Moulds and their secondary metabolites associated with the fermentation and storage of two cocoa bean hybrids in Nigeria

https://doi.org/10.1016/j.ijfoodmicro.2019.108490Get rights and content

Highlights

  • Diverse fungi including toxigenic strains were isolated during cocoa processing.

  • Eight secondary metabolites were found for the first time in the fungal cultures.

  • Strains of Penicillium citrinum produced up to 372 mg/kg citrinin in media.

  • 44 fungal metabolites were quantified in cocoa beans from various processing stages.

  • We present first report of the nephrotoxic citrinin in fermented cocoa beans.

Abstract

Fungi and mycotoxin contamination of cocoa beans during fermentation and storage may constitute a hazard in the cocoa value chain and risk to consumers of its products. In this study, fungal profile and secondary metabolite patterns in two cocoa bean hybrids, F and T series, during fermentation and storage were determined. Additionally, secondary metabolite production by the recovered fungi in the beans was examined in culture media. Fungal isolates spanned six genera and eight species: Aspergillus niger, A. tamarii, Paecilomyces variotii, Penicillium citrinum, Pseudopithomyces palmicola, Simplicillium sp., Talaromyces atroroseus and Talaromyces sp.. In both hybrids, Aspergilli (38%) dominated the other fungi while more than one half of all the fungal isolates were from the beans in storage. Among the diverse secondary metabolites produced in media by the isolates were uncommon compounds, e.g. aspulvinone E produced by A. niger, aspterric acid by P. variotii, scalusamid A and sydowinin A by P. citrinum, norlichexanthone and siccanol by Simplicillium, and fallacinol and orsellinic acid by Talaromyces. The strains of P. citrinum produced up to 372 mg/kg citrinin. Forty-four fungal metabolites were quantified in both bean hybrids across the various processing stages, with about 86% occurring in the fermented beans stored for 30 days. The nephrotoxic citrinin, which was not previously reported in cocoa beans worldwide, was the only mycotoxin found in the fermented beans at overall mean concentration of 368 μg/kg. Additionally, its metabolite, dihydrocitrinone, was detected in fermented and stored beans. Consumption of freshly fermented cocoa beans may result in citrinin exposure. Appropriate fungal and mycotoxin control measures are proposed.

Introduction

Globally, cocoa (Theobroma cacao L.) is an important food source in many homes due to the frequent consumption of its processed/finished forms such as biscuits, chocolate, chocolate drinks, cocoa powder and sweets (Sanchez–Hervas et al., 2008). It is a cash crop predominantly cultivated in the West and Central Africa regions where annual optimal temperatures are in the range of 28–32 °C. According to FAOSTAT (2017), Nigeria ranked fourth on the global scale for cocoa production at 328,263 tons; thus, this crop is highly prized as a major export crop to other parts of world. In Nigeria as well as other cocoa-producing countries, harvested cocoa pods are broken open to obtain the beans which are then fermented, dried and stored prior to industrial processing into cocoa-based products or exportation to other countries (Kamphuis, 2009). In these countries, these initial processing steps (fermentation, drying and storage) are often performed under poor conditions that favour fungal contamination and proliferation in the samples due to the relatively poorly developed food processing systems (Copetti et al., 2010; Gutiérrez, 2017; Hatmi et al., 2015). Furthermore, the seasonal production of cocoa makes the storage over a long period (up to six months) essential. In Nigeria, the dried fermented beans are normally stored in wooden baskets or jute bags for a period of one to three months.

Fermentation of cocoa seeds is an absolute requirement for the development of chocolate flavor precursors; however, fungi may threaten the outcome of the process when they proliferate during this production step (Copetti et al., 2014; Hatmi et al., 2015). Several studies have reported fungal and/or mycotoxin contamination of different cocoa products (beans, powder or chocolates) at different stages of processing (farm, fermentation and on shelve/market) in various countries, including Nigeria (Adebayo, 2016; Aroyeun et al., 2007; Aroyeun et al., 2009; Copetti et al., 2010; Copetti et al., 2011; Egbuta et al., 2013; Fapohunda et al., 2018; Hatmi et al., 2015; Naz et al., 2017; Ogundeji and Olufolaji, 2015; Pires et al., 2019; Raters and Matissek, 2005; Sanchez–Hervas et al., 2008). However, the studies from Nigeria mostly included farm, stored or market samples (Adebayo, 2016; Aroyeun et al., 2009; Egbuta et al., 2013; Fapohunda et al., 2018; Ogundeji and Olufolaji, 2015) with limited information on samples during fermentation (Aroyeun et al., 2007). Thus, there is paucity of information on the diversity of fungi and multiple secondary metabolites including mycotoxins that could contaminate cocoa beans during processing/fermentation of cocoa in Nigeria. Furthermore, there is a gap in extensive secondary metabolite profiling of fungal contaminants of cocoa processing globally. Understanding these fungal contaminants and their secondary metabolite patterns using robust analytical techniques such as the liquid chromatography tandem mass spectrometry (LC–MS/MS) may aid the prediction of possible food safety risks to consumers of cocoa and its products. Therefore, this study assessed the fungal profile and fungal metabolite patterns in cocoa beans processed in Nigeria with a view to determine the safety of cocoa beans during fermentation and storage. The potential of the fungi recovered from the beans to produce secondary metabolites in culture media was also examined.

Section snippets

Sampling of cocoa pods

Cocoa pods of two hybrids, F series and T series, were collected between late December 2016 and end of January 2017 from the cocoa plantation of the Cocoa Research Institute of Nigeria, Ibadan. Only fully ripened pods were harvested; physically damaged or diseased pods with lesions were sorted out and discarded. The remaining healthy pods were retained in the open field for no >48 h prior to processing.

Cocoa beans fermentation and storage

Fermentation and storage of the cocoa beans were performed at the facilities of the Cocoa

Moisture, pH and fungal species during cocoa fermentation and storage

The moisture content of the two cocoa hybrids ranged from 8.19% to 18.72%, with significantly (p < 0.05) higher levels in bean samples collected before fermentation (F series: 18.40 ± 0.32; T series: 15.21 ± 0.11) than in sun-dried fermented beans (F series: 8.43 ± 0.24; T series: 8.72 ± 0.12). The moisture content level for cocoa (8%; Guehi et al., 2007; Legrand, 1999) reported to limit fungal invasion was exceeded in the dried samples, suggesting that the drying process was insufficient due

Conclusion

This study assessed the fungal profile and fungal metabolite patterns in cocoa beans processed in Nigeria with a view to determine the safety of cocoa beans during fermentation and storage. The potential of the fungi recovered from the beans to produce secondary metabolites in culture media was also examined. Fungi in the samples were diverse and included toxigenic species such as those capable of producing citrinin – a nephrotoxic compound. Inadequate drying and poor storage of the beans were

Declaration of competing interest

Authors have no conflict to declare.

References (56)

  • P. Mounjouenpou et al.

    Filamentous fungi producing ochratoxin A during cocoa processing in Cameroon

    Int. J. Food Microbiol.

    (2008)
  • R.A. Samson et al.

    Diagnostic tools to identify black aspergilli

    Stud. Mycol.

    (2007)
  • M. Sanchez–Hervas et al.

    Mycobiota and mycotoxin producing fungi from cocoa beans

    Int. J. Food Microbiol.

    (2008)
  • B. Sarkanj et al.

    Ultra-sensitive, stable isotope assisted quantification of multiple urinary mycotoxin exposure biomarkers

    Anal. Chim. Acta

    (2018)
  • J. Smedsgaard

    Micro–scale extraction procedure for standardized screening of fungal metabolite production in cultures

    J. Chromatography A.

    (1997)
  • N. Yilmaz et al.

    Polyphasic taxonomy of the genus Talaromyces

    Stud. Mycol.

    (2014)
  • O.A. Adebayo

    Prevalence of fungi and mycotoxins in cocoa beans from cocoa producing areas of Ogun state, Nigeria

    J. Exp. Food Chem.

    (2016)
  • O.A. Adebo et al.

    Review on microbial degradation of aflatoxins

    Crit. Rev. Food Sci. Nutr.

    (2017)
  • S.O. Aroyeun et al.

    Effect of fermentation and storage on mycotoxigenic fungi, ochratoxin A and aflatoxin B1 in cocoa beans from southwestern Nigeria

    Malaysian Cocoa J

    (2007)
  • S.O. Aroyeun et al.

    Grading of fermented and dried cocoa beans using fungal contamination, ergosterol index and ochratoxin A production

    Mycobiology

    (2009)
  • J.C. Basílico et al.

    Characterization and control of thread mould in cheese

    Lett. Appl. Microbiol.

    (2001)
  • Commission of the European Communities

    Commission regulation (EC) no 824/2000 of 19 April 2000 establishing procedures for the taking-over of cereals by intervention agencies and laying down methods of analysis for determining the quality of cereals

    Off. J. Eur. Communities

    (2000)
  • P.W. Crous et al.

    Fungal planet description sheets: 785–867

    Persoonia

    (2018)
  • EC (European Commission)

    Commission Decision 2002/657 of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results

    OJ. EU

    (2002)
  • M.A. Egbuta et al.

    Co–contamination of Nigerian cocoa and cocoa–based powder beverages destined for human consumption by Mycotoxins

    Ethno Med.

    (2013)
  • P. Fandohan et al.

    Infection of maize by Fusarium sp. and contamination with fumonism in Africa

    Afr. J. Biotechnol.

    (2003)
  • S.O. Fapohunda et al.

    Isolation and characterization of fungi isolated from Nigerian cocoa samples

    Curr. Life Sci.

    (2018)
  • O. Filtenborg et al.

    Simple screening method for moulds producing intracellular mycotoxins in pure cultures

    Appl. Environ. Microbiol.

    (1983)
  • Cited by (19)

    • Mycotoxins in complementary foods consumed by infants and young children within the first 18 months of life

      2023, Food Control
      Citation Excerpt :

      Copious levels (16,800 μg/kg and 51,195 μg/kg) of CIT was previously detected in maize from Nigeria (Ezekiel et al., 2021; Okeke et al., 2015). Furthermore, recent reports found Penicillium citrinum at high prevalence in food crops such as cocoa, dried ready-to-eat foods, cereals and cassava-based flour in Nigeria, with one of the P. citrinum strain producing up to 372 mg/kg of citrinin (Akinfala et al., 2020; Ekpakpale et al., 2021; Ezekiel et al., 2020). In addition, CIT was recently detected in stool of Nigerian infants (Krausová et al., 2021), suggesting frequent human exposure to this nephrotoxic mycotoxin (EFSA, 2012) in the country.

    • Advances in understanding the enzymatic potential and production of ochratoxin A of filamentous fungi isolated from cocoa fermented beans

      2022, Food Microbiology
      Citation Excerpt :

      In particular, ochratoxin A and aflatoxins are the most common mycotoxins in cocoa beans (Delgado-Ospina et al., 2021a). Recently, Akinfala et al. (2020), also reported the presence of citrinin in samples from Nigeria. Some researchers have been found that species of belonged to the genera Aspergillus, Penicillium are frequently found in cocoa beans fermentation and some of them are OTA producers, in particular those belonged to Aspergillus section Nigri are the most relevant (Lima Serra et al., 2019; Mounjouenpou et al., 2008).

    • Mycotoxin exposure biomonitoring in breastfed and non-exclusively breastfed Nigerian children

      2022, Environment International
      Citation Excerpt :

      The concentrations of DHC in breast milk samples analyzed in the present study were low, and as much as 10 times lower than the concentrations found in urine. However, the presence of DHC in both biological fluids compel us to hypothesize that CIT intake is common in Nigeria (Akinfala et al., 2020; Ezekiel et al., 2020; Ojuri et al., 2018, 2019). Furthermore, it agrees with the suggestions of Ali and Degen (2019) who retrospectively estimated high CIT exposure assessment/intake above the “level of no concern for nephrotoxicity” of 0.2 μg/kg bwt/day set by EFSA (EC (European Commission), 2014; EFSA (European Food Safety Authority), 2012) in participants in a previous urinary biomarker study in Nigeria (Šarkanj et al., 2018).

    View all citing articles on Scopus
    View full text