Regulated and non-regulated metabolites were detected and quantified in South African commercial maize in varying amounts. In all, 5 Aspergillus non-regulated metabolites (Kojic acid, Orsellinic acid, averufin, sterigmatocystin, seco-sterigmatocystin) and 3 regulated metabolites (3-Nitropropionic acid (NPA), AFB1 and AFB2) were detected in SA commercial maize. Both regulated and non-regulated metabolites detected in this study are similar to those reported by Gruber-Dorninger et al. [9] in SA maize as well as in other sub Saharan Africa [19–23], emphasizing that mycotoxin contamination of foods is an important food safety challenge that is yet to be solved in sub-Saharan Africa. For example studies conducted in Nigeria revealed that stored maize samples from five Agro Ecological Zones were contaminated with 25 regulated mycotoxins and 37 non-regulated fungal microbial metabolites [24]. Furthermore, Ezekiel et al. [25] also detected 26 major mycotoxins and 121 other microbial metabolites in maize samples from Nigeria. In this study, only the aflatoxin B toxins (AFB1 and AFB2) were detected in the maize samples, implying none of the sample was contaminated with the aflatoxin G toxins (AFG1 and AFG2). Firstly, this might be because high temperatures within the optimal range (20–35 ˚C) favor the production of aflatoxin B (B1 and B2), in contrast, low temperatures which favors the production of the G-aflatoxins [26]. South Africa experienced high temperatures and very low rainfall during the 2015 maize farming season which led to a severe and prolong drought [27]. This increase in temperatures and reduced rainfall coincided with conditions favorable for the growth of A. flavus and production of aflatoxins in maize as outlined by researchers [28–31]. Secondly, A. flavus was the most dominant Aspergillus species (47.15%) isolated in the maize sample and they produce only the aflatoxin B toxins unlike A. parasiticus which was among the least species isolated (0.81%), which produces both the aflatoxin B and G toxins. This implies that local climatic conditions could thus influence fungi diversity and mycotoxin productions.
A range of Aspergillus species synthesizes the AF precursor sterigmatocystin (ST), which is also a carcinogenic compound. Sterigmatocystin is a metabolite produced mainly by Aspergillus fungi, and is an intermediate in the biosynthesis of aflatoxin B1. It is a potentially health hazardous mycotoxin and is usually detected in food and feed as a natural contaminant [32, 33]. Sterigmatocystin is a regulated metabolite (with no regulatory limits in SA as per the time of this study) and it is classified by IARC as a group 2B carcinogen [34] due to its association with immunotoxic, immunomodulatory activities as well as mutagenic effects.
3-Nitropropionic acid (3-NPA) is a natural potent environmental toxin and mitochondrial inhibitor [35], toxic to both humans and animals. It is produced by a number of fungi, including Aspergillus species and commonly found in cereals and caused mostly by extreme weather, stressed crop growth and storage conditions [36, 37]. 3-NPA which induces cellular energy deficit and oxidative stress-related neurotoxicity which is characterized by cognitive and motor dysfunctions leading to a disorder known as Huntington’s disease. It is has been reported in SA maize before and it is a regulated metabolite (with no regulatory limits in SA as per the time of this study).
In addition, other non-regulated metabolites such as; Kojic acid is an organic acid secreted by several species of Aspergillus, especially A. oryzae [38]. It is non-hazardous, with weak acidic properties, biodegradable, making it a good alternative for the development of biologically active compounds by its derivatives [39]. Orsellinic acid is a common salicylic acid unit in the biosynthesis of secondary metabolites in actinomycetes, fungi and lichens [40], and an important polar co-metabolite present in many fungi.
Major aflatoxins were detected using HPLC while LC-MS/MS detected other fungal metabolites. The sensitivity of the liquid Chromatographic method in detecting metabolites even at the lowest concentration is enhanced by the detection of a single metabolite per analysis using the liquid chromatographic method coupled with a fluorescence detector [41]. HPLC is used mainly for chromatographic identity of the components with the use of standards that preprogrammes the system for the targeted metabolites. These standards did not exist for the other undetected metabolites to be preprogrammed for HPLC analyses, therefore could not be detected by the method [24, 42, 43]. Therefore, a multi-analyte detection technique of metabolites in food is necessary because of the co-occurrence of multiple toxins in agricultural products. Recently, LC-MS/MS based methods are suitable options as this method has the ability to simultaneously determine several low molecular weight contaminants and residues at trace levels [41].
For food safety concerns, both techniques are needed; the LC-MS/MS been a multi-analyte technique will be recommended because of its ability to identify multiple unknown constituents [44, 45]. The major disadvantages of the LC-MS/MS method is that analytes present in traces are not detected or are suppressed by metabolites with higher concentrations, and also, the inability of this method to detect major mycotoxins. Most of the metabolites detected in this study (averufin, sterigmatocystin and seco-sterigmatocystin) are active precursors for the formation of aflatoxins [46], having same retentions and this may allow the class of compound to be identified in most cases with HPLC technique as just aflatoxin (either AFB1 or AFB2) as it is not efficient to separate and identify minute components [47]. On the other hand, the HPLC technique coupled with fluorescence detector enables the detection of toxins at low concentration levels [41]. Hence, the HPLC coupled with fluorescence detector is a better method for the detection of mycotoxins in maize than LC-MS/MS method as it was able to detect major mycotoxins (AFB1 and AFB2) in maize when separate standard was used for each metabolite as compared with the LC-MS/MS method where multimix standards were used.
In this study, the LC-MS/MS method developed led to the identification and quantification of 3-NPA and sterigmatoystin which are regulated metabolites. Limited or no toxicological data are available for these minor metabolites [48, 49]. These two secondary metabolites were earlier on reported in SA maize by Gruber-Dorninger et al. [9]. The LCMS/MS method developed in this study was suitable for the identification and quantification of minor mycotoxins of Aspergillus species while the HPLC method enabled the identification and quantification of major mycotoxins of Aspergillus species. Hence, both methods are suitable for the monitoring of maize quality.