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KrishnaPrasadRai*,ShivSagar Chaudhary,Sovita Shrestha,Som Kanta Rijal,Bal Kumari

SharmaandJiwan P.Lama

DepartmentofFoodTechnologyandQualityControl,CentralFoodLaboratory,Kathmandu,

Nepal

Introduction Researchdesignincludingsampling Results&Discussionwithkey

findings Conclusions Recommendations

PresentationLayout

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ATotalDietStudy(TDS)hasbeenusedasatoolforestimatingthelevelofdietaryexposuretocertainchemicalcontaminantscomingfromdifferentfoodsourcesamonggeneralpopulationsince1960s.

ItisrecognizedasacosteffectiveapproachtoestimatingdietaryexposureofcontaminantsbytheWorldHealthOrganization(WHO)in1978.

WhatisTotalDietStudy(TDS)?

AflatoxinsB1,B2,G1andG2hasbeendefinedastotalaflatoxins(TA)byCodexAllementarius Commission(CAC),whicharenaturallyoccurringtoxincompoundsproducedasmetabolitesbythefungiAspergillus flavusandAspergillus parasiticus growingonavarietyoffoodandfeedproductsunderfavorabletemperatureandhumidity.

Likewise,aflatoxinsM1andM2arethesecondarymetabolitesofG1andG2,whicharefoundinanimalsmilkandmeat.

Similarly,theothermostcommonmycotoxins areochratoxins,tricothecenes,zearalenone andfumonisins.

WhatisTotalaflatoxins?

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Aflatoxinsarepotentiallyhazardoustohumanandanimalshavingstrongimmunosuppressive,mutagenic,teratogenicandcarcinogeniceffects(HusseinandJeffrey,2001).

ChemicalStructuresofAflatoxins

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Agency Food MRL ReferenceGoN, Nepal Peanuts, beans and other

cereals products20g/kg Food Standard,

(2067)CAC Peanuts 15g/kg Codex Standard 193-

1995CAC Brazil nut, Almond,

Pistachio10g/kg Codex Standard 193-

1995EU Peanut/Groundnut 15g/kg for

unprocessed(EC) No 1881/2006

4g/kg for proceesed

EU Almond 10g/kg for directhuman consumpton

(EC) No 1881/2006

PFA , India Beans and cereals 30g/kg PFA, (2004)

FDA All products except milkdesigned for human

20g/kg Cornell University(2013)

Country Commodities Mycotoxin Level of presence Reference

Syria Different milks AFM1 >20 to 765 ng/l. Ghanem & Orfi,(2009)

Iran Human breast milk AFM1 0.326.7 ng/kg). Sadeghi et al.,(2009)

Tunisia Tunisian foods OTA 3.5 5.3 ng/g Ghali et al., (2008)Zearalenone 10.4 11.8 ng/g.

Athens Breakfast cereals AFB1 mean 1.42 ng/g Villa and Markaki(2009).OTA Mean 0.18 ng/g

Iran Pistachio AFB1 9- 45.7ng/g Sheibani,Ghaziaskar, (2009).AFB2 ND- 8ng/g

Nepal Ground nuts AFB1 3.78-39.69ppb Rai and Chaudhari,(2011)

Pakistan Buffaloesmilk

AFM1 0.027 g/ L Hussain et al.,(2008)

Cows milk 0.044lg/L

Mycotoxinscontaminationinsomefoods

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Themajorobjectiveofthisworkwastofindoutthecontaminationleveloftotalaflatoxinsinsomedietaryrawfoods(viz.maize,peanutsandtheirproducts)aswellastoassessthecrudedietaryexposureofthosecontaminantstoNepalesepeople.

GeneralObjective

SamplecollectionfromdifferentdistrictsofNepal

Quantitativeanalysisfortotalaflatoxinsincollectedfoodsamples

Assessmentofcrudedietaryexposurelevel

SpecificObjectives

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Thisstudydidnotincludetheeffectsofstorageconditionforrawfoodsinsamplingarea.

Theresultisintheformofcombineaflatoxins(B1,B2,G1andG2)termedastotalaflatoxinssothattheindividualaflatoxin wasnotdeterminedinfoodsamples.

Limitationsofstudy

Maize Peanuts Maizebasedproducts(e.g.cornflakes,suzi grits,Lito,

popcornetc.), Peanut/nutbasedproducts(e.g.Dalmoth,peanutbutter,

nutcracker,proteaseetc.), Legumes(e.g.soybean,beansetc.)and Others(e.g.Uwa,wheat,buckwheat,etc.).

FoodSamples

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SamplingmethodClustersampling SamplingDistricts:32 Totalno.ofsamples322 Sampleweight:500gto1kg AllthesampleswerepackedinPEbagscarefullyand

storedindrystorageatroomtemperaturetilltheanalysiswascompleted.

Samplingplan

Commoditywisesamplingplan

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RegionwiseSamplingplan

Region

Food commodities

TotalMaize Peanut

MaizeBasedproducts

Peanut/Nutsbasedproducts

Legumes

Others

Eastern 26 30 9 5 3 2 75

Central 38 36 12 0 1 1 88

Western 18 16 13 5 0 21 73Mid-western 19 10 7 1 0 8 45Farwestern 13 10 9 6 0 3 41

Sub total 114 102 50 17 4 35 322

EnzymelinkedImmunosorbentassay (ELISA)HELICATotalAflatoxinsAssay(USA),wasusedfortheQuantitativedetectionforaflatoxinsB1,B2,G1andG2.

LOD

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ELISAKitReader

Readtheopticaldensity(OD)ofeachmicrowellwithamicrotiterplatereaderusinga450nmfilter

Allowtheparticulatemattertosettle,thenfilter5 10mloftheextractthroughaWhatmanNo1filterpaperandcollecttheclearfiltratetobetested.Thesample

isnowreadyfortesting

Allthesamplesweremixedandstirredmanuallyforaminimumof2minutes.

Weight20gofsievedportionofthethenadd100mloftheExtractionSolvent(70%methanol:30%DW)ina250mlstopperedVolumetricflask

Grindsampletotheparticlesizeoffine(50%passesthrougha20meshscreen).

Sampleextractionandanalysis

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Samplepreparation

S.N. TDS foods Contamination No. % of Contamination

1 Maize 80 70.17

2 Peanuts 74 72.54

3 Maize based

products

40 80

4 Peanut based

products

14 82.35

5 Legumems 3 75

6 Others 8 22.85

PresenceofTotalaflatoxinsinfoodsamples

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Regions Maize Peanut

MaizedBased Pdts

Peanut/nuts based pdts Legumes Others

Eastern nd to 17.4nd to35.7 1 to 13.4 6.3 to 16 1 to 2.6 nd

Centralregion nd to 30

nd to24.5

1.1 to23.1 - nd nd

Westernregion nd to 30 nd to 30 nd to 30 nd to 30 -

nd to12.3

Midwestern nd to 27 nd to 5.8 nd to 9.6 10.2 - nd to 4.5Farwestern 1 to 30 1.9 to 21 nd to 25 nd to 20.5 -

nd to22.1

TotalaflatoxinsindifferentregionofNepal

Averageaflatoxinsindifferentfoodcommodities

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TAexceedingthemandatorylimitofGoN

Food Commodities

ICMR RDA (g/d)

Total average aflatoxin (g/ kg of food)

Crude aflatoxin consumption (g/ day)

Reference weight in Kg

Male (g/kg b.w./day)

Female (g/kg b.w./day)

60kg*** 50kg***Maize 183 7.89 1.44 0.024 0.029Peanut* 40 8.11 0.32 0.005 0.006Maize based products 183 6.39 1.17 0.019 0.023Peanut based products* 40 8.65 0.34 0.006 0.006Legumes 26 0.54 0.014 0.0002 0.0003Others** 183 2.53 0.463 0.008 0.0093

CrudedietaryexposureofTA

*Takenfrombalanceddietfornormalwomen(highcostdiet)(Swaminathan,1991)**Othersproductsmeansmostlycerealbasedotherthanmaize***ReferenceweighttakenfromGopalan etal.,(2004)

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Food Commodities

ICMR RDA (g/day)

Region***Eastern Central Western Midwestern Far western

Male Femal

e Male Femal

e Male Femal

e Male Femal

e Male Femal

e

60kg 50kg 60kg 50kg 60kg 50kg 60kg 50kg 60kg 50kgMaize 183 0.012 0.014 0.024 0.029 0.024 0.028 0.025 0.029 0.045 0.054Peanut* 40 0.007 0.009 0.003 0.003 0.007 0.009 0.0005 0.0006 0.007 0.009Maize based products 183 0.027 0.032 0.013 0.016 0.013 0.016 0.015 0.018 0.029 0.036Peanut based products* 40 0.007 0.009 0 0 0.005 0.006 0.007 0.008 0.004 0.004Legumes 26 0.0006 0.0008 0 0 0 0 0 0 0 0Others** 183 0 0 0 0 0.016 0.019 0.001 0.001 0.008 0.009

CrudedietaryintakeofTA(Regionwise)

*Takenfrombalanceddietfornormalwomen(highcostdiet)(Swaminathan,1991)**Othersproductsmeansmostlycerealbasedotherthanmaize***ReferenceweighttakenfromGopalanetal.,(2004)0=Nototalaflatoxinfoundinsamplesornosampletaken^Resultsarein(g/kgb.w./day)

Among322rawfoodsamples,TAwasfoundin68.01%ofsamples;however,theaverageTAleveldidnotcrossedthemandatorystandardforfood(20ppb)orforfeed(30ppb).

Over80%ofpeanut,maizeandtheirproductswerefoundtobecontaminatedwithtotalaflatoxins.

Keyfindings

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Interestingly,18.32%ofrawfoodsampleshowedhigherconcentrationthantheMRLofGoN,wheremaize,peanut,theirproductsandothercontributedas15.78%,11.76%,11.76%,2.86%respectively.

CrudedietaryexposureofTAwasfoundtobehighestinFarwesternregion(0.045g/kgb.w./dayformaleand0.054g/kgb.w./dayforfemale),whileEasternregionshowedthelowestinrawmaize.

Keyfindingscontd..

Theaveragecontentoftotalaflatoxinsindifferentfoodsamplesandtheirdietaryexposuretohumanwasfoundtobewithinthesafelimit;however,68%sampleswerefoundtobecontaminatedindifferentconcentrations.

Moreover,about18%werefoundtobeheavilycontaminatedwithTA.

Therefore,astrategicinterventionprogramisrequiredatpolicylevelofgovernmenttocontrolsuchcontaminantsinfoodcommodities.

Conclusions

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NationalReferenceRecommendedDietaryAllowance(RDA)shouldbeestablished.

thegovernmentsideaswellasUNagencysuchasWHO,FAOcouldworkjointlyinfuturetoestablishtheNationalRDAinNepal.

Since,thisisapreliminaryworkonTDSinNepal,scopesforothercontaminantsaswellasotherfoodcommodities(rawandcookedstate)shouldbecoveredinfuturestudiesplan.

Recommendations

AnalyticalcompetencyismostvitalpartofTDSwork;thereforegovernmentaswellasdevelopmentpartnersshouldprioritizeonbuildingupoffacilityandcompetencyforinstrumentalanalysisforallkindsofcontaminantsincountry.

Recommendationscontd..

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WewouldliketoacknowledgeWHO,countryofficeforNepalforsupportingthisprogramin2012.

WearealsogratefultoDFTQCforgivingthisopportunitytoourteam.

Acknowledgements