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Using Rhizopus sp. against the growth of Aspergillus carbonarius as a potential biological control method of ochratoxin A contamination in raw cocoa beans

a Department of Food Science and Technology, University of Nangui Abrogoua, 02 Bp 801 Abidjan 02, Côte d'Ivoire b UMR Qualisud, CIRAD - TA-B 95/16, 73 rue JF Breton, 34398 Montpellier Cedex 5, France

Didier Kra Brou KEDJEBOa, Tagro Simplice GUEHIa, Maï Koumba KONEa, Pascaline ALTERb, Noël DURANDb, Renaud BOULANGERb, Angélique FONTANAb, Didier MONTETb

Ochratoxin A (OTA) is a carcinogenic and nephrotoxic mycotoxin produced by fungal species belonging to

Aspergillus and Penicillium genus. OTA occurrence in foodstuffs depends on the climate during harvest

and on the post-harvest storage conditions. Among known ochratoxins, OTA currently contaminates

crops. Due to its prevalence and toxicity, OTA occurrence development conditions in raw cocoa beans

were previously studied during post-harvest treatments. This research aimed to investigate on the

biological control of OTA excretion in cocoa.

Material and MethodsCocoa CocoapodsusedwerethevarietyofcloneCLM99harvestedatTrinidadTobagoin2013.

Analyzes •Cocoapodinocula-onbyRhizopusRhizopussp.isolatedfromroAencocoapodsopenedaBer7daysdelayandA.carbonariusisolatedfromcoffeecherrieswereseparatelygrewonPDAagarfor5daysat25°C(Figure1).Differentconidiainoculawerepreparedfromcalibratedsuspensionsat102,104and106conidiapermillileter.Then1mLofeachpreparedinoculumwasasepPcallyinjectedusingasyringeinsidehealthcocoapods.InoculatedcocoapodswereincubatedinaclimaPcchamberat30°C,60%relaPvehumidityfor4or8days.•OchratoxinAcontentmeasurementOTAwasextractedfromcocoabeanssourcedfrominoculatedpodsandthenseparatedbyimmuno-affinity,idenPfiedbyHPLCandquanPfiedbyspectro-fluorescencedetector.

ResultsTableIpresentsthephotographsofbeansinsidecocoapodsaBerinoculaPonwithvariousconcentraPonsofA.carbonariusandRhizopussp.conidiasuspensions.Figure2indicatesOTAcontentforcocoabeanssourcedfrom:

•Podsinoculatedwith104conidiapermLofA.carbonariusand104conidiapermLofRhizopussp.-incubatedfor4days:noOTAproducPonwasobserved- incubatedfor8days:OTAproducPonof37µg.kg-1washighlightedwhencontrolpresentednoOTAproducPon.

•Podsinoculatedwith104conidiapermLofA.carbonariusand106conidiapermLofRhizopussp.-incubatedfor4days:noOTAproducPonwasnoPced-incubatedfor8days:OTAproducPonof5.6µg.kg-1wasobtainedagainst0µgperkgforthecontrol.

•Podsinoculatedwith104conidiapermLofRhizopussp.and106conidiapermLofA.carbonarius-incubatedfor4days:36.3µg.kg-1ofOTAwasrecorded-incubatedfor8days:6.2µg.kg-1ofOTAwasobserved

Conclusion

Durand,N.,2012.

Dynamiquedesp

opulationsmicrobi

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au cours du trai

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interspécifiques e

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Rhizopus sp appeared as a promising biological control fungal agent for OTA

production in raw cocoa beans. As the fungal growth in raw cocoa beans constituted

a serious qualitative default, further investigation must be done on the identification

of the metabolites leading to the control of OTA excretion in cocoa.

Figure 1. Sampling during post-harvest processing of cocoa

Figure 1. Cultures of Aspergillus carbonarius (A) and Rhizopus sp. (B) on PDA agar used for cocoa beans inoculation inside pods

Table I. Photographs of beans inside cocoa pods inoculated with suspensions of various concentrations in A. carbonarius and Rhizopus sp. conidia

Corresponding author: [email protected]

Concentration of fungal conidia (conidia.mL-1)

Incubation

time (days)

A.carbonarius Rhizopus sp A.carbonarius Rhizopus sp A.carbonarius Rhizopus sp A.carbonarius Rhizopus sp

0 104 106 104 104 106

4

8

Figure 2. OTA content of cocoa beans from pods inoculated with various

concentrations of A. carbonarius and Rhizopus

sp conidia suspensions of