k grainsar estore d injut e sacks inope ncemen twarehouses .Th e locationo fconcret ean dbuty l silos storage facilities inGhan a forcerea lgrain s andanima l feedhav e been reviewed ina recen trepor t (Anon, 1979).Th estorag efacilitie s inth ecountr y was considered inadequatebecaus e functional concrete silos couldstor e a69,00 0 metrictonne s capacity ofcerea lgrain swhils tbuty l silocoul dcontai n 130,544 metric tonneso fcerea l grains.Meanwhile ,th e total amountof'importe dcereal s andpulse sneeded tob e storedb y 1976wa s 635,819metri c tonnes.Suc ha situa tionha s favoured thehandlin go fove r60 1o fth egrai nsurpluse so rsupplie sb y farmers (majoritybein g tradition farmers)outsid e thosespecialize d storage fa cilities. Insectan d fungaldeterioratio no fgrain sunde rsuc hcondition sbecom e the rulerathe r thana nexception .Studie shav e indicated thathermeti co rair tightstructure s arebette r thannon-ai r tightstructure s forgrai nstorag e (Hydean dOkley , 1960). Buti nsilo s andstorag ebin sunde r tropical conditions, evenwit h 101initia lmoistur e content,thermogenesis ,discolouratio n ofgrain s and fungal growthdevelo pwithi nweek so fstorage .Turnin g ofth emaiz e grains topromot e greateruniformit y inmoistur e distribution,blowin g dry airthroug h thegrai nmass ,an dothe rmechanica l handlingar e required thatar e difficult andexpensive . Importedbul k grains arebagge d injut ebag s andthe nsen tt o opencemen twarehouse swit h appropriate roofing.Bu tth egrain sar esoone ro r laterexpose d to thehig h ambient temperatures (>28°C )an dhumidit y (>80$R.H .) and theyi nn o time attaina ^whic hpromot ebot h insectan d fungal growth.T o preventdeterioratio n instorag ewarehous e agents andprivat e farmers carryou t regularprophylacti c sprayingwit hrecommende d insecticides andfumigants . Chemical control Theus eo fchemica l sterilants forth econtro lo fpost-harves t diseases of insectsan d fungii swel lknown .Fo rlarg escal efumigation ,methy lbromid e or mixtures ofethylen e dibronddean dmethy lbromid e (1:3a t3 2g/ m) ha sprove d tob eeffectiv e against insects andfung iwhe n appliedunde rtropica l conditions. Innon-airtigh twarehouses ,fumigatio n sheetsmad eo fballoo n film,Pol yViny l Chloride (PVC)coate d aluminizedfabri co rhig hdensit ypolyethylen e laminated withnylon ,ar e sufficient toretai n these fumigants during exposureperiods . Phosphine generating fumigantproduct s ofDetia* - ' X are alsowidel yuse d in Ghana. Inorde r tocontro l any infestationpresen teithe ri nth ewarehous e or inth e stacks ofjut e sacks,prophylacti c sprayingwit hActelli c 25^ ' (ICI, England)i s recommended. Theus e ofmethy lbromide ,picrin ,ethylen e dibromide: methy lbromid e (1:1) besides ammoniaan dsulphu r dioxide forcontro l ofgrai n fungihav ebee nre ported (Majumdere t al., 1955;Ragunatha n et al., 1969;Tsurant a and Ishirava, 1966). Aswit h insects,th esusceptibilit y ofresistanc e of fungi toa fumigant canb e attributed to threemai n factors:th e stage of thefunga l life cycle,it s location in the grain and environmental fumigationmethod .Fo rexample ,Yana i et al. (1964)showe d thatmyceli ao f Pénicillium islandicum was more susceptible toethylen e oxide thanth espore .Studie swit h several Aspergillus spp.showe d thatspore s aremor e susceptible thanconidiophore s tomethy lbromide . In Asper gillus ochraaeus, sclerotia required 32mg/1 ,doubl e theamoun t required to control conidia.O n theothe rhand , Rhizoctonia solani producing sclerotia could notb e controlledwit hpentachloronitrobenzen e (PNCB) (Sharia andSinclair , 1965).Althoug hethylen e dibromide andchloropicri n killed sporeso f Aspergil lus flavus Link and A. nigev LDgs at 10_mg.l inlaborator y tests,th esam e fumigantcoul dno tcontro l all the fungi ifi ti spresen t inside the grain (Narasimhan andRangaswamy , 1968). Other attempts tous echemica l preservation forinhibitin g fungal growtho nstore dgrai nha sbee nreviewe db yMajumde re t al. (1963). Nono f the treatmentswer e found tob ecompletel y successful in preventing fungal growth ingrains . Indeed,Odamtte n (1982)showe d that fumiga tiondi dno tkil l fungiwhe nuse da prophylacti c spray inth ewarehouse . Mycotoxins seem, unaffectedb y commonchemica l fumigants (Brekkee t al., 1978) orpreservative s (Tuite, 1979)excep t forammoni awhic h isuse d todestro yaf - latoxin (Brekkee t al., 1975;Anon , 1977). Fumigation alsopose s ahealt h hazard toworker s infoo dprocessin g factory through the formation ofchloro - hydrins (LDr0 equal to 0.07 g/kg testanima lbod yweight )a possibl e direct toxineffec t (VanKooij , 1981). Invie wo f this toxiceffec to f fumigants,th eEnvironmenta l Protection Agency (EPA)o f theUS Aha s announced thecancellatio n andphase-ou to fal l majorpesticid e useso fethylen e dibromide (EDB). Theus eo fED B forquarantin e fumigationo f fruits,vegetables ,cereals ,etc .phase dou to n 1stSeptembe r1984 . TheEP Aha dearlie ro nordere d the immediate emergency suspensiono fED B assoi l fumigant foragricultura l cropso n 30th September 1983 (Anon, 1983). This action underscores the increasingcos to fchemical s for thirdworl d countries andth e outcryagains tcontinuou s useo fchemica lsterilant s owingt o their residue problem.Publi c HealthAuthoritie s throughout theworl dhav ebecom e stringent inthei r regulations tocurtai l chemicalburde n inhuma n food.Th e application ofionizatio n radiation asa proces smetho doffer s abette ralternativ e tochem ical insecticide andfumigant sbecaus e application ofionizin g radiation leaves no chemical residue andn o insecto r fungusha sbee n found todevelo p immunity toradiation .Applicatio n ofgamm airradiatio n incombinatio nwit hmil dhea t treatment tocontro l growthan d toxinproductio n of A. flavus inmaiz e forms thebasi so f the investigations asoutline dbelow .A suitable packaging material forpr e andpos tirradiatio n storageo fgrain swa s investigated. Aim and sequence of the investigations Insufficientwor kha sbee n carried outt oexamin eGhanaia nmould y maize forth epresenc e ofpotentia lmycotoxin-producin gfunga l species although there havebee n sporadic reports ofacut e foodpoisonin g (andsometime s death) arising 10 fromingestio no fmaiz e contaminatedb ymixture so fgrowin gfungi .Th e situation issuc h thatan ygrai n lotswit ha nobjectionabl e aesthetic look attributable tofunga ldiscolouratio ni sinadvertentl ypasse do nfo rus ea singredien ti n poultryan dlivestoc k feed.Th edetrimenta l consequencei stha tmycotoxi ncon taminationo fmeat ,eg gan dmil ka tfar mi simminen tafte r feedinganimal swit h contaminatedrations. Transmissio no faflatoxin s andOchratoxi nA fro mcontam inated rationshav ebee ndemonstrate di ndair y cattle,pig s andpoult y (Elling etal. , 1975;Krogh , 1977;Rodrick s andStoloff , 1977).Th ewate ractivit y aw and temperature conditions underwhic h some toxigenicfung iproduc epoten t mycotoxins (aflatoxins,Ochratoxi nA ,patulin ,penicilli c acid)hav ebee nex haustively studiedb yNorthol t (1979), inmaiz eb yGoug han dBateman ,1977 ; Koehler, 1938;Lope zan dChristensen ,1967 ;Ciegle ran dKurtzman , 1970;Ciegler , 1972; Kurtzmanan dCiegler , 1970).Thes econdition sar eaki nt oth eenvironment alcondition s thatoccu r.i n our tropicalregio nan di sresponsibl e forth e continuous survivalo ffunga lspore si nfoo dan dth e attendant lossesi nnutri tive andbiochemica lvalue so ffoods . Theobjectiv e inArticl e1 wa st oupdat e thestorag e fungi associatedwit h maize grainskep tunde r ambientGhanaia n tropical conditions forsi xmonth san d toacquir e informationo nth einfectio npattern so findividua l fungi.Onc e form ed themycotoxi ni simparte dt oth efoodstuf fan dalthoug h the fungusma yno t be isolatedafte ra certai n lengtho ftime ,th emycotoxin ,bein gstabl ema ypas s industrialprocessin go fth eproduc ewithou t largereductio nan d enter intocon sumerproducts .Secondly ,th eparticula r toxigenic fungishoul db e identified ifth eexten to fth eproble mi st ob eappreciate d andeffectiv e controlmeasur e ist ob edesigne dt opreven t growthan dtoxi n formationb ycombinatio n treatment ofhea tan dgamm airradiation . Sufficient informationo ncontaminatin g species offung ian dthei rinfectio npatter nwithi n sixmonth s storagewa s acquired. Toxin-producing species. Aspergillus flavus Link (aflatoxin B,an d B-), A. ochra- aeus Wilhelm (OchratoxinA an dpenicilli cacid) , Fusarium moniliforme Sheldon (Moniliformin), Paecilomyces varioti, Pénicillium expansion(Patulin )an dsevera l other Pénicillium sp. wereencountered .Thi s informationwa suse di nth enex t experiments. A. flavus was chosena sth etes torganism st oascertai n theefficac yo fth e combination treatmento fhea tan dgamm airradiatio ni npreventin g fungal growth andmycotoxi n formationfo r the following reasons: (i)aflatoxin s frequently occuri nmaiz ean dothe r foodgrai nproducts ,cerea l grains,whol ewheat , rye bread, cheese,meat ,groundnu t andnu tproducts ,frui t juice,copr aan dcotto n 11 seed and cocoabeans ; (ii)it s importancea s themos tpoten t and carcinogenic mycotoxino fhuma nan danima lhealt himportanc e and (iii) A. flavus as themos t ubiquitous fungus frequently encountered instore d food grain is also themos t radioresistant fungalspecies .Previou s studies byOdamtte n (1979)wit hmaiz e andAmoake-Att a et al. (1981)o ncoco abean s showed thata complet e control of A. flavus couldno tb eobtaine dwit h ados eo f5. 0 KGyo fgamm aradiatio nbe causehig henvironmenta l storage R.H. (>8(H)increase d the radioresistance of the spores of A. flavus bymakin gmor e freewate ravailabl e tospore s forre sumed growth after theradiatio n treatment.Therefore ,an yeffectiv e control method for A. flavus growth ando ftoxi nformatio n couldb e extended to cater for theothe r radiosensitive fungi infectingmaiz e grains instorage . InArticl e 2a ne w apparatus isdescribe dwhic h enables the application of either dryhea t (60 C for 30min )administere d under lowhumidit y (£45!R.H. ) conditions ormois thea tunde rhig h (>_8S$R.H. )ambien t conditions tospore s of fungi ina heat-treatmen t chamberprio r togamm a irradiation.Thi s combination treatment gives asynergisti c effectb y allowing greater inactivation of spores ata lowerdos e of: radiation,o rhea t applied alone.Result s ofthi s feasibil itystud ywa suse di nth esubsequen t investigation. InArticl e 3, 'invitro ' studieswer e carriedou t to investigate thefun gicidal and synergistic effecto fth e combination treatment ofhea t andirradi ationo n thesurviva l ofwe tan ddr y conidiao f A. flavus LinkNRR L 5906.Re sultspresente d indicate thatinactivatio no fgrowt ho f A. flavus sporesb y the combination treatment is feasible. Articles 4an d 5demonstrate ,i nstati cliqui d culture,maiz emea lbrot h thatbot h growth andaflatoxi n B- andB 2 formationb y A. flavus canb e attenua tedb ycombinatio n treatmento fmois thea t (60 C for3 0min )applie dunde rhig h humidity (_>85!R.H. ) conditions incombinatio nwit h 4.0 KGyo fgamm a irradiation. Article 5present s results illustrating theeffec t ofinoculu m size of A. flavus sporeso naflatoxi nproduction .Aflatoxi n levels formed inculture s tend tob e inversely related toth e inoculumsiz eo f A. flavus used in inoculating flasks.Result shelpe d in.elucidatin gwh y someworker s obtained enhanced afla toxin formation viagamm a irradiation. Itals oprovide dpertinen t datao nho w aflatoxin B..level sproduce db y A. flavus instore dgrain smigh tb e related to the inoculumsiz ewit hwhic h grain is infected. Someworker s areskeptica l about the reproducibility of 'invitro ' results 'invivo 'o n theproduc t itself,s o fara smycotoxi n production is concerned. Article 6demonstrate s positivepreventio no faflatoxi n B formationb y A. flavus 12 artificially inoculated onto the product maize and then subjected to the same combination treatment that attenuated the production of aflatoxin B and B_ in articles 4 and 5. Apart from preventing recontamination of irradiated cereals (maize, sorghum, millet, rice, etc.). a suitable packaging material should not harbour and support 'de novo' growth of fungal spores when the average tropical R.H (>80°O is attained and thus act as a springboard for infecting enclosed food grains. Owing to the robust handling of packed cereal grains e.g. maize in transit and in the warehouse, it would be ideal to have a packaging material with high tensile strength which would be able to withstand such rough handlings Investigations in Articles 7, 8 and 9 were designed to provide pertinent infor mation on the mycological, radiation response and physical parameters (tensile strength) respectively that would assist in the selection of the most suitable of two packaging material compared in these series of studies namely the in cumbent and conventional jute sack and the new introduction: synthetic woven polypropylene sack. In the last experimental sections of the thesis the questions of (i) whether woven polypropylene sacks will also be suitable for storage of other food products e.g. groundnut (Arachùs hypogene L.) bambara beans (Voandzeae subterranea), sorghum [Sorghum vulgare), cowpeas {yigna unguioülata) Walp and cocoa beans was investigated. These food products are presently stored in jute sacks with attendant heavy (>30l) losses and are often kept in the same ware house. To prevent cross-infestation by insects and cross-contamination with fungi, it would be ideal to use same packaging material for food products with common storage pests and microbial contaminants. If woven polypropylene is more suitable than jute sack even with no irradiation, its protective properties would be augmented by radiation treatment. Finally, the effect of the combination treatment of heat and gamma irradi ation on some physical and chemical properties of maize grains, i.e. sensory evaluation and quality of product is examined. Findings of Articles 1-9 in the thesis are discussed in the chapter on Discussion in the light of the results obtained and practical implications with regard to safeguarding combined-treated cereal grains from re-contamination during storage, by using the recommended storage sack is highlighted. It is con sidered that good management practices and clean warehouses would augment the radiation process. 13 REFERENCES Adams,J.M . (1977).A revie wo fth eliteratur econcernin g lossesi nstore d cerealsan dpulse spublishe d since 1964. Trop. Sei. 19, 1-28. Agrawal,S.N. ,Christensen ,CM. ,an dHodson ,A.C .(1957) .Grai nstorag efung i associatedwit hgranar yweevil . J. Econ. Ent. 50, 659-663. Alpert,E .Hüft ,M.S.R. ,Wogan ,G.N. ,an dDavidson ,CS . 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PhD Thesis.Agricultura l University,Wage ningen,Th eNetherlands . Nyanteng,V.K . (1972). Storage of foodstuffs inGhana .ISSE R Technical Publica tion No. 28, University of Ghana,Legon. . Odamtten,G.T . (1979). Control of fungi causing deterioration of maize in storageb ygamm airradiation . Paper' No. 16, 11th Biennal Conference ofth e G.S.A.Universit y of Science and Technology, Kumasi,Ghana , 30thApri l - 4th May 1979. Odamtten,G.T . (1982). Studies on the comparative efficiency ofwove n polypro pylene and jute sacks as possible packaging material from pre and post ir radiation storage of some cereal grains. African Biological Network, ABN Workshop on post-harvest losses and their prevention Aept.27-30 ,1982 , Dept. of Zoology,Universit y of Ghana,Legon . Odamtten,G.T . and Langerak, D. I. (1980).Moistur e sorption of two maize varieties kept underdifferen t ambient relative humidities. IFFIT Report No. 9, Wageningen,Th e Netherlands, 13pp . 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Rept., Purdue,Wes tLafayett e Indiana USA. Tuite, J. and Foster,G.H . (1979). Control of storage diseases in grain. Ann. Rev. Phytopathol. 17, 343-366. Tuite,J . andFoster , G.H. (1963). Effect of artificial drying on thehygros copic properties of corn. Cereal Chem. 40, 630-637. Tuite, J., Haugh,CG. , Isaacs,G.W ., an d Huxcoll,C.C . (1967). Growth and effects ofmold s in thestorag e ofhig h moisture corn. Irans. ASAE 10, 730-732, 737pp . Van Egmond,H.P. ,Paulsch ,W.E. ,Veringa ,H.A. , and Schul1er,P.L . (1977). The effect of processing on the aflatoxin M. content ofmil k and milk prod ucts. Arch. Inst. Pasteur Tunis 3-4, 381-390. Van Kooij, J.G. (1981). Food preservation by irradiation. EAEA Bull. 23 (3), 33-36. Widstrom,N.W . (1979).Th e role of insects and other plantpest s in aflatoxin contamination of corn, cotton and peanuts -A Review. J. Environ. Qual. 8 (1), 5-11. Yanai, S.,Matuno , S.,an d Matsura,S . (1964). Researches on gas sterilization effect upon microbes in stored grain.5 .Fungicida l activity ofmethy l bromide to the spores and mycelium ofPénicilliu m islandicum on an inhull- ed rice. Ann. Phytopathol. Soo., Japan, 29, 43-47. Zuber,M.S .an d Lillehoj,E.B . (1979). Status of the aflatoxin problem incorn . J. Environ. Qual. 8 (1),1-4 . 17 ASURVE Y OF THEMYCOFLOR A OF MAIZEGRAIN SSTORE D AT 26±3 °CAN D 75±5% R.H. Abstract Twenty-sixdifferen t fungibelongin g to twelve generawer e isolated and identified onmaiz e {Zea mays L.) stored forsi xmonths .Ninetee n ofthes e are recorded forth efirs t timeo nmaiz e inGhana .Member s ofth e genera Aspergil lus and Pénicillium weremos t frequentlyencountered . Aspergillus flaws Link and Rhizopus oryzae Went andPrinse n -Geerling swer e themos tcommo n species isolated ateac hmonthl y sampling. The storage fungishowe d fourdistinc tpattern s ofinfectio n over theen tireperio d ofexamination .Th e importance ofsom eo fth e isolated fungia s potentialmycotoxi n -producin g species is discussed. Keywords: survey,mycoflora , Zea mays L.,maize . 1.1 Introduction Maize,a n important staple foodcro p inWes tAfrica ,i susuall y stored overlon gperiod s forbot hhuma nconsumption ,an da s component feedfo rpoultr y and livestock. Post-harvest storage losseso f themaiz e grainswithi nWes t Africa due to insects attack andmicrobia l spoilage isestimate d at 301 (1,40 ) ofannua lharvest .Th e role ofstorag e fungi inqualit y deterioration of grains althoughwel l documented (4, 7, 18)i ndevelope d countries,ther e is limited information regarding these fungio nmaiz e inWes tAfric a and therol e such fungiplay .Th e objectives ofthes einvestigation s are toupdat e the listo f storage fungiassociate dwit hmaiz e grains andals o toacquir e information on the infectionpatter no f thesemould s aswel l as todetermin e ifther e are any toxin-producing fungio nmaiz e inGhana . 1.2 Materials and methods Maize [Zea mays L.) grains stored injut ebag sunde rambien t laboratory conditions C75±S%R.H . and 26±3°C )wer e sampledmonthl y forsi xmonths .A t eachmonthl y sampling,50 0 go fgrain swer e takenfro m the top,middl e and bottomo fth ejut eba g andpoole d together.Th emycoflor ao nth e grains 18 was assessed using the method of Tenpe (37) and Limonard (19), washing test and solid medium. 1.2.1 Blotter method A modified method of Tempe (37) and Limonard (19) was employed. Ten grains, surface-sterilized with 2% sodium hypochlorite for 15 min. and ten non-surface sterilized grains were placed on Whatman's filter paper in 9.0 cm Petri dishes. There were 50 replicates (500 grains) for each treatment and the plates were incubated for up to 14 days (14) at 26±3 C. The following monthly quantitative assessments were made for blotter test: a) the percentage of grains infected with fungi; b) the percentage of grains infected with a particular fungus species; c) the total number of fungal colonies appearing on the grains. 1.2.2 Washing method The 2%sodiu mhypochlorit e solution usedfo rsurfac e sterilizationwa s centrifugeda t2,50 0g fo r1 0min .i na nM.S.E . SuperMino rCentrifuge . The supernatantwa s discarded and the residue (with fungal spores) resuspende di n 10m ldistille d formicroscopi c examinationan d identification usinga Reicher t Nr. 4253binocula rmicroscope . 1.2.3 Solid medium method Ten surface-sterilizedan d tennon-sterilize d grainswer eplace do n Sabouraud'sAga ri n9. 0c mPetr i disheswithou tan yfurthe r treatment.Ther e were5 0replicate s (500 grains)fo reac h treatment andth eplate swer e incu bateda t26± 3° Cunti l fungi grew. Sodiumhypochlorit e treatmentwa s usedwit h the aimo freducin go rre moving completely external saprophyteswhic h competewit hpathogens . 1.2.4 Estimationo fviabl e fungal colonies Exactly5 0go fsampl ewa sweighe d into 100m l0.1 1Pepton ei n25 0m l Erlenmeyer flasks andwer e shakeni na Gallenkam pMode lOrbita l Shaker(14 0 rev./min.)fo r3 0min .Fro m this stock suspension serial dilutionmetho dwa s employed and sporeswer e raisedo nSabouraud' sAgar . 19 (* 1 2 3. 4. S 6- 7 8 storage?ftmfr (wowths ) Fig. 1. Fungal growth and kinds of fungi (N- of species) recorded on maize grains stored for six months. Fungal colonies: A A Control; o o Surface-sterilized. Kinds of fungi: A A Control; o—-*-o Surface-sterilized. 20 1.2.5 Moiste r content determination Moisture contento fgrain sa teac hsamplin gwa s determined using theove n drymethod . Sampleso fth egrain swer e groundi nWarin g Blenderan d1 0g por tionso fthei r flourwer e driedi na nove nfo r2 4h a t10 5 Can dthe n cooled ina desiccato r and reweighed. 1.3 Results Therewa sa rapi d increasei nfunga l colony countsi nstore dmaiz e grains with timewhils t colonieso nsurface-sterilize d grains showed suppressed growth comparedt oth euntreate d grains;tota l fungal colonyo nth esurface - sterilized grains remaining constant afterth e4t hmont h (Fig. 1).Th ekind s offung i (numbero fspecies )isolate d showeda ninitia l increasean dcontinue d upt oth e3r dmont h (24differen t kinds)an dthereafte r declined (Fig. 1). Thesam e trendwa s exhibitedb yfung i appearingo nsurface-sterilize d grains which reached thesam epea knumbe ro f2 4differen tspecie so nth e 3rdmont h (Fig. 1).Th emould swhic hwer e recordedo nuntreate d grainswer e thesam ea s thosewhic h appearedo nth e surface-sterilized grains.Th etwenty-si x different fungibelongin gt otwelv e genera isolated frommaiz e grainsi nthes einvesti gationsar eliste di nTabl e1 .Seed-born e fungalspecie s recorded forth e first timeo nmaiz ei nGhan awer e Aspergillus aandidusLink ,A. flavus Link,A. fumi- gatus Fresenius,A. niger van Tiechem, A. ochraceus Wilhelm,A. restictus G. Smith, A. temarii Blochwitz, A. ustus (Bainier)Thor n& Church , A. wentii Wehmer, Cladosporium herbarumLin ke xFr. , Neurospora sitophila Shear& Dodge , Paecilo- myces varioti Bain,Pénicillium digitatum Sacc,Pénicillium expansumLin ke x Fries, P. verrucosumvar . ayclopium, Pénicillium sp. , Phoma glomerata, Rhizoc tonia solani Kuhn,an d Rhizopus oryzae Wentan dPrinsen-Geerlings .Member so f thegenu s Aspergillus were themos tpredominan t followedb y Pénicillium. Asper gillus flavus infection countswer ehighes ta teac hmonthl y sampling (Figs.2 and3) . Onlynin e different fungal sporeswer eencountere di nth ewashin gtest , namely Aspergillus amstelodamiMangi nT & C ,A. flavus, A. janus, A. ruber Spieck& Bremer ,A. sydowi (Bain& Sart )Thor n&Church ,Aspergillus sp., Fusa rium sp., Neurospora sitophila and Phoma glomerata. Ofthese ,onl y A. flavus, N. sitophila and P. glomerata appearedi nth eblotte r testan dsoli dmediu m method andwer e thusviabl e spores.Mould s isolatedb yth e solidmediu mmetho d were samea sthos e obtainedb yth eblotte r method. Four distinctpattern so finfectio nwer eexhibite db yth estorag e fungi 21 100 -o—o 90 - Rhizopus 80 - Aspergillus oryzae 70 fumigatus 60 50 40 30 / Aspergillus 20 A flavus 10 j i i_ 80 70 Pénicillium Aspergillus Cladosporium 60 - sp. 2 ochraceus nerbarum 50 |- 40 30 |- Ol 20 UI 10 P-- Fig. 2.Selecte d fungal species identified onmaiz e grains showing four differ entpattern s of infection, o oControl ;o o Surface-sterilized. 22 during the six months period (Fig. 2). The percentage number of grains infect ed by A. flavus, A. fumigatus and Bhizapus oryzae increase with storage time whilst the percentage number of grains infected with Pénicillium digitatum and A. wentii decrease within the same period (Fig. 2). In contrast with this, there was an initial increase in percentage of grains infected by Pénicillium expansum. • {Pénicillium sp. 2), A. ochraceus and Cladosporiwn herbarum up to 2-3 months followed by a sharp decline (Fig. 2). Finally, the percentage of grains infected by A. niger, A. tamarii and Rhizoctonia solani remained fairly constant, after three months storage period up to the sixth month (Fig. 2). Selected six fungal species that showed the first two patterns of infection il lustrated in Fig 2 is presented in Fig. 3. The fungi A. wentii, C. herbarum and Fusarium moniliforme could not be isolated after four months from the non- sterilized maize grains (Figs 2 and 3). The fungus A. flavus infected 90-1001 of the surface-sterilized grains after three months while A. ochraceus and F. moniliforme were present in about 61 of the grains (Figs 2 and 3). The moisture content of the maize grains varied from an initial 12.11 to 15.11 in six months (Table 2). 1.4 Discussion Small amounts of fungal spores present on the surface of the grains or as dormant mycelia under the pericarp grow fast under favourable conditions. The number of fungi developing on surface-sterilized grains was reduced as compared to the untreated grains (Fig. 1) because sodium hypochlorite treatment reduced or removed completely external saprophytes which compete with the pathogens. The moulds which grew on the untreated maize were the same as those obtained on the pre-treated ones and were thus seed-borne. This observation confirms that of Lichwardt and co-workers (17) for maize. Danquah (9) isolated twenty-two different seed-bome fungi on maize in Ghana. In these investigations, nineteen new species have been identified as seed-borne fungi on maize in Ghana (Table 1). This extends to forty-one, the number of seed-bome fungi recorded on maize in Ghana. The fungi encountered in this paper belonged to twelve genera. The new genera added are Cladosporiwn, Neurospora, Pascilomyces and Rhizoctonia. Broadbent and co-workers (2) provided an extensive list of fungi associ ated with maize in Nigeria. Later, other workers (28, 29) added eleven differ ent fungi to this list. Koubasher et al. (22) isolated and identified thirty- 23 100 80 "Aspergillus Cephalosporium Drechslera 60 .ustus acremonium maydis 14 0 "S 20 -»- <_» 2 0 c Fusarium Neurospora Paecilomyces 'ë 60 moniliforme sitophila varioti 40 20 / \ 0; i-r^ï-r* 1 2 3 4 5 6 0 1 2:3 4 5 6 0 1 2 3 4 5 6 storage time (months) Fig. 3. Selected fungal species showing Che first two patterns of infection illustrated in Fig. 2. o o Control; o o Surface-sterilized. 24 fourspecie so ffung ibelongin gt othirtee ngener ao nstore dmaiz ei nEgypt . Memberso fth egenu s Aspergillus wereth emos tpredominan tfollowe db y Pénicil lium (22).Result sobtaine di nthi swor k (Table 1)agre ewit h this.Th e methodo fisolatio nma y influence thespecie s offung iencountere dalthoug hi n these investigationsn odifferenc ewa s foundbetwee nkind s (species)o ffung i isolatedb yth eblotte r testan do n Sabouraud'sAgar .Th ewashin gtes treveale d sporeso fnin e fungiwhic hwer eloosel yborn eo nth egrains .Ou to fthes eonl y A. flavus, N. sitophila and P. glomrata appearedi nth eblotte rtes tan dsoli d mediummethod . Iti seviden ttha talthoug hth ewashin gtes tgive ssom einfor mationo f fungalspore sassociate dwit h grainlot sconclusion s cannotb emad e fromth eresult so fa washin g testbecaus espore sencountere dma yno tb e viable(25) . Thequalit yo fgrain suse di sdependen to nit swholenes sa swel la sth e presencean ddominanc eo fcertai nmicroorganism swhic hma yb e detrijiientalt o itsqualit y (24).Th ekind so ffung ipresen to nan ygrai nstoc kwoil dals ode pendo nth egeographica l location,prevailin gweathe rcondition s inth elocali tyan dth epost-harves tstorag econditions .Fou rdifferen tinfectio npattern s havebee nobserve d inth eseed-born e fungio nmaiz e inGhana .Thi s-suggest s thatth emycoflor atha twoul db epresen ti nan y grainstoc k ata particula r timewoul d dependo n thelengt ho fth estorag eperiod ,fo rindeed , Aspergillus wentii, C. herbarum and F. moniliforma couldno tb e isolatedfro mth euntreate d grains,afte rfou rmonth s (Figs2 & 3). Thealteratio ni nbiochemica lqualitie s ofth ebasa lmediu m (maize)owin gt oth emetaboli cactivitie s ofth emycoflor a probablybecom e selective favouringth e growtho fsom ean ddeleteriou st o others.I ti sindee ddocumente d (43)tha tn ogrowt han daflatoxin s formation by A. flavus were detected inmaiz e grainswhe n A. niger NRRL641 1 or Triaho- derma viride NRRL641 8wer epaire dwit h A. flavus ininoculate dgrains . Foreac ho fth ecommo nspecie s ofstorag e fungi,ther ei sa minimu mmois tureconten ti ngrain sbelo wwhic h thefungu scanno tgrow .Thes eminimu mmois turecontent shav ebee ndeterminate d formos to fth ecommo nstorag efung i growingo nstarch ycerea l grains (7).Dave yan dElcoat e (10)pu tth esaf e moisture contentfo rstorag eo fmaiz ea tabou t 13$whils tLute yan dChristense n (20)suggeste d thatviabl e fieldan dstorag e fungima yb e generally reducedo r eliminated fromgrai n lotsstore da tmoistur e content 12-14$an da temperatur e of 20-30 °Cwithou tseriou s deterioration tograins . Thispartl yexplain swh y themoistur e contento fth egrain srisin gabov e thestipulate dsaf elevel ,permitte dheav yinvasio nan d growtho fstorag efung i 25 Table 1.Seed-born efung irecorde do nmaiz ei nthi sinvestigation sunde r ambient(75±5 %R.H .an d26.0± 3°C )conditions . »Aspergillus aandidus Link Dreahslera maydis (Nisikado)Subrai m & Jain *A. flavus Link Fusarium moniliforme Sheldon »4. fumigatus Fresenius Fusarium sp. xA. niger vanTieghe m tNenrospora sitophila SheardDodg e *A. oohraaeus Wilhelm *Paeailomyces varioti Bain *A. restrictus G.Smit h uPenicillium digitatum Sacc. *A. tamarii Blochwitz *P. expansum Linke xFr . «A. ustus Wehmer «P. verruaosum var. ayolopium *A. wentii Wehmer Penioillium sp.4 Aspergillus sp. aBeniaillium sp.5 Cephalosporium acremonium Corda xPhoma glomerata. iCladosporium herbarum Linke xFr . ^Rhizoctonia solani Kuhn Curvularia lunata (Wakker)Boedji n nRhizopus oryzae Wentan dPrinsen-Geer - lings. * Newrecor do nmaiz ei nGhana ;unmarked :recorde db yDanqua h(9) . Table2 .Comparativ emoistur econten to f maizegrain sdurin gsi xmonth sstorag e perioda t26± 3° Can d75+5 %R.H . Storageperio d %Moistur econten t (months) (mean± S.E. ) 1 12.1± 0. 1 2 14.3± 0. 1 3 14.6± 0. 2 4 14.7±0. 3 5 14.6± 0. 3 6 15.1± 0. 2 26 (Table 2). Noübasheran dco-worker s (22),indee dpresente devidenc et osho w that thehighes tpopulatio no ffung iwa sobtaine d fromgrain swit ha moistur e contento f15.2 %store da t3 0° Cfo rfou rmonths .Dat afro mFig .1 an dTabl e2 support theirobservation . Inthi sstudy ,potentia l toxin-producing fungi,namel y A. flavus, A. ochraeeus, F. moniliforme, Paecilomyces varieti and Pénicillium expansion were encountered.Muc hpertinen t literature existso nmycotoxLcose si nanimals , in cludingman ,cause d chieflyb yA . flavus (3,13 ,21 ,32 ,etc.) , A. ochraeeus (6,39 ,41 ,42) , Fusarium (16,26) , Pénicillium (3,30 ,42 ,44) ,an d Paecilo- myaes varioti (8). Themycotoxin s aflatoxin B-an dB .ar eproduce di nmaiz e (11,16 ,28 , 33, etc.)b y A. flavus. Brookean dWhit e (3)reporte d thati twa sno tnecessar y forth eproduc tt ob ethoroughl y andheavil yovergrow nb yA . flavus before toxinwil lb epresent .I f3-5 1o fth egrain sar einvaded ,aflatoxin swil lb e detected.Abou t 401o fth emaiz egrain si nthi spape rwer e invadedb yA . flavus afteron emont hstorag ean dth enumbe rincrease dt o95 1tw omonth s later (Fig. 2). Somestrain s of A. ochraeeus produce ochratoxin (38,41) .Isolate s capable ofproducin g ochratoxinar e commoni nblac kpeppe ran dmaiz e (5,6) ,dr yfis h (39), rice (23)an dpeanut s (12).Ochratoxi nA i sa poten tnephrotoxi ci nex perimental chicks (15),rat s (31,34) ,dog s (35)an dswin e (36).Ochratoxi nA basedo n50 1letha ldos edetermination s andminima l growth- inhibitor yconcen tration,i sth emos tpoten tmycotoxi nstudie di nchicken st oda t(15) .A n8-15 1 infectiono fgrain sb ythi s fungusbetwee n2- 6month s (Fig.2 )i ssufficien t towarran t concern. Differentkind so fextremel ypoten tmetabolite s areproduce db yspecie s of Fusarium (16,27) .Th efungu s F. moniliforme isolatedi nthi swor kproduce d toxinunde rlaborato y conditionsbu ti ti sno tknow nwhethe ri tdoe ss oi n nature.Presently , therei sn oinformatio n regardingth econcentratio no fth e toxin thatwoul d rendermaiz e toxic forhuma nan danima l consumption.I twoul d be interestingt oinvestigat e thisi norde rt oascertai nwhethe r thepopulatio n of F. moniliforme presenti n20 1o fth e grainsafte r 2-3month s storagewa s sufficientt oproduc e enoughtoxin ,sinc e F. moniliforme couldno tb eisolate d afterfou rmonths . Thepercentag eo fgrain s invadedb y Paecilomyaes varioti washighes t (81) onth e thirdmont han dthereafte rdecline d (Fig. 3). Accordingt oCovenc yan d co-workers (8) P. varioti doesproduc ea poten ttoxin . 27 Eighty-three out of 196 Peniailliim sp. screened for possible toxic meta bolites caused death, in less than seven days, to animals to which these were fed (3). Five different Peniailliim sp. have been recorded on stored maize and is reported in this paper. Their potential for producing toxic metabolites re main to be assessed. Because of the presence of toxin-producing fungi on stored maize grains, indiscriminate use of contaminated grains for poultry feed and human consump tion should be discouraged or discontinued. Improved quality of food is as important as increased quantity. Hence, programmes to control fungal deterio ration of maize grains in storage should aim at preventing growth of fungi and thus the production of toxic mycotoxins. Acknowledgement The author is grateful to Dr. R.A. Samsom of the Centraal Bureau voor Schimmelculfures,Baarn, the Netherlands for confirming some of the identifica tions of the fungi. My thanks areals o due to Prof. E.H. Kampelmacher, National Institute for Public Health, Bilthoven, the Netherlands and Prof.Dr. J. Farkas, Project Director, International Facility for Food Irradiation Technology, IFFIT, Wageningen, the Netherlands for their critical comments on the manus cript. References (1)Adams ,J.M ., 1977 .A review of the literature concerning losses in stored cereals and pulses published since 1964.Trop . Sei. (19) I: 1-28. (2)Broadbent ,J.A. ,Oyeniran ,J.O. ,an d Kuku,F.O. , 1969.A listo f fungi associated with stored products inNigeria .Nigeria n Stored Products Research Institute Technical Report No. 9: 57-66. (3)Brook ,P.J. , and White,E.P. , 1966.Fungu s toxins affectingmammals . Ann. Rev. Phytopathol. 4: 171-194. (4)Christensen ,CM. , 1957.Deterioratio n of stored grainsb y fungi.Bot . Rev. 23:108-134 . (5)Christensen , CM., Fanse,H.A. , Nelson,G.H. , Bates,F. , and Mirocha, CJ., 1967. Microflora ofblac k and red peppers.Appl .Microbiol . 15:622 - 626. (6)Christensen ,CM. , Nelson,G.H. ,Mirocha ,C.J. ,an d Bates,F. , 1968. Toxicity toexperimenta l animals of 943 isolates of fungi.Cance r Res. 28:2293-626 . (7)Christensen ,CM. , and Kaufmann, 1969.Grai n storage.Th e role of fungi in quality loss. University ofMinnesot a Press,Minneapolis . (8)Coveney , R.D.,Peck ,H.M. , and Townsend, R.J., 1966.Recen t advances in mycotoxicoses. In:Microbiologica l deterioration inTropics . Society of Chemical Industry (London),Monograp h No. 23:31-43 . 28 (9)Danquah ,A.-O. , 1973.Surve y and importance of seed-borne fungi ofrice , sorghum,maize ,cowpe aan dbambarr a groundnut of Ghana.MSc .Thesis , University ofGhana . (10)Davey ,P.M. , and Elcoate,S. , 1965.Moistur e content/relative humidity équilibrao f tropical stored produce.Par t 1\ Ceriab.Trop . Stored Prod. Inf.II :439-467 . (11)Davis ,N.D. ,an dDiener ,U.L. , 1979. Screening corn for aflatoxin:a ne w approach.Highlights ,Agricultura l Research Auburn Univ.Agric .Exp . Sta. 26 (1):11 . (12)Doupnik , B., Jr.,an d Peckham,J.C. , 1971.Toxicit y to chicks ofAsper gillus andPénicilliu m species isolated frommold y pecans.Appl . Microbiol. 21: 1104-1106. (13)Enomoto ,M. , and Saito,M. , 1972.Carcinogen s produced by fungi.Ann .Rev . Microbiol. 26A:279-312 . (14)Gemawat ,P.D. , 1968.Fusariu mmoniliform e inmaiz e seeds from India.A report submitted for the examination of seed pathology held by the Govt. Inst, of seed Pathol.,Copenhagen ,5 2pp . (15)Huff , W.E., Wyatt,R.D. , Tucker,T.L. ,an dHamilton ,P.B. , 1974.Ochra - toxioses in thebroile r chicken.Poult . Sei. 53:1585-1591 . (16)Joffe ,A.Z. , 1965.Toxi n productionb y cereal fungi causing alimentary aleukia inman . In:Mycotoxin s in foodstuffs.G.N .Woga n (Ed.),M.I . T.Press ,5 0Ame s Street,bridg e Mass.02142 :77-85 . (17)Lichtwardt ,R.W. ,Barron ,G.L. ,an dTiffan ,L.H. , 1958.Mo w floraassoci atedwit h chelled corn in Iowa. Iowa St.Coll .Journ . Sei. 1(33) : 1-11. (18)Lillehoj ,E.B. ,Fennel ,D.I. ,an d Hesseltine,C.W. , 1976.Aspergillu s flavus infection and aflatoxin production inmixture s ofhigh-nnois - ture^and drymaize . J. Stored Prod. Res. 12: 11-18. (19)Limonard , J., 1966.A modifie d blotter test for seed health.Neth . J. Pathol. 72:319-321 . (20)Lutey ,R.W. ,an d Christensen, CM., 1963.Influenc e ofmoistur e content, temperature and length of storage upon fungi inbarle ykernels . Phytopathology 53: 713-717. (21)Martin ,P.M.D. ,an d Gilman,G.A. , 1976.A consideration of themycotoxi n hypothesis with special reference to themycoflor a ofmaize , sorghum, wheat and groundnut.Rep .Trop .Prod . Inst.G . 105,vii : 112pp . (22)Noubasher ,A.H. ,El-Naghy , A.M., and Abdel-Hafez, S.I.,,1972 .Studie s on the fungus flora of three grains inEgypt .Mycopath .Mycol . Appl.4 7 (3): 261-274. (23)Natori , S., Sakaki,S. ,Kurata ,H. ,Udagawa ,S. , Ishinol,N. ,Saito ,M. , and Umeda,M. , 1970.Chemica l and cytotoxicity survey on theproduc tion of ochratoxins and penicillic acidb y Aspergillus ochraceusWilh . Chem. Pharm. Bull. 18:2259-2268 . (24)Neergaard , P., 1972.Metho d of assessment. Seed pathology.Manuscrip t in use at theDanis h Govt. Institute of Seed Pathology,Copenhagen . (25)Neergaard , PI,Lambat ,A.K. , andMathur , S.B., 1970.See dhealt h testo f rice. III.Testin g procedures for detection ofPyriculari a oryzae Cav. Int. Seed TestAssoc . 35:157-163 . (26)Nelson ,G.H. , Christensen, CM., andMirocha ,C.J. , 1965.A vetenarian looks atmould y corn.Proc . 20thAnn .Hybri d Corn Industry Research Conf.America n Seed TradeAssociation ,Executiv e Building Suite 1964, Washington D.C 200s:86-91. (27)Northolt ,M.D. , 1979.Th e effect ofwate r activity and temperature on the production of somemycotoxins .PhD .Thesis ,Agricultura l University, Wageningen,Th eNetherlands . 29 (28)Oyeniran ,J.O. , 1973a.Microbiologica l studies onmaiz e used on poultry and livestock feed at tworesearc h farms at Ibadan,Wester n State, Nigeria. Rep.Nigeria n Stored Prod. Res.Inst . 1970Tech .Rep .No .6 : 47-56. (29)Oyeniran ,J.O. , 1973b.Microbiologica l examination ofmaiz e from various sources soon afterharvest .Rep .Nigeria n Stored Prod. Res.Inst . Tech,,Rep .No . 3: 27-32. (30)Purchase ,J.F.H. ,an d Theron,J.J. , 1967.Researc h onmycotoxin s in South Africa.Internationa l Pathology 8: 3-7. (31)Purchase ,J.F.H. ,an d Theron,J.J. , 1968.Th e acute toxicity of ochra- toxinA in rats.Foo d Cosmet. Toxicol.6 :479-483 . (32)Tabor ,R.A. , and Schroeder,H.W. , 1967.Aflatoxin-producin g potential of isolates of theAspregillu s flavus-oryzae group frompeanut s (Arachis hypogea.L.) .Appl .Microbiol . 15:140-144 . (33)Ross ,I.J. , Loewer,O.J. ,an d White,G.M. , 1979.Potentia l for aflatoxin development in low temperature drying systems.Trans .Amer .Soc . Agric. Eng. 22 (6):1439-1443 . (34)Suzuki ,S. ,Kozute ,Y. , Satoh,T. , and Yamasaki,M. , 1975.Studie s on the nephrotoxicity of ochratoxin A in rats.Toxicol .Appl .Pharmacol .34 : 479-490. (35)Szezech, G.M. , Carlton,W.W ., an d Tuite,J. , 1973a.Ochratoxicosi s in beagle dogs. I. Clinical and clinico-pathological features.Vet . Pathol. 10:135-154 . (36)Szezech ,G.M. , Carlton,W.W. ,Tuite ,J. , and Caldwell,R. , 1973b.Ochra toxinA toxicosis in swine.Vet .Pathol . 10:347-364 . (37)Tempe ,J . de, 1967.Routin e investigation of seedhealt h condition in the Dutch Seed Testing Station atWageningen . Proc. Int.See dTes tAssoc . 22: 1-12. (38)Theron ,J.J. , Van derMerwe ,K.J. , Lieberngerg,N. ,Joubert ,H.J.B. , and Nel, W., 1966.Acut e liver injury induckling s and rats as a result ofochratoxi n poisoning.Jour .Pathol .Becteriol .91 :512-529 . (39)Udagawa , S.,Ichinol ,M. , and Kurata,H. , 1970.Occurrenc e and distribu tion ofmycotoxi n producers inJapanes e food. In:M .Herzber g (Ed.): Toxic Microorganisms,Ü.J.N.R . Joint Panels onToxi c Microorganisms. U.S. Department of Interior,Washington ,D.C. : p. 174. (40)unite d Nations:Foo d andAgricultur e Organisation (based on Rawnsley,1969 ) Ghana:Cro p Storage Rep.Foo d Agric.Organ .No .P L SF/GH, 7,IX : 88pp . (41).Vani'de r Merwe,K.J. , Steyn,P.S. , Fourie,L. , Scott,B . de,an d Theron, J.J., 1965.Ochratoxi n A, a toxicmetabolit e produced by Aspergillus ochraceus Wilh.Natur e (London)205 : 1112-1113. (42)Va n Walbeek,W. , Scott,P.M. , Harwig,J. , and Lawrence,J.W. , 1969.Péni cillium viridicatumWestling : ane w source of ochratoxin A. Can. J. Microbiol. 15: 1281-1285. (43)Wicklow ,D.T. , Hesseltine,C.W. , Shotwell,O.L. , andAdams ,G.L. , 1980. Interference Competition and aflatoxin levels in corn.Phytopatholo gy 70 (8):761-764 . (44)Wilson ,B.J. , 1965.Toxi c substances formed by filamentous fungi growing on feed stuffs.In :Mycotoxin s in foodstuffs.G.N . Wogan. (Ed.),M.I . T. Press,5 0Ame s Street Cambridge Mass.02142 :147-149 . 30 2. STUDIES ONTH ETECHNOLOGICA L FEASIBILITY OFTH EAPPLICATIO N OFDR YO R MOIST HEATT OGRAIN S ANDGRAI N PRODUCTS PRIOR TO GAMMA IRRADIATION G.T. Odamtten,V .Appiah , andD.I .Langera k SUMMARY A simple apparatus has been designed that enabled us to heat maize grains and grain products sustained at 60 C for at least half an hour at precise humidities, low (« k$% R.H.) or high (> 851) during the heat treatment. The process is more efficient and temperature and humidity required are reached within 5 min if the grains and grain products are heated in open containers. It was advisable toallo w the chamber to reach 'equilibrium' (required humidity and temperature) before introducing the product into the climate chamber. The practical implications of these findings are discussed bearing in mind that the heat treatment at high humidity does not significantly increase the m.c of the grains after the treatment. INTRODUCTION Heat treatment of food is a well established and accepted method of processing food for consumption whilst ionization radiation has only re cently been proven as a method of killing or reducing microbial load of stored products. In some instances, such as cocoa (AMOAKO-ATTA et al., 1980), and maize (ODAMTTEN, 1980), irradiation.alon e up to 5-0 kGy could not give complete control of pathogenic moulds because the fungistatic effect of the radiation was dependent on the storage humidity. Radiation treatment combined with heat treatment (KISS 6 CLARKE, 1969; ROY et al., 1972; SOMMER et al., 1972; LANGERAK £ CANET-PRADES, 1979 etc.) was more effective than radiation alone. 31 According to WEBB and co-workers (1969) and SOMMER et al. (1972), the moisture content of spores or conidia prior to irradiation, as well as in the developmental stage of the mycelia, affect the radiation re sistance of fungi. In the opinion of NYERGES-ROGRÖN (1973) conidia ir radiated in the wet state proved to be more radiation-sensitive than those treated in the dry state. It is the aim of the treatment with high (> 85%R.H. ) humidity during heat treatment of grains to enhance the kil ling effect of heat on the pathogenic moulds. The question of the possibility of providing high humidity (> 85$ R.H.) conditions as compared to low humidity (< ^5% R.H.) conditions during the heat treatment spured us on to examine the use of a simple apparatus that would enable us monitor the heat penetration into the product and ambient humidity in the chamber during the heat treatment. MATERIALS AND METHODS The heat treatment was initial carried out in a large climate room with polystyrene lining (320 m long x 160 m wide x 207 m high) at the Founda tion Institute for Atomic Sciences in Agriculture - ITAL, Wageningen, where an evaporator (Defensor 505) provided the requisite ambient humidity and the inbuilt heater (Contardo Model PAI2 ,Milano , Italy) in the wall of the room provided the desired temperature of 60 °C. The thermocouples were inserted into small holes (0/mm) bored at the radicle end of the grains and in the case of the apples 2 mm below the peel of the apples. Apple was used to find out if the process will be applicable to fruits. The tem perature was recorded as the potential difference between the cold (ice) point and hot point (internal portion of grains) on a potentiometer 5 cri- ber caliberated on temperature scale (0-100 °C). The humidity was recor ded by a digital Psychrometer (PTM). The samples were placed in a poly styrene container (25 x 20 x 20 cm) and opened just before experiment com menced. The temperature was regularly recorded (5mi n intervals) for 1h and then after 1i h. The volume of the large climate room did not allow, temperature and humidity to rise above 55 °C and 75% R.H. respectively. Therefore, a smaller chamber, an incubator (Marius, Utrecht, The Netherlands), was re sorted to using the same ancillary apparatus as in the larger climate room. 32 Mgj_sture_çontent_determj^nat22Q Two ten (10 g) gram portions of the flour of maize were kept at 105 °C for ïk h or until constant weight was obtained as suggested by AOAC (I960), RESULTS AND DISCUSSION When low humidity (< k$%R.H. ) was provided during heating, the grains attained a temperature of 53 °C, slightly inferior to that recorded by the thermistor probe (PT 100) which measured the ambient temperature of 55 °C. The maximum temperature of 55 °C was attained after about 50 min heating period (Fig. 1a). In the case of apples, penetration of heat was poor and a temperature difference of about 15 °C between the PT 100 rea ding'an d the thermocouple reading was obtained (Fig. 1b). Itwa s clear that this mode of heat treatment was not suitable at least for apples since the heat penetration was wel1 inferior to what obtained in the maize grains. Furthermore, there was browning of the skin of the apples thereafter. When humidity provided during heating was increased to 7^.3% R.H., pe netration of heat through the woven polypropylene bags (a and b) was about 25 °C and 15 °C respectively lower than that recorded by the thermistor probe, PT 100. The ambient temperature of 56 C was never attained by the grains in the bag even after 2 hours heating (Fig. 2a). In contrast to this, grains heated in open shallow trays (c and d) attained the required temperature about 15 min and thereafter remained constant. Treatment of apples with higher humidity proved impracticable (Fig. 2b). It was evident that to obtain good heat penetration during heat treatment, the grains should be heated in open containers before putting in bags for irradiation. This will permit maximum penetration, under high humidity in less than 15 min. In practice, a higher humidity (> 85% R.H.) and higher temperature (60 C for 30 min) will be required. It was then worthwhile to decrease the volume of the climate chamber to be used and thus improve upon the humidity and temperature distribution to ensure maximum efficiency. Thus a smaller simulated climate chamber was designed, using the same apparatus as in the bigger room but instead using an incubator as the climate cham ber. Plates 1, 2, 3 illustrates the external and internal arrangement of the apparatus. The heat treatment was carried out with and without putting 33 601 •X X X x x X X X X X X 50 te 40 -- o o - 30 —x— thermocouple (P T 100) Q. E -A— b OJ —A— c ~ 20 MAIZE GRAINS 10 - R.H. 43.6 (±3.8%) i i i i i i i i i 10 20 30 40 50 60 70 80 90 100 time (min) FIG. Ia. Graph showing temperature measurements in maize grain during heat treatment at low humidity (A3.6 + 3.8%) in large climate chamber. 34 o —x— thermocouple (PT 100) Q. E —o— b -2 20h --o-- c APPLES 10 R.H.A3, 6 (±3.8%) 1 ! 1 1 1 i i i .i i 0 10 20 30 40 50 60 70 80 90 100 time (min) FIG. lb. Graph showing temperature measurements inapple s during heat treatment atlo whumidit y (A3/6 + 3-8%) inlarg e climate cham ber. 35 70 60 B==3FK$™rjf^^^ thermocouple ( PT 100) 20 •A— o (in bog ) A— b I in bag ) MAIZE A— c A-- d 10 R.H.74.3 % 10 20 30 40 50 60 70 80 90 100 110 time (min) FIG. 2a. Graph showing temperature measurements inmaiz e grains during heat treatment athig h humidity (7^.3%) in large climate cham ber (Note thela gi ntemperatur e rise ingrain s that were treated inbag s aan db) . 36 «— thermocouple (PT 100) o— Q o- b APPLES •D— C 10 R.H. 74.3% 0 10 20 30 Ü0 50 60 70 80 90 100 110 time (min) FIG. 2b. Graph showing temperature measurements inapple s during same heating period and humidity as indicated in FIG. 2a. in large climate chamber. 37 PVöSe 1. Photograph of the external and internal arrangement of the heat treatment chamber employed in these investigation. 38 Plate 'S- Close-up photograph of the internal portion of the heat- treatment chamber. 1.Hangin g mercury thermometer. 2. A TPM Digital Phychrometer which records the ambient humidity and temperature. 3. Petri dishes with lids eccentrically placed on the lid. k. An eletric fan which together with another smaller one (at top left corner) provides a uniform conventional current of heat and humidity. 5. Control knob for the Defensor 505 Evaporator. 6. A heater to boost the heat provided by the incubator. 39 Plata;,3«. Close-up of the external arrangement of the heat-treatment apparatus. 1.Th e control Knob for the PTM Phychrometer 2. Control knob for the thermocouples 3. The Potentiometer scriber which converts the "current" from the thermocouples to temperature and record; this is recorded on the chart. 40 TABLE 1. Comparative moisture contents of maize grains after heat treatment at high (> 85% R.H.) and low humidity (< A5* R.H.). Temperature (°C) and Moisture content of grains humidity during (%) (Mean + S.D.) heat treatment 20 L 12.6 + O.k 20 H 12.7 + 0.1 60 L 12.9 + 0.4 60 H 12.9 + 0.1 L - Low humidity (< >*5% R.H.) H - High humidity (> 85% R.H.) 41 on the incubator heating system and ventilator (Electric fan).Th e fan improved the ventilation and provided a uniform convectional current of heat and humidity within the chamber. Results presented in Fig. 3 with a heating humidity of 77.3% R.H. showed that it required one hour before the grains in open tray 'a' attained the required 60 C whilst thermo couple 2 in tray b was below 60 C. The incubator and ventilator were off in this instance. Putting on the incubator heating system to 60 C and the heater at the base as well as the fan to improve the convectio nal current in the chamber permitted a uniform temperature and humidity distribution. A maximum temperature of 60 °C and humidity (high > 85%; low < hS%R.H. ) was attained in 5mi n after introducing the product into the chamber (Fig. k). The incubator was prior to this, allowed to'equi librate' to attain a temperature of 60 °C and required humidity before putting in the maize grains. Happily the process did not appreciably increase the moisture content of the maize grains after the heat treatment at high humidity (> 85% R.H.) (Table 1),thu ssettlin gth e fears that the process might increase the moisture content of the grains to the detriment of the grains during stor age thereafter. The primary objective of application of high humidity during heating is to augment the killing effect of heat on mould spores since this provides increased penetration of heat on target organisms. The increased moisture of spores during the heating at high humidity (> 85%R.H. )render s the spores more radiation-sensitive and the subsequent irradiation of the grains should be carried out not more than 30 min af ter the heat treatment in order not to lose the synergistic effect of the combination treatment. ACKNOWLEDGEMENTS The authors are grateful to Mr. J.G. de Swart for persmission to use the climate chamber and to Mr. A. Boom for his excellent technical as sistance. 42 70h :ë=^&~^^é^^^^i —x— thermocouple (PT 100) —A— Qf OfM l trojj > —A— b(i n open troy) 20 —A~ c (i n bag, polyprop.) MAIZE 10 - R.H. 77.3% 10 20 30 40 50 60 70 80 90 100 110 time (min) FIG. 3- Graph showing temperature measurments inmaiz e grains during heat treatment athig h humidity (77-3% R.H.) inth esmalle r incubator without additional ventilation andheating . 43 70 . higvh humidit y n 100 _X X \ _x-""" 60S x—"~ 90 •«—'temp . ~ 50 80 2. t_j p o < oj 40 70 3re* 3 C -4— a a. 60 JT S.3 0 E low humidity 35 *" 20 r - 50 û -ft A A 10 - 40 -, 1 1 1 1 1 10 15 20 25 30 35 time interval (min) FIG. k. Graph showing temperature and humidity measurements during heat treatment in the chamber when^adequate heat and ventilation (convectional current) was provided. 44 REFERENCES AMOAKO ATTA, B., G.T. ODAMTTEN and VICTORIA APPIAH (198O). Influence of relative humidity on radiosensitivty on Aspergillus flavus Link, in fecting cocoa beans. Presented at the INTERNATIONAL SYMPOSIUM ON COM BINATION PROCESSES IN FOOD IRRADIATION, Columbo, Sri-Lanka, 24-28 November 1980. KISS, I.an d D.I. CLARKE (1969). Elesztók pusztulâsânak vizgâlata be- sugSrzâs, hokezelés es ezek kombinâciojanak hatâsâra (A study of the death of yeasts upon heat treatment, irradiation and the combination of both). ÉLELMISZERTUDOMÂNY, 3 (2),115-126 . LANGERAK, D.IS. and F.M. CANET-PRADES (1979). The effect of combined treat ment on the inactivation of moulds in fruits and vegetables. TECHNI CAL AND PRELIMINARY RESEARCH REPORT NO. 88, ITAL, WAGENINGEN, THE NETHERLANDS. NYERGES-ROGRÜN, E. (1973). Morphological characteristics of the spore head of Pénicillium purpurogenum as affected by gamma irradiation. ACTA ALIMENTARIA, VOL. 't(1) ,8l-9*t . ODAMTTEN, G.T. (1980)j Control by fungi causing deterioration of maize in storage by gamma irradiation. (Unpublished). ROY, K., M.S. CHATETH and P.M. MUREWAR (1972). Gamma radiation in the ex tension of shelf-life of apples infected with Aspergillus niger Van Tiegham. PHYT0"ATH0L. ZEITSCHRIFT, 25, 31-37. SOMMER, N.F., R.J. FORTLAGE, P.M. BUCKELY and E.C. MAXIE (1972). Compa rative sensitivity to gamma radiation of conidia mycelia and sclerolia of Botrytis cinerea. RADIATION BOTANY, 1_2,99-103 - WEBB, B.D., H.D. THIERS and L.R. RICHARDSON (1969). Studies in feed spoilage. Inhibition of mould growth by gamma irradiation. APPLIED MICROBIOLOGY, 7, 329-333- 45 Acta Alimentaria, Vol. 14 (2),pp. 139 —150 (1985) 3. IN VITRO STUDIES ONTH E EFFECT OF THE COMBINATIONTREATMEN T OF HEATAN D IRRADIATION ONSPORE S OF ASPERGILLUS FLAWS LINK NRRL 5906 G. T. ODAMTTEN3'0, V. APPIAH34 andD . IS.LANGERAK b,e a International Facility for Food Irradiation Technology c/o Pilot Plant for Food Irradiation, P.O.Box 87, 6700AB Wageningen. The Netherlands b Research Institute 1TAL, P.O.Box 48, 6700AA Wageningen. The Netherlands. d Department of Biology, Food andAgriculture , National Nuclear Research Centre, Ghana Atomic Energy Commission, P.O. Box 80 Legon/Accra. Ghana • State Institute for Quality Control in Agricultural Products, P.O. Box 230,670 0 AE Wageningen. The Netherlands (Received: 15 July 1983; revision received: 14|Decembe r 1983;accepted : 23 December 1983) Studies have been carried out toinvestigat e the effect of the combination treatment ofhea t and subsequent gamma irradiation onth e survival of conidia of A. flavus Link NRRL 5906. The mould spores were heat-treated either in aqueous suspension (containing 0.2% Tween-80) or harvested dry and heat- treated byho t airi na climate ohamber at low humidity (^45% R.H.) or high humidity (^85% R.H.). Heat treatments ofth e aqueous suspensions were per formed at 20, 40, 45, 48, 50, 52, 53,55 , 58, 59an d 60 °Cfo r 2.5,5. 0an d 10 minutes, respectively. Hot-air treatment ofdry-harveste d spores was done for3 0 minutes at 60 °C. Suspensions were given 0.25, 0.50, 0.70, 1.0an d 1.5 kGy of gamma radiation, while dry harvested spores were exposed to 3.5 and 4.0 kGy. Irradiation was carried out not later than 30 min after heat treatment. In aqueous suspension the adverse effect of heating on the spores generally started after being at thetemperatur e of 50 °C or higher fora t least 2.5min . Heating fora t least 5 min at 59 °C inactivated the moistened spores completely (at least 5 log cycles reduction inth e viable count onmaiz e meal agar). When heating of the suspensions was combined with a subsequent irradiation treatment, a synergistic effect was observed ata temperature above 50 °C andth e synergism increased with increasing heat damage (increasing the heating time and/or tem perature). Heating of dry conidia at 60 °C for 30 min athig h humidity (^85% R.H.) reduced their viable count by 3 logcycles , whilst a parallel treatment at low humidity (^45% R.H.) didno t appreciably reduce the viable mould spores. However, when the spores were stored at 80% R.H. for4 day s before plating, a considerable fraction recovered from the heat damage. Radiation treatment was more effective after exposing the spores toho tai rwit h high humidity (^85% R.H.) before irradiation than when low humidity (^45% R.H.) was applied. The practical implications ofthes e findings are discussed. Keywords: Combined treatment, irradiation of spores, survival of A. flavus The contamination of food consumed by humans andanimal s alike by fungi has raised much concern, particularly those fungi which produce potent mycotoxins (ENOMOTO & SAITO, 1972; ATTSTWICK, 1975; TTTTNSTRA et al., 1975; SMITH et al., 1976; HARWIG et al., 1979; HJJTE et al., 1979; ZITBBB "Present address: Department of Botany, University of Ghana, P.O.Box 55, Legon/Accra. Ghana 46 ODAMTTEN et al.: COMBINATION TREATMENT ON A. FLAVUS SPORES & LUXEHOJ, 1979; ABALAKA & ELBGBEDE, 1982). Prominent amongst these is Aspergillus flavus which produces the noxious and carcinogenic metabolites known as aflatoxins. The use of gamma radiation alone for disinfestation of cereals and control of fungi infecting cereals and grains is well documented (KTTUK & JUSTICE, 1966; POISSON & CAHAGNIEE, 1974; Kiss & FARKAS, 1977; ODAMT- TEN, 1979, 1982) but it is desirable to undertake in vitro studies pertaining to the radiation response of the spores of A. flavus in combination with pre vious heating. There are references in the pertinent literature on the novel approach for controlling fungal infection of stored products by the combination treat ment of heat and irradiation. Radiation treatment combined with heat treat ment (Kiss & CLAEKE, 1969; Roy et al., 1972; SOMMEE et al., 1972; BRODRICK et al., 1976, 1977; LANGEEAK & CÂNET-PEADES, 1979) was more effective than radiation treatment alone. Recently, PADWAL-DESAI and co-workers (1976), showed that different strains of A. flavus (toxigenic and non-toxigenic) responded differently !to heat treatment alone and to the combined treatment with gamma irradiation. This phenomenon had previously been reported by SOMMEE and co-workers (1964). It is likely that the strain A. flavus NRRL 5906 infecting maize grains may show different response to heat treatment alone and to combined treat ment with gamma irradiation. Such in vitro studies on this fungus using a model medium, maize meal agar (MMA), closely related to the product will provide the most relevant information necessary for the subsequent in vivo application studies on the product, maize grains. These experiments reported here were designed to provide pertinent information on the effect ofmil d heat (20-60 °C) alone and the combination treatment with gamma irradiation on the survival of spores of A. flavus NRRL 5906. 1. Materials and methods 1.1. Treatment of spores 1.1.1. Treatment in suspension (A). Spores of A. flavus Link NRRL 5906 were used in these studies. The method employed was essentially a modification of the procedure of LANGERAK and CANET-PRADES (1979). Spores of A. flavus were suspended in sterile Tween solution (0.2% Tween 80). The suspension was centrifuged at 4000 g for 15 min, the supernatant discarded with subsequent resuspending of the pelleted spores in fresh Tween. The centri- fugation was repeated. The resultant suspension was then adjusted to a spore density of 106 to 107 spores per cm3 with the help of a Hawksley B. S. 784 Haema cytometer (Hawksley and SonsLtd. . Sussex, England). Heating tubes 47 ODAMTTEN et al.: COMBINATION TREATMENT ON A.FLAVTJ S SPORES (0 2.5 em, 16c m long) containing 27cm 3o f sterile Tween solution used for each heating temperature (20, 40,45 , 48, 50,52 ,53 , 55an d6 0 °C) were main tained at the respective stated temperatures in water bath (P.M. Tamson N. V., Holland) and 3 cm3 aliquots of the stock suspension of spores were added. After heating for the pre-determined time intervals of 2.5, 5.0 and 10 min, the tubes were transferred to room temperature (cca2 0°C) . Tubes which contained unheated spores (20 °C) were regarded as controls. 1.1.2. Treatment at different R. H. (B).The spores were harvested dry from a maize medium (200 g blended maize in 500 cm3 Erlenmeyer flask) and 0.1 g weight of spores transferred into sterile petri dishes (9.0c m diameter). The spores were heated at 60 °C for 30 mini n a forced heat micro-climatic chamber that enabled us to give the spores either a low humidity (<|45% R. H.) orhig h humidity (]>85 % R.H. ) during the heating period (ODAMTTEH et al., 1980). Thelid s of the petri dishes were eccentrically placed onth e bot toms to allow maximum exposure of the spores to the prescribed treatment. The humidity and temperature during the heating period was monitored from a remote position (ODAMTTEN et al., 1980). After irradiation in the dry state, the spores were suspended in 30 cm3o f sterile Tween solution before plating on the agar medium. Other combined treated spores in petri dishes with nipples were kept at 80%R .H . for 4 and 8 days, respectively, before plating on Maize Meal Agar (MMA, 200g maiz e powder in 1 dm3 distilled water) and Oxytetracycline Glucose Yeast Extract Agar Medium (OGYE, Oxytetra cycline 0.5g ;glucos e 20g ; yeast extract 5g ; distilled water 1dm 3). 1.2. Radiation treatment The irradiation wascarrie d out not more than 30 min after heat treat ment inth ePilo t Plant forFoo d Irradiation at the International Facility for Food Irradiation Technology (IFFIT) Wageningen, The Netherlands.Th e dose rate was 2.6 kGyh _1. Theabsorbe d dosewa s checked by Fricke's dosimetry and clear perspex dosimeters. Test tubes containing 4.0cm 3aliquot s of heat- treated moist spores (Treatment A) were given 0.20, 0.50, 0.70 and 1.0, 1.5 kGy of gamma irradiation, resp., whilst the dry spores (Treatment B) were exposed to 3.5 and 4.0 kGy ofgamm a radiation. 1.3. Assessment of survivors in treated spores Aliquots of 1.0 cm3o f the combination treated spores were diluted at the ratio 1: 9 an dwer e plated outo n MMA and OGYE media employing the conventional microbiological technique of serial dilution. After an incubation period of 3 days at 28 °C, thelo gsurviva l wasplotte d on linear graph paper against radiation dose. The D10 values and correlation coefficients of the combination treated samples were calculated where appropriate. 48 0DAMTTEN et al.:COMBINATIO NTREATMEN T ON A. FLAVUS SP0EE8 2. Results 2.1. Effectof heating andin combination withradiation onspores inTreatment A The effects of the various temperatures and heating timesi nTreatmen t A areillustrate d in Fig. 1.Ther e was no difference in the surviving population inth erang eo f20-4 5° Cafte r 2.5, 5.0 and 10 min heating periods. Subsequent increase in heating temperature from 45° C to 49° C for 2.5 and 5.0 min did not reduce the surviving population of spores. The adverse effect of heating on the spores generally started after the spores had been treated at about 52° C for at least 2.5 min. At temperatures higher than 50°C , a reduction in the surviving population was recorded when the heating time was increased from 2.5 to 10 min. The spores were, however, completely killed after heating them for 5mi n and 10mi n at 59° Can d 58°C ,respectively .Thu s heating alone Si. 56 62 Temperature (°C) Fig. 1.Grap hshowin g effect ofheatin g temperature and duration of heating on the sur vival ofspore s of A. flavus in suspension. (Medium used was MMA). -#-: heating for 2.5 min; — A —: heating for 5 min; O : heating for 10 min 49 0DAMTTEN et al.: COMBINATION TREATMENT ON A. FLAVUS SPOKES for at least 5 min at 59° C is considered sufficient to inactivate the spores of A. flavus treated in suspension. When irradiation was (0.25-1.5 kGy) combined with heating, a syner gistic effect was obtained even at the sub-lethal temperatures from 50° C to 53 °Cwher eheatin g aloneha d asligh t effect on thespore s(Figs .2-4) . Generally 5 min heating at 53° C in combination with 0.75 kGy of gamma radiation resulted in complete inhibition of spore germination (Fig. 2). The D10 values obtained are presented in Table 1.Th e correlation coeffi cients were in the range of 0.930 to 0.999. There was, generally, a decrease in Dl0 values when the sub-lethal heating temperature of 50° C was exceeded and similar D10 values of 0.19 and 0.18 kGywer eobtaine d after combination treatment with 0.75 kGy for 5 and 10mi n heating times, resp., at 53 °C. 0.25 0.50 0.75 1.25 1.50 1.75 Dose (kGy) Fig. 2. Effect of 2.5 min heating of spores of A. flavus in suspension at the indioated temperatures, in combination with gamma radiation doses (0.50-1.50 kGy) on the survival ofspores .(Incubatio n mediumwa sMMA.)-#- : 20°C ; —O—:40°C; -A-: 45°C ; —A—: 48 °C; -X-: 50 °C; -D-: 52 °C; -•-: 55 °C 50 ODAMÏTEN et al.: COMBINATION TEEATMENT ON A. FLAVUS SPOEES 025 050 1.0 1.25 Dose (kGy ) Fig. 3. Effect of 5 min heating of spores of A. flavus in suspension at the indicated tem peratures, in combination with gamma radiation doses (0.25-1.0 kGy) on the survival of spores. (Incubation medium was MMA.) —•—: 20 °C; —O—: 40 °C; —.A— : 45 °C; —A—: 48 °C; —X—: 50 °C; —D—: 52 °C; —•—: 53 °C Table 1 Comparative D10 values (Wty) of combination treatment of heating (different temperatures and times) and gamma radiation, reap. (Spores of Aspergillus flavus) CO 2.6min * COmi n 'U min 20 0.43 0.29 0.25 40 0.52 0.28 0.28 45 0.51 0.27 0.29 46 0.44 0.27 0.25 50 0.50 0.29 0.25 52 0.42 0.29 0.19 53 — 0.19 0.18 55 0.32 — — * Data for 2.5 min were obtained from a separate experiment 51 0DAMTTEN et al.: COMBINATION TREATMENT ON A. FLAVUS SPORE& 0.25 0-50 075 125 1.50 Dose (kGy) Fig; 4. Effect of 10 min heating of spores of A. flavus in suspension at the indicated temperatures, in combination with gamma radiation doses (0.25-1.0 kGy) on the survival of spores. (Incubation medium was MMA.) —•— : 20 °C; —O—: 40 °C; —A—: 45 °C; —A—: 48 °C; —x—: 50 °C; —C— : 52 °C; —•—: 53 °C 2.2. Effect of heating only and in combination vnth radiation on dry spores in Treatment B The effect of heating alone (60 °C for 30 min) and the combination treat ment with radiation (0, 3.5 and 4.0 kGy) on the dry spores is illustrated in Figs. 5 and 6. The efficacy of heat in inactivating spore germination depended strongly on the ambient humidity during heat treatment. Heating alone at 60 °G for 30 min at high humidity (^85% R. H.) reduced the mould count on OGYE (Fig. 5) and MMA (Fig. 6) by 3 log cycles whilst a parallel treatment at low humidity ( <[45% R. H.) conditions did not appreciably reduce the 52 ODAMTTEN et aL:COMBINATIO N TREATMENT ON A. FLAVUS SPORES 8 days 0 3.5 4.0 0 25 4.0 0 3.5 4.0 Dose (kGy) Fig. 5. Effect of irradiation of the survival of unheated (control) spores of A. flavus (top) kept after treatment at 80% R.H. ambient humidity for 0,-4, and 8 days, resp ectively, before plating on OGYE medium; and (bottom) the effect of the comb ination of heat (60 °C for 30 min) with irradiation on the survival of spores of A. flavus stored at 80% R.H. for 0, 4 and 8 davs, respectively, before plating on OGYE. L: humidity ^45% R.H.; H: humidity ^85% R.H. spore population. However, when the combination treated spores were stored at 80% R. H. for 4 days before plating, spore germination was considerably reduced (by 3 log cycles) even under low-humidity heating conditions. The lethality of the combined treatment was evident after 4 days but thereafter, the sporesrathe r curiouslyrecovere d after 8day sstorag e at 80%R . H. Again, similar results were obtained on the two solid media, namely OGYE and MMA. 53 ODAMTTENe t aL: COMBINATION TREATMENT ON A. H.AVUS SPORES 8 days -v/—" I 1*« - 3.5 4.0 8 days) \ \ c* 3 \ \ o \ 0 9 I- '< \ I \ *£ i .H —// i 1— JS 4.0 0 3.5 40 3.5 40 Dose tkGy) Fig. 6. Effect of irradiation on the survival of unheated (control) spores of A. flaws (top) kept, after treatment, at 80% R.H. ambient humidity for 0, 4 and 8 days, resp ectively, before plating on MMA medium; and (bottom) the effect of the comb ination of heat (60 °Cfo r 30 min) with irradiation on the survival of spores of A. flavua stored at 80% R.H. for 0, 4 and 8 days respectively before plating on MMA medium. L: humidity ^45% R.H.; H: humidity ^85% R.H. The unheated (20°C ) spore population remained the same with a slight increase on the 4th day at 80% R. H. There was no distinct difference between unheated and unirradiated spores treated under low and high humidity condi tions, respectively. In all instances, the size of colonies was larger on OGYE than on MMA. Radiation treatment was more effeotive after exposing the spores to hot air of high humidity (^>85% R. H.) before irradiation than when low humidity (<|45% R. H.) was applied (Figs. 5 and 6). 54 ODAMTTEN et al: COMBINATION TREATMENT ON A. FLAVU S SPOEES 3. Conclusions The report of MOYHUDDIN and SKOBOPAD (1975) stated that the syner gistic effect of heat and gamma radiation on tne oonidia of A. flaws Link var. columnaris Rapper and Fennel increased with an increase in temperature (35-55 °C applied for 20 min) and radiation dose (0.25-1.25 kGy) and that this increase was more pronounced at temperatures above 45 °C. They found that heating the conidia at 50 °C for 20 min followed by irradiation was most effective. At 1.0 and 1.25 kGy gamma radiation, there were no survivors. The conidia of A. flavus Link (toxigenic) were more resistant to heat treatment at 50 °Cfo r 5mi n than conidia of A. flavus, A. oryzae (non toxigenic). However, survival of both strains was not affected by being exposed to 50 °C for 75 min (PADWAL-DESAI et al., 1976). Heat treatment at 55 °C for 5 min reduced the conidial population to 6.5% and 0.12% in the toxigenic and non-toxigenic strains, respectively. Our results showed that the adverse effect of heating on the spores (conidia) of A. flavus NRRL 5906 began at a temperature greater than 52 °C when the heat was applied for 2.5 and 5 min, resp., (Fig. 1) and that heat ing at 59 °C for at least 5 min is considered sufficient to inactivate spore germination. Generally, 5 min heating at 53 °C in combination with 0.75 kGy of gamma radiation resulted in complete inactivation of spores of A. flavus (Fig. 3). The fact that different strains of a species of fungus may vary in sensitivity to heat and irradiation combination treatment has been shown by other workers (SOMMEE et al., 1964; MoYHTJDBrN & SKOEOPAD, 1975; WEBB et al., 1969). From the economic point of view, lowering the radiation dose by the combination process is advantageous and desirable for the effective control of germinating spores of fungi. This type of in vitro studies will be relevant for products likefruit s and vegetables which can beimmerse d directly in moder ately warm water without any adverse effect on the plant tissue. It would, in this instance, be superfluous to immerse the product for more than 5 min in the warm water since no significant difference was found between the D10 after 5 and 10 min heating at 53 °C (Table 1). Our experimental results show that spores of A. flavus NRRL 5906 irradiated in suspension (Treatment A) were more radiation sensitive than those treated at different humidity conditions (Treatment B). Whilst a sub lethal temperature of 53 °C applied for 5 min in combination with 0.75 kGy inactivated spores suspended in Tween solution, a dose of 4.0 kGy was required in combination with prior heating under high humidity conditions (]>85% R. H.) at 60 °C for 30 min to achieve the same purpose. These findings are in agreement with the report of NYEEGES-ROGEÜK (1975) whose test fungus was Pénicillium purpurogenum Stoll; Strain No. 787. 55 ODAMTTENe t al.:COMBINATIO NTREATMEN T ON A. FLAVÜS SPORES We recorded a higher killing effect when thespore s were heat-treated under high humidity (<^85% R. H-) conditions before irradiation than when low humidity ( This,pape r is based on IFFIT Beport No. 11 of the International Facility for Food Irradiation Technology, Wageningen, The Netherlands and ispublishe d with kind per mission of the Project Director and the Project Management Committee. The authors are deeply indebted to Mr. A.Boo m formerly of the Institute for Atomic Sciences in Agriculture ITAL, Wageningen, The Netherlands, forhi s excellent assistance. Wear e grateful to the Director of ITAL for the excellent facilities pro vided at the Institute. The financial support ofth e Dutch Government duringth e tenure of the International Atomic Energy Agency IAEA Fellowship of the first and second authors at IFFIT is gratefully acknowledged. Finally we wish to expressou r gratitude to the Director ofIFFI T Wageningen, The Netherlands Prof. Dr. J. Farkas for his keen interest and helpful criticisms ofth e manusoript. Our due thanks got o Dr. Swan Ko of the Department of Food Science, Agricultural University. Wageningen, the Netherlands for providing us with the culture ofA. flavus NRRL 5906 for the experiments. Literature ABALAKA, J. A. & ELEGBEDE, J. A. (1982): Aflatoxin distribution and total microbial counts ina nedibl e oil extracting plant. — Part I. Preliminary observations.Fd Chem. Toxicol., 20, 43-46. AUSTWICK, P.K .(1975) : Mycotoxins. Br. med. Bull., 31, 222-229. BRODRICK, H.T. , THOMAS, A. C, TONDER, A. J. V. & TERBLANCHE, J. C.(1977) : Combined heat and gamma irradiation treatments for the control of strawberry disease under market condition. Report. Atomic Energy Board, Pelindaba, Pretoria, Republic ofSout h Africa. 56 ODAMTTEN et al: COMBINATION TREATMENT ON A. FLAVÜS SPORES BRODRICK, H. T., THOMAS, A. C, VISSER, F. & BEYER, M. (1976): Studies on the use of amma radiation andho t water treatments for shelf-life extension of papayas. fn. Dis. Reptr., 60, 9. ENOMOTO, M. & SAITO, M. (1972): Carcinogens produced by fungi. Rev.Microbiol., 26A. 279-312. HARWIG, J., SCOTT, P. M., STOLTZ, D. R. & BLANCHJTELD, B. J. (1979): Toxins of molds from decaying tomato fruit. Appl. Environ. Microbiol., 38, 267-274. HAWKER, L. E., LINTON, A. H., FOLKES, B. E. & CARLIE, M. J. (1952): Introduction to the biology of microorganisms. St. Martin's Press, New York, p. 452. HUFF, W. E-, CHANG, C. F., WARREN, M. F. & HAMILTON, P. B. (1979): Ochratoxin A — induced iron deficiency anemia. Appl. Environ. Microbiol., 37, 601-604. Kiss, I. & CLARKE, D. I. (1969): Élesztök pusztulasanak vizsgälata besugârzas, hôkezelés es kombinàciojuk hatasàra. (A study of yeast destruction as affected by radiation treatment, heat andthei r combination.) Élelmiszertudomdny, 3, (2), 115-126. Kiss, I. & FAREAS, J. (1977). Thestorag e of wheat and corn of high moisture content as affected by ionizing radiation. Acta Alimentaria, 6, 193-214. KOLIK, M. M. & JUSTICE, O. L. (1966): Radiation botany. — réf.: GOLUMBIC, C. & DAVIS, D. F.(1966) : Radiation disinfection ofgrai n and seed. Fd Irradiai., No. STI/PUB/127 IAEA, Vienna. LANGERAK, D. IS. & CANET-PRADES, F. M. (1979): Tlie effect of the combined treatment on the inactivation of moulds in fruits and vegetables. Technical and preliminary research reports, No. 88.Foundatio n ITAL, Wageningen, The Netherlands. MOYHUDDIN, M. & SKOROPAD, W. P. (1975): Radiation heat synergism for inactivation of conidia of Aspergillus flavus. Radiai. Bot., 15, 185—189. NYERGES-ROGRÜN, E. (1975): Morphological characteristics of the spore head of Pénicil lium purpurogenum as affected by gamma irradiation. Acta Alimentaria, 4, 81-94. ODAMTTEN, G. T. (1979): Control of fungi causing deterioration of maize in storage by gamma irradiation. 11th Biennal Conference of the Ghana Science Association. Pa per No. 16. University of Science and Technology, Kumasi, Ghana. ODAMTTEN, G.T . (1982): The status ofreservatio n of maize: Prospects of the application of irradiation inth e extension ofth e shelf-life ofmaiz e inGhana . African Biological Network (ABN), Workshop on post-harvest losses and their prevention. Department of Zoology, University of Ghana, p. 19. ODAMTTEN, G.T. , APPIAH, V. & LANGERAK, D. IS. (1980): Studies on the technological feasibility of the application of dryo r moist heat to grains and grain products prior to gamma irradiation. Short communication. IFFIT Report, No. 10. Wage ningen, The Nerherlands. PADWAL-DESAI, S. R., GHANEKAR, A. S. & SREENTVASAN, A. (1976): Studies on Asper gillus flavus. — Part I. Factors influencing radiation resistance of non-germinating conidia. Environ. Bot., 16, 45-51. POISSON, J. & CAHAGNIER, B. (1974): La microflore du caryopse de blé: son evolution pendant le stockage en atmosphère renouvelée de l'irradiation. Quatrième jour nées de phytiatue et de phytopharmacie circumtnediterranéennes. Communications. ROY, K., CHATETH, M.S . & MUREWAR, P. N. (1972): Gamma radiation in the extension of shelf-life of apples infected with Aspergillus niger Van Tiegham. Phytopath. Z., 75, 31-37. SMITH, R. B. JR., GRUTIN, J. N. & HAMILTON, P. B. (1976): Survey of aflatoxicosis in farm animals. Appl. Environ. Microbiol., 31, 385-388. SOMMER, N. F., CREASY, M., ROMANI, R. J. & MAXTE, E. C. (1964): An oxygen-dependent post-irradiation restoration of Rhizopus stolonifer sporangiospores. Radiât. Res., 12, 21-28. SOMMER, N. F., FORTLAGE, R. J., BUCKLEY, P. M. & MAXIE, E. C. (1972): Comparative sensitivitv to gamma radiation ofconidia , myoelia and sclerotia of Botrytis cinerea. Radiât. Bot., 12, 99-103. TuiNSTRA, L. G. M. TH., VERHULSDONK, C. A. H., BBONSGEEST, J. M. & PAULSCH, W. E. (1975): Aflatoxin B, in compound feedstuffs containing citrus pulp. Procedure for Screening and semi-quantitative determination. Neth. J. agric. Sei., 23, 10-17. WEBB, B. D., THIERS, H. D. & RICHARDSON, L. R. (1969): Studies in feed spoilage. Inhibition of mould growth by gamma radiation. Appl. Microbiol., 1, 329-333. ZUBER, MS. S. & LILLEHOJ, E. B. (1979): Status of the aflatoxin problem in corn. J. Environ. Qual., 8, 1-11. 57 i i InternationalJournal of FoodMicrobiology, 00 (1986) JFM00114 Elsevier JFM 00114 4. Preliminary studies of the effects of heat and gamma irradiation on the production of aflatoxin Bx in static liquid culture, hyAspergillus flavus lin k NRRL 5906 G.T. Odamtten,1* V.Appiah 2 and D.I. Langerak l 1 InternationalFacility for FoodIrradiation Technology, c/o Slate Institutefor Quality Controlof AgriculturalProducts Bomsesteeg 45, 6708PD Wageningen, TheNetherlands and 2 GhanaAtomic Energy Commission, Department ofBiology, Food and Agriculture, P.O. Box SO, Legon/Accra, Ghana. (Received 31 May 1985; accepted 6 May 1986) Aflatoxin B, production by a strain of Aspergillusflams NRRL 5906 was examined in static liquid culture in maize meal broth (MMB) and maize meal broth supplemented with 256 glucose and 2% peptone (AMMB). Erlemmeyer flasks were inoculated with 1.0 ml aliquots of fungal spores which had been heat-treated (60° C for 30 min) under low humidity ( < 45% R.H. dry heat) or high humidity conditions ( > 85% R.H., moist heat) followed by gamma irradiation with either 0.0, 3.5 or 4.0 IcGy. AMMB supported 6-17 times more vegetative growth (depending on the heat and dose combination) than spores incubated in MMB alone. High inoculum size of control unheated spores Oog CFU/g, 6.9) yielded the least aflatoxin B, in flasks containing AMMB (8.2-19.3 fig/ml). A dose of 3.5 IcGy reduced by 3-2-3.8 log cycles the viable inoculum of control unheated spores, resulting in 2-5 fold increase in aflatoxin B, formed in flasks containing AMMB. Increasing the applied load to 4.0 kCy, however, reduced aflatoxin B, levels formed in AMMB to similar or lower levels than found in flasks inoculated with control unirradiated spores. Combination treatment of A.flams with dry heat and 3J kGy reduced the spore inoculum size by about 4 log cycles and yielded the highest amount (41.1 (ig/ml) of aflatoxin B, in AMMB. However, moist heat treatment of spores receiving the same dose (3. 5 kGy) reduced toxin level formed by 25%. Aflatoxin B, formation by A. flams spores incubated in AMMB was completely prevented by a combination treatment of moist heat and 4.0 kGy of gamma irradiation. This same treatment attenuated anatoxins Bj, G, andG 2 production which are formed with B, byA. flaous NRRL 5906. Spores raised in all flasks containing MMB did not form aflatoxin except when the medium MMB was autoclaved twice at 121°C for IS min. Keywords: Heat, moist and dry-. Gamma irradiation; Aflatoxin B,; Aspergillusflaous NRRL 5906 * Present address: Department of Botany, University of Ghana, P.O. Box 55, Legon/Accra, Ghana. Correspondence address: Department of Botany, University of Ghana, P.O. Box 55, Legon/Accra, Ghana. 0168-1605/86/J03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division) 58 Introduction Aflatoxins are secondary metabolites of the mould Aspergillusflavus an dAsper gillus parasiticus that produce a toxic reaction in humans and other mammals (Tulpule et al., 1964; Wogan,1973) . Experimentally, aflatoxins have been produced on numerous food products including cereals, grains, whole wheat and rye bread, cheese, meat, groundnut and nut products and fruit juice (Frank, 1968; Detroy et al., 1971;Zube r and Lillehoj, 1979), copra and cotton seed (Christensen and Schneider, 1950; Ashworth et aL, 1971; Simpson et al.1973) . A practical approach to protecting food against aflatoxin contamination is by preventing the'fung i from forming mycotoxin. Gamma irradiation of food for preservation has been proposed and studied for the past twenty-five years as an alternative means of food preservation. This has led to a series of investigations on the safety of food in relation to aflatoxin production by residual mycoflora after gamma irradiation treatment. Studies by Jemmali and Guilbot (1970),Schindle r et al.(1980 ) and Pnyadarshini and Tulpule (1976) indicated an increase in aflatoxin production after gamma irradiation. Several closely related papers by Applegate and Chipley (1974a,b ) also presented evidence of increased aflatoxin production by A. flavus after gamma irradiation. A novel approach to control of fungal contamination of stored products is by combination treatment of heat and irradiation. This enables one to use a much lower dose in combination with mild heat treatment to achieve very high level inactivation of fungal spores. Radiation treatment combined with heat has been found to be more effective than radiation treatment alone (e.g. Roy et aL, 1972; Langerak and Canet-Prades, 1979; Odamtten et al., 1985, etc.). Our objective in these investigations was to provide data on the effect of the combination treatment of heat (60° C for 30 min) applied under low ambient humidity (< 45% R.H.) or moist heat applied under high ambient humidity (> 85% R.H.)an d gammairradiatio n (0.0,3. 5 and 4.0kGy )o nproductio n of aflatoxin B, in submerged static culture medium prepared from blended maizegrains . Materials and Methods Treatment ofdry spores Fungal spores of A. flavus Link NRRL 5906 were harvested dry from a previously sterilized (121° C for 15 min) Dutch variety yellow maize grains (200 g blended maizei n 500m lErlenmeye r flasks). The seven days old spores incubated at 28°C were separated from themaiz emediu m by sievingwit h aplasti c seivean d 0.1 g spores transferred into sterils Petric dishes (90 mm diameter). The spores wereheate d at 60° C for 30min .i n achambe r that enabled usexpos e the spores to either dry ,(545 % R.H.) or moist (> 85% R.H.) heating conditions 59 (Odamtten et al., 1980). Fungal spores that were maintained at 20° C during the prescribed humidity treatments served as controls. The lids of the Petri dishes were eccentrically placed on the bottom to allow maximum exposure of the spores to the prescribed treatment. After irradiation (see below) in the dry state, the spores were washed with 30 ml of sterile Tween solution (2 g Tween-80, 11 ,distille d water) to get the spores into suspension. Aliquots of 1m l from the stock suspension of each treatment combination were used in inoculating 250 ml Erlenmeyer flasks. Each flask contained 25 ml of either maize meal broth MMB (200 g maize; blended, strained and supernatant made up to 11 with distilled water) or ammended maize meal broth AMMB (200 gmaize ,blended , strained and supernatant made up to 1 1 with distilled water and added 20 g peptone and 20 g glucose). There were five replicates for each treatment combination and theflask s wereincubate d in darkness at28°C for 4days . Radiation treatment Irradiation, as a rule, was carried out not more than 30 min. after the heat treatment (in order not to lose the synergistic effect of the combination treatment) in the Pilot Plant for Food Irradiation at the International Facility for Food Irradiation Technology (IFFIT) Wageningen, the Netherlands. The dose rate was 5.5kra d min-1 and theabsorbe d dosewa schecke d by clear Perspex Dosimetry.W e applied doses of 0, 3.5 and 4.0 kGy of gamma radiation to the spores. Assessment ofinitial survivors intreated spores Aliquots of 1.0 ml of the stock suspension of the treated spores were diluted up to 1:105 and plated out on maize meal agar, MMA (200 g maize grains, blended, strained and made up to 1 litre with distilled water, 20 g agar was added) and ammended maize meal agar, AMMA (200 g maize grains, blended strained and made up to 11 with distilled water, 20 agar, 20 g glucose, 20 g peptone) using the serial decimal dilution technique. After incubation at 28°C for 3 days, the log survival of the treated spores was determined. Extraction ofaflatoxin from the culturefiltrates A modification of the method of Ko, (1974) was followed. With a volumetric pipette, 50 ml chloroform was added to each of the flasks containing the culture filtrate, from which mycelium had been harvested. The extraction of aflatoxin into the chloroform layer was promoted by shaking (100 rev min-1) for 30 min. In a Gallenkamp model orbital shaker. The chloroform extract phase was simply sep arated from the rest of the medium by mean of a separating funnel. After this, the chloroform extract was evaporated to dryness in a Rotavapor vacuum rotation evaporator (Buchi, Switzerland). The residue was redissolved in 5.0 ml of chloro form and the contents of the five replicate flasks were pooled and kept in 250 ml Erlenmeyer flasks covered with cotton wool and aluminium foil at 2°C for one 60 night ormor ebecaus e determination of aflatoxin content of the filtrate by thin layer chromatography could not be completed on the same day. Quantitativeestimation of aflatoxin B} in culture filtrates Samples of the extracted aflatoxin from the culture filtrate of treated spores were spotted on MN-KIESEGEL G-HR thin layer chromatography (Machery, Nage and Company, Duran, F.R.G.) using a Desaga Microdoser (Heidelberg, F.R.G.) with acetone : chloroform (1:9) and developing solvent. The intensity of the fluorescing spots was measured with a Vitraton TLD-100 densito-meter (Vitraton Scientific Instruments, Dieren, The Netherlands) equipped with an integrator recorder. The Amount of aflatoxin Bj was calculated by comparison of the integration units between the peak area of spots óf unknown samples and those of known standard spots on the same plate. Assessment of dry weightof mycelium andpH ofcultures After incubating the cultures for 4 days at2 8° C in darkness, growth in the liquid medium was assessed by estimating the dry weight of the harvested mycelium. Mycelium collected on a previously weighed and dried filter paper was dried in an oven at 75° C for 24 h (until constant weight was attained). The filters paper carrying the dried mycelium were then re-weighed after cooling in a desiccator. The dry weight of the mycelium was estimated by difference. The pH of each of the culture filtration was determined by a Marius pH Meter Type 52A. Results Effect of thecombination treatmenton vegetativegrowth andsporulation Vegetative growth of A. flavus in flask containing AMMB (Fig. 2) was about 6-17 times greater than in MMB (Fig. 1), depending on the heat and dose combination (Figs. 1 and 2). In both types of media the lowest dry weight of mycelium was recorded in flasks containing spores treated with a combination of moist heat (> 85%R.H. ) and4. 0 kGy. Generally, the final pH of culture filtrates of A. flavus incubated in MMB was lower than the initial pH of 5.5, with the attendant restriction of both growth and toxin formation by the fungus. In flasks containing AMMB however, the final pH of the filtrate shifted from initial pH 5.3-5.5 to become less acid (6.3-6.8) in flasks containing control spores (unheated 20°C , and heat-treated 60° C under both low, < 45% R.H. and high, > 85% R.H. humidity conditions). Culture filtrates from control spores exposed to 4.0 kGy became more acidic (pH 3.9-4.1) except with moist heat-treated spores, where there was only a slight shift from the initial pH of 5.3-5.6. The lowest mycelial dryweigh t was recorded in the latter flasks. The spore inoculum used in inoculating the flasks containing either MMB or AMMB was largest in the controls. These inoculum sizes shown in Table I were 61 Maize meal broth (MMB) 10 4 days 8 20» c. 20» C. <45"/. R.H. • 6 S ~« 4 's o li 12 60» C, 60» C. <45«/. R.H. >85#/. R.H. w V,?A tamm 3-5 4-0 0 3-5 4-0 dose( kGy ) Fig. 1. Comparative mycelial dry weight yield of cultures of A. floous incubated in maize meal broth (MMB) for 4 days at 28°C . subsequently reduced by 3.3-5.8 log cyclesb y4. 0 kGy of gamma irradiation. Moist or dry heating in combination with 4.0 kGy also reduced the inoculum size by 3.4 and 5.6lo gcycle srespectivel y (Table I).I nflasks containin g largeinocul a (unheated and unirradiated controls), vegetative growth predominated over sporulation and cultures were whitish in colour as compared to the abundant green to dark green spores formed in flasks inoculated with combined-treated spores. Aflatoxinformation As seen from Table I, no aflatoxin was formed in MMB irrespective of the treatment given to the spores. Sporulation was generally poor in these flasks. The toxin was however formed freely into the medium when MMB was sterilised twice at 121°C for 15 min (results not included). Table I shows that maize meal broth supplemented with 2%glucos e and2% peptone (AMMB) supported theformatio n of low(8. 2 jug/ml) aflatoxin B, in flasks 62 Amended maize meal broth (AMMB) 4 days 20» C, >85*/. R.H. 3-5 4-0 0 3-5 40 dose( kGy) Fig. 2. Comparative mycelial dry weight yield of cultures of A. flaous incubated is amended maize meal broth (AMMB) for 4 days at 28°C. inoculated with unirradiated A. flaous spores heat treated under dry humidity conditions (<45% R.H.). Control (20°C, unirradiated) spores heat-treated under high humidity (>85%R.H.) for 30 min formed 19.3 fig/raL aflatoxin B, into AMMB. Dry and moist heat-treated spores under low (<45% R.H.) and high (> 85% R.H.) ambient humidity conditions formed higher levels of aflatoxin Bj (28.8 Mg/ird and 40.0 Mg/ml respectively) than the unheated spores and the resulting cultures were green to white in some flasks. Dark green sores banded the periphery of the mycelial mat. Irradiation alone with 3.5 kGy increased by 4-5 3-fold the aflatoxin Bj formation by the control unheated spores treated under low (< 45% R.H.) humidity conditions. The parallel treatment under high humidity conditions (>85% R.H.) doubled the toxin level. Dry heat-treated spores in combination with 3.5 kGy formed aflatoxin B, about one and a half times that formed by heating alone. The only exception was in flasks containing moist 63 + + + + + + + + ++ + + + + + + + I++ + + + + + -û S 3 s i 3 + + 2 S + + + 0 C/5 + + I++ + + + + + Ma 3 a «s o fi CI "8 «uu s s U U s u >• s s Ë ë 00 'S" \ 00 00 „ =0 00 loi o X •* •* .; J< Jl U S< o 3 I- t. £ « « ^ « s o Û a OC. e e ? a û O '2 S s z s s s e 13 S s fi 'S u S >• § u CS „ »0 00 00 "S 2 •- "ï -ä •S — -* -* es J t. ha O £ Q û < ? O û + + + o » n P; ON NO r^ — ON r- ae ge M O* m **" «0 ri IM NO M r>> «•i •" ö e '3 o a p-j O ON °! **; 1 O — r« o C NO' ei s s s U ^ eft Il ' MM » **• v e 3 ï o g.= 3 s z •= 64 II u ö Ml S S » 5 « 2a ï E u a 3 •S « Ü ifl 2 oi heat-treated spores (60°C for 30mi n at > 85% R.H.)wher e toxin level was reduced by 25%. Increasing the applied dose to 4.0 kGy in all cases decreased level of toxin formed to concentrations nearly the same or lower than that obtained in the unirradiated samples (Table I).Th e best treatment combination topreven t aflatoxin formation wasmois t heat (> 85% R.H.)a t 60° C for 30mi n in combination with4. 0 kGy (Table I). Wherever aflatoxin Bj was formed, Bj, G, and G2wer e formed as well. The treatment that prevented aflatoxin Bj formation (moist heat, 60° C for 30 min, > 85% R.H.) in combination with 4.0 kGy attenuated the formation of aflatoxins 63, Gj and G2 aswell . Discussion The abundant vegetative growth of A. flams NRRL 5906 on AMMB as compared to MMB could be attributed to the addition of 2% glucose and 2% peptone to the basal medium. Glucose appears not only to serve as the carbon source for aflatoxin synthesis but also to play a role in regulation the induction of the enzymic pathway responsible for the biosynthesis of the mycotoxin (Abdoullahi and Buchanan, 1981). Mateles and Adyes (1965) demonstrated that a correlation exists between increasing concentration of glucose from 1-4% and the increase in amount of aflatoxin Bl formed from 25 mg/1 to 58 mg/1. Peptone is an excellent source of amino acids that can be catabolised to pyruvate or acetyl-coA for use by the fungus. However, good vegetative growth by A. flavus is not always with abundant aflatoxin formation. The heavy inoculum used in the controls resulted in vegetative growth predominating over sporulation i.e. the cultures were within, very low aflatoxin Bjwa sforme d and thecultur e filtrate wasclea r to slightly reddish. Culture filtrates of MMB, where aflatoxin Bj was not produced were also clear and sporulation was sparse to nil. Detroy et al. (1971) showed that aflatoxin synthesis occurs during the period of intense sporulation of the fungus and that good aflatoxin production correlates with yellowing of mycelium and medium. In flasks which failed to yield aflatoxin, the mycelium remained white and the medium was either clear or slightly reddish (Mateles and Adye, 1965). This is confirmed by our findings. Reduction in inoculum size by 3.0-4.0;lo g cycles after gamma irradiation (3.5 kGy) followed was by abundant sporulation and enhanced aflatoxin B, formation in flasks containing AMMB after 4 days of incubation, indicating that the forma tion of aflatoxin B: is inversely related to the spore concentration. Aflatoxin B, formation was completely inhibited by a combination treatment of moist heat (60° C for 30 min) applied under high ambient humidity (> 85% R.H.) conditions and 4.0 kGy of gamma irradiation. This treatment could also prevent production of aflatoxins Bj, G, and G2 byA. flavus NRRL 5906. Recent findings by Odamtten et al. (1985) confirm that in addition to the temperature and duration of treatment, humidity strikingly affects the lethality of heat applied. 65 Flasks containing spores incubated in MMB failed to form the toxin irrespective of the type of treatment the spores received. However, after double sterilization of the medium MMB, aflatoxin B-,wa s formed in detectable quantities in all cultures but that spores treated with moist heat (60° C for 30min , under humid > 85%R.H . conditions) in combination with 4.0 kGy. Lillehoj et al. (1974) observed the production of aflatoxin byA. fleams o n various autoclaved com media, whole corn being an excellent substrate, whilst Detroy et al. (1971) demonstrated maximum -1 yield of aflatoxin Bx (700-900 mgkg ) onautoclave d wheat,rice, cotto n andcorn . Several environmental, nutritional and genetic factors considerably influence the type and amount of aflatoxins produced by moulds of the genus Aspergillus (Maggon et al., 1977;Zube r and Lillehoj, 1979). For A.fleams an d A. parasiticus, the optimal nutritional availability of certain trace metals in a crucial factor (Maggon et aL 1977).Th estimulator y effect of zinc on aflatoxin production iswel l documented (Lee et al., 1966; Maggon et aL, 1977; Lillehoj et al., 1974; Guptaan d Venkitasubramanian, 1975; Obidoa andNdubuisi , 1981). Trace elements in maize occur predominantly in theger m fraction (Garcia et al., 1972). Phytic acid in the germ strongly binds other elements in addition to zinc (O'dell and Savage, 1960). Theboun d elements are not available biologically.Th e failure of A. flavus NRRL 5906 spores to form aflatoxin B, on MMA could probably be attributed to the binding action of phytic acid in maize with zinc. Although wedi d not estimate theamoun t of Zn+ present in theautoclave dMMB , we surmise that on autoclaving of MMB,zin c is released but is not enough to permit thesynthesi s of detectable amounts of aflatoxin Bv However by autoclaving twice, phytic acid ispresumabl y destroyed byth eexcessiv e heat releasing sufficient zinc for aflatoxin synthesis.I n future studies,othe r local maizevarietie s willb euse d as cultural media to compare them with the results reported in the present paper. Wehav e shown here that combination treatment with moist hotai r(60 °C for3 0 min) administered at high humidity (> 85% R.H.) in combination with 4.0kG y prevented not only aflatoxin B, production but also attenuated Acknowledgements We thank Prof. E.H. Kampelmacher, Head, Laboratory for Food Microbiology and Hygiene,Agricultura l University,Wageninge n forpermissio n tous eth eVitatro n TLD-100 Densitometer. Thefinancia l assistance of the Dutch Government for this project isgratefull y acknowledged. Finally theauthor s aregratefu l toProf .J . Farkas for hishelpfu l criticism of themanuscript . References Abdoullahi, A. and RA. Buchanan, 1981. Regulation of aflatoxin biosynthesis: Induction of aflatoxin production by various carbohydrates. J. Food Sei. 42(2), 633-635. Applegate, KA. andJ.R . Chipley, 1973a. Increased aflatoxin production byAspergillus flavus via cobalt irradiation. Poultry Sei. 52,1492-1496. 66 Applegate, K.L. and J.R. Chipley, 1973b. Increased aflatoxin G, production by Aspergillus flauus via gamma irradiation. Mycologia 65,1266-1273. Applegale, K.L. and J.R. Chipley, 1974. Daily variation in the production of aflatoxins by Aspergillus flavus MRRL 3145 following exposure to Co60 irradiation. J. Appl. Bacteriol. 37, 359-372. Applegate, K..L. and JJR- Chipley, 1974. Effects of Co60 gamma irradiation of aflatoxin B, and Bj production by Aspergillus flavus Mycologia 66, 436-445. Ashworth, LJ., J.L. McMeans, JX. Pyle, CM. Brown, J.W. Osgood and R.E Ponton, 1968. Aflatoxin in cotton seed. Influence of weathering on toxin content of seeds and a method for mechanical sorting. Phytopathology 58,102. Ashworth, LJ., J.L. McMeans, B.R. Houston, £. Whitten and CM. Brown, 1971. Mycoflora and free fatty acids in California cotton seed during 1967-68 J. Am. Oil Chem. Soc. 48,129-133. •Bassii , O. and AA. Adekunle, 1972. Production of aflatoxin B, from defined natural cultures of Aspergillus flavus Link. Mycopathol. Mycol. Appl. 46, 241-246. Brodrick, H.T., A.C Thomas, AJ.V. Tonder and J.C Terblanche, 1977. Combined heat and gamma irradiation treatments for the control of strawberry disease under marked conditions. Atomic Energy Board, Pelindaba, Pretoria South Africa. Feb. 1977. Christensen, JJ . and C.L. Schneider, 1950. 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Gupta and TA. Venkitasubramanian, 1977. Biosynthesis of aflatoxins. Bacteriol. Rev. 41, 822-855. Mateles, R.I. and J.C Adye, 1965. Production of aflatoxins in submerged culture. Appl. Microbiol. 13(2), 2008-211. Obidoa, O. and I.E. Ndubuisi, 1981. The role of sine in the aflatoxigenic potential of Aspergillus flavus NRRL 3251 on foddstuffs. Mycopathologia 74, 3-6. Odamtien, G.T., V. Appiah and D.Is. Langerak, 1980. Short communication: Studies on-the technologi cal feasibility of the application of dry or moist heat to grains and grain products prior to gamma irradiation. International Facility for Food Irradiation IFFTTRepor t No. 10.15 pp. Wageningen. The Netherlands. Odamtten, G.T., V. Appiah and D.Is. Langerak, 1985. In vitro studies on the effect of the combination treatment of heat and irradiation on the spores of Aspergillus flavus Link. NRRL 5906. Acta Aliment. Vol. 14(2) 139-150. 67 O'dell, B.I. and J.E Savage, 1960. Effect of phytic acid on zinc availability Proc. Soc. Exp. Biol. Med. 103, 304-305. Pnyadarshini, E and P.G. Tulpule 1976. Aflatoxin production on irradiated foods. Fd. Cosmet. Toxicol. 14, 293-295. Roy, K., M.S. Chateth and P.M. Murewar, 1972. Gamma irradiation in the extension of shelf-life of apples infected with Aspergillus niger van Tiegham. PhytopathoL Z. 75, 31-37. Schindler, A.F., A.N. Abadie, and R.E Simpson, 1980. Enhanced aflatoxin production by Aspergillus flavus and Aspergillus parasiticus after gamma irradiation of spore inoculum. J. Food Protect. 43, 7-9. Simpson, R.E, E Marion, P.B. Marsh, G.V. Merola, RJ. Ferretti and EC. Filsinger, 1973. Fungi that infect cotton seeds before harvest. Appl. Microbiol. 46(6), 608-613. Tulpule, P.G., T.G., T.V. Madhavan and C Gopalan, 1964. Effect of feeding aflatoxins to young monkeys. Lancet i, 962. Wogan, G.N., 1973. Aflatoxin carcinogenesis. In Methods in Cancer Research. Edited by H. Busch. Academic Press. New York P. 309. Zuber, M.S. and EB. Lillehqj, 1979. Status of the aflatoxin problem in corn. J. Environ. Quality. 8,1-11. 68 5. INFLUENCE OF INOCULUMSIZ E OF ASPERGILLUS FLAWS LINK ONTH E PRODUCTION OFAFLATOXI N Bj INMAIZ E MEDIUMBEFO BEFORR EAN DAFTE R EXPOSURE TO COMBINATION TREATMENTO F HEATAN DGAMM A RADIATION G.T. Odamtten, V.Appiah ,an d D.I.Langerak . Abstract Thispape rexamine dth einfluenc eo finoculu msiz eo f Aspergillus flavus LinkNRR L 5906o ngrowt han dproductio n ofaflatoxi nB. .i nstati cculture , douHe-autoclavedmaiz emea lbrot h (MMB)an dmaiz emea lbrot hammende dwit h 21pepton ean d 2%glucos e (AMMB).Dr yspore so fth e funguswer eheat-treate d ina heat-treatmen t chamber thatenable du st oappl yprecisel y6 0 C for3 0 min. ata nambien thumidit y ofeithe r<45 lR.H . (dryheat )o r >85SR.H . (moist heat)durin g theheatin gperiod .Control swer emaintaine da t2 0° Cdurin gth e humidity treatment andsample swer e irradiatedwithi n 30min .afte rth etreat ment,wit h 0.0, 3.5 or4. 0 KGyo fgamm airradiation . Vegetative growtho funtreate dcontrol s incultur ewa s independento fin oculumsiz ewit hwhic h flaskswer e inoculated and reduction ininoculu msiz e '„ • by serial dilution (3-4lo gcycles )resulte d in 3-12 fold increase inaflatox inproduction .Mois thea treduce dvegetativ e growthb y two-thirds andsubse quent 3-4 logcycle sreductio no f inoculumsiz e impaired toxin-formationpoten tialo fmos tsurvivin g fractiono fhea ttreate dspores .Irradiatio nwit h 3.5 KGYreduce d inoculumsiz eb y 3-5 logcycle s andresulte d inenhance d aflatoxin B.productio nbu t thiseffec twa s similart owha texiste dwit hseria ldilutio n ofcontro lspores .Irradiatio nan dit s combinationwit hhea tma yno thav ean y directeffec to naflatoxi nB. .productio nbu t on thesiz eo finoculu mi nrela tion toviabl e spores.Maximu maccumulatio no faflatoxi n B.wa s generally in4 days inAMM Ban d 8day si nMMB .A combinationo fmois thea t (60° Cfo r 30min . under >85lR.H.) , 4.0 KGyo fgamm a irradiation attenuated aflatoxinsproductio n in flasks containingbot hMff ian dAMMB . Keywords : Inoculumsize ; Aspergillus flavus LinkNRR L 5906;vegetativ e growth andaflatoxi nB ,formation ;maizm emea lbroth ;combinatio n treatment ofhea tan dgamm a irradiation 69 5.1 Introduction Amongth e fieldan dstorag emycoflor aassociate dwit hcerea l grainpro ducts,member s ofth egenu s Aspergillus areth emos tpredominan t followedb y Pénicillium (Christensen andKaufmann ,1969 ;Nfoubashe re tal. , 1972;Odamtten , 1981).Tw oeconomicall y important aflatoxinproducin gstrain s Aspergillus fla- vus and A. parasiticus areubiquitou s associatedwit hothe rwid evariet yo f storedcommoditie s (Dieneran dDavies , 1977). Over thepas t twodecades ,considerabl e importanceha sbee nattache dt o thepresenc eo faflatoxin s in foodan dfeed sbecaus eo fthei rcarcinogenic , mutagenic andteratogeni cnatur e (Goldblatt,1969 ;Tawe se t al., 1972;Enomot o andSaito ,1972 ;Austwick , 1975;Heathcoat ean dHibbert , 1978). The factors influencing aflatoxinproductio nhav ebee nwidel yevaluate di nbot hnatura l andsyntheti c substrates (Sharmae t al., 1980).Aflatoxi nproductio n isknow n todepen dupo n thestrai no fth eorganis man dfactor s sucha scompositio no f themediu m (Mateles andAdvel , 1965;Davi se t al., 1966;Schroeder ,1966 ; Detroye t al., 1971;Abdoullah i andBuchanan , 1981), temperature (Schroeder andHein ,1967 ,1968 ;Eldridge ,1968 ;Stult zan dKrumperman , 1976), moisture level (Lopezan dChristensen , 1967;Tren k andHartman , 1970;Diene ran dDavis , 1977; Beheree tal. , 1978), oxygen tensionan d timeo fincubatio n (Landerse t al., 1967;Maggo ne t al., 1977). Syntheticmedi ahav e routinely supportedminima laflatoxi nproductio n (1-60m go fB 1 perk go fmedium )wherea smaximu myield s (700-900m go fB 1 per kg)occu ro nsuc hcommoditie sa s autoclavedwheat ,rice ,cotto nsee dan d com (Detroye t al., 1971).Hesseltin e andco-worker s (1966)i n theirrevie wo faf latoxinformatio nb y A. flavus regardedmaiz ea sa satisfactor y substrate for aflatoxinproduction . Interest incontro lo fmicrobia lspoilag e offoo db y gammaradiation ,le dt oa serie so fstudie so fth eeffec to firradiatio no n aflatoxinproductio npotentia lo f A. flavus inirradiate d foods (Jemmalian d Guilbot, 1969b;1970a,b ;Applegat e andChipley , 1974a,b;Schindle re t al., 1968)etc .Som eo fthes eworker s(e.g .Jemmal i andGuilbot ,1969b ,1970a,b ) reportedo fa n increase inaflatoxi nproductio nafte rgamm airradiatio no f A. flavus spores,whils t reporto fApplegat ean dChiple y (1973)state dtha tir radiationdi dno t induceproductio no faflatoxi no neithe rwhea to rsyntheti c medium.Sharm ae t al. (1980)showe d thatfo r A. parasiticus NRRL 3145,th e reduction inth enumbe ro fspore sb y 4-5 logcycle seithe rb y serial dilution orb ygamm airradiatio ncause da two-foldincreas e inth eaflatoxi nproduction . Itwa s apparent thatth e irradiationeffec twa ssimila rt o thatobtaine db y 70 dilution ofth e inoculum (Sharmae t al. (1980).However ,ther e ishardl y any information inth eliteratur e relating toth eeffec t ofinoculu m sizeo f A. flavus onaflatoxi nproductio n in liquidculture . A novel approach tocontro l fungal contamination ofstore dmaiz epro ducti sb y combination treatment ofmois thea t (administered underhigh , >85% R.H. conditions)an dgamm a irradiation,thi senable s one tous e amuc h lower dose incombinatio nwit hmil dhea t treatment toachiev e aver yhig h level of deactivationo f fungalspore s (Odamttene t al., 1980a).Th epurpos e ofth e presentstud y is toinvestigat e theinfluenc e ofvaryin g spore inoculum size on theproductio n ofaflatoxi n B..b y A. flavus NRRL 5906 intomaiz emea lme diumprio r toheatin g ofspores ,afte rheatin g andit s combinationwit h gamma irradiation.Thi s information B..productio nb y irradiated fungal spores of A. flavus aphenomeno n reportedb y some earlierworker s (e.g.Applegat e and Chipley, 1974).Thi sstud y canals oprovid epreliminar y datao nwhethe r cereal grains treatedb yhea tan d gammaradiatio nmigh t leadt oenhance d aflatoxinB. . formationb y A. flavus ornot . 5.2 Materials and methods The stock culture of Aspergillus flavus NRRL 5906wa sprovide db yK oSwa n Djien, Laboratory ofFoo dMicrobiolog y andHygiene ,Foo d ScienceDepartment , Agricultural University,Th eNetherlands .Th e funguswa s maintained onmaiz e meal agar (200g blende dmaize ,straine d andmad eu p to 1litr ewit h distilled water,2 0g plai n agar added). 5.2.1 Treatment ofdr y spores of A. flavus The sporeswer eharveste d dry fromth esterilize d (121 °Cfo r2 0min. ) crackedmaiz emediu m (200g moistene d crackedmaiz e in50 0m lErlenmeye r flask). The seven daysol d sporeswer e separated fromth emaiz emediu mb y sievingwit h aplasti c meshan d 0.1 gweigh to fspore s transferred intosteril epetr i dishes. The spore sampleswer e unheated (20°C )o rhea t treated at6 0° Cfo r3 0 min. ina heat-treatmen t chamberdescribe db yOdamtte n et al. (1980b)tha t enabledu s toexpos e thespores ,durin gth ehea t treatment,t oeithe r alo w humidity (<45lR.H. ) ambient conditions orhig hhumidit y (>85lR.H. ) ambient conditions.Th e lidso fth epetr i disheswer e excentricallyplace do nth ebot tom toallo wmaximu minflu xo fmois t ordr yheat .Th eheated-treate dspore s were irradiatedwithi n 30min. afte rth ehea t treatment inorde rno t tolos e 71 the synergistic effect. After irradiation in the dry state, the spores were washed into suspension using 30 ml sterile 2% Tween-80 solution. An aliquot of 1.0 ml from the stock solution and the serially diluted stock 1:106, suspension of spores (of known concentration) from each treatment combination was used in inoculating 250 ml Erlenmeyer flasks containing 25 ml of liquid medium, either maize grains, strained and made up to 1 litre with distilled water and steri lized twice at 121 °C for 20 min.) or ammended maize meal broth, AMffi (200 g blended maize grains, strained and made up to 1 litre with distilled water, 20 g peptone, 20 g glucose were added). When solid medium was required 20 g agar was added to 1 litre liquid medium. The pH of the cultures was determined by Marius pH meter type 52A. 5.2.2 Radiation treatment The irradiation was carried out not more than 30 min. after the heat treatment (to ensure that the synergistic effect was not lost). The dose rate was 5.5 Krad/min. (0.06 KGy min" ) and the absorbed dose was checked by clear perspex Dosimeters (fWP, 1.0 mm). Doses applied were 0.0, 3.5, and 4.0 KGy respectively. 5.2.3 Assessment of initial survivors and effect of serial dilution of stock suspension on aflatoxin B, formation Dilutions were carried out of 1.0 ml of stock suspension of the untreated (control) and heat-treated spores (under both low, <45l R.H. and high, >85% R.H. ambient humidity conditions) up to 1:10s and were plated out on maize meal agar (WA) and ammended maize meal agar (ANMA). The number of colonies were counted after 3 days incubation at 28 C from which the colony forming units per gm c.f.u.g" were calculated. Erlenmeyer flasks (250 ml) containing 25 ml ANMB were inoculated with 1.0 ml spore suspension of each dilution level up to 1:10s stock suspension. There were five replicates for each dilution level. Dry weight of mycelium and amount of aflatoxin B1 released by the myce- lia into the flasks were determined, as detailed below, after 4 days incubation at 28 °C. 5.2.4 Effect of varying inoculum size of control, heated and combined treated spores onaflatoxi n Bj production This was essentially similar to the above experiment but differed in that the actual viable inoculum size of spores of A. flavua NRRL 5906 before heat 72 20-C «orSOmln, « 4S*R.H. tO*C forWmln, * S51.(Ut. so A / / 2-0x10' / I1 / • 40 / •"7 1 / / WrtO7/ /* 20 H / 22-i 1 1 . / I* ° S ' 4 1 1 1 ! 1 1 1 1 1 i -À 1 i sr SO*C lor JOmi nj .»46VR.H. 80*C tor lOrnln, .»>(•% R:H. 1 so / / I / «ML • «OH 1 14»K) 6 / - ft>. 40 • *-y—r" "^S-N. /; .^9. SKtO » d.U.«-' / i 20 '\ \ \ 0 -1 .3 -5 -7 0 -1 -J 10 10 10 10 10 10 10 K swa DlUJTIO N FACTOR Fig. 1.Relationshi p between inoculum size (by serial dilution), vegetative growth and aflatoxin B.productio n by A. flavus NRRL 5906 spores incubated inAMM B for k days at 28 C. 73 treatmentwer e determinedb ybot h thedilutio npou rplat e technique andals o confirmedb y sporedensit yestimatio nusin ga Hawksle yCristalit e B.S.74 8 Haemacytometer (Hawksleyan dSon sLimited ,Lancin gSussex ,England) .Viabl e spores after combination treatmento fhea tan dgamm a irradiationwa sascer tainedb y thespor e countafte r 3day s incubationa t2 8 Co nAMvl Aan d WA and c.f.u.g" samplewer e thereaftercalculated . Erlenmeyerflask s containingeithe r2 5m lo fdouble-autoclave dMM Bo r ANMBwer e inoculatedwit h 1.0m l sporesuspensio no fth epre-determine dinoc ulumlevel so fspor e concentrations (control,heat-treate d and combinedtreat mento fhea t andgamm aradiation) .Th e conicalflask swer e incubateda t 28° C for4 an d 8day s respectively afterwhic h thedr yweight s ofmyceliu man dto talamoun to faflatoxi n B,produce dwer e determined. Flasks containingmaiz e mealbroth ,MM Bwer e autoclavedtwic e to induce formationo fhighe r levels of toxin (Odamttene t al., 1980c).Ther ewer e sevenreplicate s foreac h treatment and inoculum sizeused . 5.2.5 Assessmento fdr yweight s ofculture s Afterth epre-determine d incubationperiod s of4 and 8day s respectively, growthi nth e liquidmediu mwa s assessedb yestimatin g thedr yweigh t ofth e harvestedmycelium .Myceliu mcollecte do na previousl yweighe d anddrie d Ederol filterpape rwa s drieda t 75 C for2 4h and thenreweighe dafte rcool ingi na desiccator .Th e culturefiltrate swer e retained forp H determination andextractio no faflatoxin . 5.2.6 Extraction ofaflatoxin s Weuse da modificatio n ofth emetho do fK O (1974).T oestimat e theamoun t of toxin releasedb ymyceliu mint ocultur e filtrates,a naliquo to f5 0m lchlo roformwa s added toeac ho f four flasks (fromwhic hmyceliu mha dbee nhar vested). Th e aflatoxinextractio nfro mth echlorofor m layerwa senhance db y shakinga t 100rev .min ~ ina Gallenkam p shaker for 30min .Th e chloroform extractphas ewa s simplyseparate db ymean so fa separatin g funnel.Th echlo roformextrac t containing theaflatoxin swa sevaporate d todrynes susin g Rotavapor1- ' vacuumrotatio nevaporato r (Buchi,Switzerland) .Th edrie dresi dueswer e redissolved in5. 0m l chloroforman dkep t in 250m lErlenmeye r flaskscovere dwit h cotton,woo lan d aluminium foilan dthe nkep ta t 2° C for onenigh to rmor esinc eaflatoxin s contentdeterminatio no f thecultur e fil trateb yThi nLaye rChromatograph y (TLC)coul dno tb ecomplete do nth esam eday . 74 22 - AMMB unirradiated 4doys 10u 101 102 103 104 10s 10° 10' spore cone.(colony formingunits ,cfu ) Fig.2 .Effec to fspor edensit yo f Aspergillus flavus andambien t humidity duringhea ttreatmen to nth eproductio no f aflatoxinB .i ncultur eflask scontainin gammende dmaiz e mealbroth ,AMM Bincubate d at2 8 C for4 days .L - lo w humidity,<45 %R.H. ;H - hig hhumidity ,_>85 %R.H . (Noteth einvers erelationshi pbetwee nhig hspor e densityan dlo waflatoxi nB .production. ) 75 Myceliumo ftoxi nproducin g fungi usually retain someo fth e toxin insideth e cytoplasmo fth emyceliu m dependingo nincubatio n period andothe r environment alconditions .T oestimat eth etota l amounto faflatoxi n formedb yA . flavus themyceliu m andcultur e filtratei nthre e flaskswer ehomogenise di na nMS E Blendoran d thenwashe dwit h7 5m lo fchloroform .Th eflask s containingth e homogenised mycelium, culture filtrate andchlorofor mwer e shakena t10 0 rev. min for3 0min .an d thensubsequentl y treatedi nth esam ewa ya sdescribe d abovet oobtai n dried residueo ffiltrat e anddissolve di n5. 0m lchloroform . 5.2.7 Quantitative determinationo flevel s aflatoxin formed Quantitative estimationo faflatoxi n B..wa scarrie d outb yusin g Thin LayerChromatographic , TLCTechnique . Sampleswer e spottedonMJ-Kieselge lG-H R plates (Machery,Nagel-SCo .Dure nW .Germany )usin ga Desag aMicrodoser . Acetone:chloroform (1:9)wa s useda sdevelopin g solvent.Th eintensit yo fth e fluorescing spots was measuredwit ha vitatro nTLD-10 0Densitomete r (Vitatron Scientific Instrument,Dieren ,Th eNetherlands ) equippedwit ha nintegrato r recorder.Amount so faflatoxi n B- formedwer e calculatedb ycompariso no fth e integration unitsbetwee n thepea k areaso fth e spotso fth eunknow n sample and thato fth e known standard spotso nth esampl eplate . 5.3 Results 5.3.1 Effect of the serial dilution of spore suspension on mycelial dry weight and aflatoxin B, formation The results of the effect of serial dilution of spores of A. flavus NRRL 5906 on dry matter production and aflatoxin B.. formation are summarised in Figs 1 and 2. After 4 days incubation at 28 °C in AMMB, dry matter production was independent of the inoculum size used in oculating flasks because serial dilution by 4-5 log cycles did not significantly change the dry weight of mycelium harvest from the flask containing unheated control spores (20 L, 20 H) and the heat-treated spores under low humidity conditions (60 L). However, a 2 log cycles reduction, 1:102 in spores that had been heat-treated (60 °C for 30 nun.) under high humidity (>85% R.H.) conditions 60 H, resulted in the re duction of the mycelial dry weight by more than two-thirds (Fig. 1). A 3-4 log cycles reduction in inoculum size by serial dilution resulted in a twelve-fold increase in aflatoxin B. formed by control 20 L) spores; ten-fold increase in toxin formed by control (20 H) spores and a three-fold increase in aflatoxin B.. 76 ou X>u |:2•u o> e N O •rf o m e •c c o •i-I -H 4-1 C « z 0 .H •H -O 4J n . w« u ui >• S h . •0o •* es i a E m X$ E œ e —to A z ce oo c T> ^ y •rt c *J 8 x m -H 0 T3 u u.j B o E rt U 5 «•i X. J= 10 <" X COM O -H •" JC m C ai a) i h E OUS a to a tu •- u • œ •" as S C pi ö 0 T<1 .^ B^ «K « m to •», .Cv Ä *j; -rf ^ J3 " e U-l .g 4J 0 0 C *t* cj g) N M 4J i •^ ai tO W 4J QJ U-t ^ E to JJ 3 1„ >-l "O >> o S C JJ O tO .H s o -o C 41 -H •H h E O 3 tU IW£ O 01 4J * »0 Ü ^ —1 I m 77 •a •H JS x o M-< " o m en uCU 4-1 o s a. o CO »H CO CO s a s -H CU CW -UO O MH co O M so co § § . IS o o o u O CU CU N O I Eb 78 produced by heat-treated spores under low-humidity (<45% R.H.) conditions (60 L). Interestingly, no toxin was formed in flasks containing spores heat- treated under high ambient humidity (>85% R.H.) conditions (60 H) and serially diluted by 3 log cycles. Generally high inoculum level of spores yielded the least amount of aflatoxin B.. i.e. toxin formation was inversely related to the inoculum size (Fig. 2). When no toxin was formed in flasks (in high inoculum size conditions) the mycelium remained white or yellowish and culture filtrate was reddish in colour but reduction in inoculum size via serial dilution, heating, radiation or combination of heat and radiation resulted in green cul tures with abundant spores and culture filtrates were yellow in colour. 5.3.2 Influence of inoculum size on the formation of anatoxins by control and combined-treated spores Figures 3-6 summarise the effect of radiation treatment and its combina tion with heat on the production of aflatoxin B.. by A. flavus spores cultured in double autoclaved M4B. The effect is similar to what existed with the un- heated control spores in Fig. 1. High inoculum size of spores yielded the least aflatoxin B,. Reduction of the inoculum size by 3-5 log cycles via 3.5 KGy enhanced that aflatoxin B.. formed by dry heat-treated spores (60 L) fifty- fold but nearly doubled (1£ times) aflatoxin B. formed by spores that had been moist heat-treated under high humidity conditions prior to applying 3.5 KGy. No aflatoxin B.. was formed by spores that had been heat-treated under high humidity conditions prior to applying 4.0 KGy. Figures 7-10 summarises the effect of radiation treatments and its combi nation with heat on the formation of aflatoxin B.. in flasks containing AM^B. Results are similar to what existed in double autoclaved KMBexcep t that great er amounts of toxin were formed in AMMBtha n in MMB and the mycelial dry weight were 3-8 times greater than that in MMB. Again, combination treatment of moist heat (60 C for 30 min., applied under high humidity conditions, >85% R.H.) and 4.0 KGy of gamma radiation completely prevented aflatoxin B. formation by the spores in the culture medium. Aflatoxins B2, G, and G2 were formed along side B-|. But when aflatoxin B, formation is attenuated by a combination of moist heat and 4.0 KGy of gamma radiation, aflatoxins B2, G, and G2 could not be detected either. 79 5.4 Discussion The data fromthes e studies indicate thattota lgrowt ho funirradiate d sporeswa s independento finoculu msiz e insam evolum e ofmedium .Th eonl yex ceptionwa s thespore s thatha dbee nheat-treate d (60° C for 30min. )unde r highambien t (>853 R.H.)humidit y conditionswher e serial dilution reducedth e dryweigh tb y two-thirds.Mois thea t thereforeha s an intrinsic lethality.Th e stresso fhea to nessentia lbiologica lmolecule s of the spore isaugmente db y highhumidit y conditions.Hea t isals o transferredmor e readily inwe t air (Hawkere t al., 1952). Iti swel l documented thati nadditio nt o temperature andduratio no f treatment,humidit ystrikingl yaffect slethalit yo fhea tap plied.Th e gradual increase inaflatoxi nB. .productio nwa s related to thede crease ininoculu msiz eb y serialdilution .Mois thea t treatmentcoul d reduce thepopulatio no fspore sexponentiall y andaffecte d theirtoxigeni cpotentia l after 3lo gcycle s reduction inpopulation . The growtho ffung ii s restricted to theapica l regiono fth emyceliu m andth eape xi sconstantl ysupplie dwit hnutrient s fromth e resto fth emyce liumfo rcel lmultiplicatio n andbranchin g (Burnett, 1968). Studiesb yJink s (1969)an dSharm ae t al. (1980)sugges ttha taflatoxi nproductio n ina mediu m mightb e associatedwit hmycelia lbranchin g anddifferentiation .Hypha l fusion, common inth eDeuteromyce s (Park& Robinson , 1966;Raper ,1952 )wil lb e faster ina largepopulatio no fspore s and fasterreleas eo f certainsubstance sma y also influence thelatera lbranchin g (Burnett,1968 ;Gottlieb, 1971;Par kan d Robinson, 1966).Wit hsmalle rpopulations ,thes e limitations arereduce dt o affordmor e lateralbranchin go rsecondar ymycelia l growth,whic hma y intur n result inhighe ryiel do faflatoxi n in thesecultures . Indeed,w e found that dilutedspore s inculture sproduce d thehighes t amounto faflatoxi nB- ,an dtha t culturesdevelopin g fromdilut einocul abranche dan dsporulate dprofusel y and fasterafte r 4day sincubatio n inAMM Bo rMMB .Ther ewa sn o sporulation incon trolunheate dspore sdevelopin g fromlarg e inoculasuggestin g the involvement ofmycelia l differentiation inaflatoxi nB 1 production in A. flavus NRRL5906 . Thisobservatio ncontrast s the findingso f Sharmae ta l (1980)wh o reported thatculture s developing fromdilut e inoculao f A. parasitions NRRL 3145hardl y sporulatedeve nafte rprolonge d incubation,wherea s sporulationwa smuch faster andprofus e inculture s developing fromlarg e inocula.Ou r findings confirm thato fD etro ye t al. (1971), thataflatoxi n synthesisoccur sdurin g theperio d ofintens e sporulationo fth e fungus andtha t goodaflatoxi nproductio ncor relatedwit hyellowin g togreenin go fth emyceliu man d themedium . Inflask s 80 which failed toyiel daflatoxi nB.. ,th emyceliu mremaine dwhit e andth emediu m was either clearo rslightl yreddis h (Matelesan dAdye , 1965). Irradiation alone (3.5KGy )o rit s combinationwit hheatin g reducedth e inoculum sizeb y 3-5 logcycle swhic h resultedi nenhance daflatoxi nB ,syn thesis.Th eeffec twa ssimila rt owha texiste db yseria ldilutio no fth eun treated large inoculumo fspores .Thu s irradiationma yno thav ean ydirec t effecto naflatoxin-producin g abilitybu to n thesiz e ofinoculu m inrelatio n to theviabl e spores.However ,whe nheat-treatmen ti sadministere d (60 Cfo r 30min. )unde rmois thumi dcondition s (>85%R.H. ) incombinatio nwit h 4.0 KGy ofgamm aradiation , A. flavus NRRL 5906 growthan daflatoxin s formation iscom pletely impaired.Th eproductio n ofaflatoxi nb y A. flavus NRRL 3145wa sre ported tob emonophasi ci nth eculture sexpose d togamm airradiatio nb yApple - gate andChiple y (1977).W e didno t traceaflatoxin-producin gpatter no f A. flavus NRRL 5906beyon d 8days .However ,maximu maflatoxi nB - formationb y treated sporeswa s generally 4day s inAM^ Ban d 8day s inhWB .Greate ramoun t of toxinwa s formed inANW B thani nNMB ,presumabl y fortw oreasons : (i)th e fastergrowt ho fspore s inANMB ,an d (ii)th epresenc e of 2%pepton e and 2% glusoce inANW Bwhic har erequire di nth esyntheti cpathwa y foraflatoxi nbio synthesis. We conclude thata combinatio n treatment ofmois theat ,6 0° C for 30min . (>85%R.H . ambient)an d4. 0 KGyo fgamm a irradiation canpreven tbot h growth andaflatoxi nproductio nb y A. flavus NRRL5906 .Aflatoxi nB. .formatio ni sin versely related toth e inoculumsiz eo f A. flavus NRRL 5906.Th e apparenten hancement ofaflatoxi nB. .formatio nb y acombinatio n ofheatin gan d3. 5 KGy is adirec tresul to freductio n ininoculu m sizeo f 3-4 logcycles .Thu sirradi ationan d itscombinatio nwit hhea tma yno thav e anydirec teffec to nafla toxin-producing abilitybu t onth esiz eo fth einoculu m inrelatio n toviabl e spores.Thi seffec ti ssimila r towha texiste dwit h serialdilutio no funtreat edan d irradiation spores.Th eeffec to fthi scombinatio n treatment ofmois t heat (60 C for 30min. )an d irradiationi npreventin g aflatoxin inmaiz e grains artificially inoculatedwit h A. flavus isexamine d ina subsequen t paper. 81 Acknowledgement Ife thank Prof.Dr. J. 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Odamtten,G.T. , 1981.A survey of themycoflor a ofmaiz e grains stored at 26+3 °C and 75±5% R.H. in Ghana.Proceeding s 12th Biennal Conference of theGhan a Science Association,Legon ,Apri l 1981: 14pp . Odamtten, G.T.,V .Appiah ,an d D.I.Langerak , 1980a.Contro l ofmould s causing deterioration ofmaiz e grains in storage by combination treatment:A pre liminary model studywit h Aspergillus flavusLink .NRR L 5906. Internation alFacilit y for Food Irradiation Technology IFFIT,Rep . 12,Wageningen , TheNetherlands : 23pp . Odamtten,G.T. ,V .Appiah ,an d D.I.Langerak , 1980b. Short Communication. Studies on theTechnologica l Feasibility of the application of dry or moisthea t to grain and grainproduct s prior to gamma irradiation. Inter national Facility forFoo d Irradiation Technology IFFIT,Rep . 10,Wagenin gen, TheNetherlands : 8pp . Odamtten,G.T. ,V .Appiah ,an d D.I.Langerak , 1980c.Productio n of aflatoxin B.b y Aspergillus flavus Link in submerged static culture after combina tion treatment ofhea t and gamma irradiation. International Facility for Food Irradiation Technology, IFFIT,Rep . 15,Wageningen ,Th eNetherlands : 37pp . Park, D., and P.M. Robinson, 1966.In :E.G . Cutter (Ed.): Trends inplan t morphogenesis.Longmans ,London ,p . 24-44. Paper, J.R., 1952.Chemica l regulation of sexual processes inThalophyte s Bot. Rev. 18:447-545 . Schindler,A.F. ,A.N .Abadie ,an d R.E. Simpson, 1980.Enhance d aflatoxinpro duction by Aspergillus flavus andAspergillu s parasiticus after gamma ir radiation of spore inoculum. Jour.Foo d Protection 43 (1):7-9 . Schroeder,H.W. , 1966.Effec t of corn steep liquour onmycelia lgrowt h and aflatoxin production inAspergillu s parasiticus.Appl .Microbiol . 14(3) : 381-385. Schroeder,H.W. , and H. Hein Jr., 1967.Aflatoxi n production of the toxins in vitro inrelatio n to temperature.Appl .Microbiol . 15:441-445 . Schroeder,H.W. , and H.Hei n Jr., 1968.Effec t of diurnal temperature cycles on theproductio n of aflatoxin.Appl .Microbiol . 16:988-990 . Sharma,A. ,A.G . Behere, S.R. Padwal-Desai and G.B.Nadkarni , 1980.Influenc e of inoculum size ofAspergillu s parasiticus spores on aflatoxin production. Appl. and Environ.Microbiol . 40 (6):989-993 . Stultz,H.K. , and P.H. Krumperman, 1976.Effec t of temperature cycling onpro duction of aflatoxinb y Aspergillus parasiticus.Appl .an dEnviron . Microbiol. 32 (3):327-332 . Tawes III,J.W. , G.D. Hoke,an d G.C.Llewellyn , 1972.Contraception , fertili zation and teratology in rats incubated with themycotoxin ,aflatoxi n B.. Div. Ind.Microbiol . 18:711-715 . 83 Trank,H.L. , and P.A. Hartman, 1970.Effect s ofmoistur e content and tempera ture on aflatoxin production in corn.Appl . Microbiol. 19 (5):781-784 . 84 STUDIESO NTH E COMPARATIVE EFFICIENCYO FWOVE N POLYPROPYLENEAN DJUT E SACSA SPOSSIBL E PACKAGING MATERIALFO RPRE -AN DPOST-IRRADIATIO N STORAGE OF SOME CEREAL GRAINS G.T. Odamtten. Abstract The importanceo fusin g the correct storageba g forextendin g theshelf - lifeo fstore d food,i sth eprimar yconcer no fth eproduce r andbuye r alike. Studies reportedher ewer e carried outt ocompar eth e efficiencyo ftw o sacs Cjutean dwove npolypropylene )i npreservin g grainqualit y from fungalde teriorations otha ti tcoul db e used forpre -an dpost-irradiatio n storageo f cereal grains.Afte r onemonth' s storage andusin g Student's "t"test , the differencei nviabilit y (% germination)o fmaiz e grains inbot h sacswa sgene rally not statistically significant (P= 0.0 1o r0.05 ) irrespective ofwher e sacswer eplace di nth ewarehouse .However ,afte r2- 4 months storage,th eef ficiency ofwove npolypropylen e sacsbecam e apparentwhe n theviabilit yo f maize grainskep ti nsuc h sacswa s statistically higher (P=0.0 1o r0.05 ) than thatkep ti njut e sacs. Correspondingly, therewa sa lowe r fungal contamination (initial 1.2x102 c.f.u.g" to7.6x10 2 c.f.u.g" in4 months )o nth e maize grainskep t inwove npolypropylen e sacs thano nmaiz e grainsstore di njut e sacs (initial 1.2x102 c.f.u.g to7.0X10 1*c.f.u. g in4 months) .Ric e grains kepti nth etw odifferen t sacsunde r investigation didno t differ significantly (P= 0.0 1 or0.05 )i nthei r fungal contamination after 1,2 an d4 month s storage periods.Bu tric e grainskep ti njut esac swer eheavil y infestedb y Corcyra eephaloniea (Stantion),th elarva e constructing silken tunnels inwhic h they lived, causing aggregationo fth e rice grains. Ephestia aautella (Walker) in fested ricei njut e sacst oa lesse rextent . Certain fungiwer e persistently isolatedi nth eweekl y airsamplin gfo r mycoflorai nth ewarehouse ,despit eth esporadi c aerial sprayingwit h Actellic (R) 25 .Thes ewer e Aspergillus flavus3 Aspergillus niger, Fusarium moniliforme, Muaor pusillus, Pénicillium verrucosum var. ayolopiuman d Bhizopus oryzae. Fumigants, therefore,d ono tkil l fungi.Thes e listed fungi also infected greaterpercentag eo fmaiz e grains kepti nbot h jute sacs andwove n polypropy- 85 lenesacs .Ne wspecie so ffung i recorded for the firsttim eo nmaiz ei nGhan a are Aspergillus glauous, Aspergillus japonicus, Fusarium nivale, F. oxysporium, F. solani, Muco? pusillus and Myoelia sterilia. Thepractica limportanc eo f these findingsar ediscusse dan d futurewor k suggested. 6.1 Introduction Sanitationrequirement s forpreventin g food infectionan dfoo dpoisonin g arecontaine di nfoo dlegislatio n throughoutth eworl d(1) .Th eobjectiv eo f choosinga prope r foodpackagin gmateria li st oprovid e safetyfo r foodcon sumedb yth e generalpopulation . InGhana ,th eus eo fjut esac s forstorin g fooditem s sucha scowpeas , rice, groundnut,maize ,millet ,sorghu man dcoco ai swel lknow nan dth eatten dantlos so ffoo dowin gt oinsect san dfung ii sestimate da tove r 30%o fth e annualharves t(2) .Jut e sacs allowinflu xan defflu xo fai ran dmoistur ean d thepenetratio no finsecticide sdurin g routineprophylacti capplications .I n additionjut esac shav e lowtea rresistance ,lo wburstin gstrength ,ar ewate r permeable andhav ehig hwate rvapou rtransmissio n rate.Despit e thesemishaps , theus eo fthes esac si sstil lpopula rbecaus ei ti scheape r than thesuggeste d substitutes sucha swove npolypropylen esacs . The first commercialus eo fpolypropylen ea sa sa cmateria lwa si n195 8 forpackagin go f2 5k gquantitie so ffertilize ran dpolyethylen e resins(3) . Fromthe non ,progres si nth eus eo fheavy-dut ypolypropylen e sacswa s fairly rapidi nEngland ,Canada ,unite d Stateso fAmeric aan dSout hAfrica ,mainl y forfertilize rpackaging .Withi n theEuropea nEconomi cCommunit y (EEC)an dUSA , theus eo fwove npolypropylen e sacs,whic har emuc h lighteran dstronge rtha n jutesac shav ebecom e therul e rathertha na nexception ,fo rpackin g fooditem s sucha smaize ,rice ,etc .whic h aresen tt oAfric aa sfoo daid . Among the factors thatar eo fma jo rconcer ni npackagin gproblem sconnecte d with radiationpreservatio no ffoo dar e (i)th e capacityo fth econtaine rt o preserveth eirradiate d fooddurin ghandlin gan dstorag e (ii)imperviousnes s ofth econtaine ran dth econtaine rmateria lt oentr yo fspoilag eorganism s(iii ) thecapacit yo fth econtaine rmateria lt oaccept ,without undu e injuryt oit s chemicalcompositio n andphysica l structure,period so fexposur et ogamm aray s ofhig henerg yelectron san d(iv )th ecapacit yo fth econtaine rmateria lt o submitt oradiatio nwithout impartingoff-odour so rflavour st oth e contents ofth e foodpackag e(4) .T oasses sa sa c forit ssuitabilit y forpackin go fir radiatedcerea l grainsa sspel tou ti n(i )an d(ii )th emajo rtype so flosse s 86 àtmm ««• "W L_— i J J i *« r~"1 T——— 1 L J ! *» J 1 1 _i 1 _ ,1 *f i I J Vi 1 ! ! **• i UZ2 i i i ** 1 W ,L_J Fig.1 . Photographshowin gth elayou to fth e storedmaiz egrain swithi nth eGrain s Warehouse. Theposition so freplicat e sampleso nto po fth estack sar eindicate d byshadedtriangl e ( A )• 87 causedb y fungicontaminatin g stored cereal grains shouldb eexamined ;namel y (a)decreas e ingermination ,ft) discolouratio n ofpar to ral lo f the graino r kernel, (c)heatin go rmustiness , (d)variou sbiochemica l changes,( e) produc tiono fmycotoxins ,an d (f)los s inweigh t(5) . Inrecen tstudie s(6 ,7 )maiz e grains and animal feedwer e artificially inoculatedwit h Aspergillus flavus Link,an d thesamples ,wer e treatedwit h a combination treatment ofmois thea t (60° C for 30min .unde rhig h humidity conditions (>85$R.H. )an d gammaradiatio no f4. 0 KGybefor ebein g stored in wovenpolypropylen e sacs.Th esample swhic hwer e stored at 28° Can d 80%R.H . for4 month s stillha dgoo dkeepin gqualit y (i.e.n oenterobacteriaceae ,n o fungal growth,n o increase in free fatty acid,an dn o aflatoxin B..production) . There isn o information inth epertinen t literature oncomparativ e studies with jutesac s inthi s regard. Studies reportedher ewer e designed toprovid e informationo nth ecomparativ e abilityo fjut e andwove npolypropylen e inkeep ingcerea l grains (maizean drice )i ngoo dqualit y conditions duringprolonge d storageunde rpractica l fieldconditions . 6.2 Materials and methods Themaiz e grains andric ewer eprovide db y theGrain sWarehousin g Company, Tema.Eac hpalle tboar dcarrie d6- 9 jutesac s containing 50k gmaiz e andar rangedhorizontally .Twent y to twenty-one suchsac swer e stacked oneo nto po f another.Twelv ewove npolypropylen e sacswer e thenfille dwit h 50k gw t of maize grains and thesac swer e randomlyplace do n topo fth estack s inth e positions indicated inFig . 1.On ewove npolypropylen e sacwa splace d adjacent toon eo f thejut esac sa teac ho fth e indicated locations.Th eWarehous e has a storage capacity for 75,000 tons. The ricestorag e roomwa s aboutone-quarte r thesiz e andcapacit y ofth e Warehousewher e themaiz e grainswer ekept .Eac hpalle tboar d againcarrie d 6-9 jute sacs containing 50k go fric ean d arrangedhorizontall y on theboard . Twenty to twenty-one suchsac swer e stacked on topo fon e anothero n thepalle t board.Fou rwove npolypropylen e sacs containing 50k gw t foric ewer e placed on foursuc hstack so fjut e sacs randomly selected inth eRic eWarehouse . 6.2.1 Measurement ofambien t RelativeHumidit y and Temperature A thermohygrograph (Wilh.Lambrech tK.G. ,Göttingen) ,THG ,wa splace do n topo fth estack so fsac si nBa y 8.Thi s automatically recorded simultaneously 88 12 4 OIS STORAGE PERIOD IN MONTHS Fig.2a .Change s inviabilit y( % germination)o fmaiz e grainskep ti nth eposition s intw odifferen t indicated storage sacskep t for 1,2 an d4 months . (Datarepresent s averageresult so f5 0petr iplate s eachcontainin g 10grains. ) * P= 0.0 5 SEX P= 0.0 1 Unmarked- no tsignificantl ydifferent . 89 2 4 O 12 STORAGE PERIOO IN MONTHS Fig.2b . Changesi nviabilit y( %Germination )o fmaiz e grainskep ti nth e positionsi ntw odifferen t indicatedstorag esac skep tfo r 1#2 an d4 months . (Datarepresent saverag eresult so f5 0petr iplate s eachcontainin g 10grains) . * Ps 0.0 5 ***. P5 0.01 unmarked - Notstatisticall ydifferent . 90 the daily ambient relative humidity (R.H.)an dtemperatur e ona weekl y chart. The chartwa s changed everyweek .A t the endo fever ymonth ,th eR.H . and tem perature within the twodifferen t storagebag s (jute andwove npolypropylene ] were ascertained, using anElectroni cThermometer/Hygromete r Model HT-1 (Delm- horst Instrument Co.,Boonton ,Ne wJersey ,USA )designe d tomeasur e tempera ture andrelativ e humidity simultaneously ontw ometre s caliberated for direct reading.Th e instrument isprovide dwit h three aluminiumprob e rods,eac hon e metre longwit h a terminal SN-1 Sensorwhic hwa s inserted into the sacs. Six readingswer e taken aton eminut e intervals foreac hba g anddat aobtaine dre present average ofsi x successive readings. 6.2.1 Sampling of airmycoflor a Sterilepetr iplate s containing Oxytetracycline glucoseyeas t extract agar (OGYE)wer emomentaril y exposed (for 5min. )i n theWarehous e atBa y 3 . (lAo, 1AB; 1Ba, IBß); Bay 8 (2Aa,2Aß ;2Ba ,2Bß )an dBa y 9 (3Aa, 3AB; 3Ba,3B6) . Sixteenreplicat e petriplate s (i.e.fou rpetr iplate s ineac h ofth e a andß positions)wer e exposed ateac hBa y forsixtee nweeks .Durin g the 7th,11th , 14th and 16thweek s samplings,petr iplate s containing Sabouraud'sAga rwer e included (fourpetr iplate s ineac h ofth e a and ßposition s inFig . 1)t ose e ifan ydifference s exist in thekind so ffung i andno .o f fungal colonies iso lated on the twomedi a (OGYE,an d Sabouraud's Agar). Thepetr i disheswer e in cubated at 28 Cunti l colonies appeared in3- 5 days.Th e totalno .o fcolonie s and thespecie s offung i isolatedwer e recorded ateac hweekl y interval.Ther e wasprophylacti c application of insecticideActelli c 25* • J during the sampling of the airmycoflora . Theseperiod swer enote d and taken into consideration during analysis ofresults . 6.2.3 Determinationo f viability of grains Themaiz e grainswer e sampledmonthl y for 1,2 an d fourmonths .A t each samplingperio d 500 go fgrain swer e taken fromth e top,middl e andbotto m of the jute andwove npolypropylen e sacs and thesample s fromeac hsa cwer e then pooled together.Ther ewer e twelvepoole d samples foreac h type ofstorag esac . Themycoflor a on thegrain s and theirpercentag e germination (viability)wer e assessed using theblotte r testo fTemp e(8 )an dLimonar d(9) .Te n grains from either jute saco fwove n polypropylene sacwer eplace d on sterile Whatman's filterpape r in9 cmpetr i dishes.Ther ewer e 50replicate s (500grains )fo r each of the 12sampl e grains fromjut e sacs andwove npolypropylen e sacsres - 91 Table 1.Recor d of fungal contamination on maize grains kept inwove n poly propylene sacs for the indicated periods. Storage pe riod in months Location Replicates 0 1 2 4 (Initial) lAo 5.4X101 1.6xl02 2.8xl02 Uß 7.8X101 3.0xl02 3.2xl02 Bay 3 1.2xl02 IBa !.2xl02 6.4xl02 2.8xl02 IBB 8.6X101 1.4xl02 2.6xl02 2Aa 4.2X101 2.8xl02 3.5xl02 2A6 3.4x1a1 5.0xl02 2.5xl02 Bay 8 1.2xl02 2Ba 2.2xlOx 5.8X101 5.2xl02 2Bß 6.5X101 2.lxl02 7.6xl02 3Aa 2.8X101 - - 3A6 3.7X101 Bay 9 1.2xl02 3Ba 4.2X101 6.4X101 8.0X101 3BS 5.3xlOx 1.4xl02* 3.2xl02* Microbial count determined from samples in adifferen tba g (for 3Bß only); original bags having been inadvertently carried away. Data quoted are in colony forming units per gmsampl e (c.f.u.g ). 92 Table 2. Record of fungal contamination on maize grains kept in jute sacs for the indicated periods. Storage period in months Location Replicates 0 1 2 4 (Initial) lAo 7.8xl02 2.0x10" 2.0xl0lf 1A6 1.2xl02 6.2xl03 4.4xl03 Bay 3 1.2xl02 IB o 4.0xl02 1.6xl03 3.6x10" IBB l.lxlO3 6.8xl03 6.6xl03 2Ao 5.2xl02 3.6x10" 2.8x10" 2Aß l.lxlO2 6.4x10" 5.2x10" Bay 8 1.2xl02 2Bo 1.4xl03 1.4xl03 1.4x10" 2B8 !.7xl02 1.6xl03 7.0x10" 3Act 1.2xl03 - — 3A6 2.2X102 - - Bay 9 1.2xl02 3Bo l.lxlO2 4.2xl03 5.4x10" 3BB 1.3xl02 1.8xl02* 2.4x10"* Microbial count determined from samples in a different bag (for 3B6 only); original bags having been carried away. Data quoted are in colony forming units per gm sample (c.f.u. g~'). 93 Table 3. Record of fungal contamination of rice grains kept in named sacs for the indicated periods. Storage period in months Location Replicates IA 7.6X101 7.4XI01 7.6xlOx Rice Room 7.9x1t)1 (Jute sac) 2A 3>2xlQl 5_4X1Q2 8>2X1Q1 Rice Room IB 1.ôxlO1 6.8X101 1.9X101 (Woven poly- x propylene sac) 2B 4.0X101 7.2xlOx 7.6X101 rungal contamination not statistically different at both, P =0.0 I and P= 0.05 level of significance. 94 pectively and theplate swer e incubated foru pt o1 4day s(10 )a t 28±3°C . The followingmonthl yquantitativ e assessmentswer emad e forth eblotte rtest : (a) thepercentag eo fgrain s germinating.An ygrai nproducin g rootso rcoleop - tilewa s consideredt ohav e germinated; (b) thepercentag eo fgrain s infectedwit ha particula r fungusspecies . 6.2.4 Procedure for preparationo fhomogenat eo fspore s formicrobia l load determinationo fmaiz e grains Exactly2 5go fth e samples (maizeo rric e grains)wer eweighe d into sterile 250m lErlenmeye r flasks containing 100m lsteril e 0.1°*pepton e solu tiona sdilutio nblanks .Th econtent swer e shakeni na Gallenkam p Orbital Shakera t14 0rev.min " for3 0min .Th eresultin ghomogenat ewa s regardeda s initial stock spore suspension fromwhic h dilution serieswer eprepared . The plateswer e incubateda t2 8° Cfo r3 day s afterwhic h thecolonie sap pearingwer e counted andhenc eth ecolon y forming units (c.f.u.)pe rgra m samplewer e determined. 6.2.5 Statistical analysis Data, where appropriate were analysed using Student's "t" test and the re sults were tested for significance at P= 0.0 1 or 0.05 levels of significance. 6.3 Results After one month's storage and using Student's "t" test, the difference in viability (I germination of maize grains in both jute sacs and woven polypro pylene was generally not statistically significant (P = 0.01 or 0.05) irrespec tive of where the sacs were placed in the Warehouse (Fig. 1). The only excep tion for the first month's germination data was at site 2Awher e the differ ence in the %germinatio n of maize grains kept in woven polypropylene sac was significantly higher (P =0.05 ) than grains stored in jute sac (Figs 2a and 2b). After 2-4 months storage period, the efficiency of woven polypropylene sacs became apparent, when the viability of maize grains kept in such sacs were statistically higher (P= 0.01 or 0.05) than those stored in jute sacs (Figs 2a and 2b). Correspondingly, there was a lower fungal contamination (initial, -1 —1 1.2x102 c.f.u.g to 7.6x102 c.f.u.g in 4 months) on the maize grains kept in woven polypropylene sacs than on maize grains stored in jute sacs (initial, 1.2x102 c.f.u.g"1 to 7.0X101* c.f.u.g-1 in 4 months (Tables 1 and 2). Rice 95 ~2—4—6 à l4 12 14 16 Ó 2 4-4 3 10 13 14 16 SAMPLING PtniOO IWEÏKSI Fig. 3.Grap h showing occurrence ofmicoflor a in the Warehouse illustrated inFig . 1.S represent s spraying with insecticide. 96 :>40 12 *o 7g O a •16 O. i s l u. O 4 m u D UJ 3 £ soi 4 6 « 10 12 SAMPLING PERIOD (WEEKS) Fig.4 Graphshowin goccurrenc eo fmycoflor ai nth eRic e StorageRoa mfo rth eindicate dperiods . 97 9» O 0 9 è eC06*G* I j KL MNötoRU?v*xn FUNGI RECOR0E0 Fig.5 a Scattergrap hshowin g occurrence,o ffung ia tth e indicatedposition s (1A,2A ,3A )i nth eGrain swarehous e (Fig. 1) (Readgrap hverticall y alongeac hindicate d fungus) ,unbroke nlines ; representconsecutiv eweck(s ) isolationo findicate dfungus . isolatedspots ; sporadic isolationo f indicatedfungus . FUNGIRECORDED : Aspergillus Candidas M. Fusarium moniliforce B. A. cameus 0. J?F., nivalnivale C. A. flavus P. J?F., oatvsporiuoxvsjsprium 0. A. qlaucua Q. MuMucoee rr jDusillupusilluss E. A. japonicus R. Mvcelia sterilisterilia F. A. niger S. Neurospora sitositophilp a G. A. ochraceus T. Pénicillium digitatudigi m H. A. tamarii a. P. verrucosuverrucosum vava r cvclopium I. Asceraillus sp. v. Pénicillium spsp. J. Cephalosporium acrenonium . w. Rhizoctonia solsolana i K. Curvularia lunata X. Rhizopus orvzae L. £L' semitectian y. Trichoderma virida H. Drechslara maydi s 3. paecilomycea varioli 98 TsfrfTtrrtttdttrtAtm*«! FUNGI RECORDED Fig.5 b Scattergrap hshowin goccurrenc eo ffung ia tth eindicate d position (IB,2B ,3B )i nth eGrain sWarehous e (Pig. 1). (Readgrap hverticall yalon geac hindicate dfungus ) Unbroken lines; represents consecutive v/eek(s) isolation of the indicated fungus. (b) 0; isolated spots, sporadic isolation of indicated fungus. FDHSI RECORDED: A. Aspergillus candidua N. Fusarium monilifoane B. Ä. carneus 0. X. nivale C. ,&. rlavus D. Â. qlaucus 2. Muoor ousillus E. K. japonicu s R. a/celiô s ter ilia F. A. niqer S. Heurespor a sitophila T. Pénicillium digitatum G. A_. cchraceus U. P. verrueo sur n var cvclopium H. A. taroarii V. Pénicillium sp. I. Aspergillus sp. W. Rhizoctonia solani j. Cephalosporium acreiaoniuni X. Rhizopus orvzae K. Curvularia lunata X. Trichoderna viride L. £.• semltec&tum Z. paecilomvces varioti. K. Prechslera maydjs 99 grains (polishedrice )kep ti nbot h jutean dwove npolypropylen e sacsdi dno t differ significantly (P=0.01 or0.05 )i nthei rfunga lcontaminatio nafte r1 , 2,an d4month s storageperiod s (Table 3).However ,ric egrain skep ti nth e jutesac swere ,relatively ,heavil y infestedb y Corayra aephalonioa (Stantion), thelarva econstructin g silkentunnel s inwhic h theylived ,causin g aggregation of theric egrains . Ephestia aautella (Walker)infeste dric ei njut e sacst oa lesserextent . Therecor do ftota lnumbe ro ffunga lcolonie san dspecie so ffung i iso latedi nth eaeria lsamplin gi nth eWarehous e (Fig.1 )an dRic eStorag eRoo m are illustratedi nFig s 3an d4 ,respectively .Aeria lprophylacti c spraying with fumigantActelli c 25^ ,whic hha sbee na routin epractic ei nth eWare house,coul dno tb eprevente dan dthi sexercis ewa s always followedb ya de clinei nth enumbe ro fspecie so ffung iisolate dan dals oth enumbe ro ffunga l colonies.Bu tthi swa s followedb ya nincreas ei nth eai rmycoflor ai n2- 4 weeks (Fig.3 )i nth eWarehous ewher emaiz e grainswer ekep tan d1- 3week s (Fig. 4)i nth eRic e StorageRoom .Althoug haeria lsprayin gwit h insecticides mightkil l insects,i tdi dno tcompletel yeliminat e fungalspores .Also ,i n betweenth estack so fjut esac s wasfumigatio npreparatio n"Deti aGa sEx-B " (Phosphinegas )i nbag s (6x8cm) .Thi s didno tkil lth efunga lspore san dthe y were isolated throughoutth e4month s samplingperiod . Duringth eai rmycoflor asamplin gperio d thespecie so ffung itha twer e isolatedwer enote dan d theiroccurrenc ei nth evariou sselecte dsectio no f theWarehous e (Fig.1 )wa srecorde d (Figs5 aan d5b) .I na simila rmanner , the fungalspecie s thatwer e isolatedi nth eRic e StoreRoo mar erecorde di nFig .6 . Certainfung iwer e frequently isolatedi nth eweekl yai rsamplin g forpresenc e ofmycoflor ai nth eWarehouse .Thes ewer e Aspergillus flavus, Aspergillus niger, Fusarium moniliforme, Mueor pusillus, Penieillium verruaosumvar . ayalopium and Rhizopus oryzae (Figs 5a,b).Thes e listed fungials o infecteda greate rper centageo fmaiz e grainskep t inbot hjut e andwove npolypropylen esac s (Tables 4a-e).Th efung iwhic hpredominate di nth eai rmycoflor ai nth eRic eStorag e Roomwer e Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium moniliforme and Pénicillium digitatum (Fig.6) .Ver y fewspecie so ffung iwer e foundo nth eric egrain swhe n themicrobiologica l loado nthe mwa sdetermined . Themould swer e Curvularia lunata, Aspergillus niger and Fusarium moniliforme, inorde ro fabundance .Funga lspecie s recordedfo rth efirs ttim eo nmaiz ei n Ghana,durin gthi sinvestigation ,ar e Aspergillus glauaus, Aspergillus japoni- ous, Fusarium oxysporium, Fusarium solani, Muoor pusillus and Myaelia sterilia. Therewa sn osignifican tdifferenc ebetwee nOGY Ean dSabouran dAga ra sa 100 FIG-6 RICE STORAGE ROOM O e (> o H e r <> Î o. 2< en ui FUNGI RECORDED Fig.6 Scatter graphsharin goccurrenc e offung ii nth eRic e StoreRoo mdurin ga twelvewae k period. (Readgrap hverticall y alongeac hindicate d fungus) (a) Unbroken lines; represent consecutiveweek(s ) isolationo findicate d fungus. (b) isolatedspots ; sporadic isolationo f indicated fungus. FUKCTRECORDED ; A. Aspergijlu sc-anctidu s o. S~ nivale-. D. A.carneu s p. J". esr»'sporiupi C. A.flawf e Q. tfccor pusillus D. A.g lau eu s R. tfrrcciia scerilta E. A. japonicus fi. F. A.nige r T. G. A.ochrace-o s n. P. verrucosum var evelooiuni H. A.tawari i V. I. Aspergillussp . Ejhizccto&ia aaiani J. Cephalosporinacremoniu m X. Hhizopus jQEXSâÊ K. Curvularia 1unat a Y. L. C. semitectu m ?.. Paecilon^yces variotl M. Drechslera inaydis bacteria (unidentified) N. Fusarium monilif orate CH. Cladoaporium herbarum. 101 medium forisolatin g themycoflor a (Fig.3) . Recordso fambien ttemperature s and relativehumiditie s takensimultaneous lyusin ga thermohygrographplace d inth eWarehous e (Fig. 1)ar e represented inFig . 7 (9thDecembe r -29t hDecembe r 1981), Fig. 8 (1stJanuar y -26t hJan uary 1982), Fig.9 (February 1982),Fig . 10 (3rd- 23r dMarc h 1982), andFig . 11 (24thMarc h -5t hApril 1982). There are large fluctuations inrelativ e humidities (25-901R.H. ) and less sowit h temperature (25-40°C )record s in theWarehouse .Th e lowestR.H . recordedwa s 25$,o n the 5tho fJanuary ,1982 , during theharmatta n season (lateDecembe r -earl yFebruary) .Th e lowestR.H . was always recordeda tmidda y (12.00h r CM1). The relativehumidit y inth e Warehousewa shig h during theearl ypar to fth eda y (1.00-6.00h rOff )an d thereafterdecreased .A secondhig hhumidit ywa s recordeddurin g theperiod 18.00-24.00h rGM T (midnight). Conversely,th ehighes t ambienttemperatur ewa s always recordeda tmid day (12.00h r GMT),wit h the lowestdurin gth eearl ypar to fth eda y andi n theevening .Th e twocommunicatin g doors inFig . 1wer e openeddurin g theda y (07.30-16.30h rGMT )an d thereafterremaine dclose d till the followingday . These results indicate thatEquilibriu mRelativ e Humidity (E.R.H.)wa sno tat tained in theWarehous e during theentir e storageperiod o f4 months .I ncon trastwit h this,a correspondin g recordo fR.H . and temperature taken fromou r laboratory,wher esom eo fth emaiz e grainsan d ricewer e stored forth eperiod 24thMarc h - 14thApri l 1982,showe d that theR.H . andtemperatur e remained fairlyconstan t throughout theperio d (Fig. 12).Unde rsuc h conditions.E.R.H . isattaine d in 12days .Th emoistur e content (m.c.)o fgrain s dependo n the storagehumidity .Th ehighe r thestorag ehumidity ,th ehighe r theequilibriu m m.c. ofgrains .Bu tth em.c .o fmaiz e grainsdoe sno t increase appreciably at E.R.H. 80$an dbelow ,afte r 12day s storageperio d (10).I fther e are large diurnal fluctuations inambien tR.H . and temperature,suc h asobtaine d inth e Warehouse during these investigations,m.c .o f the grainswil lno t reachequi libriumwit h theambien tR.H .Th em.c .o fth e grainswil lb e closely related toth eprevailin gR.H . atth e time andda yo fsamplin g (Table 5). Them.c .o f grainskep ti njut esac swa shighe r than thosestore di nwove n polypropylene sacs (Table5) . 102 Table 4a.Tabl e showing species of fungi isolated frommaiz e grains and their percentage on grains they contaminated in the indicated sacs. % Occurrence on grains kept in Location Polypropylene Fungi recorded on grains Jute sacs for code sacs for 2 months 4 months 2 months 4 months lAa Aspergillus flavus Link 47.5 27.5 40.0 55.0 A. glauaus 2.5 A. niger Van Tieghem 52.5 35.0 22.5 25.0 A. ochraceus Wilhelm 7.5 2.5 A. ustus Wehmer 2.5 Drechslera maydis (Nisi- kado) Subr a i m & Jain 20.0 Fusarium moniliforme Sheldon 100.0 22.5 82.5 75.0 Fusarium oxysporium 20.0 Neurospora sitophila Shear &Dodg e 5.0 25.0 — - Paeailomyaes varioti Bain - 2.5 — - Pénicillium verrucosum var. ayalopium 30.0 25.0 90.0 72.5 Rhizoctonia solani Kuhn 5.0 - 25.0 — Rhizopus oryzae Went and Prinsen Geerling 100.0 17.5 7.5 32.5 1A6 Aspergillus flavus Link 40.0 27.5 52.5 47.5 A. glauaus 2.5 7.5 5.0 12.5 A. japonicus - 2.5 - - A. niger 15.0 27.5 17.5 22.5 A. oohraceus 12.5 10.0 2.5 - A. ustus 2.5 - 5.0 2.5 Drechslera maydis 7.5 - 12.5 - Caldosporium herbarum Link ex Fr. - - 2.5 - Curvularia lunata (Wakker) Boedjin 2.5 - 5.0 - Pénicillium verrucosum var. cyclopium 17.5 20.0 30.0 77.5 Rhizopus oryzae 10.0 12.5 5.0 50.0 Fusarium moniliforme 15.0 27.5 57.5 30.0 Fusarium oxysporium 2.5 - 7.5 - 103 Table 4b. % Occurrence on grains kept in Location Polypro pylene Fungi recorded on grains Jute sacs for code sacs for 2month s 4 months 2 months 4 months 2Aa Aspergillus flavus Link 40.0 97.5 62.5 27.5 A. glauous 2.5 - 2.5 - A. niger 15.0 70.0 45.0 35.0 A. oehraceus 12.5 - - - A. ustus 2.5 - - - Fusarium moniliforme 15.0 - 75.0 22.5 Fusarium oxysporium 2.5 Neurospora sitophila 25.0 Paeailomyces varioti 2.5 Cladosporium herbarum Cephalosporium acremonium 2.5 Dreohslera maydis 7.5 50.0 Pénicillium verruaosvm ayclopium 17.5 22.5 60.0 25.0 Rhizopus oryzae 10.0 50.0 25.0 17.5 Myaelia sterilia - 62.5 - 2.5 Rhizoctonia solani 25.0 Curvularia lunata 2.5 2A8 Aspergillus flavus Link 37.5 65.0 55.0 50.0 A. fumigatus - 12.5 - - A. glauous - 20.0 2.5 A. niger 12.5 32.5 67.5 35.0 A. japonicus 2.5 - - - A. oehraceus 5.0 2.5 - 5.0 Curvularia lunata 7.5 - - - Fusarium monili forme 65.0 25.0 92.5 40.0 Fusarium oxysporium 22.5 - - - Dreohslera maydis - - 15.0 32.5 Penioéllium verruoosum var. ayelopium 10.0 22.5 25.0 32.5 Rhizopus oryzae 82.5 75.0 17.5 25.0 Rhizoctonia solani - - 7.5 - Neurospora sitophila 12.5 - - - Mycelia sterilia - 25.0 25.0 - 104 Table4c . % occurrence on grains kept in Location Polypropylene Fungi recorded on grains Jute sacs for code sacs for 2 months 4month s 2month s 4 months IBa Aspergillus flavus 47.5 100.0 50.0 35.0 A. glauous - 2.5 - 5.0 A. japonicus - - - 5.0 A. niger 52.5 70.0 82.5 52.5 A. ochraaeus 7.5 — — — A. ustus Drechslera maydis _ _ 5.0 62.5 Fusarium moniliforme 100.0 7.5 75.0 67.5 Fusarium oxysporium 5.0 32.5 — 7.5 "Pénicillium verrucosum var. eyclopium 30.0 40.0 50.0 42.5 Rhizoctonia solani - 52.5 7.5 - Rhizopus oryzae 100.0 92.5 35.0 15.0 Aspergillus fumigatus 7.5 32.5 ~ "" 1B6 Aspergillus flavus 55.0 70.0 57.5 70.0 A. fumigatus - 22.5 - - A. japonicus - 10.0 - - A. ochraaeus 7.5 - - - A. ustus 5.0 - - - A. niger 42.5 50.0 47.5 27.5 Cephalosporium aoremonium Corda 5.0 12.5 Pénicillium verrucosum var. eyclopium 45.0 17.5 55.0 60.0 Fusarium moniliforme 42.5 37.5 45.0 75.0 Fusarium oxysporium 5.0 - 2.5 5.0 Rhizopus oryzae 40.0 95.0 82.5 75.0 Aspergillus glauous 2.5 - - - Rhizoctonia solani - 22.5 - - 105 Table 4d. % Occurrence on grains kept in Location Polypropylene Fungi recorded on grains Jute sacs for code sacs for 2month s 4month s 2month s 4month s 2Ba Aspergillus flavus Link 45.0 65.0 67.5 50.0 A. fumigatus 2.5 A. glauaus 2.5 7.5 A. nigev 37.5 52.5 15.0 32.5 A. japoniaus 2.5 2.5 A. oohraaeus 5.0 2.5 5.0 A. ustus 5.0 17.5 Dreahslera may dis 7.5 30.0 17.5 Curvularia lunata 5.0 5.0 Fusarium moniliforme 40.0 5.0 100.0 52.5 Fusarium oxysporium 2.5 10.0 5.0 Neurospora sitophila 5.0 Pénicillium verruoosum var. ayolopium 5.0 5.0 37.5 17.5 Rhizopus oryzae 30.0 7.5 Myaelia sterilia 25.0 Cladosporium herbarum 2.5 2BB Aspergillus aandidus Link 2.5 2.5 A. flavus 47.5 62.5 47.5 45.0 A. glauaus A. niger 37.5 47.5 37.5 50.0 A. ustus 2.5 2.5 A. japoniaus 2.5 - 2.5 2.5 A. oahraaeus 10.0 Cephalosporium aaremonium 15.0 15.0 Dreohslera may dis 27.5 Penioillium verruoosum var. ayolopium 67.5 17.5 67.5 40.0 Rhizopus oryzae - 20.0 47.5 37.5 Rhizoctonia solani - 2.5 - - Fusarium monili forme 80.0 27.5 80.0 90.0 Fusarium nivale - 10.0 - - Fusarium oxysporium 7.5 5.0 7.5 - Fusarium solani 7.5 - 7.5 — 106 Table4e . % Occurrence on grains kept in Location Polypropylene Fungi recorded on grains Jute sacs for code sacs for 2 months 4month s 2month s 4month s 3Ba Aspergillus flavus Link 35.0 45.0 40.0 40.0 A. glauaus - 5.0 2.5 - A. niger 32.5 45.0 72.5 92.5 A. fumigatus - 17.5 - - A. oohraoeus Cladosporium herbamm 2.5 - - - Fusarium moniliforme 20.0 22.5 55.0 95.0 Fusarium oxysporium Paeailomyoes varioti 32.5 2.5 - - Penieillium verruoosum var. ayalopium 20.0 25.0 60.0 40.0 Rhizopus oryzae 50.0 17.5 12.5 75.0 Myoelia sterilia 5.0 2.5 - - Rhizoctonia solani 25.0 107 6.4 Discussion Decreasei ngerminatio ni son eo fth emos tsensitiv e indicatorso fincip ientspoilag eo fstore dseeds ,includin gcerea lgrains .Althoug h abilityo f grainst ogerminat ema ydecreas ewithou tattendan tdecreas ei nprocessin g quality,thi sreduce dviabilit ycertainl y shows thatgrai n lotsar ebein g slowlyinvade db ystorag efungi .Ther ei sinformatio ni nth epertinen tlitera ture,o nth edrasti c reductiono fgerminatio no fgrain sb ystorag e fungi. For example,Lope zan dChristense n (11)store dmaiz ea t19-20 1m.c .an d20-2 5 °C, some samples freeo ffungi ,other s inoculatedwit h Aspergillus flavus. After 74days ,th esample s freeo ffung iaverage d97 1 germination,whils t thosein oculatedwit h A. flavus averaged 131.Al lspecie so fth e A. glauous groupa s wella sA . aandidus and Pénicillium invadevariou spart so fth eseed ,includin g theger man ddirectl y causeo rcontribut et oreductio ni ngerminatio n(12) . Presentdat acanno texplai nwh yi nsom e instances fungirecorde do ngrain skep t injut ean dwove npolypropylen esac swer esimila r (Table4a-e )bu tviabilit y (Igermination )o fgrai ndiffere d (Figs2 aan d2b) . Thebette rstorag esac , wovenpolypropylene ,maintaine da lowe rfunga ldeterioratio no fmaiz e grains kepti nthem ;germinatio no fgrain swa s significantlyhighe r (ascompare dwit h thosekep ti njut e sacs)afte r2- 4month s storage (Figs2 aan d2b )an dth efun galcoun tincrease donl yslightl y (initial 1.2x102- 7.6x10 2c.f.u. g )i n4 months.Presumably ,th ehighe rm.c .o fgrain si njut esac s (14.4-15.61m.c. ) permittedmor e fungaldeterioratio nactio no nth eger mo fth egrain s resulting inlowerin go fgerminatio nfro mabou t98 %t oaveragel y 351i nfou rmonths . Corresponding fungalcoun tincrease d froma ninitia l 1.2x102- 7.0x10**c.f.u. g (twolo gcycles )ove rth esam eperiod) . Additionalwor ki sneede dt oelucidat e theeffec to fth esaprophyti cac tivityo fth esi xmos tpredominan t fungiisolated ,namel y A. flavus, A. niger, Fusarium moniliforme, F. oxysporium, Pénicillium verrucosum var. ayalopiuman d Rhizopus oryzae, onth etensil estrengt ho fth etw otype so fstorag e sacs. Ricedoe sno tsee mt ohav eseriou s storageproblem swit hfunga lcontami nation (Table 3)excep t thatth epresenc eo finsect s sucha s Corcyra aephalo- nica and Ephestia cautella would require gammaradiatio nt oeliminat eth ein sects.I fthi si sno tdone ,th einsect sma ygna wth ewove npolypropylen esac s andcaus eperforation si nthem .I tha sbee n showntha ta dos eo f8 0Kra dwil l disinfestgrain so fal linsect sunde rth eGhanaia ntropi c conditions (13). Theus eo fchemica l fumigantsfo rth econtro lo fpost-harves t losses causedb yinsect san dfung ii swel lknown .Th eus eo fmethy lbromide ,picrin , 108 ,-^VMM Ficj. 7 Record of dally ambient Relative Humidity and Temperature in the Warehouse (Fig.1) during the period 9th-29th December, 1981. 109 Fig. 8.Grap h showing daily record of ambient Relative Humidity and temperature in theWarehous e (Fig. 1) during 30thDecember , 1981 through 26th January, 1982. (Note the low 25%R.H . recorded on the 22nd January, 1982durin g theHarmatta n Period.) 110 TIME OF 9A Fig.9 Graphshavin gdail y .recordo fanbien tR.H .an d Temperaturei nth eWarehous e (Fig.1)durin g February 1902. Ill 1* O IS3 4O Fig. 10.Grap h showing daily record of ambient R.H.an d temper ature in theWarehous e (Fig.1 )durin g 3rd- 23r dMarch ,1982 . 240 13 J*O Fig. 11.Grap h showing daily record of ambient R.H.an d temper ature in theWarehous e (Fig.1 )fro m24t hMarch-5t h April,1982 . 112 Î40 lî 240 Fig. 12 Graphshowin gdail yrecor do fambien tR.H .an d Temperaturei nth eLaborator y from24t hMarc h- 14thApril ,1S32 . (Noteth enearl yconstan tR.H .an dTemperatur e obtained). 113 ethylenedibromd e: methy lbromid e (1:1)beside s ammoniaan dsulphu rdioxid e forcontro lo fgrai n fungihav ebee nreporte d (14,15 , 16). Forexample ,ethylen e dibromide andchloropicri nkille dspore so f A. flaws and A. niger (LDgr at 10m g 1~) i nlaborator y testbu t the same fumigant couldno t control all fungi ifth e fungiar einsid eth e grain (17)suc h as in thisinvestigation . Inhibition ofmoul d growth andformatio no fmycotoxin sha s beenreporte d (17, 18, 19)fo r fumigantsmethy lbromide ,ethylen e oxide,sul phurdioxide ,chloropicri n andmixture s ofethylen e dibromide andmethy lbro mide.Conversely ,grain s(wheat )treate dwit hphosphin e caused significantin crease inyiel do faflatoxi n B,b y A. flams NRRL 3251 and asignifican tde creaseo faflatoxi n B1 andG 1 by A. flavus NRRL 3145(20) . Inman ypublication s (14-20,etc. )effec to finsecticid e treatmento n some strainso f fungiwer e strain dependenta swel l as insecticide dependent. Inaddition ,i ti sestablishe d (21)tha tvolatil e substances interferewit h respiratory mechanisms ofspores .Th e cellwal l composition andwal l contents varyno tonl y inspecie s of fungibu t also the stages such ashypha econidio - phores andspores ;myceli abein gmor esusceptibl e thanspores .Durin g this studypublishe dhere ,aeria lprophylacti c sprayingo fWarehous ewit hActelli c fRl 25^ ' andPhosphin e resulted inth e reductiono f fungalcolon y counts and eli mination ofsom e fungal species (Figs 3-6) butthi swa s followedb y anin creasei n thesubsequen t samplingweek .Additiona lwor k isneeded toelucidat e theeffec to fActelli c 25^ ' andPhosphin e gaso n the fungiencountered .Th e continuous useo fth e insecticides isobjectionabl ebecaus e of theresidu e they leavei nou rfoo dsource .Th eus eo fgamm a radiationoffer sabette ralter native (22)an dstorag e sacsuc ha swove npolypropylen e saci s suggested aspo tential replacement forjut e sac inorde r topreven t reinfection afterth e terminal irradiation treatment. 6.5 Conclusions Grainskep t inwove npolypropylen e sacs,unde rpractica l fieldconditions , were significantly (P=0.0 1 or0.05 )mor e viable after 2-4 months storage than thosekep t injut esacs .Decreas e ingerminatio n isa nindicatio no f thecer tainty thatgrai n lotsar ebein g slowly invadedb ystorag e fungi resulting in reducedquality .Th ehighe rrecord so ffunga lpopulatio no nmaiz e grainskep t injut e sacs confirm this. Evidence ispresente d tosho w thatprophylacti c sprayingwit h insecticides neitherkil l the fungio n and inside thegrain sno r inth e surrounding air,i n 114 CM co —• o o +i y3 f> ro m m tn m m m — es) co m r** r» m O o O O O +1 +1 +1 +i +1 1^1 O CM — m m -3- m •V e m CM m •P-I — O O O o a 01 co — r^ CM u o o d o +! +1 +1 +1 tri \£> r*» 00 to O 00 c S -S -OTO ca H m (1) OD 01 Ol CO e I c ê r-l M o> 1 o> 0 u r—>4, 0 i-i CO 4J >o 0 u >, H Cfl TOCO 3 a a a 115 theWarehouse .Therefore ,othe rmeans ,notabl y gamma radiationi srequire dt o kill sporeso ffung i contaminating cereal grainsan d thus improve themicro biological qualityo fth egrains . The objectiveo fgamm a radiation treatmenti st oprolon g the shelf-life of these grains fora perio d exceeding oneyear .Th eprove n abilityo fgamm a radiationt ocontro l mould growthwil lb eaugmente db yth eus eo fwove n poly propylene saca sa packagin gmateria l forpre -an dpost-irradiatio n storageo f cereal grains.Wove npolypropylen e sacs aremuc h lighteri nweigh t than jute sacs andweigh t forweight ,wove n polypropylene sacsar esignificantl y strong er thanjut e sacs (23).Unlik e the jute sacs,the yar e unaffectedb ywater ,d o not roti ndam p conditions,d ono t absorb moisture,the y areclea nan dd ono t impart any odouro rtain tt othei r contents.A surve y conductedb yTrip p(4 ) confirmed that radiation levelo flMra d (10KGy )induc e onlymino r changesi n themechanica l andchemica l propertieso fpackagin gwove npolypropylen ean d thatn omeasurabl e radioactivity isinduce db y60 Co, 137Cso rb yelectron su p to 10MeV ,i nth enormall y presentelement so fth epackagin g polypropylene. Thissa ctherefor e offers many advantages over the traditional jutesa cfo r usei ntropica l areas likeGhana .I tremain st ob eestablishe dwhic ho fth e two sacswoul dharbou r andsuppor t saprophytic activityo ffung ian dthu s acts as springboard for infectiono fcerea l grains kepti nit . Acknowledgements The authori sdeepl y indebtedt oth eManagin gDirector ,Grain s Warehousing Company,Toma , forgrantinghimpermissio nt ous e his"premise s for the studies reported here.H eappreciate sth ewillin g helpo fth e Staffa tth eWarehous ean d thato fth e SeniorWarehous eManage r forprovidin g samples andusefu l informa tiono nfumigatio nhistor yo fthes e samples.Th eTechnica lAssistanc eo fMr . M. Ofori-Appiah andMr .E .Yanke yi sals o gratefully acknowledged. Finally,hi s thanksar eals odu et oMis sM .Wayo mfo rhe r excellent typingo fth emanuscript . References (1)Elias ,P.S. , 1979.Foo d irradiation and food packaging. Chemistry and Industry, 19Ma y 1979,p .336-341 . (2)ANON , 1970.FA O Report,Rom e 1970. (3)Flatman ,D.J. , 1977.Sack s made fromplasti c film. Chapter 4.Th e pack aging media,F.A . Paine (Ed.), Blackwell &Son s Ltd.,Londo n W2 1EG. 116 (4)Tripp ,G.E. , 1959.Packagin g for irradiated foods.Internationa l Journal ofApplie d Radiation and Isotopes.Vol .5 . (5)Christensen , CM., and Kaufmann,H.H. , 1969.Grai n Storage.Th e role of fungi in quality loss.Minneapolis ,Minnesota , 153pp . (6)Odamtten ,G.T. ,Appiah ,V. , and Langerak,D.I. , 1980.Contro l ofmould s causing deterioration ofmaiz e grains in storage by combination treatment:a preliminar y model studywit h Aspergillus flavus Link NRRL 5906. IFFITRepor tNo . 12,Wageningen ,Th e Netherlands:2 3pp . (7)Odamtten ,G.T. ,Appiah ,V. , andLangera k D.I., 1980.Preliminar y studies on the effect of the combination treatment ofhea t and gammairra diation on thekeepin g quality of animal feed and cottonseeds . IFFIT Report No. 16,Wageningen ,Th eNetherlands : 24pp . (8)Tempe ,J . de, 1967.Routin e investigation of seedhealt h condition in the Dutch seed testing station atWageningen . Proc. Int.See d TestAsso . 22: 1-12. (9)Limonard , J., 1966.A modified blotter test for seed health.Neth .J . of Path. 72:319-321 . (10)Odamtten ,G.T. , andLangerak ,D.I. , 1980.Moistur e sorption of twomaiz e varieties kept under different ambient relative humidities.IFFI T ReportNo . 9,Wageningen ,Th eNetherlands . (11)Lopez ,L.C. , and Christensen, CM., 1967.Effec t ofmoistur e content and temperature on invasion of stored corn byAspergillu s flavus. Phytopathology 57:588-590 . (12)Tuite ,J.F. ,an d Christensen, CM., 1955.Grai n storage studies 16:In fluence of storage conditions upon the fungus flora ofbarle y seed. Cereal Chemistry 32: 1-11. (13)Amoako-Atta , B., 1981.Simulate d radiation disinfestation of infested maize in Ghana. Symposium on Combination Processes inFoo d Irradia tion. IAEA-SM-250/8, SriLanka . (14)Majumder , S.K., Sharangapani,M.V. ,an d Pingale,S.V. , 1955.Chemica l con trol of spoilage caused by microbes in stored grain.Bull .CFTR I5 : 47-50. (15)Raghunathan ,A.N. ,Muthu ,M. , and Majumder, S.K., 1969.Contro l of inter nal fungi of sorghumb y fumigation. J. Stored Prod.Res .5 :389-392 . (16)Tsurunta ,0. , and Ishirava,A. , 1966.Studie s on fumigant ethylene oxide VII. Sterilization ofmould s injurous to the stored cereals and in fluence on germination of plant seeds.Foo d Sanitation 7:298-303 . (17)Narasimhan ,K.S. ,an d Rangaswamy,G. , 1968.Effec t of some fumigants on themicroflor a of sorghum seeds.Bull .Ind .Phytopath . 5:57 . (18)Majumder , S.K., Narasimhan,K.S. ,an d Parpia,H.A.B. , 1965.Microecologi - cal factors of microbial spoilage and theoccurrenc e of mycotoxins on stored grains.Mycotoxin s Foodst.Proc . Symp.MI T 1964:27 . (19)Raghunathan ,A.N. ,Muthu ,M. , andMajumder , S.K., 1969.Contro l of inter nal fungi in sorghumb y fumigation. J. Stored Prod. Res.5 :389 . (20)Vandegraft ,E.E. ,Shotwell ,O.L. , Smith,M.L. ,an d Hesseltine, C.W., 1973. Mycotoxin formation affected by fumigation ofwheat .Cerea l Science Today Vol. 18,No . 12:412-414 . (21)Hawker ,L.E. ,Linton ,A.H. ,Folkes ,B.F. , and Carlie,M.J. , 1952.Intro duction to the Biology ofmicroorganisms. St.Martin' s Press,N.Y. : 452pp . (22)Odamtten ,G.T. , 1982.Th e status of preservation ofmaize :prospect s of the application of irradiation in the extension of shelf-life of maize inGhana .Africa n Bio Sciences Network (ABN)Worksho p onpost - harvest losses and theirprevention . Sept. 27 - 30Dept .o f Zoology, Legon. (23)Paine ,F.A. , 1977.Th e packaging media. Blackie &Son s Ltd,London ,W 21EG . 117 InternationalJournal of Food Microbiology:2(1985)151-15 8 Elsevier JFM 00058 7 Studies on the selection of a suitable packaging material for pre- and post-irradiation storageo f cereal grains in Ghana G.T.Odamtte n' and E.H. Kampelmacher 2 ' Department of Botany, Universityof Ghana. P. O. Box 55, Legon/A ccra. Ghana, and • Laboratoryfor Food Microbiologyand FoodHygiene. Department of FoodScience. Agricultural University; 'Biotechnion'. De Dreijen 12.6703 BC Wageningen.The Netherlands. (Received 30 July 1984; accepted 7 February 1985) Twenty different species of fungi belonging to the genera Aspergillus. Cephalosporin Dreschlera, Fusarium, Mucor. Neurospora.Rhizoctonia, Rhizopus. Pénicilliuman d Trichoderma were isolated from jute and woven polypropylene sacks using the blotter test, solid medium test and the decimal serial dilution technique. There was a highly significant difference (Students' /-test, P < 0.05) between the larger number of fungal colonies associated withjut e sacks than with woven polypropylene sacks. Correspondingly, more fungal species (16) were isolated from jute sacks than from woven polypropylene sacks (9). The blotter test showed that in the absence of an exogenous supply of nutrients. 88% of the sections of jute sacks supported in vivo growth of fungal spores whilst woven polypropylene sacks could not support the growth of contaminating spores. Evidence is presented that mould and yeast counts on sections of new-woven polypropylene sacks incubated at 80 and 90% relative humidity (R.H.) for four months increased by less than 1 log cycle, whilst in similar sections hung on a line under ambient conditions (75 ± 10% R.H. and 28± 3°C) viable counts of spores decreased. Sections of freshjut e sacks similarly treated supported 1 log cycle increase in mould and yeast counts at 80% R.H. and 2 log cycles increase at 90% R.H. after four months of storage. Gamma-ray irradiation(4. 0 kGy) reduced the mould and yeast counts on newjut e and new woven polypropylene sacks by 1 and 2 log cycles, respectively, but post-irradiation storage at 80% R.H. allowed moulds like Aspergillus floats, A. niger, Fusarium nivale and Pénicillium verrucosum var. cyclopium to commence growth on jute sacks. This presumably, may act as a springboard for infecting grain contenu of jute sacks. Inert woven polypropylene sacks failed to support fungal growth. Keywords: Packaging materials: Cereal grains: Mycoflora; Jute and woven polypropylene sacks; Gamma-ray irradiation Introduction Flatman (1977) suggested that jute sacks should be replaced by synthetic sacks such asthos ewove n from polypropylene for storageo f cerealgrains ,grai n products, cocoa, animal feed etc.i n the tropics. However,warehousin g agents and the Ghana Cocoa Marketing board continue to usejut e sacks because they are cheaper. The existing standard practice in Ghana of bagging cocoa, rice, maize, sorghum, groundnuts etc. in jute sacks causes defects and loss of over 30% of the annual 0168-16O5/85/SO3.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division) 118 harvest due to insects and fungi. Recent 1983 estimates by the Ministry of Agricul ture showed that stored maize worth US$ 2 million waslos t due to insect and fungal activity. A search for alternative packaging materials for cereal grains and grain products is desirable if maximum benefits are to be derived from radiation decon tamination of mould on cereal grains and grain products. Amoako-Atta (1979) showed that radiation insect disinfestation of cocoa and other seed grains using 0.8 kGy was feasible when kept in woven polypropylene sacks. But this dose was insufficient for killing fungi. Odamtten et al. (1980a) reported that gamma irradiation (4.0kGy )i n combination with heating (60°C for 30 min) administered under high ambient humidity conditions (>85% R.H.) was effective in preventing both growth and aflatoxin B, formation byAspergillus flavus NRRL 5906 in maize stored in woven polypropylene sacks for 4 months at 80% R.H. and 28°C. This same combination treatment decreased aflatoxin B, formation and eliminated Enierobacleriaceaei n animal feed and cotton seeds (Odamtten et al., 1980b).Ther ei sn o information in the literature on the comparison of the efficacy of jute and woven polypropylene sacks in curtailing the re-contamination of grains by fungi. Since maize grains and other cereals are stored over long periods, as buffer stocks before use, it is important to evaluate the performance of the two packaging materials microbiologically because any possible benefit from radiation decon tamination of cereal grains would be negated if the packaging material should support 'quiescent' growth of residual fungal spores after radiation treatment, possibly allowing influx of spores through its lattice work to re-contaminate the contents. Hueck (1965) stated that the mechanical penetration of fungi into a storage sack could be an initiating mechanism for chemical and mechanical damage and soiling. Such mechanical penetration would eventually manifest itself in the reduc tion of theintrinsi c tensile strength of thepackagin g material, lowering the ability to withstand rough transit and warehouse handling. Our objectives in this paper are firstly to survey the natural mycoflora associated with jute and woven polypropylene sacks and find out the effect of gamma irradiation on the moulds. Secondly, the evaluation of pre- and post-irradiation microbiological quality of the sacks and contents after extended storage under ambient warehouse conditions (75 ± 10% R.H. and 28± 3°C) and also representa tive storage conditions similar to tropical high humidity conditions. (80 and 90% R.H.) conducive for growth of moulds. Materials andMethod s Mycoflora ofjute and wovenpolypropylene sacks Three different methods were used; a solid medium test, a blotter test and a serial dilution technique. Solid medium test Sections (5x5 cm) of either fresh jute or woven polypropylene were placed in sterile petri dishes (9.0 cm diameter) containing Sabouraud's maltose agar. Each of 119 the fourteen replicate petri dishes contained only one section. The plates were incubated at 28± 3°C for 5-7 days.Th e species of fungi appearing after 7days , the percentage of each fungus and the total number of fungal colonies appearing were recorded. The experiment was repeated four times. Blotter test In the blotter test, the sections (5x5 cm) of either fresh jute or woven polypro pylene sacks were placed, one in each of fourteen sterile 9.0 cm diameter petri dishes each containing Whatman's filter paper moistened with sterile distilled water without any further additional treatment. The dishes were then incubated for up to 14 days at 28± 3°C (Gemawat, 1968). The experiment was repeated four times and the percentage number of sections showing visible fungal infection viewed under a Prior binocular microscope (W.R. Prior & Co. Ltd., Herts, England) were noted. Serialdilution technique In the third series of tests, twelve sections of fresh or used sacks ofjut e and woven polypropylene (4 x 18 cm) were enclosed in desiccators (with inner environment) adjusted to relative humidities of 80o r 90% with potassium hydroxide solutions after the method of Solomon (1952). There were thus four dessicators with ambient R.H. 80% each containing either 12 sections of fresh jute, fresh woven polypropylene, used jute or used woven polypropylene sacks. In a parallel experiment, four dessicators at 90% R.H. contained the same series of cut sections as mentioned above. Similar sections (4x18 cm) of both fresh and used woven polypropylene sacks were hung on a line outside the laboratory exposed to ambient environmental conditions (75± 10% R.H. and 28 ± 3°C) for 4 months. We exposed 12 sections of each packaging sack in the fresh or used state. The initial and final mould and yeast counts of packaging materials were determined by placing samples into 100 ml 0.1% Peptone as diluent in 250 ml Erlenmeyer flasks shaken in a Gallenkamp orbital shaker at 140 rev./min for 30 min. From this stock suspension, the serial decimal dilution technique was employed and the spores were raised on Oxytetracycline glucose yeast extract agar (OGYE, Oxoid CM 545) for 3 days at 28°C. The number of colonies appearing were counted and the number of cfu/72 cm2 (area of strips) was calculated. Effect ofgamma irradiation onthe natural mycoflora Cut sections (4 x 18 cm) of both used and fresh jute and woven polypropylene sacks were irradiated with 0.0, 3.0 and 4.0 kGy of gamma radiation from a "Co source (Gamma Cell 220, Atomic Energy of Canada Limited) with a dose rate of 2.13 kGy/h. There were 10 cut sections per dose for each type of sack in the used and fresh state.Th e mould and yeast counts/72 cm2o f the sections were determined in the same manner as above. Concurrently, 12jut e and 12 woven polypropylene sacks were each filled with 50 kgo f maize and stored in the warehouse of the Grains 120 WarehousingCompany , Tema. Wecompare d the fungal population of the grains in the two types of packaging materials initially and after 4 months storage. Results Results of the survey of the mycoflora of jute and woven polypropylene are presented in Fig. 1 and Table I. Twenty different fungi were isolated in the solid medium test (Fig. 1). There was a highly significant (Student's /-test, P < 0.05) difference between the higher number of fungal colonies associated with the in cubatedjut e sack sections and the lower numbers counted on the sections of woven polypropylene sacks. Correspondingly, more fungal species were recorded on jute than on wovenpolypropylen e sacks(Tabl e I). Results of theblotte r test showed that in the absence of an exogenous supply of nutrients, 88%o f the section of jute sack supported growth of colonies of Aspergillusflavus, A. niger, A.japonicus, Fusarium R.I R.3 i • Jule @ W»ven polypropylene i k.Pî • • «' '/Fl H »m in rn m R4 0 » 1» 1 A I* i mlJm 'L! !..»Ill . Iyf ki SPECIES OF FUNGI RECORDED Fig. 1. Histogramsshowin g association of fungi with cut sectionso f jute, and woven polypropylene sacks. placed on Sabouraud'smaltos e agar for 7 days at 28±3°C. R.1-R. 4 are replicate experiments. Code: a. Aspergillus niger h, Mucorpusillus o. Mycelia sterilia b. Aspergillus flavus i. Rhizopus oryzae p. Fusarium semitecium c. Drechslern maydis j, Rhizoctonia solani y, Cephalosporium acremonium d. Fusarium moniliforme K Fusarium spp. u. Pénicillium digitatum e. Fusarium oxysporium 1. Aspergillusparasiticus V-,Pénicillium verrucosum f. Fusarium nivale m.Aspergillus japonicus var. cyclopium g. Neurospora sitophila n. Aspergillus ochraceus 4*. Pénicillium expansum. 121 TABLE ! Number of fungal species and lotal number of colonies recorded on the indicated sacks placed on Sabouraud's maltose agar at 28± 3°C for 5day s Replicates Numbero f fungal Total numbero f fungal species recorded colonies recorded on WP' J" wp J R, 10 16 42 100 R, 9 16 44 100 9 R3 14 33 95 R4 9 12 40 115 Woven polypropylene sack. Jute sack. moniliforme. Pénicilliumverrucosum var. cyclopium and Curvularialunata which could be identified after 21 days of incubation. No fungal growth however, was recorded on the woven polypropylene sections in the blotter test. This material, therefore, could not support de novo growth of fungal spores. There was a slight change (less than 1lo gcycle ) in the mould and yeast counts on sections of fresh woven polypropylene sacks incubated at 80 and 9056 R.H. for 4 months whilst fungal spores on used woven polypropylene sacks hung outside the laboratory decreased after 4 months of incubation (Table II). Freshjut e supported 1 log cycle increase in viable counts of mould and yeasts at 80% R.H. and about 1.3 logcycl e increase at 90% R.H. after 4 months. Both fresh and used jute and woven polypropylene sacks carried appreciable amounts of viable fungal spores (Table III), which were reduced by about 2-3 log cycles by 4.0 kGy of gamma radiation. Although irradiation lowered the microbial burden on the two types of sacks, spores of A. flaws, A. niger, Fusarium nivale and Pénicillium verrucosum var. cyclopium remained viable after exposure to 4.0 kGy of gamma irradiation (Fig.2) . TABLEI I Mould and yeast counts on sections of jute and woven polypropylene sacks kept under the indicated humidity conditions for four months at 28± 3°C 2 Storage Mouldan d yeastcount s(Iog 10 cfu/72 < :m ) on: humidity Fresh WP' UsedWP Fresh J b UsedJ (% R.H ) Initial 4 mnth. Initial 4mnth . Initial 4 mnth. Init. 4 mnth. 80 1.00 1.30 3.73 2.0 2.8 3.8 5.6 5.9 90 1.00 1.80 3.73 2.1 2.8 4.1 5.6 5.4 Openai r 1.00 0.60 3.73 2.9 3.8 2.7 5.6 5.6 (75±10%R.H.) * Woven polypropylene sacks. b Jute sack. 122 TABLEII I Theeffec t of gammairradiatio no n the fungal contaminationo f fresh(new )an d usedpackagin gmaterial s Typeo f Mouldan dyeas tcoun t Mouldan dyeas tcoun t (cfu/72 cm-1 ) ' packaging (cfu/72 cm2) onfres h on usedsack s material sacks 3.0 kGy 4.0 kGy 4.0kG y 0.0kG y 0.0 kCy 3.0kG y Jute 13x10' 1.3x10' 9.3x10* 9.2x10s 5.9x10' 4.3xl0: Woven 1.1 X103 9.3X102 "7.3 1.5X10' 6.7x102 6.0x10' polypro pylene Data represents the mean of four replicates. * Sackstha tha dbee nuse d for storingmaiz e for4 monthswer euse d for assessing theeffec t of irradiation on contaminating moulds. G VfcC» JV»t •M M JWII G * 9_ • O * G e G 0 G e ^e A -mM _ *M»M •OM« »otv*«e»*i.i«t w»ift vovt* «oi**ao»*L<«i « G e G * ^ G e G G POU <*Gf1 Fig. 2. Scatter graph showing absence or presence of fungi onnon-irradiate d (0 kGy) and irradiated (3.0 and 4.0 kGy) fresh and used storage sacks. Code: 1. Aspergillus candidus 7. Fusarium moniliforme 2. Aspergillus flavus 8. Fusarium nivale 3. Aspergillus glaucus 9, Fusarium oxysporum 4. Aspergillus niger 10, Pénicillium verrucosum var. cyclopium 5. Aspergillus usius 11, Rhizopm oryzae 6. Curvularia lunaxa 12. Trichodermo viride. 123 TABLEI V Mould and yeast counts of maizegrain sstore d in the fresh sacks for 4 months in theGrain s Warehousing Company's Warehouse at Tema Typeo f packaging Initial mould andyeas t Final mould andyeas t material count onOGY E count on OGYE (cfu/g) (cfu/g) Jute 1.2X102 7.6X104 Woven polypropylene 1.2x10* 6.8xlO 2 Data represents the mean of six readings. During the four months storage of maize grains in fresh jute sacks, the mould and yeast counts increased by more than two log cycles (from 1.2 x 102 to 7.6 x 104 cfu/g). On the other hand, there was only a slight increase (1.2 x 102 to 6.8 X 102 cfu/g) in the mould and yeast counts on maize grains stored in woven polypro pylene sacks for the same period in the warehouse. Discussion This paper reports the first record of fungi associated with jute and woven polypropylene in Ghana (Fig. 1) and thus provides information which warrants further research on the effect of the mycoflora on the decomposition rate of jute fabric during storage under local warehouse conditions. Fungi and insects alike continue to take a heavy toll of our stored cereal grains due to the persistent use ofjut e sacks by our warehousing agents.Jut e sacks contain sufficient nutrients to support fungal growth on 88%o f the sections placed on sterile filter paper. This implies that fungi can attack this packaging material and cause mechanical and chemical damage and soiling as stated by Hueck (1965). Gamma irradiation (4.0 kGy) could reduce the viable mould and yeast counts on both jute and woven polypropylene sacks by 2-3 log cycles (Table III) but subsequent storage of jute sacks at high ambient relative humidity (> 80% R.H.) resulted in resumed growth (Table II) of fungi such as A.flavus, A. niger, F. nivale, P.verrucosum var cyclopium and Curvularia lunata. The presence of fungal spores on the fabric ofjut e sacks not only causes soiling but also alters the appearance of the sacks by their coloured metabolites. Secondly, spores may presumably pass through the lattice work of the sack and act as a spring-board for contaminating grain contents. This partly explains why viable mould and yeast counts of maize grains stored injut e sacks increase by 2.8 log cycles (from an initial 1.2 X10 2 to 7.6 X10 4 cfu/g) in 4 months. On the other hand, woven polypropylene sacks did not support fungal growth. Maize grain contents were of better microbiological quality (6.8 x 102 cfu/g) than those kept in jute sacks for 4 months. According to Beuchat (1978) fungi often decrease germination capacity and cause discolouration of grains and seeds. Using 124 thecriterio n of viability (percentage germination) as an index of fungal deterioration of maize grains, Odamtten (1982) demonstrated that maize grains stored in woven polypropylene sacks under field conditions were significantly (P <0.05 ) more viable than grains stored in jute sacks after 4 months storage period. Based on the microbiological evidence that jute sacks support fungal growth, complementary studies are in progress to (a) confirm that at sufficiently high tropical ambient humidities (> 80% R.H.) fungi contaminating jute packaging sacks could reduce drastically the intrinsic tensile strength of the sacks due to their chemical activity (b) single out fungi that are only surface contaminants of sacks and have no cellulolytic activity. Spores of such fungi can cause soiling and presumably pass through lattice work of sack and subsequently contaminate sack contents. Findings of these studies will be reported in a subsequent paper. Acknowledgements The authors are grateful to Mr. J,K. Edwards, Managing Director of the Grains Warehousing Company, Tema, Ghana for permission and personnel to assist in the field studies in their warehouse at Tema.W e are alsoindebte d to Prof. Dr. J. Parkas. Project Director, International Facility for Food Irradiation Technology, IFFIT, Wageningen, The Netherlands, for his critical comments on the manuscript. Finally, we thank Mr. E. Barko of the Botany Department, University of Ghana. Legon, for his technical assistance. References Amoako-Aua, B.. 1979. Simulated radiation disinfestaiion of cocoa beans in Ghana. Radiât. Phys. Chem 14, 655-662. Beuchat. L.R., 1978. Microbial alteration of grains, legumes, and oilseed. Food Technol. May 1978. 193-198. Flatman, D.J.. 1977.Sack smad efro m Plastic film. Thepackagin gmedia .Chapte r4 ,edite d by F.A. Paine. Blackwell and Sons. Ltd.. London, pp. 3.47-3.93. Gemawat, P.K., 1968. Fusarium monilo/orme in maize seeds from India. A report submitted for the examination of seed pathology held by the government institute of seed pathology. Copenhagen. Denmark, 52pp . Hueck. HJ.. 1965. The biodeterioration of materials and part of hylobiology. Mater. Org. 1. 5-34. Odamtten, G.T.. 1982. Studies on the comparative efficiency of woven polypropylene and jute sacks as possible packaging material for pre- and post-irradiation storage of some cereal grains. Proceedings. African biological network ABN workshop on post-harvest losses and their prevention. International Union of Biological Sciences, September 27-30th, Department of Zoology. University of Ghana. Legon. 32 pp. InPress . Odamtten. G.T., V. Appiah and D. Is. Langerak. 1980a. Production of aflatoxin B| during storage of maize grains subjected to the combination treatment of heat and gamma irradiation. International facility for food irradiation technology, IFFIT. Report No. 13. Wageningen.Th e Netherlands. 37pp . Odamtten, G.T.. V. Appiah. and D. Is. Langerak, 1980b. Preliminary studies on the effect of beat and gamma irradiation on the keeping quality of animal feed and cotton seeds. International facility for food irradiation technology, IFFIT, Report No. 16. Wageningen. The Netherlands. 24pp . Solorrno. M.E.. 1952. Control of humidity with potassium hydroxide, sulphuric acid and other solutions. Bull. Eni. Res. 42, 543-544. 125 InternationalJournal of Food Microbiology, 2(1985 ) 227-237 Elsevier JFM 00065 8.Mycologica l and physical parameters for selecting suitable packaging material for pre- and post-irradiation storage of cereal grains in Ghana G.T.Odamtten ' and E.H.Kampelmache r 2 ' Departmentof Botany. Universityof Ghana, P.O. Box 55, Legon/Accra, Ghanaand ' Laboratoryf or Food Microbiologyand Food Hygiene, Department af FoodScience, Agricultural University, 'Biotechnion', De Dreijen12, 6703 BC Wageningen, The Netherlands (Received 30Jul y 1984; accepted 22 March 1985) Owingt o theroug hwarehous e handlingo f storagesack si n tropical areas inAfrica , a suitablestorag e sack should not support de novo growth of fungal spores because this would reduce the tensile strength of the packaging material and act as a springboard for infecting grain contents. This paper reports the effect of activity of saprophytic fungi on the tensile strength of jute and woven polypropylene sacks. New woven polypropylene sacks carried lower levels of fungal spores (1.3X10 ' cfu/72 cm2) thanjut e sacks(3. 0 X103 cfu/72 cm2) . The natural mould penetration and growth wasexamine d on sections(4x 5 cm) of bothjut e and woven polypropylene after previous incubation at relative humidities of 55, 60,65. 70, 75, 80. 90 and 95% for 10week s by placing themo n Sabouraud'sAgar . Therewa s a significant difference (P - 0.05 level of significance) between the higher penetration of mould growth on jute sacks and that obtained on woven polypropylene sacks. Saprophytic fungi (Aspergillus candidus, A. flams, A. fumigotus,A. niger, A. japonicus. A. parasiticus, A. ustus, Fusarium oxysporium, F. moniliforme. Pénicillium oerucosum var. cyclopium, Rhiiopus oryiae and Trichodermaoiride) isolated from jute sacks reduced tensile strength, measured by an Instron Model 1026. by 50-75% after 10 weeks at 90% R.H. Same fungal species on woven polypropylene sacks did not alter the tensile strength. Woven polypropylene sacks did not absorb moisture whilst the moisture content of jute sacks increased by 5.3-6.0% in 10 weeks at 90% R.H. with concomitant increase in mould and yeast counts by 1-2 log cycles. Evidence is presented to show that there was a positive correlation between the final mycoflora on jute sacks and loss in tensile strength. No correlation, however, was found between the total aerobic bacteria on jute sacks and the concomitant reduction in tensile strength. Fungi therefore play a major role in the reduction of tensile strength of jute sacks. Sterilization by gamma irradiation (8.0 kGy) of jute and woven polypropylene sacks did not affect their intrinsic tensile strength. Woven polypropylene sacks therefore have many microbiological and physical advantagesove rth e traditionaljut e sacks to merit theirus e for grainstorag e in tropical areas like Ghana. Key words: Fungi; Jute sacks, tensile strength; Polypropylene sacks, woven; Cereal grains Introduction The sanitary requirements for preventing food infection and poisoning are embodied in food legislation throughout the world. Selection of the most suitable 0168-1605/8S/S03.30 ° 1985 Elsevier Science Publishers B.V. (Biomedical Division) 126 packaging material can ultimately result in improved shelf-life and better quality while reducing costs, particularly by avoiding undue food losses and waste (Elias, 1979). Packaging research has therefore aimed at developing flexible, light-weight containers capable of withstanding rough handling and storage while retaining their protective properties(Elias , 1979; Küloran and Wierbicki, 1966). The measurement of extent of fungal growth and extent of deterioration of food including cereal grains and grain products is promising and experimentally useful (Tuite, 1978). In this regard, fungal activity on grains contributing to post-harvest losses and quality deterioration has been well documented (Christensen, 1957; Christensen and Kaufmann, 1969;Odamtten , 1981;Oyenira n 1973a,b ;Quase man d Christensen, 1958, 1960; Wallace and Sinha, 1962). But there are limited studies on the effect of contaminating moulds on storage sacks. The tropical environment is amply laden with spores of microorganisms which require the availability of ap propriate substrate, humidity, temperature and pH to commence growth. Ayerst, (1969) stated that some fungi are indeed able to make mycelial growth at relative humidities (R.H.) well below that of other groups of microorganisms. In addition, fungal hyphae can penetrate into a substrate, exploiting substrate by their ability to translocate nutrients. According to Eggins and Allsopp (1975) such growth of fungi into substrates often enables growth and breakdown to continue even when adverse humidities may prevail at the surface. Hueck (1965) stated that the mechanical penetration of fungi on astorag esac kcoul d be aninitiatin g mechanism for chemical damage because fungi can attack materials and cause mechanical and chemical damage and soiling. Chemical damage is caused by all four major groups of fungi. The presence of fungal spores and mycelium on cellulose material, plant fibers such as cotton (Gossypium hirsutum L.), hemp(Cannabis satioa), sisal (Agave spp.) textiles andjut e (Corchorus capsulons, C.olitorius) is objectionable because of the soiling of material caused by their coloured spores and metabolites. Secondly, several fungi are able to cause damage to materials due to their ability to produce extracellular cellulases. Chemical assimilatory damage by fungi mainly manifests itself, in economic terms, as either weight loss (as in food products) or by loss in tensile strength of sack or fabric. A good packaging material should not harbour and support growth of con taminating fungi. It is important, therefore, to evaluate the packaging material microbiologically under adverse and favourable conditions that would promote growth after exposure of the material to gamma radiation. Bothast et al. (1979) reported that fungal growth during transit shipment of corn soya milk in conven tional multiwall paper bags was confined to the bag surface and was not severe. However, when the conventional bags were subjected to conditions conducive to growth,moul d penetrated all layers. Incontrast , polyethylene coated bagswer emor e resistant to moisture and fungal penetration unless the polyethylene coat was fractured. Once penetrated, the polyethylene-coated bags were no better than the conventional ones.W eaime d at measuringchange si n tensilestrengt h and microbio logical quality of jute and woven polypropylene sacks exposed to environmental conditions conducive to mould growth. Our paper provides the first microbiological 127 and physical information which would assist in the eventual selection of the more suitable of the two types of sacks for packaging of cereal grains in humid tropics after irradiation treatment. Materialsan d Methods New and unusedjut e sacks were obtained directly from the Grains Warehousing Company, Tema, Ghana. Jute sacks are presently the conventional packaging material for storing of 50 kg weight of bulk maize in Ghana. Woven polypropylene fabrics (40 warpsx 35 tex, 100 tex: the lattice pattern) were purchased from Polyproducts* Factory, Ring Road Industrial Area, Accra. Qualitative estimation of theinfluence ofstorage relative humidity on the development ofthe natural mycoflora in sacks To examine the influence of relative humidity on the development of mycoflora on the sacks, ten sections (4 x 18 cm) of eitherjut e or woven polypropylene sacks were stored in dessicators with inside R.H. adjusted to 55, 60, 70, 75, 80, 85, 90 or 95% by the method of Solomon (1952). The set up was kept at 30± 2°C for 10 weeks. Thereafter, approximately, 4 x 5 cm sections of eitherjut e or woven polypro pylene sacks stored at the preselected R.H. values wereasepticall y cut and placed on Sabouraud's Maltose Agar in 9.0 cm diameter Petri dishes. There were twenty replicates for each storage humidity and each plate carried only one cut section of the sack. After 3 days incubation at 30± 2°C, a subjective evaluation was made to estimate the extent of fungal penetration. A modification of the method of Bothast et al. (1979) wasemployed . A value of 0 was assigned to sections yielding no fungal growth, 1t o sections one-quartercovere d with fungi, 2 to sections half covered, 3 to sections three-quarters covered with fungi and 4 to sections completely covered. Quantitative assessment ofmicrobiological loadon jute andwoven polypropylene sacks Approximately 72 cm2o f the remaining sectionso fjut e and woven polypropylene sacks kept at R.H. conditions of 55-9556 and 30± 2°C for 10 weeks were used in assessing quantitatively, the microbiological load of the sacks. The sections were aseptically transferred into 250 ml Erlenmeyer flasks containing 100 ml of 0.1% Peptone diluent. Each flask was shaken at 140 rpm for 30 min in a Gallenkamp Orbital shaker, and serial dilutions were plated on either Oxytetracycline glucose yeast extract agar (OGYE), (Oxoid CM 545) for mould and yeast count or plate count agar (PCA) (Oxoid CM 325) for assessment of the total number of aerobic bacteria. All plates were incubated at 30± 2°C and colonies were counted after 3 days of incubation. The predominant moulds were identified based on cultural and morphological characteristics. 128 Effect of thenatural mycoflora on tensile strengthof packaging materials To evaluate the effect of the natural mycoflora on the tensile strength of both packaging materials kept under conditions conducive to fungal growth, two equal sections(4x1 8 cm) of eitherjut e or woven polypropylene were hung on a hook held by cotton wool plug inside one liter Erlenmeyer flasks. About 250 ml potassium hydroxide solution (11.8/6 w/w) provided an ambient R.H. of 90%(Solomon , 1952). Six Erlenmeyer flasks containing eitherjut e or woven polypropylene were incubated at 30± 2°C for 10 weeks. Samples which served as control were sterilized with 8.0 kGy of gamma radiation from a MCo source (Gamma Cell 220, Atomic Energy of Canada Limited). Thereafter approximately 72 cm2 of the sections were assessed microbiologically in the same manner as described above. We measured the extent of mould deterioration (if any) of the two packaging sacks using the criterion of percentage change (loss) in tensile strength measured on an Instron Universal Testing Instrument, Table Model 1026 (Instron Limited, High Wycombe, Bucks,U.K.) . Sections of thejut e and woven polypropylene sacks (4 X 18 cm) werehel d taut in the sample-holding column of the crosshead. The samples were given a full-scale load range of 0-50 kg on a scale factor 5. Record of controls were superimposed on the deteriorating samples to enable us to measure the difference in tensile strength directly. The calculation of the percent loss in tensile strength was obtained from the mathematical relationship: j"=(l- b/a) XIOO Where a is the peak extension for the applied load on control sample, b the peak extension for the applied load on test sample and y the percent loss in tensile strength. We assumed initially that all fungi appearing after the incubation period contrib uted to the decrease in tensile strength. As a follow up. we studied the role of selected individual fungal species in decreasing the tensile strength of jute sack. Sections of jute sack (4x18 cm) were first sterilized by 8.0 kGy of gamma radiation, and the sections (six replicates for each fungus) were inoculated by smearing section using a micropipette containing 1.0 ml aliquot of 2% Tween-80 solution suspending 7.2 x 105 spores ml"1 of either Aspergillus flavus, A. niger, A. parasiticus or Rhizopus oryzae. A Hawksley Haemacytometer Cristalite B.S. 748 (Hawksley and Sons Limited, Sussex, U.K.) was used in estimating the spore density. The inoculated sections were then incubated in 1 1Erlenmeye r flasks on hooks hanging from the cotton wool plugs with an ambient R.H. of 90%provide d by potassium hydroxide solution (Solomon, 1952). The change in tensile strength after 10 weeks incubation at 30± 2°C was estimated as above. Moisture content This was determined in duplicate. Jute sack samples (10 g) were dried at 103°C for 16 h and then reweighed after cooling in a desiccator. Results are expressed on wet weight basis. 129 Results Less than half (mean score < 2) of the sections of woven polypropylene sacks were covered with moulds when they were placed on Sabouraud's maltose agar after 10 weeks incubation at R.H. 55-75% and the moulds never covered three quarters (mean score - 3) even at storage humidity 80-95% R.H. (Fig. 1). In contrast to this, more than half of the sections of thejut e sack were covered with sporulating fungi when placed on the same medium after 10 weeks storage at 60% R.H. and this growth increased to total coverage (mean score= 4) of sections at R.H. 90 and 95%. respectively. Statistical analysis (/ test) showed that differences observed in the extent of fungal penetration between jute sack sections and woven polypropylene sections at each storage humidity were significant (P <0.05) . The quantitative assessment of microbiological load measured in cfu/72 cm2 of surface using the dilution plate method confirmed the trend we obtained employing the hedonic infection scale (Fig. 1).Ther e were more fungal species associated with STOHAOC «CLATIVC HUUfOITV <«fcl Fig. 1. Qualitative and quantitative estimationo f the influence of storage relative humidity (R.H) on the extent of fungal development and penetration into sections of jute and woven polypropylene sacks incubated at S5-9S1 R.H. for 10 weeks at 30±2°C. Qualitative by infection hedonic scale (1-4). 2 Quantitative by dilution plate method (log,0 cfu/72 cm ): O O, jute: A *,, woven polypro pylene; line indicating section half-covered with fungal growth. Inset A: Relationship between number of species isolated from indicated sack material and the storage humidity. 130 Extension Fig. 2. Chan records of tensile strength characteristicso f control (UT) and infected (Inf) samples ofjut e during measurement on an lnstron Table Mode) 1026. A-D arereplicates . Inset Ei s an example of same measurement with woven polypropylene. Crosshead speed -100 mm/min; chart speed - 50 mm/min; Load range0-5 0 kg(ful l scale); scale factor » 5. jute sacks than with woven polypropylene sacks at each storage humidity investi gated (Fig. 1Ainset) . Saprophytic fungi isolated from woven polypropylene did not reduce significantly the tensile strength of the sections of woven polypropylene (Fig. 2E inset). Woven polypropylene sack did not absorb moisture. A loss in tensile strength of 50-75% caused by the natural mycoflora onjut e sacks namelyAspergillus candidus, A.flauus, A. fumigatus,A. niger, A.japonicus, A. parasiticus, A. ustus, Fusarium moniliforme, F. oxysporium,Rhizopus oryzae and Trichoderma viride was recorded. The sections of jute sacks absorbed moisture (from initial 11.4-11.6% to final 17.1-17.6%) with a concomitant increase in mould and yeast count by about 1-2 logcycle s in 10weeks . There was a positive correlation between the final fungal population on jute sack and the extent of reduction in tensile strength jute sack (Fig. 3a) whilst no correlation was found between the total aerobic bacteria count and the percentage reduction in tensile strength (Fig. 3b). Test of significance of the regression coeffi cient showed only the effect of fungi to be significant (P <0.05) . Gamma irradiation (8.0 kGy) did not change significantly the intrinsic tensile strength of jute and woven polypropylene sacks. However, inoculation of the jute sack sections with 7.2 x 105 spores ml"' of selected individual fungi namely A. flavus,A. niger, A. parasiticus and R. oryzae followed by incubation at 90% R.H. for 10 weeks resulted in a comparatively smaller percentage decrease (10.5-17.1%) in tensile strength in the order A. flaous >R. oryzae > A. parasiticus > A. niger (Table I)- 131 TABLE 1 Reduction in tensile strength of jute inoculated with the same initial number of spores of indicated fungi and incubated at 90% R.H. and 30±2°C for 10 weeks Fungal Replicate ^Moistureconten t Inoculum size % Loss in tensile species (ml) strength Initial Final Initial Final (mean± S.E. " Aspergillus 1 11.6±0.3 17.8±0. 2 flavus 2 18.6±0. 3 3 17.6±1. 2 7.2 x10s 3.8x10' 17.1 ±3.1 4 18.2±0. 7 (5.9)* (7.6) 5 16.8±0. 8 6 17.4±0. 5 Aspergillus 1 11.6±0.3 17.5±0. 7 tiger 2 18.1 ±0.3 3 17.3±0. 4 7.2 x10s 4.2X10' 10.5±6. 0 4 17.1 ±0.3 (5.9) (7.6) 5 18.8±0.1 6 18.2±0.2 Aspergillus 1 11.6±0.3 17.5±0. 3 parasiticus 2 17.7±0. 7 3 17.8±0. 4 7.2x10s 3.3x10' 11.3±5. 4 4 17.6±0. 2 (5.9) (7.5) 5 17.6±0. 3 6 18.1 ±0.1 Rhizopus 1 11.6±0.3 18.2±0. 7 oryzae 2 17.6±0. 3 3 17.7±0. 2 7.2 x10s 5.8X10' 12.7±8. 2 4 17.3±0. 5 (5.9) (7.8) 5 17.1 ±0.1 18.1 ±0.1 ' Logl0 cfu/ml are in parentheses. b Value represents mean of six replicates. Discussion In the absence of an exogenous supply of nutrients,jut e sacks could support a de novo growth of contaminating mycoflora on 88%o f the cut sections whilst no fungal growth occured on the woven polypropylene sections under the same conditions (Odamtten and Kampelmacher 198S). The growth of fungi on the sections of jute and woven polypropylene placed on Sabouraud's maltose agar is not unique but the striking difference is the ability of the moulds to grow well even after being at adverse relativehumidit y of 55% for 10 weeks. Eggins and Allsopp (1975) stated that such quiescent growth of fungi into substrates enables growth and breakdown to continue even under adverse prevailing humidities at the surface. The significant difference (P <0.05 ) observed in the greater extent of fungal contamination and 132 penetration ofjut e than of woven polypropylene sections for each storage humidity (55-95% R.H.) confirms our previous findings (Odamtten and Kampelmacher. 1985). Siu (1951) summarised research findings on cellulolytic activity of fungi and he placed Aspergillusfumigatus an d Trichoderma viride among the strongly cellulo lytic group, the moderately cellulolytic ones wpre Fusarium moniliforme, F. oxysporiuman d F. solani. The moulds Aspergillusjaponicus and A. ochraceus were placed in the weak cellulolytic category of fungi whilst A. candidus, A.flaous an d R. oryzae were classified as non-cellulolytic. Basu (1948) reported that his strain of A. niger on jute fibre was non-cellulolytic and A. ustus,A. terreus and A.fumigatus were cellulolytic. Reese and Downing (1951) classified twelve Aspergillus spp. according to their cellulolytic ability and he placed A. flavus, A. ochraceus and A. niger, also isolated in the present study, in the non-cellulolytic group. On the other hand, Flannigan (1970) reported A. fumigatus and A. niger isolated from barley kernels as cellulose decomposing and Mazen (1973) found A. flavus isolated from soil of low cellulolytic activity. Presumably, cellulolytic activity of fungi might be species and strain specific as well as depending on the type of substrate which is being metabolised by the fungus. Several members of the genus Aspergillusappea r to be capable of hydrolysing the /}-(l-4)-glucosidic linkage in the cellulose chain (Alexander, 1961; Raper and Fennel, 1972; Mazen, 1973; Reese and Downing. 1951; Steward and Walsh, 1972). But this does not itself determine whether they can attack cellulose or not since many non-cellulolytic microorganisms possess the Cx enzyme carrying out that reaction (Reese et al., 1950). Judging from our findings, we surmise that the decrease in tensile strength of 50-75% we obtained after 10 weeks injut e sacks could be attributed to the possible cellulolytic activity of A.fumigatus, A. ustus, Fusarium oxysporium and T. viride.I n vitro cultural studies arepresentl y in progress to confirm the cellulolytic potential of these fungi isolated from thejut e sack. Reese and Downing (1951) considered as inactive those organisms which effect a loss in tensile strength of fabrics of less than 15%i n two weeks. Our findings on the reduction in tensile strength of 10.5-17.1% in 10 weeks by A. flavus, A. niger, A. parasiticus and R. oryzae would place them in the category of inactive species (non-cellulolytic) according to Reese and Downing (1951). This implies that their growth and penetration ofjut e sack (without substantial decrease in tensile strength) would be a springboard for infecting grain content of sack. From the mycological point of view,wove n polypropylene is a superior packaging material since it neither supports fungal growth nor absorbs moisture and its tensile strength was not altered by fungi during 10 weeks storage at 90% R.H. Flatman (1977) stated that polypropylene sacks are much lighter in weight than jute sacks and the former has good tensile strength. Sacks made from polypropylene are clean and do not impart any odour or taint to their contents. There is also absence of contamination of loose hair fibres that support a de novo growth of fungi. Jute sacks on the other hand, absorb moisture rapidly and support prolific growth of fungi at high ambient humidity conditions (Fig. 1) to the detriment of the intrinsic tensile strength which was reduced by 50-75% in 10 weeks at R.H. 90% (Fig. 3). This implies thatjut e sacks under such high ambient R.H. conditions would not be able 133 6 rg K cc u . a. <3 O Correlation coofficitnr r'0-912 Corrtlorion coefficient r • 0*126 t 0iSS 'calculâto d •4-44 coleuloro« * P i 0-05 '257 P S 009 «-2-S7' 50 54 58 62 66 70 T« 78 0 50 $4 56 62 66 70 74 78 LOSS IN TENSILE STRENGTH (%) Fig. 3. Relationship between percentage loss in tensile strength of jute and mould and yeast counts (A) and total aerobic plate counts (B) after 10 weeks incubation at R.H. 90% and 30+ 2°C . to withstand rough warehouse handling and might break with increasing load. Indeed, this breaking or rupturing of jute sack is a common phenomenon in our warehouse facilitieswher egrai n foods arekep ti njut esacks . Open sacks with spilled food then become good food bait for fungi which shorten the shelf-life of the grains and alter the chemical quality of the grains by imparting mycotoxins to the food. Both woven polypropylene andjut e sacks can withstand a single gamma steriliza- rion up to 1.0 Mrad (10 kGy) but woven polypropylene is one of the interesting materials for consideration because of its combination of inertness and mechanical properties making it useful for biomedical applications as well (Piester. 1970). Furthermore, for radurization in which the radiation dose does not exceed 1.0 Mrad (10 kGy), the Food and Drug Administration of the U.S.A. has approved the use of several packaging materials (polystyrene, wax coated paper board, nitrocellulose- coated cellophane, copolymer-coated cellophane etc.) including polypropylene for packaging of gamma-irradiated food (Anon, 1967). Currently, woven polypropylene sacks are in use for packing potatoes, vegetable seeds, metallic abrasives, fertilizers, sugar, coffee, rice, wheat and other crops exported to and imported from Europe and America. In Peru, woven polypropylene is used in fish meal trade for export markets. From ourstudies , thepackagin go f cereal grains and animal feed is another obvious future use of woven polypropylene sacks for storage of cereal grains and grain products in Ghana, and in other tropical countries. 134 However, theeffectivenes s of thewove npolypropylen e sack could be augmented by good storagepractice s and cleanlinesso f thewarehouse . Acknowledgements We are grateful to Dr. K. Agyemang and Mr. W.E. Gbedemah of the Physics Department, University of Ghana, Legon, for their invaluable assistance in the measurement of the tensile strength on the Instron instrument. The authors are indebted to Prof. Dr. J. Farkas, Project Director, International Facility for Food Irradiation Technology, IFFIT, Wageningen, The Netherlands for his helpful com ments on themanuscript . References Alexander, M., 1961. Introduction to Soil Microbiology. John Wiley and Sons. New York. Anon, 1967. United States of America, Food and Drug Administration Title 21, Food and Drugs. Chapter 1. Pan 121, Paragraph 121. 2543. Quoted by Killoran. J.J. (1967) Packaging of irradiated foods. Modern Packaging. April, 179-183. Ayerst, G., 1969. The Effects of moisture and temperature on growth and spore.germination in-some fungi. J. Stored Prod. Res. 5.127-141. Basu. S.N., 194S. Fungal decomposition of jute fiber and cellulose. Part 1. A preliminary survey of commonly occurring species. J. Text. Inst. Marchr 39. 232-237. Bothast, RJ., R.F. Rogers and C.W. Hesseltine. 1979. Fungal deterioration of bags during international transport of com soya milk: a test shipment. J. Food Sei. 44. 411-415. Christensen, CM., 1957. Deterioration of stored grains by fungi. Bol. Rev. 23. 108-134. Christensen, CM. and H.H. Kaufmann, 1969. Grain storage, the role of quality loss. University of Minnesota Press. Minneapolis. MN. Eggins. H.O.W. and D. Allsopp. 197S. Biodeterioration and biodégradationb y fungi. In:Th e filamentous fungi, Vol. 1, Industrial mycology, edited by J.E Smith and D.R. Berry. Edward Arnold. Englandpp . 301-319. Bias. P.S., 1979. Food irradiation and food packaging. Chem. and Ind. 19. 336-341. Flannigan, B., 1970. Degradation of arabinoxylan and carboxymethyl cellulose by fungi isolated from barley kernels. Trans. Br. Mycol. Soc. 55 (2). 277-281. Flatman, DJ., 1977. Sacks made from plastic film. In: The packaging media, edited by F.A. Paine, Blackwel l and Son Ltd.. London. Hueck. HJ.. 1965. The biodeterioration of materials as part of hylobiology. Material Organ, 1. 5-34. Killoran,J J . and E Wierbicki, 1966. Development of flexible,container s for irradiated foods: I. Screening of commercially available plastic laminates. Institute of Food Technology. IF T Meeting. Portland. Oregon, May 1966. Mazen, M.B.. 1973. Ecological studies in cellulose decomposing fungi in Egypt. Ph.D. Thesis Botany Department. Faculty of Science, Assiut University. Odamtten, G.T., 1981. A survey of the mycoflora of maize grains stored at 26±3°C and 75±5$ R.H. Proceedings, 12th Biennial Conf. of the Ghana Sei. Assoc.. University of Ghana, April. 1981. Odamtten, G.T. and E.H. Kampelmacher. 1985. Studies on the selection of suitable packaging materials for pre- and posi-irradiation storage of cereal grains in Ghana. Int. J. Food Microbiol. 2, 151-158. Oyeniran,J.O. , 1973a. Microbiological studieso n maize used aspoultr y and livestock feed at two research farms at Ibadan, Western State. Nigeria. Rep. Nigerian Stored. Prod. Res. Inst. 1970 Tech. Rep. No. 6,47-56 . 135 Oyeniran. JO., 1973b. Microbiological examination of maize from various sources soon after harvest. Rep. Nigerian Stored Prod. Res. Inst. 3, 27-32. Plester. D.W. 1970. The effects of sterilization processes on plastics. Biomed. Eng. 5, 443. Quasem. S.A. and CM. Christensen. 1968. Influence of moisture content, temperature, and time on the deterioration of stored corn by fungi. Phytopathology 48, 544-549 Quasem. S.A. and CM. Christensen, 1960. Influence of various factors on the deterioration of stored corn by fungi. Phytopathology 50, 703-709. Raper, K.B. and D.I. Fennell. 1972. The genus Aspergillus. Williams and Willems. Baltimore. U.S.A. Reese. ET., R.G.H. Siu and H.S. Levinson. 1950. The biological degradation of soluble cellulose derivatives and its relationship to the mechanism of cellulose hydrolysis. J. Bacteriol. 59, 48S-497. Reese, E.T. and M.H. Downing. 1951. Activity of the Aspergilli on cellulose, derivatives and wool. Mycologia 43, 16. Siu. R.G.H., 1951. Microbial decomposition of cellulose. Reinhold Publishing Corp. New York, 531 pp. Solomon. M.E.. 1952. Control of humidity with potassium hydroxide, sulphuric acid and other solutions. Bull. Ent. Res. 42. 543-544. Steward. C. and J.H. Walsh, 1972. Cellulolytic activity of pure and mixed cultures of fungi. Trans. Br. Mycol. Soc. 58. 527. Tuite. ].. 1978. Use of fungi and their metabolites as criteria of grain quality and storage systems. Proc. 1977 Com Quality Conference, University of Illinois, AE 4454. Wallace. H.A.H. and R.N. Sinha. 1962. Fungi associated with hot spots in farm grain. Can. J. Plant Sei. 42. 130-141. 136 InternationalJournal of Food Microbiology, 3(1986 ) 57-70 Elsevier JFM 00086 9. Influence of packaging material on moisture sorption and the multiplication of some toxigenic and non-toxigenic Aspergillus spp.infectin g stored cereal grains, cowpea and groundnut G.T. Odamtten ' and E.H. Kampelmacher 2 ' Departmentof Botany, Universityof Ghana, PostOffice Box 55,Legon /A ccra, Ghana, and ' Laboratoryof Food Microbiology and Hygiene, Departmentof Food Science, Agricultural University, DeDreijen 12,6703 BC Wageningen, The Netherlands. (Received 19Decembe r 1984; accepted 16Novembe r 1985) The moisture sorption isotherms of jute sack were determined at 12, 22, and 32°C by a standard procedure usingglycerohwate r mixtures providingequilibru m relativehumiditie s (ERH) of 65. 70. 75,80 , 90 and 95%. The equilibration period of jute sack kept at 65-85% was found to be between 6-8 days whereas themoistur e content (MC)an d weight ofjut e sack kept at 90-95%continue d rising. Theamoun t of moisture absorbed at 12°C was significantly greater (F < 0.05) than at 32°G Analysis of variance applied to collected data of adsorption at ERH 85% (aw 0.85) and desorption ERH 20% ( Keywords: Moisture sorption isotherms; Jute sack; Toxigenic fungi; Aspergillusspp. . non-toxigenic; Cereal grains; Legumes Introduction In considering storage potential, the ambient equilibrum relative humidity (ERH) is an important parameter as it determines the amount of water available to microorganisms and hence is an indication of the biological activity or potential 0168-1605/86/S03.S0 © 1986 Elsevier Science Publishers B.V. (Biomedical Div)sion) ] 37 activity of the product (Ayerst, 1965). Above 75% RH, products absorb moisture rapidly and fungi develop rapidly during storage, and heating of the product would produce subsequent deterioration and loss of quality. RH below 75% is accepted as 'safe' for storage of food commodities e.g. cereal grains. A packaging material for cereal grains and legumes could either restrict passage of water vapour through the sack or may itself absorb and transfer moisture to its contents. A packaging material that would restrict passage of water vapour would also curtail increase and decrease in weight or moisture content of the enclosed product and could, therefore, extend the shelf-life of the food commodity. The persistent use of jute sacks in packaging of cereal grains, pulses and other food commodities in Ghana has been attended by the loss of > 30% of annual harvest of crops due to insect infestation and growth of fungi. The pertinent literature has rarely any information on the moisture sorption of jute sacks under ambient tropical conditions and no comparative studies have been carried out to elucidate how water sorption of food commodities stored in jute sacks differ from the same commodities stored in other packaging materials such as woven polypro pylene sacks. The objectives of these studies were (a) to determine the moisture sorption isotherm ofjut e sack kept under pre-selected ERH values (65,70 , 75, 80, 85,9 0 and 95% RH) (b) to examine the influence of packaging materials on moisture sorption at 85% ERH and desorption at 20% ERH of cowpea. groundnut (shelled), maize (white), millet, and sorghum stored in either jute or woven polypropylene sack - a suggested replacement for jute sack in tropical areas like Ghana (Odamtten and Kampelmacher, 1985a,b) (c) to study the multiplication of five potential mycotoxin- producing fungi (Aspergillus flavus, Aspergillus ochraceus,Fusarium moniliforme, Pénicillium digitatum and Pénicillium expansum) and five non-toxigenicAspergillus spp. (A. candidus, A. niger, A. terreus, A. sulphweus and A. panamensis) under the moisture sorption humidity (85% RH) and desorption (20% RH) humidity condi tions during storage for six months. Materials and Methods White maize (Zea mais L) millet (Pennisetum americanum) shelled grouridnut, variety florispan runner (Arachis hypogeae L),cowpe a red variety (Vigna unguiculata Walp.)an d sorghum, local Accra red (Sorghumbicolor subsp. bicolor) were obtained from the market whilst unusedjut e and woven polypropylene sacks were supplied by the Grains Warehousing Company, Tema and the Polyproducts* factory, Accra, Ghana, respectively. Moisture sorption isotherms ofjute sack This was determined by a standard method using glycerol:water mixtures to provide ERH values of 65, 70, 75, 80, 85, 90 and 95%. Seven desiccators (diameter 22 cm) were used to hold the humidity solutions. Forty sections of jute sack each 138 weighing 5.0 g were placed at each RH inside the desiccators and were allowed to start moisture sorption at 32°C in a Freas 816 low temperature incubator (GCA/Precision Scientific Co., U.S.A.). The weights and moisture content of the samples were determined at regular intervals (2, 4, 6, 8, 10, 21,28 , 32 and 52 days) until they reached equilibrum. The moisture content was determined at each predetermined incubation period as outlined below. Moisturecontent determination The moisture content (%) of jute sack samples stored at ERH values between 65 and 95% were determined in duplicate before and after every indicated incubation period. The samples were dried at 103°C for 16 h. For maize, millet and sorghum, the International Standards Organisation method was employed (ISO 2291). Dupli cate 10 g ground samples were dried at 103°C for 72 h. Cowpea and groundnut samples were dried at 113°C for 4 h in a mechanically ventilated Gallenkamp oven (Oxley et al., 1960). Effect of temperature on moisture sorption byjute sack To find out the effect of temperature on moisture sorption, quadruplicate 5 g samples of jute sack were stored at ERH of 65-95% and at temperatures of 12, 22 and 32°C for 14 days. The internal ERH values of the desiccators were adjusted by glycerol:water mixtures. After the equilibration period of 14day s the weight and the percentage moisture content of the jute sack strips were determined as outlined above. Influenceof packaging materialon moisture sorption and desorption byfood commod ities Four replicate samples (50 g) of cowpea, shelled groundnut, white maize, millet and sorghum were placed in satchets (10 cm X 5 cm) of either jute or woven polypropylene. The listed commodities in the two different packaging materials were then equilibrated to either ERH 85% for absorption, or to 20% for desorption at 32°C in desiccators where the ambient ERH was controlled by glycerol:water mixtures. The course of the moisture changes was followed by regular weighing of samples for up to 30-34 days. Effect ofpackaging material andstorage humidityon microbiological quality Mould and yeast counts on naturally contaminated samples, initially, and after 2 and 6 months storage were estimated by pouring 10g o f the appropriate sample into 100 ml 0.1%pepton e solution in 250 ml Erlenmeyer flasks which were subsequently shaken in a Gallenkamp Orbital shaker for 30 min at 140 rev/min. Serial dilutions were made from the stock suspension up to 1:106. 1 ml spore suspension was cultured on Oxytetracycline glucose yeast extract, OGYE (Oxoid CM 545). Char- 139 acteristic colonies appearing after 3 days incubation at 28°C were counted. From this data the microbial loads, (log CFU g~' sample) were calculated. Estimationof germination capacity and length of emerging radicles As an index of incipient deterioration owing to fungal activity in food commod ities, the germination capacity of the samples was assessed using the blotter method of de Tempe (1967) and Limonard (1966). Seeds and grains with emerging radicles were considered to have germinated. The lengths of the emerging radicles of germinating seeds were measured for each storage sack stored at ERH values 20 and 85%. About 250 grains or seeds were used from each sack and storage humidity combination for this assessment. Speciescomposition and relative occurrence offungi Quantitative data on the number of fungal species isolated from the commodities and their relative occurrence was noted for all food commodities in the two types of packaging material.Th e relative occurrence of five toxigenic fungi (Aspergillus flavus NRRL 5906, A. ochraceus NRRL 6514, Fusarium moniliforme NRRL 6413, Pénicil lium digitatum Sacc. and P. expansum D19) and five non-toxigenic (Aspergillus candidus. A. niger,A. terreus. A. sulphureusan d A. panamensis from the Centraal Bureau Schimmelcultuur, Baarn The Netherlands) was studied. These strains were similar, in cultural characteristics, to the local Ghanaian isolates. Results Moisturesorption isotherms ofjute sack In Fig. 1, results of the moisture sorption of jute sack are summarised. The equilibration periods of samples kept at ERH 65-85% were found to be between 6-8 days.Th e moisture content ofjut e sack kept at 90-95% ERH continued rising. Effect of temperature onmoisture sorption byjute sack Fig. 2 summarizes the results obtained. Temperature significantly influenced the amount of moisture absorbed byjut e sack (P <0.05) . Both the moisture content and weight of the sack at the lowest temperature used (12°C), were significantly higher (P <0.05 ) than at the highest temperature used (32°C). Weight of sack and moisture absorption at 22°C lay between the two temperature extremes. Influenceof packaging material on moisture sorption byfood commodities The moisture sorption (85% RH) and desorption (20% RH) isotherms of cowpea and groundnut are summarised in Fig. 3, those for maize in Fig.4 and for millet and 140 16 20 24 26 32 36 PERIOD OF INCUBATION IN DAYS Fig. 1. Moisture sorption isotherms ofjut e sack stored at 32°C and the indicated humidities for 52days . sorghum in Fig. 5. Application of analysis of variance in order to examine the influence of incubation period (A), type of commodity (B) and type of packaging material (C), as well as the interaction of these factors showed that A, B and C 7-0 60 65 70 75 80 85 90 95 60 65 70 75 80 85 90 95 RELATIVE HUMIDITY (%) Fig. 2. Effect of storage temperature on weight and moisture content ofjut e sacks incubated for 14days . 141 20% R. H. (oaoaw) GROUNDNUT A B ! ., • a - / jr-""* 85% R. H. Jjr (0-850w) s ' 2 0 ^. 10 20 30 36 tu -I -VV^^ PERIOD OF INCUBATION IN DAYS o I —_, t ; o-3 -4 -5 20% R.H. (O-2O0w) -6 Fig. 3. The changes in weight of cowpea (top) and shelled groundnut (bottom) exposed to 20% and 85% ERH at 32°C for 30-34 days A a jute; • • woven polypropylene. significantly (P <0.05 ) influenced the moisture sorption and desorption by the five listed food commodities. However, interaction between A and B,A and C as well as B and C and A, B, C was highly significant (P <0.05) . Closer examination of the interaction of A and C showed that cowpea, groundnut, maize, millet and sorghum injut e sacks significantly absorbed and desorbed moisture to a greater extent than same produce stored in woven polypropylene sacks. For all food commodities water sorption differed according to the packaging material. Microbiological quality Ambient storage ERH and type of packaging material had an effect on mould and yeast count of food commodities. We found counts to be 0.5-1.0 log cycles 142 A J • W P 83% R.H. (0-860w) 10 20 30 PERIOD OF INCUBATION IN DAYS 20% R.H. (0-200w> MAIZE D B __„. - - 85% R.H. K 3 b^ ^-*^" (0850w) I u 2 ÜJ / %y S I S 0 10 20 30 36 PERIOD OF INCUBATION IN DAYS zI» -I «t-2 \• *-—•—.—. . . I °-S '—A « * t -4 - 20% R.H. -5 (0-20ow) -6 L Fig. 4. The changes in weight of maize exposed to 20% and 85% ERH at 32°C. Maize II is a repeat experiment. lower on commodities stored in woven polypropylene at 20% ERH for 2 months (Fig. 6A) than injut e sack and this difference increased to 1.0-1.5 log cycles after 6 months (Fig. 6B). Correspondingly, counts on commodities incubated at ERH 85% was 1-4 log cycleslowe r in woven polypropylene (depending on commodity) than in jute sack after 2month s storage, but this changed to 2-3 logcycle s in 6 months (Fig. 6B). Theseobserve d differences were statististically significant (P <0.05) . Both ERH and type of packaging material had a significant effect on the occurrence of toxigenic fungal species on the food commodities. Viable A. flauus spores occurred in cowpea, groundnut, maize and millet stored at both ambient 20% RH and 85% RH but not on sorghum. Groundnut was not infected by A. ochraceus 143 20% R.H. (0-20aw) 6 SORGHUM 5 4 i —"—*— d * 3 .^*— • —- 1- 2 ^ 85% R.H. X 2 1 (0-85aw ) Ui * 0 10 20 30 36 z . PERIOD OF INCUBATION IN DAYS ü-i 2 5-I-3 -4 - 20% R. H. -5 (0-200w) -S Fig. 5. The changes in weight of millet (top) and sorghum (bottom) exposed to 20% and 85% ERH at 32°C for up to 34 days. whilst F. moniliforme infected all commodities stored at both 20 and 85% ERH except sorghum, groundnut, and cowpea which were not infected at 85% ERH. Only maize grains were not contaminated by P. digitatum (Fig. 7A). Finally, Pexpansion could not be isolated from all commodities stored injut e sack at 85% ERH but only maize, groundnut and millet stored at 20% ERH contained the spores of P. expansum. In all instances, significantly greater percentages of toxigenic fungi were recorded in commodities stored in jute sacks than in woven polypropylene sacks whilst only commodities stored in jute sack harboured toxigenic fungi (Fig. 7A). Infection by toxigenic fungi was reduced considerably after 6 months.(Fig .7B) . Data for multiplication of non-toxigenic Aspergillus species on the five food commodities after 2 and 6 months storage are summarised in Fig. 7C and D. The 144 145 146 food commodities were infected variably with the different species of the non-toxi- genic fungi. Mould occurrence was significantly higher (P < 0.05) injut e sacks than in woven polypropylene sacks (Fig. 7C). The populations of non-toxigenic Aspergil lus species on the food commodities were higher at ambient ERH 85%tha n at 20%. After 6 months storage, the population of non-toxigenic moulds decreased in all instances so that A. candidus and A. niger could not be isolated from groundnut, millet and sorghum; A. sulphweus was not present on any food commodity stored at 20% ERH whilst A. panamensis could not be detected on all food commodities stored in both jute and woven polypropylene sack at both 20% and 85% ERH (Fig. 7D). Cowpoa Groundnut( »helled ) 120 80- *0- „ 160 Maize Milt»! E E i ™> <5 IT t to- x 5z tu 40 0 Î ' 6 160 Period of incubation*months) Sorghum A Wovon polypropylene 120- >aw sO-20 A Jute sack J(R.H.20V.) o woven polypropylene [a^.O-SS • JuttMCk J(RH. 85' 2 6 Period of incubation( months ) Fig. 8. Figure showing comparative changes in lengths of emerging radicles of germinating legumes and grains stored in either woven polypropylene or jute sack under ERH 20% and 85% for up to 6 months. 147 Germination capacity Results obtained are summarised in Figs. 6C, 6D and 8. Both storage ERH and type of packaging material affected the germination of seeds and grains of the five food commodities as well as the length of the emerging radicles. Application of analysis of variance in order to examine the influence of type of commodity, type of storage sack, and period of incubation showed that the germination capacity of the grains and seeds differed according to packaging material. Percentage germination in jute sacks was significantly lower (P <0.05 ) than that obtained in woven polypro pylene sacks (Fig. 6C) and the emerging radicles were shorter in length than in the same seed lots stored in woven polypropylene sacks (Fig. 8). Seed lots of commod ities stored at 20% ERH were statistically more viable than those incubated at 85% ERH (P< 0.05). Moisture content The decreases in moisture content of seed lots injut e sacks for six months at 20% ERH were as follows: cowpea (12.3± 0.7-8.2 ± 0.1%); groundnut (6.9± 0.1-5.4 ± 0.3%); maize (12.0 ±0.6-11.6 ±0.3%); millet (10.6 ± 0.2-8.4 ± 0.5%) and sorghum (9.0 ± 0.0-7.8 ± 0.2%). At 85% ERH moisture contents after six months for the five food commodities were as follows: cowpea (17.3 ± 0.1%); groundnut (10.2 ± 0.1%); maize (15.6 ± 0.3%); millet (16.8 ± 0.3%) and sorghum (17.8 ± 0.2%). Moisture con tents of seed lots kept in woven polypropylene sacks were 0.8-2.3% lower than in jute sacks. Discussion Moisture sorption characteristics of jute sack (Fig. 1) were similar to those of maize grains kept under ERH 65-95% (Odamtten and Langerak, 1980). However, the amount of moisture absorbed by jute sack at 12°C was significantly larger (P <0.05 ) than at 32°C. Hayakawa et al. (1978) also observed the same phenome non in coffee products. Different products at same ERH may have different water contents (Lacey et al., 1980). Cowpea, groundnut (shelled), maize (white), millet and sorghum stored in jute sacks desorbed moisture to a greater extent than same seed lots stored in woven polypropylene sacks and this was reflected in the lower moisture contents recorded for seeds kept in woven polypropylene. Given the fact that each food commodity behaved differently, woven polypropylene is then a semi-barrier to moisture transfer to and from the sacks and the seeds. Mould and yeast content on all food commodities stored at ERH values of 20% and 85% in woven polypropylene sacks were significantly lower than in jute sacks (Figs. 6 and 7). The viability of fungi isolated from the food commodities stored at low ERH 20%i sno t surprising. Although there was no visible growth of moulds, the spores remained viable and could commence a 'de novo' growth on suitable media. Eggins and Allsopp (1975) stated that quiescent growth of fungi into substrate 148 enable growth and breakdown to continue even under adverse prevailing humidities at the surface. The presence of greater percentages of both toxigenic and non-toxi- genic fungi on the food commodities stored in jute sacks than in woven polypro pylene makes jute an inferior packaging material for the legumes (cowpea and groundnut) and the cereals (maize, millet and sorghum). Germination is frequently referred to as one of the criteria upon which the condition of grains and seeds can bejudged . In this paper, germination and viability are taken to be the same. Christensen and Kaufmann (1969) provided evidence that storage fungi are one of the main causes of loss of viability of seeds during storage. Storage fungi like A. candidus, A. flavus, and A. restrictus reduced percentage germination drastically from 97% to 13%o r 0% in peas and maize respectively when stored for periods of 74 days up to 12 months (Lopez and Christensen. 1967; Fields and King, 1962). The critical moisture contents for safe storage of legumes and cereals in the tropics at 27°C and 70% ERH have been provided by Muckle and Stirling, (1971) as follows: cowpea, 15.0%; groundnut (shelled), 7.0%; maize(yellow) , 13.0%; maize (white), 13.5%; millet, 15.0%; and sorghum, 13.5%. Our data suggest that at 20% ERH the moisture content of the seeds was on the 'safe' side in both woven polypropylene and jute sacks. However, storage at 85% ERH reduced drastically the viability and length of radicles of germinating seeds and increased the moisture content of seeds above the 'safe' level with the attendant increase of fungal deterioration particularly in jute sacks such that 0% germination was obtained for groundnut, maize and millet after 6 months storage injut e sack (Fig. 6C & D). We conclude that, placed under the same ERH and temperature conditions, the five food commodities behaved differently in jute and woven polypropylene sacks. Each food commodity absorbed and desorbed moisture to a significantly greater extent (P <0.05 ) in jute sack than in woven polypropylene. Woven polypropylene sack offered better protection from moisture than jute sack. The microbiological quality of the grains and seeds kept in woven polypropylene sack was better than in jute sack. This was reflected in the mould and yeast counts and the occurrence of toxigenic and non-toxigenic fungi. These fungi reduced the viability and length of radicles of seeds and grains stored in jute sacks for six months (Fig. 8). Woven polypropylene is therefore microbiologically and physically a better storage material for cereals and legumes (Odamtten and Kampelmacher, 1985a,b). We surmise that application of gamma irradiation and other treatments for mould decontamination of cereal grains and legumes in tropical areas like Ghana could be augmented by post-treatment storage of the food commodities in woven polypropylene sacks. Acknowledgements We are grateful to Mr. K.K. Etsibah of the Institute for Statistics, Social and Economic Research, ISSER, University of Ghana for computer analysis of data, Dr. S. Sefa-Dedeh, Department of Food Science, University of Ghana and Prof. J. Farkas. Central Food Research Institute, Budapest, Hungary for useful comments on the manuscript. We thank Mr. E.K. Barko for technical assistance and Mr. A.A. Brown for stencilling the graphs. 149 References Ayerst, G., 1965., Water activity - its measurement and significance in biology. Inst. Biodetn. Bull. 1. 13-26. Christensen, CM. and H.H. Kaufmann, 1969.Grai n storage. The role of fungi in quality loss. University of Minnesota Press. Minneapolis, MN, 1S3pp . de Tempe, J., 1967. Routine investigations of seed health condition in the Dutch seed testing station at Wageningen. Proc. Int. Seed Test Assoc. 22, 1-12. Eggins, H.O.W. and D. Allsopp. 197S. Biodeterioration and biodégradation by fungi. In: The filamentous fungi, Vol. 1, Industrial Mycology. Chapter 5, edited by J.E. and D.R. Berry, Arnold Publishers Ltd., England, pp. 301-319. Fields, R.W. and T.H. King, 1962. Influence of storage fungi and deterioration of stored pea seed. Phytopathology, 52, 336-339. Hayakawa. K.-I., J. Matas and M.P. Nwang, 1978. Moisture sorption isotherms of coffee products. J. Food Sei. 43, 1026-1027. Lacey, I., S.T. Hill and M.A. Edwards, 1980. Microorganisms in stored grains: their enumeration and significance. Trop. Stored Prod. Inf. 39, 19-32. Limonard, J., 1966. A modified blotter test for seed health. Neth. J. Pathol. 72. 319-321. Lopez, L.C. and CM. Christensen, 1967. Effect of moisture content and temperature on invasion of stored corn by Aspergillusflavus. Phytopathology 57, 588-590. Mucicle, T.B. and H.G. Stirling, 1971. Review of drying of cereals and legumes in the tropics. Trop. Stored Prod. Inf. 22, 11-30. Odamtten, G.T. and D.Is. Langerak. 1980. Moisture sorption of two maize varieties kept under different ambient relative humidities. International Facility For Food Irradiation Technology IFFIT Report No. 9, Wageningen, The Netherlands, 13 pp. Odamtten, G.T. and EH. Kampelmacher. 1985a. Studies on the selection of asuitabl e packaging material for pre- and post-irradiation storage of cereal grains in Ghana. Int. J. Food Microbiol. 2, 151-158. Odamtten, G.T. and EH. Kampelmacher, 1985b. Mycological and physical parameters for selecting suitable packaging material for pre- and post-irradiation storage of cereal grains in Ghana. Int. J. Food Microbiol. 2. 227-237. Oxiey, T.A., S.W. Pixton and R.W. Howe, 1960. Determination of moisture content in cereals. I. Interaction of type of cereal and oven method. J. Sei. Food Agric. II, 18-25. 150 10. SENSORY EVALUATIONAN DSOM E QUALITY PARAMETERSO FMAIZ E COMBINED-TREATED WITH HEATAN DGAMM A IRRADIATION Abstract This paper presents preliminary results of a consumer acceptance test performed with a popular Ghanaian Kenkey made from combined-treated corn dough presented to twenty adult panelists. Krammer's Quick Rank Test showed that there was no significant difference (P = 0.05) in colour, flavour and taste between the control and the combined- treated maize grains. Starch viscosity measurements showed that the change in viscosity was not directly due to the combined heat and radiation treatments. Heat-treated and combined-treated maize grains yielded more reducing sug ars (as mg l" dextrose) than the unheated controls. 10.1 Introduction Many consumerspurchas eo raccep tne wproduct so nth ebasi so fsensor y experiencewhic hi tdelivers . Sensoryevaluatio nha sbee n used fora lon g time todetermin ewhethe ra ne wproduc tmatche s some goalso rtarge t (Civille,1978) . Accordingt oBlai r (1978),th e sensory evaluation processpermit s onet o forma data-base dpoin to fvie w aboutyou rproduct .I tgive syo u approximation ofoveral l acceptability. Food irradiation standsi ncompetitio nwit h other conventional meanso ffoo dpreservatio nprocesse san dther ei snee dt oasses s theacceptanc eo fcombined-treate dgrain si non eo fth e2 2foo dpreparation s made frommaiz e grainsi nGhana . 10.2Material san dmethod s Batcheso f1k gmaiz e grainswer e eitherheate da t6 0° Cfo r3 0min .i na specially designed heat-treatmentchambe r thatenable du st ocarr you tth e heating under eitherlo w(<45 %R.H. ) ambienthumidit yo rhig h (>_85$R.H. ) ambienthumidity .Th etw ocontro l sampleswer emaintaine da t2 0° Cfo r 30min . under theprescribe dhumidities . The grainswer e irradiated (within3 0min. )wit h0 o r4 KG yo fgamm a radiationwit ha sourc e dose rateo f0.0 6 KGymin . The sampleswer e soakedseparatel y (1k gi n1 litr e ofwate r for4 8hr ) and thenblende d separately into flour.Th eresultin g flourswer ewette dt o 151 Fig. 1. Sensory quality of con trol and combined-treated maize grains. (Differences in rank sums between the lowest and highest limits, 52-88, are not significant at P<_0.05.) Acceptability limit of mean score is 5. O 40 40 4« DOSE(KGy) •'•**& Te 1-4. 12 KEY Fig. 2. Comparison between 601, 0 KGy 10— viscosity measurements of -420L4.0KGy control and combined-treated 20L;O KGy samples. 04 O -O20H, 0 KOy 0-6 -A60H,0 KGy 0-1 0-2 0-3 04 0.5 0-6 CONC.O FSTARC H CM 152 forma doug han d allowed toinitiat e fermentationa t2 5 C for4 8hr .There after,KENKEY ,a popula rGhanaia nmea lwa sprepare dusin gth eloca lconven tionalmetho dan dth ekneade d doughcovere dwit h 'com leaves'boile do na gas cooker for9 0min . Coded samples representingal lth etreatmen tcombination swer epresente d toth e 20panelist swh ower e asked tojudg e thesample so na hedoni cscal e 1-10 (1-2bad ;3- 4 objectionable;S limi t ofacceptance ;6 medium ; 7-8 good; 9-10 excellent).Result swer e assessedusin gKrammer' sQuic kRan kSu mTest . Crudeextrac to fmaiz e starchwa s obtainedfro m 100g o fblende dsample s previously soaked inwate r for4 8hr .Th esample swer edissolve di n 1litr eo f distilledwate r and thenboile d for20-3 0min .befor estrainin g throughchees e fine cloth.Th emaiz e starchsolution swer e diluted tofor m0.1 ,0.2 ,an d0.5 1 starchsolution san dth eviscositie so fth estarc hwer ecompare dusin ga n UBBELOHDEviscosimete ra t 32°C .Th especifi cviscositie swer e calculated thus: nsP=^~ V inwhic h n =specifi cviscosit y tw, = time forwate r tofal lA toB 23.5sec . t = time forsampl e to fall fromfixe dpoin tA toB . n = sp nc concentration ofstarc h Thevalue so fn obtained foreac hsampl ewer eplotte d againstconcentration . Reducingsugar s (asdextrose )wer e estimatedusin gFehling sA andB so lutionaccordin g toth emetho do fLee s (1968). 10.3Result s Therewa sn o significant difference (P=0.05 )betwee ncolour ,flavou ran d taste ofcontro lan d combined-treated grains (Fig. 1). Differences inran ksum s between thelowes t andhighes t limits (52-88)wer eno t significant atP = 0.05 . The acceptability limito fmea nscor e is5 . Theviscosit ymeasurement s showed thatth ecombined-treate d grainsde crease inviscosit y (Fig.2) .Bu t thedecreas e inviscosit ywa sno t directly related toth ehea tan d irradiationtreatment . Theestimate d reducing sugarconten t ispresente d inTabl e 1.Generall y 153 combinedtreate dsample syielde dmor e reducing sugars than thecontrols . 10.4Conclusio n Maize grainscombined-treate dwit hhea tan d irradiationwoul db eaccept ablet oth econsumers .Th edecreas ei nviscosit ywhic hi sreporte dt ohav e broughtabou thighe r digestibilityo fgrain si sadvantageous .Code xAlimenta - riusWholesom e Commission's acceptancea sirradiate d food subjectedt ooveral l doseo f1 0KG y givesimpetu s toth epractica l applicationo fou rfindings . Table 1.Comparativ e reducing sugaryield s of combined-treated maize grains starch. Treatment Dose applied Calculated value of reducing (KGy) sugar as dextrose (mg.l ) 20L 0.0 30.1 4.0 31.7 20H 0.0 31.6 4.0 48.1 60L 0.0 39.3 4.0 * 66.1 60H 0.0 52.3 4.0 57.3 L - Low Humidity (<45%R.H. ) H -Hig h Humidity (>85% R.H.) Jt Combined treated samples Calculated value of reducing sugar dextrose frompur e Sample - 60.2 mg.l". References Civille,G.V. , 1978.FoodTechnol . Nov., 1978:59-60 . Blair,J.R. , 1978.Foo d Technol.Nov. , 1978:61-62 . Lees,R. , 1968.Laborator y Handbook ofMethod s of Food Analysis,Leonar d Hill Books,London . 1S4 11. MICROBIOLOGICAL QUALITY AND PRODUCTION OFAFLATOXI N BjB Y ASPERGILLUS FLAWS LINK NRRL 5906 DURINGSTORAG E OFARTIFICIALL Y INOCULATEDMAIZ E GRAINS TREATEDB YA COMBINATION OFHEA TAN D RADIATION G.T.Odamtten ,V .Appiah ,D.I .Langera k Abstract Maize grains artificially inoculatedwit h 1.3x106 c.f.u/go fspore s of Aspergillus flavus LinkNRR L590 6wer ekep ti nope ncontainer s eitherunheate d (20°C )o rheat-treate d (60° C for 30min. )unde rlo whumidi y (<45$R.H. )o r highhumidit y (>85$R.H. ) conditions andth egrain s subsequently irradiated within 30min .i nwove npolypropylen e sacks,wit h 0.0, 3.5,o r4. 0 KGy.Th e grainsample swer e storeda t65 1R.H . and2 8° C for 1mont han dthe na t80 1 R.H. forth e following 3months . Moisthea t treatment (60° C for3 0min. ,>85 $R.H. )doe sno t increasesig nificantly (P= 0.05 ) theinitia lmoistur e contento fmaiz e grains (above13 1 m.c.)bu t reduced theinitia lmoul dan dyeas t countb y0. 9 logcycle san dth e totalaerobi cbacteri a countb y 0.3 logcycles .A combinationo fmois thea t and4. 0 KGyhowever ,lowere d theinitia lmoul dan dyeas t countb y 5.1 log cycles and the total aerobicbacteri acoun tb y 4.2 logcycles .Incubatio n of 651R.H . and 28° C for 1mont haugmente d thekillin g effecto fth e radiation treatment.Afte r 3month sstorag e at 801R.H . thepopulatio no fmoul dan d yeast as well as the total aerobicbacteri ao fth ecombine dtreate dgrain s (60°C ,4. 0 KGy)remaine dnearl y thesam e (i.e.5. 0 and4. 3 logcycle sreduc tionrespectivel y fromth e initial)wherea s controlo fth emois theat-treate d grainsha dmoul d andyeas t and totalaerobi cbacteri acount slowere db y 1.5 and 1.3 logcycle s respectively after 3month s storage at 801R.H .Th e grains did notbecom e rancid. Based on thesensitivit y ofth emetho d foraflatoxi n B1 detection,w e found fromtriplicat e samples thatonl y control (20L an d 20H )grain scon tained0.8-4. 0ug/k go faflatoxi nB. .afte r3 month s storage at80 $R.H . and 28° Cexcep tth e control (20H )grain s given4. 0 KGy. 155 20°C TOTAL AEROBIC Fig. 1. Showing effect of the MOULDAN DYEAS T BACTERIA combination treatment on ( logg CFU/g ) QQo (log ^ CFU/g) microbiological quality of 6 maize grains. DOSE (KGy ) »•C Motetura Content(% ) Fig. 2.Relationshi p between moisture content, combination treatmentan dleve lo fafla - toxin B formed ingrains . «•"IMTIAL OOSE(KGy) 156 11.1 Introduction The occurrence of Aspergillus flavus and aflatoxin B. in foodstuffs in cluding maize and cassava flour has been reported in Ghana (Nartey, 1966; Lokko, 1978). Unavoidably, maize constitutes a major part of human diet in many countries. Gamma irradiation in combination with heating has been proposed as an alternative means of extending the shelf-life of maize (Odamtten et al., 1980). Many researchers reported that gamma irradiation could increase aflatoxin formation (e.g. Applegate & Chipley, 1978; Priyardashini and Tulpule, 1976) in foods. Hence, we examined the aflatoxin content of combined-treated maize grains artificially inoculated with A. flavus spores and stored under tropical ambient conditions conducive for aflatoxin formation in maize. 11.2 Materials and methods Maize grains (2 kg/beaker) were inoculated with 2.5x107 spores per ml of A. flavus by surface swabbing of grains and then covered with aluminium foil at 28 C for one day. Heating was performed in a specially designed heat-treat ment chamber that enabled us to heat-treat the grain under either high humid conditions (>85l R.H., moist-heat) or under low humidity conditions (<45% R.H., dry heat) for 30 min. at 60 C. Unheated controls were maintained at 20 C for 30 min. After the heat-treatment 1 kg weight of grains were put in sachets of woven polypropylene sacks (30x15 cm) and the irradiation was carried out not more than 30 min. after the heat-treatment. Gamma irradiation doses applied were 0.0, 3.5, and 4.0 KGywit h a source dose rate of 0.06 KGy min."1. The maize grains in the woven polypropylene sacks were stored at 801 R.H. and 28 °C for four months. After every month, the moisture content (1S0 2291), aflatoxin B1 content (Dantzman and Stoloff, 1972), rancidity (AQAC, 1960), mould and yeast (Mossel et al., 1970) and total aerobic bacteria (ISO 4833) were determined. 11.3 Results Moisthea t treatment (60° Cfo r3 0min. ,>85 lR.H. )doe sno t increasesignifi cantlyth einitia lmoistur e contento fmaiz e grains (about 131m.c. )bu tre duced the initialmoul d andyeas t countb y0. 9lo gcycle san dth e totalaero bicbacteri a countb y0. 3lo gcycle s (Fig. 1).A combinatio no fmois thea t 60H an d4. 0KG yhowever ,lowere d theinitia lmoul d andyeas t countb y5. 1 log 157 cyclesan dth etota laerobi ccoun tb y4. 2lo gcycle s (Fig.1) . After3 month sstorag ea t80 1R.H .th elog 10 c.f.u./gcoun to fmoul dan d yeastan d totalaerobi cbacteri ao fth ecombine d treatedgrain s (60H ,4. 0KGy ) remainednearl yth e same (i.e.5. 0an d4. 3lo gcycle s reduction respectively fromth e initial),wherea s controlo fth emois thea t treated grainsha dmoul d andyeas tan dtota l aerobicbacteri a counts loweredb y1. San d1. 3lo g cycles respectively after3 month s storagea t80 1R .H .Th egrain sdi dno tbecom e rancid. We found fromtriplicat e samples thatonl ycontro l (20L an d2 0H ) grains contained0.8-4. 0ug/k go faflatoxi n B1 after3 month s storagea t80 1R.H .an d 28° C(whe nsoake d for2 day sbefor eextraction )excep t the control grains given4. 0KG y(Fig .2) . 11.4Conclusion s We conclude that: (i) Acombinatio n treatmento fmois thea t (60° Capplie dfo r3 0min .unde r >85%R.H. )an d4. 0KG yca nimprov e themicrobiologica l qualityo fgrain s kepta t80 % R.H. and2 8 Ci nwove npolypropylen e sacksb y4- 5lo gcycles . (ii)Combined-treate d (60H ,4. 0KGy ) A. ftavus spores couldno t form aflatoxin B-.i nth evariet yo fyello wmaiz e used. (iii)Maiz evarieta ldifference s influence theirsusceptibilit yt oaflatoxin s contaminationan dtherefor e further studies using other local Ghanaian maizevarietie sshoul db epursued . References (1) Odamtten,G.T. ,V .Appiah ,an dD.I .Langerak , 1985.Act aAlimentaria , Vol. 2,96 . (2) Odamtten, G.T., V. Appiah, and D.I. Langerak, 1980. IFFIT Report No. 10, Wageningen, The Netherlands: 15 pp. (3) Mossel et al., 1970. J. Appl. Bacteriol. 33: 453-457. (4) Anon, 1978. International Standard Organisation ISO, 4833, Microbiology: General guidance for enumeration of microorganisms. Colony count technique at 30 C, 1st Ed., Geneva. (5) Dantzman, J., and L. Stoloff, 1972. J.Assoc Official Anal. Chem. 55: 139-141. (6) Anon, 1960.Officia lMethod s ofAnalysi sAOAC ,Horowitz ,W.P. ,p . 283. 158 12 DISCUSSION Comparison ofmycoflor a recorded onmaiz ewit h thatobtaine d fromothe ragri cultural products in relation tothei r spoilage potential InTabl e 1i ssummarize dmycoflor ao nmaiz egrain s isolated inGhan a (Article 1)a scompare dwit h thatfoun do nothe rcerea l grains,legume san d grainproducts . Microorganisms havebee nknow nt oinfec tstore d grains,legume s andoil seeds aswel l as flours andfurthe r- processe d foodproduce d fromthem .Micro bial invasiono fstore d grainsan d grainprouduct s canresul ti nvariou skind s ofdamage .Fung i decreaseviabilit y ofseed s (hencereducin gcro pproductio n potential)caus e discolouration (whichreduc e grade)an dtherefor emonetar y value ofgrain s andothe rseeds . Microbes areprimaril y responsible forth eheatin g ofmois tgrain s andlegumes . Mustiness,caking ,tota ldeca y areth efina lstage so fspoilage .Must y odours persistthroughou twhateve rprocessin g treatment theinfecte d grainsan dseed s mayb e subjected todurin g thepreparatio n offoo d (Beuchat, 1978). The carcinogenic,mutageni c and teratogeniceffect so fmycotoxin sproduce d by storage fungi isa proble mo fgrav e concern,fo rthei rproductio n in vivo isa seriou s threatt owholesomenes so f foodsprepare d frominfecte dgrains . Mould growth dependso nmoistur e contento fseeds ,temperature ,humidity , rapidityo fdrying ,aeration ,th emicrobiologica l ecosystem,insects ,mixin g ofseeds ,chaf fan ddirt ,chemica l treatment,interna l infection,accidenta l rewetting of the seedsb y condensation or leakage andth edevelopmen to fho t spots (Hesseltine, 1976). Themoul d contaminants encountered inArticl e 1an d thosei nTabl e 1be longpredominantl y to thegener a Aspergillus and Pénicillium. Minimumtempera tures forgrowt han d toxinproductio no fAspergill i arehighe r thanthos eo f Penicillia.Th ewate r activity,a ,fo rgrowt h ofPenicilli a differs fromthos e ofAspergill i (Northolt, 1979).On eo fth emos t important factors influencing fungal growth on grains,legume s andoilseed s ismoistur e content (m.c). The minimumm.c .fo rstorag e ofgrain s and legumes inth epertinen t literature is summarized inTabl e 2.Northol t (1979)state d thecondition s forgrowt han d production ofsom e mycotoxins formedb y Aspergillus spp.an d Teniaillium spp. Mycotoxins areno t fomed until themoul dha s gonethroug h logarithmic growth phase.Fo rexample ,aflatoxi n isno t formedunti l atleas t4 8h afterspore s of 159 +> s •ti Î3 Ol Û, CO t•«j» CfUf CU •H O öj R< CU a co e . co a\ oo •H fei a\ — er\ eu JG O« 4J co •> M A 0 s h eu M u ca eu J3 eu 0 J3 CJ A !• o çd CJ cd e I-t i + 1 + + E i-< i-l CU 1 ca *0 1—1 •W o CU o. (U 4J §• § a B cd CU •^. co ü cd 4J Ui O id o. co s: P3 • 33 S • W 1 M U r-«i V, + + + + + T3 £3 r- •O CO e id 00 e cd CT> • E-« cd H 3 j= H IW .H H -d 1-4 O CO O 00 r-- 00 r— co en O O 4-1 e en en -rj- en• ^ H OOI 3 - e H- 8! 160 Aspergillus flavus germinate (Hayese t al., 1966).Hig h ambientmoisture s and temperatures characteristic of the tropical climate (>8Q$R.H. ,28± 3 C)haste n themicrobia l deteriorative process. According toFig . 2i nArticl e 1ther ewer e four infectionpattern s in themycoflor a encountered. Thisimplie s that incertai n instances,th e fungus may form the toxin in the grainbu tma yno tb e isolated latero nwit h prolonged storage time.Thi smicrobia l competitionwhic h inhibits growth andmycotoxi n productionha sbee n demonstratedb y otherworker s (Ashworth et al.,1965 ; Wicklowe t al., 1980). Butonc emycotoxin s are formed,remova l or destruction inseed , grain or grainproduct s is difficult andofte nexpensive .Mycotoxin s known tooccu rnaturall y in grains,legume s oroilseed s are summarized in Table 3. Hesseltine (1976)enumerate d some ofth eproblem s associatedwit h detoxi fication ofcommodities : (a) Any chemical ofphysica l treatmenttha tremove smycotoxin s adds toth e costo f theproduc t already damagedb ymoul d growth;beside s the initial processing cost,additiona l material mayb e lost fromseparatin g the toxin-infectedpart smechanicall y or chemically; (b) Mostprocesse s thatremov emycotoxin s areno t 1005»efficient ; (c) When chemicalprocesse s areused ,extensiv e testing is required toestab lish thata secondbiologicall y active compoundwit h adifferen t mode of actionha sno tbee n formed; (d) Processes thatremov emycotoxin s may reduce the foodvalu e of the final product. Marth and Doyle (1979)update d thevariou sprocesse s used in thedegrada tiono faflatoxi n in foods andthe y concluded thatmuc h additional research is needed before thisbecome spractical . Although chemical sterilants forth e control ofpost-harves t diseases of insects and fungi arewel lknown ,the y dono tkil l fungi (Article 7),i naddi tion,th e chemical residueswhic h theyimpar t to foods leavemuc h tob e desired. Invie w of this,publi chealt h authorities theworl d overhav e stringent regu lations on the reduction oreliminatio n ofchemica lburden s in food. Itis , therefore,expedien t topreven t growth (and toxin formation),o fmould sbefor e chemical damage isdone .Th e different foodcommoditie s listed inTabl e 1shar e common storage fungian d this couldb e abasi s forextendin g results obtained formaiz e to these agriculturalproducts . 161 ON mm in p^ r*- vO ON ON ON CM O CM es) l-s C < 00 o\ o C ON tjN •H •H r*• JJ n r-t •i- 4 O U n M « c» > IJ •r-t C a U ai 4J CU tu tu en 3 en en en 14 C Z 0• •3 C C tu 01 CU a. 4-1 «3 z tu •u 4-1 en kl r-t en en •§ T5 tu M •1-4 •H 3 -*U O Cu a U M £ O C0 3 -C A CJ O h eu S u U S 3 T> C 41 •O -4 C -H 3 Ol O S. M 01 m es CO 0) O. 3 O 111 u e _ O "O m o 4-1 CU 4J CU O w CJ 3 O •U • W 00 3 r-» -o O—N rtU 3 ON C -o tu c (5 tu 4J (0 u il T3 oo - en tu V co a C0 3 u a S. O O tn tn m M o O« O CO o o u 4J O m m NO r^ U 1-4 •O X » tn 3 B -r* so fJ en S 3 O to u to r-t CU O 14-1 en Z CU L. M G u si h -^ a tu •• co 0 . 4J o < eu S O X! tu T3 3 m m •H O > S o r-m^ ooo O d d ja I <0 Ei 3 162 Interaction between storage humidity and gamma irradiation or itscombinatio n with heat Theapplicatio no fionizin gradiatio na sa processin gmetho doffer sa betteralternativ e tochemica linsecticide s and fumigantsbecaus e application ofionizin gradiatio n leavesn o chemical residuesan dn o insecto r fungusha s been found todevelo p immunity toirradiation . InFig s 1an d2 ,complet econ trolo ffung io nmaiz e couldno tb e obtainedeve nwit h ados eo f5. 0 KGybe causehig henvironmenta l storagehumidit y (R.H. >&0%)increase dradio-resist ance ofth espore s of A. flavus (Odamtten, 1979;Amoaka-Att ae t al., 1981)b y makingmor e freewate ravailabl e tospore s forgrowt hafte rth eradiatio n treatment.Radiatio nu p to5. 0 KGyonl ydelaye d theonse to finfection .Fo r example,probi t analysiso fdat ai nAppendi x 1show n inFig s 1,2, and 3 indicate thus: 841spoilag e = log10 1.5 days (=4.5days )a t 85%R.H . 841spoilag e at 851afte rapplyin g 5.0 KGy= = log1Q 1.9 days (»6.7 days). Thehighe rth estorag ehumidity ,th efaste rth erat eo fspoilag eo fgrains . Thehea t treatmentapparatu s inArticl e 2offere d thepossibilit yo faug menting thekillin geffec to firradiatio nb y firstenablin gu s toappl ymois t (at >_85%R.H. )o rdr y (at<45 tR.H. )hea t (60° C for3 0min. )t ograin sprio r toirradiation .Thi ssynergisti ceffec t lowered thegamm airradiatio n dosere quired to4. 0 KGy andth etreatmen twa smor eeffectiv e afterexposin gspore s tomois thea t (60° C for 30min. , >85%R.H .ambien thumidity )befor eirradia tion (Fig.3 )tha nwhe n dryhea t (60° C for 30min. ,<45 lR.H . ambienthumidi ty)wa sapplie ddurin g theheatin go fgrains . Experimental results inArticl e 3sho wtha tspore so f A. flavus NRRL590 6 irradiated in thewe tstat ewer emor e radiationsensitiv e thanthos e treated inth edr ystate .For ,indeed ,whils ta subletha l temperature of5 3° C applied for5 min .i ncombinatio nwit h 0.75 KGyinactivate dmoistene d spores suspended inTween-8 0solution ,a dos e of4. 0 KGywa s required incombinatio nwit hprio r heating (60°C for 30min. )unde rmois thumidit y conditions (>85%R.H. )t o achieve thesam e objectives fordr y A. flams spores.Thu s fruitsan dvegeta bleswhic h canb e immersedi nho twate rwoul d requiremuc h lowerdose s tokil l contaminating spoilage fungi thandr ygrain s infectedwit h the samefunga l species.Th ereaso ncoul db e thatth eenlarge dvolum eo fspore s inwate ri s attendedb yenhance dmetaboli cprocesse swhic har evulnerabl e tohea t andlo w gamma irradiation. Indeed,accordin g toGregor y (1966),dr yspore shav e low water contentan dslo wmetabolism ,lac kvacuole s and cytoplasmicmovement . 163 Table 3.Mycotoxin s and organisms producing toxins invariou s cereal grains andseeds . Mycotoxin Organism Foodstuff affected References Aflatoxins Aspergillus flavus Groundnut, rice, 1 A. parasiticus Maize,Cotto n seed 2 Coconut,Coco abean s 3 Wheat,Millet , Sorghum Aspergillic A. flavus Cereals 1 acid ATA toxin Fusarium Oat,Wheat ,Barle y 4 sporotriahoides Ochratoxin Aspergillus Cereals 1,5 ,6 ,7 oahraaeus 8,9 , P. viridiaatum Citrinin Pénicillium oitrinum Rice P. ahrysogenum Cereals 1,1 0 Penicillic acid P. ayolopium Maize 1 P. puberulum Patulin3 P. expansion, Maize,Ric e 1,5 ,6 P. urtioae P. patulum P. digitatum, Animal feed Byssoahlamus, A. alavatus nivea Rubratoxins P. rubrum Maize,Cereal s P. purpurogenum Meat Sterigomato- A. versicolor Cereals 11, 12,6 cystin Aspergillus nidulans Moniliformin Fusarium moniliforme Maize,Cereals , 1,5 Legumes Roquefortine Pénicillium commune Cotton seed 13,1 4 P. roqueforti Zearalenone Fusarium graminearum Maize ,Hay ,e tc . 1,1 5 Kojic acid A. flavus andothe r Cereals Aspergillus spp. PenitremA Pénicillium commune Cotton seeds 16 P. crustosum T-2 Toxin4 Fusarium tricinctvm Cereals,Maiz e F. nivale, F. poae Butenolide F. nivale Maize,cereal s Tremorgenic toxin A. flavus Maizean dothe r foodstuffs Beta-nitro A. flavus Cereals 1 propanoic acid 164 Table 3 (continued) Mycotoxi n Organism Foodstuff affected References Nivalenol Fusarium nivale Rice, Cereals 1 Deoxynivalenol Fusarium nivale Rice,Cereal s 1 Fusarenone Fusarium nivale Rice, Cereals 1 a.Mycotoxirt sdetecte d asnatura l contaminants. References: 1- FAO ,Foo dan dNutritio n PaperNo . 10,1979 . 2- Widstrom ,1979 . 3- Zube ran dLillehoj ,1979 . 4- Ciegle r etal. ,197 2 5 -Odamtten , 1981. 6 -Reiss ,1978 . 7 - Harwig et al., 1974. 8 - Krogh, 1974. 9- Pavlovi c etal. ,1979 . 10- Austwick ,1975 . 11- Hitokot oe tal. ,1978a . 12- Hitokot o etal. ,1978 b 13- Ciegler ,1969 . 14- Ohmom oe tal. ,1977 . 15- Scot tan dKennedy ,1976 . 16- Wagane re tal. ,1980 . 165 UNIRRADIATED (CONTROL) « S 1 4 <181~> 0-5 1-0 1-5 2-0 Log^ Storage Tim« (Days) IRRADIATED (3-5 KGy) 0-5 1-0 1-5 2.0 Log^ StorageTim e (Days) Fig. 1.Probi t analysis of percentage spoilage datao fmaiz e grains stored at indicated relative humidities (R.H.)afte rirradi ationwit h 0.0, 3.5,an d 4.0 KGy. (Percentage spoilage data are inparenthesi s Q ~J ; seeAppendi xFig s 1an d 2.) 166 IRRADIATED (4-5 KGy) 4> Ol W- ,2 'ö a * S-[50] .a 8 a. "^ao1**64 1 J«*e4 0-5 1-0 1-5 2-0 IRRADIATED (50 KGy) 5? in a- ! s aJÛ! JÛ 4- ^16 ^ao^tac^* 84^^11,4 0-5 10 1.5 2-0 bogjQ Storage Time (Days) Fig. 2.Probi t analysis ofpercentag e spoilage datao fmaiz e grains stored at indicated relative humidities (R.H.)afte rirra diationwit h 4.5_and 5.0 KGy. (Percentage spoilage dataar e inparenthesi s [_] 5se eAppendi x Figs 1an d 2.) 167 UNIRRADIATED (CONTROL) UNIRRADIATED*HEAT 8- IRRADIATED ( 40 KGy) HEAT(60«C,30min) COMBINED WITH 4-0 KGy 90% R.H.(Low) 75«fc R.H.( LOW) 90%R.R(HigW 75«fcR.H.(Hi 04 0-0 0-7 0-8 1-01- 1 1.3 0.7 0-9 Log-to StorageTim e (Days) Fig. 3.Prohi t analysis ofpercentag e spoilage datao fmaiz e grains stored at indicated relative humidities (R.H.)afte r heating and its combinationwit h gamma irradiation. (Note the absence of infection at storage humidity <80%. ) 168 However,the ycontai nenzyme s fora variet yo fmetaboli c activities (Knight, 1966). Before swelling (andprotrutio no fger mtubes )spore sar e 3-10 times more active thanmyceliu m (Vézinae tal. , 1968).Whe nhydrated ,ungerminate d sporeshav e theirenzyme s activated and theycontai n ifno t allth e components ofprotei n synthesis apparatus (ribosomes,t-RNA ,aminoacy lt-RN Asynthetase) . Heatingwoul d "sensitize"th espore s andrende rthe mmor evulnerabl et olo w radiation (>0.75 KGy).Radiatio nwoul d impairth em-RN A alreadypresen ti n hydrated dormant spores;whic hwoul dhav e acteda stemplat e forprotei nsyn thesisi nth eformatio no fne wwal lo fger mtub e (Bramblan dVa nEtten ,1970 ; VanEtten , 1969).W ecoul dno texplai n fromdat ai nArticl e3 th erecover yo f heated (60° Cfo r3 0min. )an dunirradiate d dryspore s of A. flavus afterstor agea t80 1R.H .fo r8 days .Furthe r investigations areneede dt oexplai n these observations. Effecto fcombinatio n treatmento naflatoxi n formation Aflatoxinsar eregarde d asth emos tpoten t carcinogenic,mutageni can d teratogenicmycotoxin s ofhuma nan danima lhealt h importance (Goldblatt,1969 ; Maggone tal. , 1977;Heathcoat e andHibbert , 1978). Twoeconomicall y important aflatoxin-producingstrain s Aspergillus flavus and Aspergillus parasiticus are ubiquitously associatedwit ha wid evariet yo fstore dcommoditie s (Dieneran d Davis, 1977).Th efungu s A. flavus isth emos t radioresistant fungalspecie s associatedwit h storedgrains ,legum e andoilseed s andthi smake si tareasonabl e choice fordeterminin g theeffec to fth e combinationproces so nmycotoxi npro duction.An yeffectiv e controlmeasur e for A. flavus growthan dtoxi n formation couldb eextende dt ocate r forothe rradiosensitiv e species.Tabl e4 summurize s pertinent literatureo nmycotoxi nproductio nb yeithe r A. flavus or A. parasi ticus before andafte rexposur et ogamm airradiation .Dose so f4. 0 RGyo rmor e prevented sporegerminatio n entirely,henc en oaflatoxi nwa sproduce d (Apple- gatean dChipley , 1974a). Resultsi nArticl e4 sho w that thenatur e andtyp eo fhea t appliedt o spores influenced growth andamoun to faflatoxi nformed .Aflatoxi n B..formatio n be A. flavus spores incubatedi namroende dmaiz emea lbrot h (AMMB)wa s complete lyprevente db ya combinatio no fmois thea t (60° Cfo r3 0min. )applie dunde r 851R.H .prio rt oexposur eo fspore st o4. 0KG yo fgamm airradiatio nbu tno t when treatedi nth esam ewa ywit h dryhea tprio rt oirradiation .Hea t treatment tooha sbee nprove d tob emutageni c (Eignere tal. , 1961;Northro pan d Slepecky, 1967; Bridgese tal. , 1969). Buti ti swel lknow ntha thig hhumidit y strikingly 169 s- 31 u •rf 80 en — 0 r-. S 3 CU O o a OO a oao a •A V o S£• ^ u •*«i oo cd (0 m •f * 3 T3 IN HJ CO « CA U col s CU 00 M e o co o. t>o S to a TJ m> ecj 3s1- •rl 00 •o co -i 6 s CD coo CJ 00 •ri Ol £< a +i Ol •ti OS S ^ CO CM « « _ SL.0A SB — eu S CcoU l 00 •£> — oo S T M S e O en en co •* o. oo iJ -a Ü3 3. cu CO C O eg eu u CS .C CU • fi, co a cu o o o o o •H c O IA CM o. "O -$• r~ -Cf — ó a ou < z a •O i I I I o o o .. acu e .O o e a S O CU Z 13 § C <3 in •* co § E CU £. co M CU o O -o 3 co o o O 4J O •08 Z O OS co O in o — en u 3 CM co r- CU t-t O O o o m a* u oo .o ca — •-! CM vß m sj- OS H X en CO s e 3 M 3 CU. co 4J s -O M M -H CU CU co u ^ a\ oo 33 ti C cu cu <•* 3 O O O — e •s a a M-4 a en I I I a co a e m m m z « iJ e o o en o O SS 'M -H CU T3 e -o O O O r* C O « eO r-i O co •S SB O, CO •H •fi .O M Ä J= c a ja e c CJ CO M r-4 •H CO TJ r-l -o a. CQ CO co ü o e o. 3 O co co <; > s4 ! •O 4J CU O co 0 co cu *• - co B co £ u C eu M u CU CU CU -H ed CU c •e 00 <-> /—\ CO co 4J •u >s •H CU u C O >N S o O rrl S u o O. CO u a -H |5 S O.T3 3CU efl •C•O u a + cd + + 170 affects lethalityo fhea tapplied .Mois thea tshow s gTeaterlethalit ybecaus e (a)enzyme sar emor e readily coagulatedan dhydrated , (b)hea ti stransferre d morereadil yi nwe tai r (Hawkere t al., 1952). Enhanced aflatoxinproductio nafte rgamm airradiatio nb y A. flavus and A. parasiticus hasbee nreporte db yman yworker s (Applegate andChipley ,1973 , 1974a,b;Bullerma ne t al., 1973;Jemmal i andGuilbot ,1969 ,1970a,b ;Priyadar - shini andTulpule ,1976 ;Schindle re t al., 1980).Thi senhance daflatoxi npro ductionha sbee nexplaine do n thebasi so fmutation sresultin g fromexposur e ofspore s toradiation .Th e generalmutationa leffec to firradiatio n evi dently causes damage ingeneti can dbiochemica lmechanisms ,creatin g impair mentso f functionan dbiologica ldemand swher enon eexiste dbefor e (Ingraman d Farkas, 1977).Whil e thisi s likely tomak eorganism smor edifficul tt ogro w andrecognize ,i tdoe sno tsee mlikel y tomak ethe mmor epathogenic ,rathe r thereverse . Irradiationsometime s increases toxinproductio n andsometime s diminishes it. Therewer en o clearindication s ast owh y theon eo rth eothe rshoul dhap pen ona particula roccasion ;generall y suchbehaviou rwa sexpecte do nth e basis ofrando mmutatio n (Ingraman dFarkas ,1977) . However,result s inArticl e 5sho w thatreductio n innumbe ro fspore so f A. flavus by 3-4 logcycle seithe rb y serial dilution,heatin go rheatin gi n combinationwit h irradiation ledt omanifol d (3-12fold )increas e inaflatoxi n B. formedb y thespores .Th e aflatoxigenicpotentia lo f A. flavus was inversely proportional to the inoculumsize :hig h inoculumsiz eo fspore syielde dth e least aflatoxin.Sharm ae t al., 1980)reporte d that for A. parasiticus spores, irradiation (andit scombinatio nwit hheat )ma yno thav e anydirec teffec to n aflatoxinproductio nbu to n thesiz e ofinoculu mi nrelatio n toviabl espores . This confirmsfinding s inArticl e 5.Mois thea t (60° Capplie d for3 0min .un der >85%R.H. ) combinedwit h 4.0 KGycompletel y impaired theaflatoxigeni cpo tentialo f A. flavus spores. Influenceo fothe r factors on the formation of aflatoxin Rather curiously, A. flavus spores failed tofor maflatoxi ni ncultur eo n maizemea lbrot h (Article4 )excep twhe n themediu mwa s autoclavedtwic ea t 121 C for 15min .Th eexcessiv eheatin gpossibl yrelease dminera lelement s required foraflatoxi nbiosynthesis . Information in theliteratur eshow stha t autoclavedmedi asuppor tproductio no fgreate r amounto faflatoxi n thannon - autoclavedsubstrate s (Guptaan dVenkitasubramanian , 1975;Lilleho je t al., 171 1974; Detroye t al., 1971). Severalenvironmental ,nutritiona l and genetic factors considerably influence thevariatio n inth e type andamoun to f afla- toxinsproduce db ymould s ofth e genus Aspergillus (Davis andDiener ,1970 ; Lillehoje t al., 1976;Maggo ne t al., 1977;Zube ran dLillehoj , 1979;etc.) . The stimulatory effecto f zinco naflatoxi nproductio n iswel l documented (Leee t al., 1966;Maggo ne tal. , 1973,1977 ;Obido aan dNdubuisi , 1981)an d theresistanc e orsusceptibilit y ofnatura l foodcommoditie s andcultura lsub strates toaflatoxi n formationb y A. flavus or A. parasiticus hasbee nattrib uted toth epresenc eo rabsenc e ofadequat e amountso ftrac eelement sespecial ly zinc (Mateles andAdye , 1972;Le ee t al., 1966;Davi se t al., 1967;Bassi r andAdekunle , 1972).However ,phyti c acidi n the germo fgrain s stronglybind s severalelement s including zinc (Garciae t al., 1972a,b;O'del l andSavage , 1960). Theboun delement s areno tavailabl ebiologically . Growtho f A. flavus andaflatoxi n levels in foodar e related toth ephyti c acid levelan d zincconcentratio n available for toxin formation.Phyti c acid levelan d zincconcentratio n in foodar eunde r genetic control (Zubere t al., 1978; Zuberan dLillehoj , 1979;Bassi r andAdekunle , 1972). Future studies willexamin e therelationshi pbetwee n concentration ofphyti c acidan d trace elements inothe r localmaiz evarieties ,an d theirsusceptibilit y toaflatoxi n contaminationsinc ebindin go ftrac eelement sb yphytate s inmaiz e germinter ferewit hbiologica l availabilityo ftrac eelement s (Garciae t al.,1972 ; O'dell et al., 1972a,b). Practical implications Results obtained in this thesissho w that thedecontaminatio n ofcerea l grains andgrai nproduct sb y thecombinatio n treatmento fhea t andgamm air radiation istechnologicall y feasible.However ,sanitatio n requirements for preventing food infectionan dfoo dpoisonin g (byusin g theappropriat epacka gingmaterials )ar econtaine d infoo d legislations throughout theworl d (Elias, 1979).Article s 6, 7,8 ,an d9 contai n information onserie so fstudie swhic h compared themicrobiologica l andphysica l qualities of theimcumben tjut e sacksuse d inth ecommercia lpackagin go fcerea l grainsan d legumes inGhan a with syntheticwove npolypropylen esacks . First,i tha sbee ndemonstrate d thatgrain skep t inwove npolypropylen e sacksunde rpractica l field conditionswer e significantly (P=0.05 ) more viable andha dbette rmicrobiologica lqualit y thansam e grainsstore d injut e sacks (Article6) . 172 Secondly,ther ewa sa significan t difference (P= 0.05 )betwee nth egreate r numbero ffunga lcolonie s associatedwit h jutesack stha n thewove npolypropy lenesacks .Correspondingly ,mor e fungalspecie s (16)wer e isolatedfro mjut e sacks thanfro mwove npolypropylen esack s(9) . Itwa s alsodemonstrate dtha ti n theabsenc eo fexogeneou s supplyo fnutrient s 88$o fth ejut esac ksection s supported growtho ffunga lspore swhils twove npolypropylen e couldno t support growtho fcontaminatin g fungalspore s (Article 7).Gamm airradiatio n (4.0KGy ) reducedth emoul dan dyeas tcount so nne wjut esack san dne wwove npolypropy lenesack sb y1 an d2 lo gcycle s respectively;bu tpost-irradiatio n storagea t 801R.H .woul d allowgrowt ho ffung io njut e sack (Article 7). These fungi couldac ta sspringboar d forinfectin ggrai ncontent so fjut esacks . Thirdly,owin gt oth erobus thandlin go fpacke d cereal grainsan d legumes in transit, itwoul db e idealan ddesirabl et ous ea packagin gmateria lwit h high tensile strengthwhic hwoul db e ablet owithstan d suchroug hhandling . Saprophytic fungio nwove npolypropylen e couldno t reduce tensile strengtho f sections storeda t90 1R.H .fo r1 0week swhils t thesam e fungio njut esac k collectivelyreduce dtensil estrengt ho fjut esack sb y50-75 $a t90 1R.H . (Article 8). Unlike jutesack swove npolypropylen e sacks dono t absorbmoisture , theyar estronge r thanjut esacks ,d ono t rotunde rdam pconditions ,d ono t support growtho ffunga lspores ,the yar eclean ,d ono timpar tan yodou ro r taint theircontents .Wove npolypropylen e sacks thushav e manymicrobiologica l andphysica l advantages over the traditional jutesack st omeri tthei rus ei n tropicalarea s likeGhan afo rgrai n storage. Fourthly,othe rcerea lgrain s (sorghum,millet )pulse s (groundnut andcow - pea)store d inwove npolypropylen e sackswer e2- 3lo gcycle slowe ri nmoul d andyeas t counttha nsam eproduc ekep ti njut e sacks for6 month sa t85 $R.H . (ora 0.85). Significantly lowerpercentage so ffiv e toxigenic (Aspergillus flavus, A. oahraaeus, Fusarium moniliforme, Pénicillium digitatum and P. expan sion)an dfiv enon-toxigeni cfung i (Aspergillus eandidus, A. niger, A. terreus, A. sulphureus, A. panamensis) were recordedo ncommoditie s storedi nwove npo lypropylene sacks thani njut e sacks (Article 9).Th etoxigeni c andnon-toxi genic fungal species collectivelyreduce d drastically theviabilit yo fseed s kepti njut esack s for6 months .Thu swove npolypropylen e sackswoul dals ob e suitable forstorin gothe r cereal grainsan dlegumes .Wove npolypropylen e sacks are includedi nth e listo fpackagin gmaterial s approved forirradiatio nb y Food andDru gAdministratio nFD Ao fth eUSA . Inth e concludingexperiment so fthi s thesis,dat aobtaine dshowe dtha t theenhance d aflatoxin B,productio nb yA . flavus spores afterirradiation , 173 theenhance d aflatoxin B productionb y A. flavus spores after irradiation, reportedb yothe rworker s onirradiate d foods (e.g.Priyadarshin i andTulpule , 1976)coul dno tb efoun do nth evariet yo fyello wmaiz e usedi nth eexperiment . Maize varietal differences influence theirsusceptibilit y toaflatoxin s con tamination (Zuberan dLillehoj , 1979;Zube re tal. , 1978). Future studies would examine other localmaiz e grain varieties inrelatio nt oaflatoxi n B. formation afterth ecombinatio n treatmento fhea tan dgamm a irradiation. Twenty tastepanelist si na sensor yevaluatio n (consumeracceptance ) test foundn ostatistica l difference incolour , flavouran dtast ebetwee n control and combined treated grains.Th ejoin t FAD/IAEA/WHOExper t Committeeo nWhole - somenesso fIrradiate dFoo d (Jecfi,1980 )recommende d acceptability, froma toxicological standpointo fan yfoo dcommodit y irradiatedu pt oa noveral l dose of1 0KGy .Th eCommitte e also statedtha t irradiated foodsu pt o10. 0KG y introducedn ospecia lnutritiona lo rtoxicologica l problems.Thi sJECF I recom mendationwa sadopte db yth eCode xAlimentariu sCommissio n inJul y 1983.Thi s establisheda ssaf efo rgenera l application, irradiationu pt oa dos eo f1 0KGy . Therefore,dat apresente di nthi s thesisca nb eapplie di npractic e inth e de contaminationo fmaiz e grains (othercerea l grainsan dlegum ea swell) . However, because irradiation doesno ttur na ba dqualit y foodint oa health yone ,onl y goodqualit y grainsan dlegume s shouldb esen tt oirradiatio n facilities, packedi nwove npolypropylen e sacks,fo rirradiation .Routin e quarantine checks shouldb ecarrie dou to nqualit yo ffoo dcommoditie s before radiation treat ment.Fo rmaxima lextensio no fshelf-lif e cleanlinesso fth ewarehous ean d good storage management practices shouldb eth erul e rather thanth eexception . References Amoako-Atta,B. ,G.T .Odamtten ,an dV .Appiah ,1981 .Influenc eo frelativ e humidityo nradiosensitivit yo f Aspergillus flavus Linkinfectin gcocoa . In: Combinationprocesse si nfoo dirradiation .Internationa lAtomi c EnergyAgenc yIAEA ,Vienna ,pp .161-168 . Applegate,K.L. ,an dJ.R .Chipley ,1973a .Increase daflatoxi nproductio nb y Aspergillus flavus viacobal tirradiation .Poultr yScienc e52 ,1492-1496 . 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Mycol.Appl .46 : 241-246. Beuchat,L.R. , 1978.Microbia l alteration of grains,legume s and oilseed.Foo d Technol.Ma y 1978:193-198 . Brambyl,R.M. , and J.L. vanEtten , 1970.Protei n synthesis during fungal spore germination. V.Evidenc e that the ungerminated conidiospores of Botrydio- plodia theobromae containmessenge r ribonucleic acid.Archive s ofBiochem . and Biophys.137 :442-452 . Bridges,B.A. ,M.J .Ashwood-Smith ,an d R.J.Munson , 1969.Correlatio n ofbac terial sensitivities to ionization radiation andmil d heating.J . Gen. Microbiol. 58: 115-124. Bullerman,L.B. ,H.M . Barnhart,an d T.E.Härtung , 1973.Us e of gammairradi ation topreven t aflatoxin production inbread . J. Food Science38 : 1238-1240. Christensen, CM., 1972.Microflor a and seed deterioration. In:Viabilit y of seeds,E.H . Roberts (Ed.). Chapman andHal lLtd. ,London . Ciegler,A. , 1969.Tremorgeni c toxin from Pénicillium palitans. Appl.Micro biol. 18:128-129 . Ciegler,A. ,D.I .Fennell ,H.J . 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M.Sc.Thesis ,Universit y ofGhana , 168pp . Mateles, R.I., and J.C.Adye , 1965.Productio n ofaflatoxin s insubmerge d cul ture.Appl .Microbiol . 13 (2):208-211 . Muckle, T.B., and H.G. Stirling, 1971.Revie w of thedryin g of cereals and legumes in the tropics.Trop . Stored Prod. Inf.22 :11-13 . Navarro, S.,an d S.Paster , 1978.Prope r aerationprevent s self-heating of stored cotton seed.Hassadeh ,58 :954-95 9 (Hebrew,wit h English summary). Northolt,M.D. , 1979.Th e effect ofwate r activity and temperature onproduc tion of somemycotoxins .Ph.D .Thesis ,Agricultura l University,Wageningen , The Netherlands, 32pp . Northrop,J. , and R.A. Slepecky, 1967.Sporulatio n mutations induced byhea t in Bacillus subtilis. Science 155:838-839 . Obidoa,0. ,an d I.E. Ndubuisi, 1981.Th e roleo f zinc in the aflatoxigenic potential of Aspergillus flavus NRRL 3251 on foodstuffs. Mycopathologia 74: 3-6. Odamtten,G.T. , and G.Y.P. Klu, 1987. 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Food Protect.4 3 (1):7-9 . Scott,P.M. , and B.C.P.Kennedy , 1976.Analysi s ofblu e cheese for roquefortine and other alkaloids form Pénicillium roque forti. J. Agric. Food Chem.24 : 865-868. Sharma,A. ,A.G . Behere.,S.R . Padwal-Desai,an d G.B.Nadkarni , 1980.Influenc e of inoculum size of Aspergillus parasiticus spores on aflatoxin production. Appl. andEnviron .Microbiol .4 0 (b);989-993 . Van Etten,J.L. , 1969.Protei n synthesis during fungal spore germination. Phytopathology 59:1060-1064 . Vézina,C , S.N. Sehgal,an dK . Singh, 1968.Transformatio n oforgani c com pounds by fungal spores.Advance s inApplie d Microbiology 10:221-268 . Waganer,R.E. ,N.D .Davis ,an d U.L.Diener , 1980.Penitre mA and Roquefortine production by Pénicillium commune. Appl.Environ .Microbiol . 39 (4):882 - 887. Nicklow,D.T. , C.W.Hesseltine, O.L . Shotell,an dG.L .Adams , 1980.Inter ference competition and aflatoxin levels in corn.Phytopatholog y 70(8) : 761-764. Widstrom,N.W. , 1979.Th e role of insects andothe r plant pests in aflatoxin contamination of corn,cotto n and peanuts -A review. J. Environ.Qual . 8 (1):5-11 . Zuber,M.S. ,O.H .Calvert ,W.F .Kwolek ,E.B .Lillehoj ,an dM.S .Kang , 1978. Aflatoxin B.productio n in aneigh t line diallel of Zea mays L. infected with Aspergillus flavus. Phytopathology 68:1346-1349 . Zuber,M.S. ,an dE.B .Lillehoj ,1979 .Statu s of aflatoxin problem incorn . J. Environ.Qual .8 (1):1-5 . 178 APPENDIX METHOD FOR DETERMINING THE INTERACTION BETWEEN ENVIRONMENTAL RELATIVE HUMIDITY AND APPLIED DOSE Ghanaian localvariet ymaiz e {Zea mays L)o f initialmoistur e content (m.c.)o f 13.61 was exposed (without any artificial infection)t ogamm air radiationdose so f0.0 ,0.8 , 1.0,2.0 ,3.0 , 3.5,4.0 ,4.5 ,an d 5.0 aswel l as the combination treatment ofmois to rdr yhea t (60°Cfor 30min. ) (Odamttene t al., 1980)prio r to irradiationwit h 3.5 or4. 5 KGy.Th e Co sourceha da mean dose rate of2. 7KG yh . Thirty grains,i neac hPetr i dish (diameter9. 0 cm)wer e transferred into environmental chambers (polyethylene hood)describe db yOdamtte n andLangera k (1980).Eac h chamber containednin e replicates.Potassiu mhydroxid e solutions provided the desiredhumiditie s 70,75 ,80 ,85 ,90 ,an d95 1R.H . (Solomon, 1952). The ambienthumiditie s above the solutionswer e checkedb y leaving a DirectReadin g Hygrometer (Bacharach InstrumentCompany ) inside theenviron mental chambers overnight.Fo reac h radiation dose investigated,ther ewer e sixhumidit y chambers representing all theR.H . regimes and these tu pwa sin cubated at 28°C .Dail y observationswer emad e ofth enumbe r ofgrain shavin g visible external infection.Fro m these readings,percentag e infection ofmaiz e grainswit h time,o feac h treatment groupwa s determined. Afteron emonth ,grain s irradiatedwit h0.0-3. 0KG y and incubated at 75-95%R.H .wer emouldy ,mouldines sbein g greatera t thehighe rR.H . regimes (80-951 R.H.). Thehumidit y chambers containing grains givendose su p to 3.0 KGywer e opened forassessment .Th e remaininghumidit y chambers containing grainsex posed to 3.5-5.0KG ywer e left intact for 100days .Th e dataafte r 100day s appear in Figs 1an d2 . 179 uttirrccdiated 3:5 kGy 4,0 kGy 4,5 kGy 5L0kG y 20: 40 60 80 100 cnoibetfion tinte (days) Fig. 1.Percentag e spoilage ofmaiz e grains scored at75 % and 85% R.H. for10 0day s after irradiation with indicated doses. 180 REFERENCES Odamtten,G.T. , and D.I.Langerak , 1980.Moistur e sorption isotherms of two maize varieties kept under different relative humidities. International Facility forFoo d Irradiation Technology (IFFIT)Repor tNo .9 ,Wageningen , TheNetherlands . Odamtten,G.T. ,V . Appiah,an d D.I.Langerak , 1980.Shor t Communication. Studies on the technological feasibility of application of dry ormois t heat tograin s and grainproduct s prior to gamma irradiation. Internation al Facility forFoo d Irradiation Technology (IFFIT)Repor t No. 10, Wageningen,Th e Netherlands, 15pp . Solomon,M.E. , 1952.Contro l ofhumidit y with potassium hydroxide, sulphuric acid and other solutions. Bull.Ent .Res .42 :543-544 . unirradiated 3.5 kGy 4.0 kGy 4.5kG y 5.0kG y 20 40 60 80 100 incubation time (days) Fig. 2.Percentag e spoilage ofmaiz e grains stored at 80%an d 90%R.H . for 100day s after gamma irradiationwit h indicated doses. 181 13. SUMMARY Cerealgrain sar e importantstapl efoo dcrop si nAfric aan dthe yar ea sa rule storedfo r longperiod sa sbuffe r stock forbot hhuma nconsumptio nan d lately,maiz ei sbein g useda singredien t forpoultr yan dlivestoc k feed. The high average temperature andrelativ ehumidit y (28±3°C ; >80'oR.H. )permi t lossesi nstorage ,du et oinsec tactivitie s andfungi ,estimate dbetwee n30 - 50%o fth eannua lharvest .Fiel d fungi infectgrain so nth eplan tbefor ehar vesto rdurin gharves t throughpur emechanica ldamage . Storage fungi seti nwhe n aerial sporeswithi n thewarehous e settleo nbul k grainsbefor ebagging .Funga l activitydiscolou r grains,deplet enutritiv ean d biochemicalvalu ean dreduc eviabilit yo fgrains .Furthermore ,fung i impart potentmycotoxin sint o storedcerea l grains.Therefore ,improve dqualit yo f grainsi sa simportan ta sincrease dquantity .Thi s thesis aimsa tth e control of fungaldeterioratio no fmaiz e grains (asa nexampl eo fa cerea l grain)b y the combination treatmento fhea tan dgamm airradiation .Th epreventio no f growthan dproductio no fth enoxiou s aflatoxinB ,forme db yAspergillus flavus Linkwa s also studied.Finally ,a packagin gmateria l thatma yexten d theshelf - lifeo fgrain sbette r thanjut e sackwa s comparedwit hjute . Inth e firstarticle ,twenty-si x (26)differen t fungibelongin gt otwelv e genera {Aspergillus, Cephalosporin, Cladosporium, Curvularia, Dreahslera, Fu sarium, Neurospora, Paeoilomyces, Penioillium, Phoma,Rhizoctonia and Rhizopus) were encountered.Thi sextende dt oforty-tw o (42)th enumbe ro fseed-born e fun gi recordedo nmaiz ei nGhana .Ninetee n (19)o fthes ear erecorde d forth e firsttim eo nmaiz ei nGhana .Member so fth egenu s Aspergillus predominated followedb y Penioillium amongth e speciesencountered .Th estorag e fungi showed fourdistinc tpattern so finfectio nove rth eentir e sixmonth sperio do fexam ination.Thi s shows thatmycoflor atha twoul db epresen ti nan y grainstoc ka t anyparticula r timewoul ddepen do nth e lengtho fth estorag eperiod .For , in deed,Aspergillus wentii, Cladosporium herbaruman d Fusarium moniliforme could notb eisolate d after4 month s storage.Potentia l toxin-producing funginamel y A. flavus (aflatoxins),A. oahraoeus (ochratoxin,penicilli cacid) , F. monili forme (moniliformin), Paeeilomyees varioti and Penioillium expansion(patulin ) were encountered. 182 Intiie secon darticle ,a ne wapparatu swa s designedwhic henable dappli cationo feithe rdr yhea t (60°C )fo r3 0min )administere dunde rlo whumidit y (<45$R.H. ) conditions ormois thea t (60 C for 30min )administere d underhig h (>85lR.H. ) ambientrelativ ehumidit y conditions.Thi ssynergisti c effectlow eredth e gammairradiatio n doserequirement s form5. 0 KGy to4. 0 KGy andth e treatmentwa smor e effective afterexposin g thespore s tomois thea t (60 Cfo r 30min ; >85%R.H. )befor e irradiation thanwhe n dryhea t (60° C for3 0min ; Inth e thirdarticle ,i twa s demonstrated thatspore s of A. flavus NRRL 5906irradiate d in thewe t statewer emor e radiationsensitiv e thanthos e treated inth edr y statebecaus ewhil e asubletha l temperature of5 3° Capplie d for5 mi n incombinatio nwit h 0.75 KGyinactivate dmoistere dspore s suspended inTween-8 0 solution,a dos eo f4. 0 KGywa s required incombinatio nwit hprio r moistheatin g (60 C for3 0min )unde r8 5 % R.H.t oachiev eth esam eobjective s fordr yspores .Fruit s andvegetable swhic h canb e immersedi nho twater woul d requiremuc h lowerdose s tokil l contaminatingspoilag e fungi thandr y grains infectedwit h thesam e fungalspores . In thefourt harticle ,th enatur e andtyp eo fhea tapplie d tospore so f A. flavus wasshow nt ohav e influenced vegetative growthan damoun to faflatoxi n B..formed .Aflatoxi nB ,productio nb y A. flavus sporesincubate d inammende d maizemea lbrot hwa s completelyprevente db y acombinatio no fmois thea t (60° C for 30min ; >85lR.H. ) appliedprio r toexposur e ofspore s to4. 0 KGyo fgamm a irradiationbu tno twhe n treated in thesam ewa ywit h dryhea t (60° Cfo r3 0 min; <45$R.H. )prio r toirradiation .Mois thea tshow s greater lethalitybe cause enzymesar emor e readily coagulated andhydrate d and,hea t istransferre d more readilyi nwe tair . The fiftharticl e contains evidence thatreductio ni nnumbe ro fspore s of A. flavus by 3-4 log cycleseithe rb yseria l dilution,heatin go r itscombina tionwit h irradiation ledt o3-1 2fol d increase inaflatoxi nB .forme db y A. flavus spores.Thus ,th e aflatoxigenicpotentia l of A. flavus was inversely proportional toth e inoculumsize :hig h inoculumsiz eyieldin g leastaflatoxin . Irradiation orit s combinationwit hhea tma yno thav e anydirec teffec to n aflatoxinproductio nbu t on thesiz e of inoculumi nrelatio n toviabl espores . Moisthea t (60° Capplie d for 30mi nunde r >85lR.H. ) combinedwit h4. 0 KGy of 183 gamma irradiationcompletel y impaired theaflatoxigeni cpotentia l of A. flavus spores. Interestingly, A. flavus spores failed to formaflatoxin s incultur eo f maizemea lbrot halon eexcep twhe n themediu mwa s autoclaved twice at 121 C for 15min .Th eexcessiv eheatin gpresumabl y releaseminera lelement s required foraflatoxi nbiosynthesis . Results inarticle s 2-5hav eshow ntha tdecontaminatio no fcerea lgrain s andgrai nproduct sb y acombinatio ntreatmen to fhea tan dgamm airradiatio ni s technologically feasible.However ,i ti simperativ e topreven tfoo dre-infec tionb yusin g theappropriat epackagin gmaterial . Inth esixt harticl e itwa s demonstrated thatmaiz e grainskep ti nwove n polypropylene sacksunde rpractica l fieldcondition swer e significantly (P<0.05)mor eviabl e andha dbette rmicrobiologica lqualit y thansam e grains stored injut esacks . The seventharticl eha sdat awhic hshow s thatther ewa s a significant difference (P<0.05)betwee nth egreate rnumbe ro ffunga lcolonie s associated withjut e sackstha nth ewove npolypropylen e sacks.Correspondingly ,mor efun galspecie s (16)wer e isolatedfro mjut esack s thanfro mwove npolypropylen e sacks (9).I twa s alsoshow ntha ti nth eabsenc eo fexogeneou s supplywhils t wovenpolypropylen e couldno t supportgrowt ho fcontaminatin g spores.Gamm a irradiation (4.0KGy )reduce d themoul dan dyeas tcount so nne w jutesack s and newwove npolypropylen e sacksb y 1an d2 lo gcycle srespectively ;bu tpost-ir radiationstorag e at 80%R.H . allowedgrowt ho f fungio n jutesacks .Thes e fungio njut esack scoul dac ta sspringboar d forinfectin g graincontent s of jutesacks . Theeight harticl ecompare d thetensil estrengt ho fjut ean dwove npoly propylene sacks.Saprophyti cfung io nwove npolypropylen e couldno treduc e tensilestrengt ho fsection s storeda t90 1R.H . for 10week swhils tth esan e fungio njut e sackcollectivel y reduced thetensil e strengtho fjut e sacksb y 50-75% at 90%R.H .afte r 10weeks . Inth eninet h article,i twa s demonstrated thatwove npolypropylen e sacks would alsob e suitable forstorin gothe rcerea l grains (sorghum,millet) , le- 184 gumesan dpulse s (cowpeaan dgroundnut) .Thes e foodcommoditie sstore di nwo venpolypropylen e sackswer e2- 3lo gcycle s loweri nmoul dan dyeas tcoun t thansam eproduc ekep ti njut e sackfo r6 month sa te.r.h . 851 (a^0.85) . Significantly lowerpercentage so ffiv e toxigenic (A. flaws, A. oahraoeus, F. monili forme, P. digitatum andP . eapansim) andfiv enon-tox igeni efung i (Aspergillus candidus, A. niger, A. sulphureus and A. panamensis')wer erecord edo ncommoditie s storedi nwove npolypropylen e sacks thani njut esacks .Th e toxigenican dnon-toxigeni c fungalspecie s collectively reduced drastically theviabilit yo fseed skep ti njut esac kfo r6 months .Wove npolypropylen e sacksrestricte dmoistur e transfert oa greate rexten ttha njut esack sreflect edb yth elowe rmoistur e absorbedan ddesorbe db ycommoditie skep ti nwove n polypropylene sacks thani njut e sacks.Wove npolypropylen e thusha sman y microbiologicalan dphysica l advantagesove rth etraditiona l jutesac kt o meritit sus ei ntropica larea slik eGhan afo rgrai nstorage . The concludingexperiment so fthi s thesissho wtha tenhance d aflatoxin biosynthesisb y A. flavus spores afterirradiation ,reporte db yothe rworker s inirradiate d foodcoul dno tb efoun di nth evariet yo fyello wmaiz euse di n theexperiment .Twent ypanelis ti na sensor yevaluatio n (consumer acceptance test)foun dn ostatistica l difference (P<0.05)i ncolour ,flavou ran dtast e between controlan dcombined-treate dgrains . Becauseo fth epassin ga ssaf efo rhuma n consumption foodirradiate dwit h anoveral ldos eo f10. 0KG y (JECFI, 1980),dat apresente di nthi s thesisca n be appliedi npractica ldecontaminatio no fmaiz egrain san dothe rcerea l grainsan dpulse sa swell . However,irradiatio ndoe sno ttur na ba dqualit y product intoa health yon ean dtherefor ei ti sexpedien tt otrea tonl ygoo d qualitygrain swit h combination treatment.Fo rmaximu mextensio no fshelf-life , therecommende d storage sack (wovenpolypropylene )shoul db euse di nbaggin g priort oirradiation .Cleanlines so fth ewarehous ean dgoo dstorag emanagemen t practice shouldb erigidl y adheredto . 185 14. SAMENVATTING Granen zijnee nbelangrij kvolksvoedse l inAfrika .Dez eworde n gewoonlijk voor langere tijdopgeslage nal svoorraa dvoo rmenselijke ,e ni ntoenemend e mateoo kvoo rdierlijk e consumptie.Bi jhog egemiddeld e temperatuure nrela tievevochtighei d (28+3° C; >80 %R.V. )worde nbewaarverlieze nveroorzaak tal s gevolgva nontwikkelin gva ninsecte ne nschimmels .Dez ebewaarverlieze nworde n op 30-50$va nd eoogs tgeschat .Besmettin gme tschimmel s treedto pvóó re ntij densd eoogst ,maa roo ki nhe tpakhuis ,vöö rhe tafvullen .Schimmelgroe ika n resultereni nverkleurin gva nkorrels ,verlie sva nnutriënte ne nverlie sva n kiemkracht.Oo kkunne nmycotoxine nworde ngevormd . Dedoelstellin gva nhe tonderzoe kbeschreve ni ndi tproefschrif ti she t beheersenva nschiimnelbeder fva nmaï sdoo rgecombineerd ebehandelin gme thitt e engammastralen .D erenmin gva nd egroe ie naflatoxin e B..produkti edoo r Asper gillus flavus Linkwer d tevensbestudeerd .Tenslott ewer dd ehoudbaarhei dva n graneni nee nnieu wverpakkingsmateriaa lvergeleke nme tdi ei nd egangbar eju tezakken. Inhe teerst e artikelworde nui tmaï s2 6schimmelsoorte n geïsoleerd,be horendto td evolgend e 12geslachten : Aspergillus, Cephalosporium, Cladospori- um, Curvularia, Dreohslera, Fusarium, Neurospora, Paecilomyces, Pénicillium, Phoma, Rhizoctonia en Rhizopus. Hetbetref toverwegen dsoorte nva n Aspergillus envoort sva n Pénicillium. Hetblee kda td emycoflor adi eo pee nbepaal dtijd stipi nee ngraanvoorraa dword taangetroffen ,afhang tva nd ebewaarduur .Z o konden Aspergillus wentii, Cladosporium herbarume n Fusarium moniliformisn a viermaande nbewaarduu rnie tmee r geïsoleerdworden .Potentiël e toxinevormers, diewerde naangetroffen ,waren : A. flavus (aflatoxine), A. ochraceus (ochra- toxine,penicillinezuur) ,F. moniliforme (moniliformine),Paeoilomyoes varioti enP . expansion (patuline). Het tweede artikelbeschrijf td eontwikkelin gva nee napparaa twaarme e hittebehandelingen (60° C, 3 0min. )bi jlag e (<45%)e nhog e (>85$)relatiev e luchtvochtigheidkunne nworde nuitgevoerd .Doo rdez ebehandelin gko nde ,voo r afdoende schimmeldodingbenodigd edosi sgammabestralin gva n5. 0kG yto t4. 0kG y verlaagdworden .Daarbi jwa sd evochtig ewarmtebehandeling ,uitgevoer dvôô rd e bestraling,effectieve rda nd edrog ewarmtebehandeling . Inhe tderd e artikelword taangetoon dda tspore nva n A. flavus NRRL590 6 innatt e toestandbestralingsgevoelige r zijnda ni ndrog evorm .Spore ngesus - 186 pendeerd inee nTwee n 80oplossin gwerde n gedoodn a eenbehandelin g van 5min . bij desüblethal e temperatuur van 53 C, i ncombinati eme t eenbestralingsdo sisva n0.7 5kGy ,terwij l4. 0 kGynodi gwa s voor droge sporen dieee n vochtige warmtebehandeling haddenondergaa n (30mi nbi j 60° C, relatiev e vochtigheid >85$).Groente n en fruit,waarvoo r eenwarmwaterbehandelin g mogelijk is,zoude n daaromme tee n veel lagerebestralingsdosi s behandeld kunnenworde n dandrog e granen. Inhe tvierd e artikelword t aangetoond datd eaar dva n de hittebehandeling invloedheef to p devegetatiev e groeie n daarmee opd eprodukti e vanaflatoxi - ne B..D evormin g vandi t toxine inee nverrijk tmaïsmee lmediu mko nworde n voorkomenme tbehul p van eenbehandelin g metvochtig ewarmt e (30mi n 60° C, >85l relatieve vochtigheid) gevolgd oorgammabestralin gme t4. 0 kGy,maa rnie t dooree n combinatiebehandeling metdrog ewarmt e (30mi n 60° C, <45 $ relatieve vochtigheid). Hetvijfd e artikelbevestig t gegevens uitd e literatuur datee n reductie vand e concentratie vanspore nva n A. flavus metdri e totvie rlogcycl i öf doorverdunning ,ö fdoo rhittebehandelin g öfdoo rhitt e gecombineerdme tbe straling leidt totee ndrie -t otwaalfvoudig e toename inaflatoxin e B..produk tie. Het vermogen omaflatoxin e teproducere nwa s dusomgekeer d evenredig met deomvan g vanhe t inoculum. Bestraling,o fd e combinatie daarvanme tee nhitte behandeling heeft danoo kwellich t geen directeffec to paflatoxine-produktie , maar slechts ophe t oorspronkelijke aantal levende sporen.N aee n gecombineer debehandelin g van A. flavus sporenme tvochtig ewarmt e (30mi n6 0° C, rela tieve vochtigheid >85%)e n 4.0 kGy gammabestraling kon geen aflatoxinevorming meer aangetoondworden . Heti s interessant dat A. flavus geen aflatoxine vormt inee n culture van uitsluitend maïsmeel medium,maa rwe l indiendi tmediu mtwe ekee r 15mi nwa s geautoclaveerd bij 121 C. Door dezehittebehandelin g wordenmogelij k mineralen vrijgemaakt,di enodi g zijnvoo rd ebiosynthes eva n aflatoxine. Ind e artikelen tweeto te nme tvi jf word t aangetoond dat decontaminatie van granen dooree n gecombineerde behandeling vanhitt e enbestralin g techno logisch uitvoerbaar is.Daarbi j isechte rhe tvoorkome nva nherbesmettin g door het gebruik van geschiktverpakkingsmateriaa l noodzakelijk. Inhe t zesde artikelword t aangetoond datmaï sbewaar d ingeweve npolypro - pyleen zakken,onde rpraktijkomstandighede n significant (P=0.05 ) kiemkrachti- gerblee f enmicrobiologisc h bezien zijnkwalitei tbete rbehiel d danmaï s in jutezakken. 187 Uitgegeven si nhe t zevende artikelblijk t dathe tschimmel-kiemgeta lva n jutezakken significant hoger is(P<0.05 )da nda t vanpolypropylee nzakken . Erwerde noo k dienovereenkomstig meer schimmelsoorten (16)geïsoleer d vanjute zakkenda n vanpolypropylee n zakken (9). Ookblee k dat,indie n geenander esub straten aanwezigwaren ,i n88 %va nd emonster sva njutezakke nd eontwikkelin g vanschimmelspore nmogelij kwas ,terwij lbi j polypropyleend egroe iva nschim melsnie tmogelij kwas .Bestralin g (4.0kGy )reduceerd ed ekiemgetalle nva n schimmelse ngiste no pnieuw e jutezakkene no pnieuw e geweven polypropyleen zakken,respectievelij k metee ne ntwe e logcycli.Maa rbi j bewaringbi j 80% re latievevochtighei dn abestralin g tradbi j jutezakken schimmelgroei op.Dez e schimmelso pjutezakke n zoudeninfecti e vand einhou dkunne nveroorzaken . Inhe t achtste artikelword td etreksterkt eva njute -e ngeweve npolypro pyleenzakke nvergeleken . Saprofytische schimmelskonde nd etreksterkt eva n monsters vanpolypropylee n zakken,tie nweke nbewaar dbi j 90'«relatiev e voch tigheid,nie t doen afnemen,terwij l datwe lhe tgeva lwa sbi j jutezakken (50- 75% verliesva ntreksterkte) . Inhe tnegend e artikelword t aangetoond datgeweve npolypropylee n zakken ook geschikt zijnvoo rd eopsla g van andere granen (sorghum,millets )e npeul vruchten ('cowpea'e naardnoten) . Kiemgetallen (gistene nschimmels )va ndez e Produktenwaren ,n a6 maande nbewarin gbi j85 % rel.vochtigheid , tweeto tdri e logcycli lageri npolypropylee n zakkenda ni njutezakken .Daarbi jwerde n tevens beduidend lagerepercentage s toxigene (4. flavus, A. oahvaaeus3 F. monili forme, P. digitatum enP . expansion)e nniet-toxigen eschimmel s (Aspergillus oandidus, A. niger, A. sulpkureus en A.. panamensis) gevondeni nProdukten ,bewaar di n polypropyleen zakken.D etoxigen ee nniet-toxigen e schimmels veroorzaaktenee n aanzienlijke verlagingva nd ekiemlcrach tva nzade nn aze smaande nbewarin gi n jutezakken.Geweve npolypropylee n zakken zijnminde rvochtdoorlaten d danjute zakken.Di tresulteer ti ngeringer e vochtabsorptiee n-desorpti e doorprodukte n inpolypropylee n zakken.Geweve npolypropylee n zakkenhebbe ndu smicrobiolo gischee ntechnisch e voordelenbove nd etraditionel e jutezakkene nverdiene n aanbevelingi ntropisch e landen alsGhana . Inee n afrondend onderzoekblee kee n verhoogde aflatoxinebiosynthes e door A. flavus nabestraling , zoalsi nd eliteratuu rvermeld ,nie t aangetoondt e kunnenworde nbi jhe tgel emaïsra sda t daarvoorwer d gebruikt.I nee n senso rische evaluatie,waarbi jme t2 0panellede n een"consume r acceptance test" werduitgevoerd ,wer d geen significant verschil (P<0.05)i nkleur ,geu re n smaakwaargenome n tussenmaï swaaro pd egecombineerd ebehandelin gwa suitge voerde nd eblanco . 188 CURRICULUM VITAE George T.Odamtte nwa sbo m on 7thJul y 1948a tKoforidu a inth eEaster n Regiono fGhana .Afte rhi sPrimar ySchoo leducatio n at thePresbyteria nPrimar y Schoolsa tSuhu man dAccr a (1952-1958),h eproceede d toth ePresbyteria nBoy s Boarding School,Osu ,Accr a fromwher eh eentere d theAccr aAcadem y forhi s Secondary Schooleducatio n (1962-1969).H eobtaine d theWes tAfrica nExamina tionCouncil' s GeneralCertificat e inEducatio n (G.C.E.,Ordinar y Level)i n 1967an dth eUniversit yo fLondo nAdvance dLeve lCertificat e in1969 . Hsha dhi sUniversit y education atth eUniversit yo fGhana ,Legon ,fro m October 1969 to 1974durin gwhic hh eobtaine d theB.Sc .Genera l degree (Botany, Zoology)i n 1973an d theB.Sc .Honour s Botanydegre e (Microbiology andPlan t Pathology)i n 1974.Fro mSeptembe r 1974t oAugus t 1975h ewa s aTeachin gAssis tanti nth eDepartmen to fBotan ywher eh e assisted inbot h researchan dteach ing.H ewa s awardeda nM.Sc . Botanydegre e inMycolog yb y theUniversit y of Ghanai n1977 . Later,i n September 1977,h e joinedth estaf fo fth eGhan aAtomi cEnerg y Commission,Departmen to fBiology ,Foo dan dAgriculture, a s aScientifi c Officer (Microbiology).Hi sapplie dresearc h interestswer e post-harvest pathology aswel la sbiologica lcontro lo fplan tpathogens .H ewa s awarded an appliedresearc h fellowshipb y the InternationalAtomi cEnerg yAgenc y topar ticipate inth e courseo fstud ya t the 1stIFFI T InterregionalTrainin gCours e onFoo d Irradiation (Wageningen,Th eNetherlands ,4t h Sept. 1979- 12thOct . 1979),durin gwhic hh eobtaine da Certificat e inFoo dIrradiatio nTechnology . Thereafter,h econtinue dhi s appliedresearc h inth econtro lo fmicrobia l spoilageo fcereals ,cocoa ,anima l feed,cotto nseed s and aubergines employing ane w technique ofcombinatio n treatmento fhea tan dgamm airradiation . Thiswor k forming thethesi swa s carriedou ta t ITAL,Wageninge n and then continuedo nhi s returnhom ea tth eUniversit y ofGhana .H e lectured inBotan y onpart-tim ebasi s at theUniversit y ofGhana ,Lego n from 1981 toFebruar y 1982 andthe nbecam e full-timeLecture r inMarc h 1982whils th e continuedhi sre searchwor k under thesupervisio n ofProf.Dr .E.H .Kampelmacher ,durin g the period 1981-1985.Th epresen taddres so fth ecandidat e isDepartmen t ofBotany , Universityo fGhana ,Pos tOffic e Box55 ,Legon/Accra ,Ghana . Aangezienbestralin gva nvoedingsmiddele nme tee ntotal edosi sva nmax . 10kG yal sveili gword tgeaccepteer d (JECFI, 1980),ko nd ei ndi tproefschrif t voorgesteldebehandelin gvoo rontsmettin gva nmaï se nander e granene npeul vruchten ind epraktij kworde ntoegepast .Bestralin gverander tee nproduk tva n slechtekwalitei techte rnie t inee ngoe dprodukt ,e ndaaro mi she t zaakdez e behandelingallee no pgrondstoffe nva ngoed ekwalitei t toe tepassen .Voo ree n maximalehoudbaarhei dmoe the tproduk t ind eaanbevole nemballag e (gewevenpo - lypropyleen zakken)verpak tworde nv66 r debestraling .Daarbi j dientme nzic h striktt ehoude naa nee nhygiënisch ebedrijfsvoerin g inhe tpakhuis . 189