1.      Introduction

Thehighincidenceofpost-harvestfoodlosses,arisingmainlyduetoinadequatefoodstorage andpreservationtechnologies,isamajorissueaffectingthequalityoffoodinWestAfrica, whereseasonalfoodshortagesanddiseasesresultingfromnutritionaldeficiency arestillamajor concern[1].Studyhasshownthatfruits,vegetables,rootsandtuberscontributetonearly50%of perishablefood commoditieswhile grainssuchasmaize,sorghum,millet,riceandcowpeas contributetoabout30%offoodlossafterharvestinWestAfrica[1].Factorsthatcontributeto theselossesmay include;inappropriatefoodprocessingtechnologies,poorharvestingand inefficientpost-harvesthandlingpractices,badroads,moribundrailsystemsandmanyothers [1].

InGhana,issuesofpost-harvestlossesarepredominantespecially wherelocally producedcrops suchascassava,yam,maize,rice,beans,andothersarehardlyprocessedleadingtowasteof cropsespecially duringbumperharvest.Inordertoextendtheshelf-lifeofsomeofthesecrops andhencereducetheincidenceofpostharvestlosses,they areprocessedintofloursandother productswhichmaybeusedby individualsathomeorsoldoncommercialbasis.Methods involvedinprocessingthese indigenousfoodstuffsmay,however,exposethemtocontamination by severalpathogensmainlyfilamentousfungiandsomebacteriainadditiontocontamination from thefarm beforeprocessing. Forinstance, most cereal grains can becontaminated by differentspeciesofmicroscopic fungusduringitdevelopmentalstages[2]andthese pathogens mayaffect thecrop resulting in a reduction ofthequalityof thegrain.

Somespeciesmay producemycotoxinsthatintoxicatebothhumansandanimalupon consumption[2].Mycotoxinclassesknownto occur incereals,includingtheaflatoxins(AFB1, AFB2andAFG1,G2),tricotecens,deoxinivalenol(DON)and(T-2toxin),the fumonisins(FB1, FB2andFB3),thezearalenone(ZON),ochratoxinA(OTA)andtheergotalkaloids[3]are knowntobecarcinogenic.Studieshaverevealedthatmajority ofthesemycotoxinsareproduced bythe genuses Aspergillus, Penicilliumand Fusariu[3].

 Sporesproducedby thesefungiareverydifficulttoeliminateduetotheirstabilitytohigh temperature and other harsh environmental conditions, hencethe presenceofthesespores in food poses threattothehealthofconsumers.Inthecase offlour,thehigh-gradetypesare treatedto containverylowornocontaminationduetouseofadvancetechnologies[4].However,locally processedindigenousfloursmay becontaminatedby differentmicrobesduetoimproperfood safety practicesandasthesefloursareusuallysoldoncommercialbasis,they may resultin exposing consumers to several health risks.

 The purposeofthisstudy,therefore,wastoevaluatethefilamentousfungiofsomeselected processed indigenousflours sold in Kumasiin Ghana.

 2.      Materialsand Methods

 2.1.  Experimental design

 Twotypesofprocessedindigenousflourswere consideredfor thisstudy;maizeflouranddry cassava(kokonte)flour.Thesetwoflourswereconsideredforthestudybecausethey arevery commonandfrequently usedinfood preparationinGhana.Sixteensamples(eightofeachflour) were selected for this study from four different markets (Ayigya market, Central market, Bantama marketandAtonsumarket)inKumasi.Foreachmarket,twoVendorswere selectedat randomfor samplecollectionandadministrationof questionnairesandfromeachVendor three samplewere bought.Sampleswere packagedintosterilesamplebagsandbroughttothe laboratory foranalysis.Sampleswereanalyzedonthesameday theywereboughtfromthe market,butthosethatwerenotanalyzedsamedaywerestoredintherefrigeratorat4^°C. Sampleswereanalyzed inbatches,foursamplesperbatch.Twosamples(maizeflourand kokonteflour) werealsoprocessed in thelaboratoryto serveascontrols.

 2.2.  MoistureContent Analysis

 Two(2) gramsample wasweighedintoa Petridishandplacedinanovenat130^°Cfor 2hours, weighingintermittentlyuntiltherewasnochangeinweight[5].Thesampleswerecooledto roomtemperatureina desiccator at eachtimebeforeweighing.The moisturecontentwas express as; (Weightloss/initial weight of flour)x100%

 2.3.  Microbial Sample Preparation

 Workingunderasepticcondition,tengrams(10 g)ofeachsamplewasweighedusingasterile weighingboatandtransferredtosterilesamplebottlescontaining90mLsterilepeptonewater [3].Eachsamplewasvortexedforabout1minuteatmoderatespeedandseriallydilutedto make fivedilutions(10^-1-10^-5)bytransferring1mLhomogenizedsampleto9mLdilutionblank, mixingwelluntilthe10^-5dilutionwasobtained.Aliquots(0.1mL)ofthesedilutionswereused forthe study.

 2.4.  MicrobialEnumeration

 Spreadplate methodof inoculationwasemployedinthe microbialexaminationof the samples. Fromtheprepared10-foldserialdilutions,enumerationofmouldswerecarriedoutbythespread platemethodonPotatoDextroseAgarcontaining100mg/L ofchloramphenicoland50mg/L Oxytetracyclinetosuppressthegrowthofbacteria[3].Theplateswereincubatedat25^°Cfor5 to7days.Aftertheappropriateincubationperiods,dilutions with20-200 colonieswereselected andmanually counted.Thenumberofcolony-formingunitspergram(cfu/g)ofsampleswas calculated bymultiplyingthe number oforganisms bythe dilution factor.

 2.5.  IsolationandIdentificationofMoulds

Threedifferentmediaforthecultivationoffungi; PotatoDextroseAgar(PDA),Oxytetracycline- GlucoseYeastExtractAgar(OGY)andDichloranRoseBengalChlortetracyclineAgar(DRBC) (eachcontaining100mg/L ofchloramphenicol)wereusedfortheisolationofthemoulds.From theprepareddilutions,0.1mloftheinoculumwasinoculatedontothedifferentmediaby the spread platemethod andthe plates incubatedat 25^°Cfor5 to 7 days.

 2.6.  IdentificationofMoulds

 Mould cultures were prepared by lifting the mycelia mat of the organism with a sterile inoculationpinintoa dropof lactophenolblueona slideteased(spreadingthemat)andcovered withacover-slip andobservedunderamicroscope.Different characteristicfeaturesofthe isolatedorganismwereobservedandused intheiridentificationusingthefourtheditionof introduction to foodborne fungi [6].

 2.7.  Total PlateCount

 Spreadplatemethodofinoculationwasusedtodeterminethetotalplatecountoforganismsin thesamples.Fromthepreparedserialdilutions,enumerationwascarriedoutby thespreadplate methodonPlateCount Agar.Theplateswereincubatedat37^°Cfor24hours.Afterthe appropriateincubation,plateswith30-300colonieswereselectedandmanually counted.The numberofcolony-formingunitspergram(cfu/g)ofsampleswascalculatedby multiplyingthe numberof organisms bythe dilution factor.

 2.8.  ColiformCount

 SterileMacConkey brothwas preparedintesttubesforthecultivationofcoliforms.Fromthe preparedserialdilution,1mLofinoculumfromthe1:10dilution(10^-1)wastransferredinto9 mL ofMacConkey brothunderasepticcondition.Incubationwasdoneat37^°Cfor24hoursand test tubes which showedchangein mediacolour from red toyellow wererecordedas positive.

 2.9.  Statistical analysis

Analysiswasdone intriplicatesinorder tominimize the error marginasmuch aspossible. ResultsobtainedweretabulatedintoMicrosoftExcel2010andforeasy interpretation,thedata was subjected to one-wayAnalysis of Variance (ANOVA) and the significance differences betweenthemeansofthevariousmarketsdetermined by usingStatisticalPackageforSocial Sciences (SPSSversion 20). P-values ≥ 0.05 were considered as statisticallynot significant.

 3.      Resultsand Discussion

 3.1.  Moisture content ofthesample

Theaveragemoisturecontentofthemaizefloursamplesrangedfrom12.37%±0.15to19.70% ±0.12whilethatofcassavaflourrangedfrom10.93%±0.27to16.90%±0.56asshownin Table1.Themoisturecontentobtainedforthevariouskokontefloursamplesisinagreement withthestudy result(10.0%to16.9%)reported byLokko,(1978).Severalresearcheshavebeen conducted toestablishanacceptablemoisturecontentofkokonteflour. Reportsshow thatfora kokonteflour toremainstable,a moisture contentof 12% isrequired[7].Apartfromsamples bought from Atonso market (AtmKF1 andAtm KF2) and thecontrol (CKF) which recorded valueslowerthan12%,theremainingsampleshadmoisturecontents higher than12%.This suggeststhatthehighmoisturecontentmay beacontributoryfactortomicrobialcontamination sincestudieshaveshownthatmicroorganismsrequiremoisturefortheirgrowth[8]. Correlation analysis revealed a positive correlation between moisture content and mould count of dry cassava(kokonte) flour.Thoughhighmoisturecontentinfoodsisknowntobe a stronginfluence for growthofmicroorganisms,theresultsforthemoisture contentofAtmKF1,AtmKF2and CKFfromthisstudysuggestthatotherfactorsapartfromthehighmoisturecontentmay account forthe contamination in the samples.

3.2.  EnumerationofMoulds

Resultsfor theenumerationofmouldsinthesampleshavebeenrepresentedinTable2.Forthe dry cassava(kokonte)flour,allthesampleswerecontaminatedwithmoulds,representing100% mouldscontamination,butthelevelsofcontaminationvariedamongthesamples.The levelofmouldcontaminationamongthesamplesexceededtheacceptablemouldslevelinfood(10³cfu/g)asreportedby AfricanOrganizationfor Standardization[9].Thisis anindicationthatthe drycassava(kokonte)floursoldonthevariousmarketsmaybeunwholesomeand,therefore, pose healthrisktoconsumers.Incomparingtheresultsfor mouldcountinthe variouskokonteflour,withresultsobtainedbyLuetal.[10],all the samplesrecordedcounthigher than6.5×10³cfu/gasthey reportedintheirstudiesexceptforthesamplesfromVendor-2inBantamamarket (BmKF2)whichrecorded1.70×10³±0.15cfu/g.The controlsamplealsorecordedcountlower than reported. Statisticalanalysisshoweda significantdifference betweenthe countsatp<0.05. Enumerationofmouldsinthe maize floursamplesrevealedvaryingdegree ofcontamination among thesamplesranging from no observablemould count as recorded byAtm MF1, BmMF1, BmMF2andCMFtohighcountof1.18×10⁶±0.18cfu/gasrecordedby CmMF1.Samples fromAymMF2,CmMF2andAtmMF2showedcountsthatwerealittlehigherthanthe tolerablelevelof10³cfu/g,recording1.67×10³±0.30cfu/g,3.40×10³±0.30cfu/gand2.97× 10³±0.30cfu/g,respectively.Extremelylowcountsofmouldinthesamplesasrecordedby AtmMF1, BmMF1, BmMF2and CMFis inagreementwithresultsobtained byAdu-Gyamfi andAppiah[11],obtainingmouldcountof5.0×10¹cfu/g.Thelowcountofmouldsreported may beduetothedehullingofgrainsbeforeprocessingintoflourasreportedbyVictoretal.[12].Itisthoughtthatthemicroorganismsareusually foundontheoutercoatofthegrainsand hencedehullingisameansofreducingcontamination.Theextentofcontaminationinsamples for Aym MF1 and Cm MF1, however, suggests that the dehulling process may not be a guaranteethatsamplesareabsolutely freefromcontaminants.Thehighincidenceof contaminationinthesesamplesmay beattributedtofactorssuchasthehighmoisturecontentof thefloursamples,thelength/periodthesampleshavebeenonmarket,theprocessingmethod,the hygienic practices employed in processing, and the conditions under which the food commodities weresold on the market.

3.3.  Identificationofmoulds

Mouldsbelongingtofivegenera wereisolatedandidentifiedfromboth themaizeflourandthe dry cassava(kokonte)flour.Thedifferent generaincludedCladosporium,Aspergillus,Mucor, RhizopusandPenicillium(Figure 1).Tables3and4,showthedifferentorganismsisolatedfrom thevariousfloursamplesfromthedifferentmarkets.Thesefindingsarecontrary tothatofLuet al.,[10]whoisolatedonly two genera;AspergillusandPenicilliumfromtheirsamples.Themost predominantorganisms isolatedfromthedry cassava(kokonte)flourwereAspergillusflavus, RhizopusstoloniferandMucorhiemaliswhilethatforthemaize flour werePenicillium crustosum,Rhizopusstolonifer,CladosporiumcladosporioidesandAspergilluswentii. The prevalenceoftheseorganismsinthevariousfloursamplesmaybeduetotheubiquitousnature oftheirsporesasmentionedbyO’Gormanetal.[13].Studieshaveshownthatsomeofthese mouldsproducemycotoxinswhichare knowntobeteratogenic,mutagenic,hepatotoxic, genotoxicandhepatocarcinogenicdependingonhowlonganindividualgetsexposedtothe toxin [14].

3.4.  Total Plate count

Resultsfromthetotal platecountisanimportantindicationofthehygienicconditions surrounding the foodandalso shows theeffectiveness andefficiencyofthefood chain process as wellas the shelf lifeof the food[12].In this study,the total plate countfrom thekokontesamples werehigher(Table5)thanthatreportedbyLuetal. [10]whorecorded16×10³cfu/g.Only samplesfromVendor-2inBantama market(BmKF2)andthe ControlKokonteFlour (CKF) recording7.8×10³±0.30cfu/gand5.63×10³±0.45cfu/g,respectively,werealittle lowerthan theresultsobtained byLuetal.[10]. Formostofthesamples,thelevelofcontaminationwas foundtobehigherthantherecommendedlevel(10⁵cfu/g)andthisisanindicationofpoor sanitationorproblemsresultingfromtheprocesscontrolorhandlingoftherawmaterial[12]. Forthemaizefloursamples,exceptforsamplesfromBantamamarketVendor-1(BmMF1) whichdidnotrecordany growthfortotalplatecount,theremainingsamplesrecordedcounts whichexceededthe tolerable level.Thisisanindicationthatfavourable conditionsexistwithin the flour to support thegrowth of various organisms.

3.5.  ColiformCount

Mostof the samplestestednegative for coliformsexceptfor maize floursample boughtfrom Vendor-2fromAtonsomarket(AtmMF2),dry cassava (kokonte)flour boughtfromtheCentral market Vendor-1(Cm KF1) andkokonteflourbought from Vendor-1(Bm KF1) (Table 6). Resultsfromcoliformtest,whichisanindicatorof personalhygienelevelof flour sellers[12], suggestthatthehighlevelofcontaminationmaynotnecessarily beasaresultofpoorhygiene practices by floursellersbutotherfactorssuchascontaminationduringharvestandstorageof cereal as mentioned byVictor et al.[12].

4.      Conclusion

 The studiesrevealedthatmostoftheflour sampleshadhighmoisture contentandthelevelof mouldcountandtotalmesophilicmicrobe presentintheflourexceededthetolerablelevel indicating that consumers maybeat health risk upon consumption ofthesesamples.

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