Temperature and pH Factor on Cocoa Bean-Based Ochratoxin-A (OTA) and Detoxification Potential of Rhizopus stolonifer

Abstract

Aim: To study the effect of temperature and pH on detoxification potential of Rhizopus stolonifer on Ochratoxin-A (OTA) from cocoa beans in Nigeria. Place and Duration of the Study: This study was carried out in the Plant Pathology laboratory of Cocoa Research Institute of Nigeria (CRIN) for the period 2013-2017. Methodology:  Stored cocoa samples were collected from Cross-River, Ondo and Osun States in Nigeria, assayed on Potato Dextrose Agar. The pure culture of the isolate of Rhizopus stolonifer used as a detoxifying agent was obtained from CRIN Mycobank. Aspergillus ochraceus, A. niger and Penicilium verrucossum cultured from cocoa bean samples were used in the study. These ochratoxigenic fungi were cultured on Yeast Extract Sucrose (YES) agar in the dark for 14 days for detection and detoxification of toxin. Ochratoxin-A (OTA) productions were detected using retention factor (RF) on simple Thin Layer Chromatography (TLC) plate of solvent system Toluene: Ethyl acetate: Formic acid (5:4;1 v/v/v) while confirmation of OTA was done using 365nm wavelength of ultraviolet (uv) light. Results: Five fungi genera: Aspergillus, Penicilliun, Fusarium, Yeast and Rhizopus were cultured from cocoa beans sample across the study locations: The tested temperature and pH indicated that Aspergillus ochraceus and Penicillium verrucosum had retention factor (Rf) value of 0.88 to 1.29 which showed green-blue colouration under Ultra Violet (UV) light confirming the presence of Ochratoxin-A (OTA) while A.nigerisolate had RF of 0.88-0.55 with yellow to olive yellow colour confirming the absence of OTA.  Rhizopus stolonifer isolate detoxify OTA when cultured with A. ochraceus and P. verucossum at pH 7.0 and 9.0 and 25°C-30°C with yellow to brownish-yellow colouration confirmed as a less toxic metabolites (Citreoviridin, Palutin and Palitantin) while no toxin was detected in A. niger plus R. stolonifer after detoxification. Conclusion: There is variability in retention factor as temperature and pH changes. Production of OTA occurred between 25-30°C and pH of 4.0 to 9.0 but peaked at pH 4.0 with Rf 1.3 at neutral pH and 25°C enhanced detoxification potential of R. stolonifer. Thus R. stolonifer can be developed to detoxify OTA from Cocoa beans at favourable eco-physiological factor.