Carbon dioxide production as an indicator of Aspergillus flavus colonisation and aflatoxins/cyclopiazonic acid contamination in shelled peanuts stored under different interacting abiotic factors
Graphical abstract
Introduction
Peanuts (Arachis hypogaea L.) also known as groundnuts, is a legume that originated in South America. Peanut plants are grown widely in China, India, Africa and the USA. Peanut world production in 2016 was approx. 43.9 M tonnes (Food and Agriculture Organisation of the United Nations, 2018). Peanut crops are very susceptible to fungal diseases, especially by mycotoxigenic fungi during the pod filling phase at pre-harvest, particularly during drought stress episodes (Paterson and Lima, 2011). Subsequently, poor drying and storage can result in further mycotoxin contamination, as peanuts are hygroscopic and absorb moisture easily. Fusarium, Penicillium, and Aspergillus species are commonly isolated from peanuts during the whole production phase from growth to storage. Aspergillus flavus infection occurs during the pre-harvest stage but colonisation can also occur during drying and storage, when the most commonly isolated species have been reported (Atayde et al., 2012, Gonçalez et al., 2008, Sultan and Magan, 2010, Zorzete et al., 2011, Zorzete et al., 2013). This results in significant contamination with toxic secondary metabolites, especially aflatoxins (AFs) and cyclopiazonic acid (CPA). Such contamination has major impacts on the quality of the product and the potential for its export from producer countries resulting in significant economic impacts, especially in Lower Middle Income Countries (LMICs) (Atayde et al., 2012, de Souza et al., 2014, Zorzete et al., 2011, Zorzete et al., 2013).
Peanuts like other edible seeds respire at very low levels under safe storage and reduced water availability conditions (water activity, aw; <0.70 aw = 8 % moisture content (m.c.). Under these conditions, while fungal contaminants remain viable, they are unable to initiate spoilage or any additional toxin contamination (Fleurat-Lessard, 2017, Paterson and Lima, 2011). However, poor storage conditions or inadequate silo hygiene can lead to the introduction of water from outside, boosting the pest and disease activity. This biological activity can lead to pockets of spoilage resulting in toxin contamination. This also results in an increase in the respiration of the stored peanuts and of the associated mycobiota. Spoilage fungi are able to utilise the lipids present, leading to a deterioration in quality and associated dry matter losses (DMLs) (Seitz et al., 1982). Saul and Lind (1958) were the first to correlate the impact the respiration (CO2 production) and DMLs due to fungal colonisation and mycotoxin production. According to Seitz et al. (1982), mould colonisation increases the DML during storage at a rate dependent on the prevailing m.c., temperature, level of kernel damage and the fungal community present on the phyllosphere surfaces of the peanuts.
Recent studies have shown that changes in CO2 production during storage of cereals, including maize, wheat, oats and rice can be used as an indicator of DML, as well as the relationship with mycotoxin contamination (Garcia-Cela et al., 2018a, Garcia-Cela et al., 2018b, Garcia-Cela et al., 2019, Martín Castaño et al., 2017b, Martín Castaño et al., 2017a, Mylona, 2012, Mylona et al., 2012, Mylona and Magan, 2011). In these studies it was found that it is possible to use the progressive increase in the aerobic respiration rate under increasingly conducive interacting abiotic factors. This is related to mould growth, as their activity leads to an oxidation of carbohydrates/lipids and hence CO2 production. Therefore, it can be linked to the quality losses as DML percentages. Respiration rates (R) using Gas Chromatography and the associated DMLs can be used to establish an “index of risk in storage” to predict overall quality changes and mycotoxin contamination in stored cereals and nuts (Magan et al., 2010).
Previously, DML values as low as 0.04, 1 and 2 % indicated impacts on seed germination and risk of mycotoxin contamination in the context of the EU legal maximum limits (Garcia-Cela et al., 2018a, Garcia-Cela et al., 2018b, Garcia-Cela et al., 2019, Lacey et al., 1994, Martín Castaño et al., 2017a, Martín Castaño et al., 2017b, Mylona et al., 2012, Mylona and Magan, 2011, White et al., 1982). However, very little data is available on nuts, especially peanuts, although studies by Mylona (2012) examined A. flavus and CO2 production in hazelnuts, showing that very small DMLs resulted in aflatoxin B1 (AFB1) contamination exceeding the EU legislative limits.
Indeed, subsequent studies have suggested that CO2 production could be a very powerful tool for the early prediction of the initiation of spoilage mould activity and therefore mycotoxin contamination of wheat, maize and rice (Mylona et al., 2012, Martín Castaño et al., 2017a, Martín Castaño et al., 2017b). This approach could have benefits in the development of sensing systems for the early indication of spoilage initiation, based on real time temporal monitoring of CO2.
Thus, the aims of our study were to (a) examine temporal respiration (R) and cumulative total CO2 production by stored naturally contaminated shelled peanuts or those artificially inoculated with A. flavus conidia stored under different conditions of aw (0.77–0.95) and temperatures (20–35 °C) conditions; (b) relate R production during storage to relative DMLs; (c) quantify AFs and CPA in all stored conditions; and (d) investigate the relationship between %DMLs and AFB1 contamination relative to the EU legislative limits for food and feed use. The potential outcomes for using such data sets and models as a predictive tool of the relative risk of AFB1 contamination during storage of peanuts by monitoring of CO2 production are discussed.
Section snippets
Fungal isolate
An aflatoxigenic type strain of A. flavus (NRRL 3357; Northern Regional Research Laboratories (NRRL) of the US Department of Agriculture USDA, New Orleans) was used in this experiment. The strain was maintained in glycerol:water (70:30, v/v) at −20 °C in the culture collection of the Applied Mycology Group, Cranfield University.
Peanuts samples
Shelled peanuts from China, were used as naturally contaminated peanuts for storage experiments. Water activity (aw) of both batches was 0.77, and they were stored at
Impact of aw and temperature on the temporal and total accumulated CO2 in naturally and contaminated shelled peanuts with A. flavus inoculum
Overall, the respiration rates of artificially inoculated shelled peanuts with A. flavus inoculum increased after two days storage, depending on the aw and temperature treatment. Fig. 1 shows the temporal (hourly) respiration rate (R) and the total accumulated CO2 (cumulative R; g CO2 kg−1 peanuts) at 30 °C for the four different aw levels. In general, respiration in the stored peanuts at 0.77–0.90 aw was relatively low, regardless of the storage temperature. The highest respiration rates were
Conclusions
This study showed that it is possible to use production of CO2 as an early indicator of the onset of fungal colonisation or perhaps pest activity in stored peanuts. Small changes in DMLs resulted in AFB1 levels exceeding the EU legislative limits in food and feed. Contamination with CPA occurred over a narrower range of conditions than that for AFB1. Boundary conditions for colonisation and mycotoxin production could be effectively utilised to develop predictive models which can be used in
Acknowledgements
This project (MyToolBox) was funded from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 678012.
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