Mycotoxigenic fungi contamination of grains and peanuts from open markets in Kelantan, Malaysia

The warm weather and high relative humidity in Malaysia are ideal for the survival and proliferation of mycotoxigenic fungi leading to a high rate of stored product contamination. This study was conducted to enumerate and characterise the mycotoxigenic fungi associated with commonly consumed food grains in Kelantan, Malaysia. The fungal bioburden and fungal identification from forty-four composite food samples comprising 11 samples each of maize, wheat, rice, and peanuts from open markets in Kelantan, Malaysia, were determined using standard mycological techniques. A total of 115 mould fungal isolates belonging to 12 species were isolated, of which Aspergillus flavus (17.39%), A. versicolor (13.04%), A. felis (12.17%), Neoscytalidium dimidiatum (11.3%), Penicillium cheresanum (11.3%) and P. chrysogenum (8.7%), were predominant. Peanuts were the most contaminated (9.7×105 ± 1.5×105 CFU/g) followed by maize (7.5×105 ± 1.8×106 CFU/g), wheat (1.9×105 ± 2.6×105 CFU/g), and rice (9.9×104 ± 1.5×105 CFU/g). The levels of the mycotoxigenic fungi in peanut, maize, and wheat were above the permissible limit of 102 CFU/g set by the Malaysian Ministry of Health and 102 to 105 CFU/g set by the International Commission for Microbiological Specification for Foods, signifying that they are unsafe for use as food or feed ingredients. Hence, there is a need for more stringent control measures.

Fatty Acids and Oxylipins as Antifungal and Anti-Mycotoxin Agents in Food: A Review

Fungal contamination of food, especially by mycotoxigenic fungi, not only reduces the quality of the food, but can also cause serious diseases, thus posing a major food safety challenge to humans. Apart from sound food control systems, there is also a continual need to explore antifungal agents that can inhibit fungal growth and mycotoxin production in food. Many types of fatty acids (FAs) and their oxidized derivatives, oxylipins, have been found to exhibit such effects. In this review, we provide an update on the most recent literature on the occurrence and formation of FAs and oxylipins in food, their effects on fungal growth and mycotoxin synthesis, as well as the genetic and molecular mechanisms of actions. Research gaps in the field and needs for further studies in order to realizing the potential of FAs and oxylipins as natural antifungal preservatives in food are also discussed.

Evaluating the effects of cellulolytic enzymes and Lactobacillus bulgaricus on mycotoxins production and the quality of maize silage

Fungal spoilage and mycotoxin contamination are two of the greatest hazards of silage. The present work was carried out to evaluate the impact of Lactobacillus bulgaricus and cellulolytic enzymes on the maize silage (MS) quality. Fungal analysis of different MS samples showed different mycotoxigenic fungi. The highest frequency (62.8%) was associated with Fusarium spp. Four species with different relative densities were found: F. graminearum (71.1%), F. culmorum (15.2%), F. proliferatum (11.2%), and F. oxysporum (2.50 %). High-performance liquid chromatography analysis showed the presence of trichothecene, nivalenol, zearalenone, and fumonisins mycotoxins in MS inoculated by F. graminearum. The inhibition % of trichothecene, nivalenol, and zearalenone synthesis was 50.2%, 47.5%, and 23.5%, respectively, in MS inoculated by Lactobacillus bulgaricus after a 30 d incubation period. Trichoderma harzianum succeeded in producing cellulolytic enzymes, i.e., carboxymethyl cellulase, manganase peroxidase, and laccase, with a maximum production of 350 µg/mL, 5.47 µg/mL, and 16.0 µg/mL, respectively, after 21 d using MS as the substrate. Treatment by the extracted cellulolytic enzyme with L. bulgaricus enhanced unfavorable conditions for MS fungal contamination, i.e., the production of lactic acid, a lowered pH, and increased L. bulgaricus colony-forming units, compared to the addition of enzyme extract or L. bulgaricus alone.

How Maize Seed Systems Can Contribute to the Control of Mycotoxigenic Fungal Infection: A Perspective

Mycotoxins are toxic secondary metabolites produced by fungi on agricultural produce. Mycotoxins can be cytotoxic, genotoxic, mutagenic, and teratogenic, and they are persistent threats to human and animal health. Consumption of mycotoxin-contaminated maize can cause cancer and even sudden death. Health hazards can also occur from consuming products from animals fed with mycotoxin-contaminated feed or forage. The main mode of spread of mycotoxigenic fungi is through air-borne spores originating from soil or plant debris, although some fungi can also spread through infected seed-to-seedling transmission, ultimately followed by contamination of the harvestable product. This perspective assesses opportunities to prevent mycotoxigenic fungal infection in maize seeds produced for sowing as an important starting point of crop contamination. A case study of Nigeria showed infection in all tested farmer-produced, seed company, and foundation seed samples. A schematic overview of the formal and informal seed systems is presented to analyze their contribution to fungal infection and mycotoxin contamination in the maize value chain, as well as to set criteria for successful control. We recommend an integrated approach to control mycotoxigenic fungal infection, including resistant varieties and other control methods during seed production, grain production, and grain storage, with an important role in maintaining seed health.

Effect of Lactic Acid Bacteria on the Fermentation Quality and Mycotoxins Concentrations of Corn Silage Infested with Mycotoxigenic Fungi

This study was conducted to evaluate the effect of lactic acid bacteria (LAB) on fermentation quality, mycotoxin concentrations, and microbial communities of whole-crop corn silages infested with mycotoxigenic fungi. Cultured spores (106 cfu/mL) of mycotoxigenic Aspergillus flavus and Fusarium graminearum were sprayed (5 mL) on corn forage on 27 July and 10 August 2018. On 21 August 2018, sprayed (FI; 3 plots) and unsprayed (NFI; 3 plots) corn forage were harvested at the 1/2 kernel milk line stage, followed by chopping and ensiling without inoculants (CON), or with Lactobacillus buchneri (LB, 1 × 106 cfu/g FW), Lactobacillus plantarum (LT, 1 × 106 cfu/g FW), or L. buchneri + L. plantarum (BT: both L. buchneri and L. plantarum applied at 0.5 × 106 cfu/g FW). After 90 d of ensiling, FI silages had a higher (p < 0.05) pH value and higher acetic acid (ACA), ethanol, and ammonia nitrogen (ammonia N) concentrations, but lower (p < 0.05) lactic acid (LA) concentrations than NFI silage. The inoculants decreased pH and increased LA concentration and LA/ACA compared with CON. The aflatoxin B1 (AFB1) was only detected in FI fresh corn and silages; ensiling decreased (p < 0.05) AFB1 concentration compared with fresh corn, and LB and BT decreased AFB1 concentration compared with CON. The zearalenone (ZEN), deoxynivalenol (DON), and fumonisin B1 (FB1) concentrations were similar (p < 0.05) for NFI silages, while ZEN concentration in BT was the lowest (p < 0.05) among all FI silages; DON and FB1 concentrations in LB, LT, and BT silages were significantly lower (p < 0.05) than those of CON in FI silages. The fungal infestation increased the bacterial and fungal diversity of silages compared with NFI silages. The FI silages had a higher relative abundance (RA) of Lactobacillus, Weissella, Wickerhamomyces, Pichia, and Epicoccum than the corresponding NFI silages. The RA of Aspergillus and Fusarium markedly decreased after 90 d of ensiling, and the inoculation expanded this trend irrespective of fungal infestation. The Penicillium in FI silages survived after 90 d of ensiling, while the inoculants decreased the RA of Penicillium. Inoculants mitigate the adverse effects of fungal infestation on corn silage quality by changing the bacterial and fungal communities.

Analysis of Stored Wheat Grain-Associated Microbiota Reveals Biocontrol Activity among Microorganisms against Mycotoxigenic Fungi

Wheat grains are colonized by complex microbial communities that have the potential to affect seed quality and susceptibility to disease. Some of the beneficial microbes in these communities have been shown to protect plants against pathogens through antagonism. We evaluated the role of the microbiome in seed health: in particular, against mycotoxin-producing fungi. Amplicon sequencing was used to characterize the seed microbiome and determine if epiphytes and endophytes differ in their fungal and bacterial diversity and community composition. We then isolated culturable fungal and bacterial species and evaluated their antagonistic activity against mycotoxigenic fungi. The most prevalent taxa were found to be shared between the epiphytic and endophytic microbiota of stored wheat seeds. Among the isolated bacteria, Bacillus strains exhibited strong antagonistic properties against fungal pathogens with noteworthy fungal load reduction in wheat grain samples of up to a 3.59 log10 CFU/g compared to untreated controls. We also found that a strain of the yeast, Rhodotorula glutinis, isolated from wheat grains, degrades and/or metabolizes aflatoxin B1, one of the most dangerous mycotoxins that negatively affects physiological processes in animals and humans. The mycotoxin level in grain samples was significantly reduced up to 65% in the presence of the yeast strain, compared to the untreated control. Our study demonstrates that stored wheat grains are a rich source of bacterial and yeast antagonists with strong inhibitory and biodegradation potential against mycotoxigenic fungi and the mycotoxins they produce, respectively. Utilization of these antagonistic microorganisms may help reduce fungal and mycotoxin contamination, and potentially replace traditionally used synthetic chemicals.

Diversity of Mycobiota in Spanish Grape Berries and Selection of Hanseniaspora uvarum U1 to Prevent Mycotoxin Contamination

The occurrence of mycotoxins on grapes poses a high risk for food safety; thus, it is necessary to implement effective prevention methods. In this work, a metagenomic approach revealed the presence of important mycotoxigenic fungi in grape berries, including Aspergillus flavus, Aspergillus niger aggregate species, or Aspergillus section Circumdati. However, A. carbonarius was not detected in any sample. One of the samples was not contaminated by any mycotoxigenic species, and, therefore, it was selected for the isolation of potential biocontrol agents. In this context, Hanseniaspora uvarum U1 was selected for biocontrol in vitro assays. The results showed that this yeast is able to reduce the growth rate of the main ochratoxigenic and aflatoxigenic Aspergillus spp. occurring on grapes. Moreover, H. uvarum U1 seems to be an effective detoxifying agent for aflatoxin B1 and ochratoxin A, probably mediated by the mechanisms of adsorption to the cell wall and other active mechanisms. Therefore, H. uvarum U1 should be considered in an integrated approach to preventing AFB1 and OTA in grapes due to its potential as a biocontrol and detoxifying agent.

Current Approaches for Advancement in Understanding the Molecular Mechanisms of Mycotoxin Biosynthesis

Filamentous fungi are able to synthesise a remarkable range of secondary metabolites, which play various key roles in the interaction between fungi and the rest of the biosphere, determining their ecological fitness. Many of them can have a beneficial activity to be exploited, as well as negative impact on human and animal health, as in the case of mycotoxins contaminating large quantities of food, feed, and agricultural products worldwide and posing serious health and economic risks. The elucidation of the molecular aspects of mycotoxin biosynthesis has been greatly sped up over the past decade due to the advent of next-generation sequencing technologies, which greatly reduced the cost of genome sequencing and related omic analyses. Here, we briefly highlight the recent progress in the use and integration of omic approaches for the study of mycotoxins biosynthesis. Particular attention has been paid to genomics and transcriptomic approaches for the identification and characterisation of biosynthetic gene clusters of mycotoxins and the understanding of the regulatory pathways activated in response to physiological and environmental factors leading to their production. The latest innovations in genome-editing technology have also provided a more powerful tool for the complete explanation of regulatory and biosynthesis pathways. Finally, we address the crucial issue of the interpretation of the combined omics data on the biology of the mycotoxigenic fungi. They are rapidly expanding and require the development of resources for more efficient integration, as well as the completeness and the availability of intertwined data for the research community.