Advances in genomics, biodiversity and rapid systems for the detection of toxigenic fungi and mycotoxins

ABSTRACTMycotoxins are the foremost naturally occurring contaminants of food products such as corn, peanuts, tree nuts, and wheat. As the secondary metabolites, mycotoxins are mainly synthesized by many species of the genera Aspergillus, Fusarium and Penicillium, and are considered highly toxic and carcinogenic to humans and animals. Most mycotoxins are detected and quantified by analytical chemistry-based methods. While mycotoxigenic fungi are usually identified and quantified by biological methods. However, these methods are time-consuming, laborious, costly, and inconsistent because of the variability of the grain-sampling process. It is desirable to develop rapid, non-destructive and efficient methods that objectively measure and evaluate mycotoxins and mycotoxigenic fungi in food. In recent years, some spectroscopy-based technologies such as hyperspectral imaging (HSI), Raman spectroscopy, and Fourier transform infrared spectroscopy have been extensively investigated for their potential use as tools for the detection, classification, and sorting of mycotoxins and toxigenic fungal contaminants in food. HSI integrates both spatial and spectral information for every pixel in an image, making it suitable for rapid detection of large quantities of samples and more heterogeneous samples and for in-line sorting in the food industry. In order to track the latest research developments in HSI, this paper gives a brief overview of the theories and fundamentals behind the technology and discusses its applications in the field of rapid detection and sorting of mycotoxins and toxigenic fungi in food products. Additionally, advantages and disadvantages of HSI are compared, and its potential use in commercial applications is reported.

Fungi are the second largest group of eukaryotic organisms, ranging from 1.5 to 5.1 million species. Mycotoxins are chemical rich, stable and tolerate secondary fungal metabolites found in all food processing. Mycotoxin contaminations emerge in the food chain due to infection of agricultural commodities with moulds. Horticultural products are economically and nutritionally important crops that contaminate their products with both toxigenic and pathogenic fungi and produce a variety of mycotoxins. Unfortunately, for taxonomic specialists, identification of closely related species is an overwhelming challenge. Using multilocus sequence analysis (MLSA), molecular identification, genome sequences and genetic variability among fungal complex creates molecular markers that solve taxonomic difficulties. In addition to food, molecular detection of mycotoxins may serve as good alternatives to conventional methods that target their biosynthetic pathway for spotting horticultural products to ensure food protraction and food security. Frequently polluted contaminants are aflatoxins (AFs), ochratoxins (OTs), patulin (PAT), citrinin (CIT), zearalenone (ZEA), fumonisisns (FUM) and trichothecenes (TRI). Because of the high public concern about chemicals/metabolites from microbes in food today, end-product consumers have to beware of their adverse effects. However, nutritional formulations for children and infants based on fruits and vegetables demonstrate the vital impact on their physiology. In fact, these horticultural products are highly contaminated with complex obscure toxins. Mycotoxin detection is another significant problem in the food industry, including fruit export and import. Nanobiotechnology has recently been developed for a wide range of applications in the diagnosis, detection and management of pathogens in agriculture. Furthermore, magnetite nanoparticles for the detection of mycotoxins are more advantageous for nanosensors. Using aptamers (Apt), the novel fast, responsive and field-deployable rapid point-of-care diagnostics precisely recognizes and binds its targets via novel nanobiotechnological approaches.

How solid is the Academy position paper on mold exposure?

Screening, identification and detection of the regulated and emerging mycotoxins in edible and medicinal products by integrating diversiform liquid chromatography/mass spectrometry systems and high-throughput sequencing of contaminated fungi

Chapter 2 - Role of nanotechnology in the detection of mycotoxins: a smart approach

Mycotoxins are metabolites and toxic substances produced by certain filamentous fungi that frequently contaminate food and agriculture commodities and it may cause disease in animals or humans. The toxigenic fungi are responsible for mycotoxin production in food that belongs to mainly three genera: Aspergillus, Penicillium and Fusarium. The contamination of food by mycotoxins is difficult to control in addition, causing economic impacts on public health, so their identification and quantifications are very necessary. Various analytical methods are developed for the detection and quantification of mycotoxins in order to control the residual contents of these toxins. Among them there is a widely used chromatography. This paper reports some chromatographic methods for the detection and quantification of mycotoxins described in patents and scientific articles.

Nutrition-rich cereal grains and oil seeds are the major sources of food and feed for human and livestock, respectively. Infected by fungi and contaminated with mycotoxins are serious problems worldwide for cereals and oil seeds before and after harvest. The growth and development activities of fungi consume seed nutrients and destroy seed structures, leading to dramatic declines of crop yield and quality. In addition, the toxic secondary metabolites produced by these fungi pose a well-known threat to both human and animals. The existence of fungi and mycotoxins has been a redoubtable problem worldwide for decades but tends to be a severe food safety issue in developing countries and regions, such as China and Africa. Detection of fungal infection at an early stage and of mycotoxin contaminants, even at a small amount, is of great significance to prevent harmful toxins from entering the food supply chains worldwide. This review focuses on the recent advancements in utilising infrared spectroscopy, Raman spectroscopy, and hyperspectral imaging to detect fungal infections and mycotoxin contaminants in cereals and oil seeds worldwide, with an emphasis on recent progress in China. Brief introduction of principles, and corresponding shortcomings, as well as latest advances of each technique, are also being presented herein.

Bioburden contamination and Staphylococcus aureus colonization associated with firefighter's ambulances

This paper presents an overview of how microsystem technology tools can be applied to the development of rapid, out–of–laboratory measurement capabilities for the determinations of toxigenic fungi and mycotoxins in foodstuffs. Most of the topics discussed are all under investigation within the European Commission–sponsored project Good–Food (FP6–IST). These are DNA arrays, electronic noses and electronic tongues for the detection of fungal contaminants in feed, and biosensors and chemical sensors based on microfabricated electrode systems, antibodies and novel synthetic receptors for the detection of specific mycotoxins. The approach to resolution of these difficult measurement problems in real matrices requires a multidisciplinary approach. The technology tools discussed can provide a route to the rapid, on–site generation of data that can aid the safe production of high–quality foodstuffs.

This study aimed to assess the occurrence of toxigenic fungi and mycotoxin contamination in stored wheat grains by using advanced molecular and analytical techniques. A multiplex polymerase chain reaction (PCR) strategy was established for rapid identification of mycotoxigenic fungi, and an improved analytical method was developed for simultaneous multi-mycotoxin determination in wheat grains by liquid chromatography-tandem mass spectrometry (LC/MS/MS) without the need for any clean-up. The optimized multiplex PCR method was highly specific in detecting fungal species containing species-specific and mycotoxin metabolic pathway genes. The method was applied for evaluation of 34 wheat grain samples collected from storage warehouses for the presence of mycotoxin-producing fungi, and a few samples were found positive for Fusarium and Aspergillus species. Further chemical analysis revealed that 17 samples contained mycotoxins above the level of detection, but only six samples were found to be contaminated over the EU regulatory limits with at least one mycotoxin. Aflatoxin B1, fumonisins, and deoxynivalenol were the most common toxins found in these samples. The results showed a strong correlation between the presence of mycotoxin biosynthesis genes as analyzed by multiplex PCR and mycotoxin detection by LC/MS/MS. The present findings indicate that a combined approach might provide rapid, accurate, and sensitive detection of mycotoxigenic species and mycotoxins in wheat grains.

The global demand for alternative sugars is rising due to their lower glycemic index and other health benefits. However, improper manufacture, processing, transport, export, and/or marketing of regional sugar products increases the risk of microbial infection upon consumption. Fungal exposure and contamination of alternative sugars may occur as a result of enhanced hygroscopicity of certain sugar forms such as coconut and palm sugars, which strongly attract water molecules to their surface and thereby fungal spores, a property affected by adulteration practices, e.g. incorporation of cane sugar. The present study highlights risks of unregulated processing of palm sugar in the form of fungal contaminants and their toxigenic potential. Palm sugar was sampled for fungal isolates which were identified as Aspergillus flavus, A. niger, A. carbonarius, A. terreus, and A. fumigatus, followed by colony enumeration. Subsequent extraction of fungal extracts by thin layer chromatography resulted in detection of mycotoxins, including aflatoxins B1 and G1, citrinin, and ochratoxin. The findings confirmed that humid conditions may be optimal for the presence of toxigenic fungi in the palm sugar and production of toxic metabolites, indicating that more stringent regulation is required for palm sugar processing, as the toxins can lead to detrimental health consequences, including acute poisoning, nephropathy, and liver cancer.

Mycotoxins, produced by fungi that colonise foods and feeds may be carcinogenic, cytotoxic, oestrogenic, immunosuppressant, nephrotoxic, neurotoxic or teratogenic compounds and pose, therefore, serious public and animal health hazards. Food and feed safety, as a major concern all over the world, is the driving force of mycotoxin research and development activity. The present study provides an overview of the major mycotoxins and mycotoxicoses including chemistry, toxicity, and detection of mycotoxins. Special attention is devoted to biodiversity, genetic variation, life cycle strategies, pathogenicity and identification of toxigenic fungi. Risk assessment and climatic models developed to predict mycotoxin contamination of crop products are considered as potential solutions of reducing the threat of mycotoxicoses. The role of storage conditions and food processing technologies in the reduction of mycotoxin concentrations are also discussed.

Mycotoxins are secondary metabolites produced by toxigenic fungi under specific environmental conditions. Fruits, owing to their high moisture content, rich nutrition, and improper harvest or storage conditions, are highly susceptible to various mycotoxins, such as ochratoxin A (OTA), zearalenone (ZEN), patulin (PAT), Alternaria toxins, etc. These mycotoxins can cause acute and chronic toxic effects (teratogenicity, mutagenicity, and carcinogenicity, etc) in animals and humans. Given the high toxicity and wide prevalence of mycotoxins, establishing an efficient analytical method to detect multiple mycotoxins simultaneously in different types of fruits is of great importance. Conventional mycotoxin detection methods rely on high performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). However, fruit sample matrices contain large amounts of pigments, cellulose, and minerals, all of which dramatically impede the detection of trace mycotoxins in fruits. Therefore, the efficient enrichment and purification of multiple mycotoxins in fruit samples is crucial before instrumental analysis. In this study, a reliable method based on a QuEChERs sample preparation approach coupled with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established to determine 36 mycotoxins in fruits. In the optimal extraction method, 2.0 g of a sample was extracted with 10 mL of acetic acid-acetonitrile-water (1∶79∶20, v/v/v) in a 50 mL centrifuge tube, vortexed for 30 s, and ultrasonicated for 40 min. The mixture was then salted out with 2.0 g of anhydrous MgSO4 and 0.5 g of NaCl and centrifuged for 5 min. Next, 6 mL of the supernatant was purified using 85 mg of octadecylsilane-bonded silica gel (C18) and 15 mg of N-propylethylenediamine (PSA). After vigorous shaking and centrifugation, the supernatant was collected and dried with nitrogen at 40 ℃. Finally, the residues were redissolved in 1 mL of 5 mmol/L ammonium acetate aqueous solution-acetonitrile (50∶50, v/v) and passed through a 0.22 μm nylon filter before analysis. The mycotoxins were separated on a Waters XBridge BEH C18 column using a binary gradient mixture of ammonium acetate aqueous solution and methanol. The injection volume was 3 μL. The mycotoxins were analyzed in multiple reaction monitoring (MRM) mode under both positive and negative electrospray ionization. Quantitative analysis was performed using an external standard method with matrix-matched calibration curves. Under optimal conditions, good linear relationships were obtained in the respective linear ranges, with correlation coefficients (R2) no less than 0.990. The limits of detection (LODs) and quantification (LOQs) were 0.02-5 and 0.1-10 μg/kg, respectively. The recoveries of the 36 mycotoxins in fruits ranged from 77.0% to 118.9% at low, medium, and high spiked levels, with intra- and inter-day precisions in the range of 1.3%-14.9% and 0.2%-17.3%, respectively. The validated approach was employed to investigate mycotoxin contamination in actual fruit samples, including strawberry, grape, pear, and peach (15 samples of each type). Eleven mycotoxins, namely, altenuene (ALT), altenusin (ALS), alternariol-methyl ether (AME), tenuazonic acid (TeA), tentoxin (Ten), OTA, beauvericin (BEA), PAT, zearalanone (ZAN), T-2 toxin (T2), and mycophenolic acid (MPA), were found in the samples; three samples were contaminated with multiple mycotoxins. The incidence rates of mycotoxins in strawberry, grape, pear, and peach were 27%, 40%, 40%, and 33%, respectively. In particular, Alternaria toxins were the most frequently found mycotoxins in these fruits, with an incidence of 15%. The proposed method is simple, rapid, accurate, sensitive, reproducible, and stable; thus, it is suitable for the simultaneous detection of the 36 mycotoxins in different fruits.

The common bean (Phaseolus vulgaris L.) is one of the most widely farmed legumes, with farmers cultivating a variety of cultivar variants in Kurdistan of Iraq. Total of 50 bean samples were tested to establish their mycological contamination and their toxigenic potential under certain conditions, a variety of fungi may develop within bean grains; some of which have the capacity to synthesize mycotoxins. In current study toxigenic fungi were studied in beans (Phaseolus vulgaris L.) Aspergillus spp., Fusarium spp. and Penicillium spp. were the most frequently isolated genera, followed by Phoma sp., Mucor spp., Alternaria spp., Curvularia spp., Rhizopus spp., Eurotium sp. Chaetomium spp, Yeasts and Drechslera spp. Among 20 Aspergillus 11 strains produced mycotoxins: 30% produced aflatoxins (AFs) ranged between 81-260 ppb; 5% produced ochratoxin A (OTA 70-100 ppb) and 26.6% of Fusarium produced Trichothecene T-2/HT-2 (50- 94 ppb). The toxigenic species were A. flavus, A. parasiticus, A. ochraceus, A. carbonarius A. niger and F. sporotrichioides.

Six Aspergillus flavus isolates out of 17 fungal isolates were sampled from diverse food and organic matter in southwest Nigeria. All the A. flavus samples produced aflatoxin and cyclopiazonic acid. These six isolates constitute a mycobank of toxigenic species for analytical research involving the safety of food, feed and the general environment. Consumption of wholesome food materials is contingent upon accurate identification of fungal contaminants, detection and quantification of potential mycotoxins and subsequent removal and prevention of fungal contamination. Thorough investigations rest on proper maintenance of a reliable mycobank. This article introduces a mini toxigenic A. flavus bank, supported by pictorial illustrations, as a preliminary project for the establishment of a permanent culture collection centre in Nigeria.