Carbon dioxide production as an indicator of Aspergillus flavus colonisation and aflatoxins/cyclopiazonic acid contamination in shelled peanuts stored under different interacting abiotic factors

doi:10.1016/j.funbio.2019.10.003

Fungal Biology

Volume 124, Issue 1, January 2020, Pages 1-7

https://doi.org/10.1016/j.funbio.2019.10.003 Get rights and content

Highlights

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Higher optimum temperature for Aspergillus flavus growth (30–35 °C) than for AFB₁ production (25–30 °C).
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Optimum cyclopiazonic acid production was at (30–35 °C), with none at 0.90 a_w.
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Dry matter losses varied from 0.3 to 17 % at 0.90–0.95 a_w in stored peanuts depending on the temperature.
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Dry matter losses can be used as a sensitive early indicator of initiation of fungal spoilage.
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Dry matter losses due to fungal activity (0.56 %) resulted in aflatoxin contamination exceeding EU limits.

Abstract

Aspergillus flavus is the main xerophylic species colonising stored peanuts resulting in contamination with aflatoxins (AFs) and cyclopiazonic acid (CPA). This study evaluated the relationship between storage of shelled peanuts under interacting abiotic conditions on (a) temporal respiration (R) and cumulative CO₂ production, (b) dry matter losses (DMLs) and (c) aflatoxin B₁ (AFB₁) and CPA accumulation. Both naturally contaminated peanuts and those inoculated with A. flavus were stored for 7-days under different water activities (a_w; 0.77–0.95) and temperatures (20–35°C). There was an increase in the temporal CO₂ production rates in wetter and warmer conditions, with the highest respiration at 0.95 a_w + A. flavus inoculum at 30°C (2474 mg CO₂kg⁻¹h⁻¹). The DMLs were modelled to produce contour maps of the environmental conditions resulting in maximum/minimum losses. Maximum mycotoxin contamination was always at 0.95 a_w although optimal temperatures were 25-30°C for AFs and 30-35°C for CPA. These results showed a correlation between CO₂ production and mycotoxin accumulation. They also provide valuable information for the creation of a database focused on the development of a post-harvest decision support system to determine the relative risks of contamination with these mycotoxins in stored shelled peanuts.

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, a_w; <0.70 a_w = 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 a_w (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 (a_w) of both batches was 0.77, and they were stored at

Impact of a_w 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 a_w 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 a_w levels. In general, respiration in the stored peanuts at 0.77–0.90 a_w 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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Carbon dioxide production as an indicator of Aspergillus flavus colonisation and aflatoxins/cyclopiazonic acid contamination in shelled peanuts stored under different interacting abiotic factors

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Fungal isolate

Peanuts samples

Impact of aw and temperature on the temporal and total accumulated CO2 in naturally and contaminated shelled peanuts with A. flavus inoculum

Conclusions

Acknowledgements

Crop Prot.

J. Stored Prod. Res.

J. Stored Prod. Res.

Int. J. Food Microbiol.

Food Control

J. Chromatogr. A

J. Stored Prod. Res.

J. Stored Prod. Res.

Food Chem.

Int. J. Food Microbiol.

Food Res. Int.

Food Res. Int.

Food Chem.

Temporal monitoring of the nor-1 (aflD) gene of Aspergillus flavus in relation to aflatoxin B1 production during storage of peanuts under different water activity levels

J. Appl. Microbiol.

The effect of soybean moisture during storage on the lipid composition of extracted crude oil

J. Am. Oil Chem. Soc.

Commission regulation (EC) No. 1881/2006 of 19 December 2006, setting maximum levels for certain contaminants in foodstuffs

Off. J. Eur. Union

Commission directive 2003/100/EC of 31 October 2003 amending annex I to directive 2002/32/EC of the European Parliament and of the council on undesirable substances in animal feed (text with EEA relevance)

Off. J. L

Impact of a_w and temperature on the temporal and total accumulated CO₂ in naturally and contaminated shelled peanuts with A. flavus inoculum