Diagnosis of Fungi Contaminating Imported Fruits and Their Metabolic Analysis Using GC-MS and PCR Techniques

Abstract

Fungi contamination due to imported fruit is a problem for food security and health because these fungi can spoil food and may create toxins that can cause illness. It was our goal to identify contamination from imported fruit using multiple methods of examining the fungi: morphology, molecular biology (PCR), and metabolites (mass spectrometry). We examined 100 imported fruit samples from commercial grocery stores; specifically, we assessed apples, oranges, bananas, grapes, and strawberries to determine whether or not they were contaminated by fungi. We cultured fungi from imported fruit using potato dextrose agar (PDA) and sabouraud dextrose agar (SDA), then conducted morphological and molecular characterizations of each individual culture using three molecular markers: internal transcribed spacer (ITS), β-tubulin, and translation elongation factor (TEF). We also completed metabolic analyses of selected fungal cultures using gas chromatography/mass spectrometry (GC/MS). The outcome from this study confirmed that 46% of the tested fruit samples had been infected by fungi. Strawberry and grape samples had the highest percentage of fungi; the lowest percentage of fungi was found in the banana samples. Molecular analysis showed that the most common fungus found in the samples was Aspergillus niger (32%), followed by Aspergillus flavus (24%), Penicillium expansum (18%), Fusarium oxysporum (14%) and Alternaria alternata (12%). PCR amplification allowed specific genomic fragments to be amplified for each species based on their expected target gene regions, showing successful amplification. Numerous metabolites produced by fungi were identified using GC–MS; these metabolites were present as fatty acids, phenolic compounds, esters, alcohols and/or ketones. Of the identified metabolites, the majority of them belonged to either fatty acid or phenolic compound classes. Additionally, principal component analysis identified three separate clustering patterns for fungal generum nucleotide variations. This study emphasizes that there are numerous microbial risks involved when dealing with imported fruits, which reinforces the need for ongoing monitoring of fungi in international food commerce. Our combined use of PCR-based molecular identification and GC–MS metabolomic profiling allows better identification of fungi and their specific metabolites. The use of newer analytical methods and technologies will ultimately improve food safety by minimizing the risk of mycotoxins to the general public.