Strategies for achieving global food security include identification of alternative feedstock for use as animal feed, to contribute towards efforts at increasing livestock farming. The presence of theobromine in cocoa pod husks, a major agro-waste in cocoa-producing countries, hinders its utilisation for this purpose. Cheap treatment of cocoa pod husks to remove theobromine would allow largescale beneficial use of the millions of metric tonnes generated annually. The aim of this study was to isolate theobromine-degrading filamentous fungi that could serve as bioremediation agents for detheobromination of cocoa pod husks. Filamentous fungi were screened for ability to degrade theobromine. The most promising isolates were characterized with respect to optimal environmental conditions for theobromine degradation. Secretion of theobromine-degrading enzymes by the isolates was investigated. Theobromine degradation was monitored by HPLC. Of fourteen theobromine-degrading isolates collected and identified by rDNA 5.8S and ITS sequences, seven belonged to Aspergillus spp. and six were Talaromyces spp. Based on the extent of theobromine utilization, four isolates; Aspergillus niger, Talaromyces verruculosus and two Talaromyces marneffei, showed the best potential for use as bioagents for detheobromination. First-time evidence was found of the use of xanthine oxidase and theobromine oxidase in degradation of a methylxanthine by fungal isolates. Metabolism of theobromine involved initial demethylation at position 7 to form 3-methylxanthine, or initial oxidation at position 8 to form 3,7-dimethyuric acid. All four isolates degraded theobromine beyond uric acid. The data suggest that the four isolates can be applied to substrates, such as cocoa pod husks, for elimination of theobromine.

BackgroundUtilization of cocoa pod husks (CPH) in animal feed is hindered by the presence of theobromine, which is variably toxic to animals. Treatment of this agro-waste to remove theobromine, while preserving its nutrient content, would allow beneficial use of the millions of metric tonnes discarded annually. The aim of this study was to assess the suitability of selected theobromine-degrading filamentous fungi for use as bio-tools in degradation of theobromine in CPH.ResultsThe candidate fungi assessed in this study were an Aspergillus niger (AnTD) and three Talaromyces spp. (TmTD-1, TmTD-2, TvTD) isolates. All the fungi eliminated CPH theobromine, 0.15% w/w starting concentration, within 7 days of start of treatment, and were capable of degrading caffeine and theophylline. The fungi decreased CPH ochratoxin A content by 31–74%. Pectin was not detectable in fungus-treated CPH whereas parameters assessed for proximate composition were not affected.ConclusionsThe data provide ample evidence that the four isolates can be applied to CPH for the purpose of eliminating theobromine and decreasing ochratoxin A content without affecting nutrient profile. Comparatively, Talaromyces verruculosus TvTD was considered as most suitable for use as a bio-tool in detheobromination of CPH for animal feed.

Aflatoxin and other mycotoxin contamination are major threats to global food security and present an urgent need to secure the global food crop against spoilage by mycotoxigenic fungi. Cocoa material is noted for naturally low aflatoxin contamination. This study was designed to assess the potential for harnessing cocoa-associated filamentous fungi for the biocontrol of aflatoxigenic Aspergillus flavus. The candidate fungi were isolated from fermented cocoa beans collected from four cocoa-growing areas in Ghana. Molecular characterization included ITS-sequencing for identification and PCR to determine mating type. Effects of the candidate isolates on growth and aflatoxin-production by an aflatoxigenic A. flavus isolate (BANGA1) were assessed. Aflatoxin production was monitored by UV fluorescence and quantified by ELISA. Thirty-six filamentous fungi were cultured and identified as Aspergillus, Cladosporium, Lichtheimia or Trichoderma spp. isolates. The isolates generally interacted negatively with BANGA1 growth and aflatoxin production. The A. niger and A. aculeatus biocontrol candidates showed the strongest colony antagonism (54–94%) and reduction in aflatoxin production (12–50%) on agar. In broth, the A. niger isolates reduced aflatoxin production by up to 97%. Metabolites from the A. niger isolates showed the strongest inhibition of growth by BANGA1 and inhibited aflatoxin production. Four of the candidate isolates belonged to the MAT1-1 mating type and 12 identified as MAT1-2. This may be indicative of the potential for genetic recombination events between fungi in the field, and finding which is particularly relevant to the risk posed by A. flavus biocontrol measures that rely on atoxigenic A. flavus strains.

Aflatoxin and other mycotoxin contamination are major threats to global food security and present an urgent need to secure the global food crop against spoilage by mycotoxigenic fungi. Cocoa material is noted for naturally low aflatoxin contamination. This study was designed to assess the potential for harnessing cocoa‐associated filamentous fungi for the biocontrol of aflatoxigenic Aspergillus flavus. The candidate fungi were isolated from fermented cocoa beans collected from four cocoa‐growing areas in Ghana. Molecular characterization included Internal Transcribed Spacer (ITS)‐sequencing for identification and polymer chain reaction (PCR) to determine mating type. Effects of the candidate isolates on growth and aflatoxin‐production by an aflatoxigenic A. flavus isolate (BANGA1) were assessed. Aflatoxin production was monitored by UV fluorescence and quantified by enzyme‐linked immunosorbent assay (ELISA). Thirty‐six filamentous fungi were cultured and identified as Aspergillus, Cladosporium, Lichtheimia, or Trichoderma spp. isolates. The isolates generally interacted negatively with BANGA1 growth and aflatoxin production. The Aspergillus niger and Aspergillus aculeatus biocontrol candidates showed the strongest colony antagonism (54%–94%) and reduction in aflatoxin production (12%–50%) on agar. In broth, the A. niger isolates reduced aflatoxin production by up to 97%. Metabolites from the A. niger isolates showed the strongest inhibition of growth by BANGA1 and inhibited aflatoxin production. Four of the candidate isolates belonged to the MAT1‐1 mating type and 12 identified as MAT1‐2. This may be indicative of the potential for genetic recombination events between fungi in the field, a finding which is particularly relevant to the risk posed by A. flavus biocontrol measures that rely on atoxigenic A. flavus strains.