Fungal Infections, Toxin Production, and Nutritional Quality in Food and Feed
Exploring Fungal Infections, Toxin Production, and Nutritional Quality in Agriculture Products (Food and Feed): Understanding Contamination Pathways and Implementing Effective Control Measures
Aflatoxins present a pressing global concern due to their profound impact on public health and economies worldwide. Originating from fungal species within the Aspergillus genus, these toxic, mutagenic, and carcinogenic metabolites consistently contaminate various food and agricultural products. Aflatoxin contamination is significantly influenced by climate and diet, with warmer climates and aflatoxin-susceptible staple foods contributing substantially to the global burden. Regulatory measures, prevention strategies, decontamination, and detoxification efforts have been employed to mitigate human exposure to aflatoxins. Detection methods leveraging aflatoxins’ chemical and structural properties play a crucial role in monitoring and quantifying their presence in foods. Beyond public health, aflatoxin contamination carries significant trade and economic implications globally. For instance, maize farmers in the United States face annual losses of $160 million due to aflatoxin-related issues, while developing countries, particularly in sub-Saharan Africa, incur losses amounting to $450 million, representing 38% of global agricultural losses attributed to aflatoxin. Effective implementation of Good Agricultural Practices (GAPs), Good Manufacturing Practices (GMPs), Hazard Analysis Critical Control Points (HACCP), proper storage, informed stakeholders, and the ability to predict on-field and in-store contamination are crucial preventive measures against human exposure to aflatoxins. Long-term strategies should focus on minimizing on-field contamination through bio-control measures or cultivating resistant crop varieties. When rigorously enforced, regulations serve as the last line of defense before food reaches consumers. While many countries worldwide have enacted aflatoxin-related regulations, a significant portion of African countries lacks such regulations. Precise, accurate, affordable, and simple aflatoxin detection methods are essential for enforcing regulations and assessing the efficacy of aflatoxin control measures. While current chromatographic, immunochemical, and spectroscopic methods are effective, ongoing efforts to develop new or improved methods aim to enhance speed, detection, and accuracy, ultimately ensuring the safety of food and agricultural products.
Aspergillus flavus Contamination
Aspergillus flavus, characterized as a saprotrophic and pathogenic fungus, exhibits a widespread global distribution. Its notable affinity lies in the colonization of cereal grains, legumes, and tree nuts. The onset of postharvest rot typically manifests during harvest, storage, and/or transit phases. The specific epithet ‘flavus’ originates from the Latin word meaning yellow, aptly describing the frequently observed coloration of its spores. Infections by A. flavus can occur both in the field preharvest and during postharvest stages, with preharvest infections often remaining asymptomatic until the postharvest storage or transport period. Beyond its ability to cause preharvest and postharvest infections, numerous strains of A. flavus produce substantial quantities of mycotoxins. These toxic compounds, when ingested, pose a threat to mammals. Notably, A. flavus also serves as an opportunistic pathogen for humans and animals, contributing to the development of aspergillosis, particularly in immunocompromised individuals.
Aflatoxin
Aflatoxins are various poisonous carcinogens and mutagens that are naturally produced by certain molds, particularly Aspergillus species (Aspergillus flavus and Aspergillus parasiticus). Aflatoxins are synthesized by many fungi spp. including Aspergillus, Penicillium, Fusarium, and Alternaria, but Aspergillus flavus and Aspergillus parasiticus are known to produce the most toxigenic strains of aflatoxins. There are mainly six different types of aflatoxins-Aflatoxins-B1(AFB1), Aflatoxins-B2(AFB2), Aflatoxin G1(AFG1), Aflatoxins G2 (AFG2), Aflatoxin M1 (AFM1), and Aflatoxin M2 (AFM2). Out of these, B1, B2, G1, and G2 are found in food crops or their products, while M1 (Metabolite of B1) and M2 are found in the animals’ by-products such as dairy products. The fungi grow in soil, decaying vegetation and various staple foodstuffs and commodities such as hay, sweetcorn, wheat, millet, sorghum, cassava, rice, chill, peppers, cottonseed, peanuts, tree nuts, sesame seeds, sunflower seeds, maize, rice, figs and other dried foods, crude vegetable oils and cocoa beans, as a result of fungal contamination before and after harvest and various spices. In short, the relevant fungi grow on almost any crop or food. When such contaminated food is processed or consumed, the aflatoxins enter the general food supply. They have been found in both pet and human foods, as well as in feedstocks for agricultural animals. Aflatoxin is the main mycotoxin that harms animal and human health due to its carcinogenic nature. AFB1 constitutes the most harmful type of aflatoxins and is a potent hepato-carcinogenic; several types of aflatoxins are produced naturally. Aflatoxin B1 is the most common in food and among the most potent genotoxic and carcinogenic. Aflatoxin M1 is a major metabolite of aflatoxin B1 in humans and animals, which may be present in milk from animals fed with aflatoxin B1 contaminated feed.
Health hazards of aflatoxins
Aflatoxins are fungal toxins known for their acute life-threatening toxicity, carcinogenic properties, and other potential chronic adverse effects. Aflatoxin B1 stands out as the most detrimental aflatoxin for both humans and animals, given its carcinogenic nature and its link to hepatocellular carcinoma, ultimately resulting in liver cancer. Children, in particular, are more vulnerable to the toxic effects of aflatoxin, heightening the risk of early infections due to compromised immunization. Beyond its carcinogenic properties, aflatoxin also exerts adverse effects on various vital organs such as the kidneys, heart, liver, testes, and brain.
The issue of dietary exposure to aflatoxins is a significant public health concern, particularly in subsistence farming communities in sub-Saharan Africa and South Asia. In these regions, dietary staple food crops like groundnuts and maize are often highly contaminated with aflatoxin due to hot and humid climates, poor storage practices, low awareness of the risks, and a lack of enforcement of regulatory limits. Biomarkers have been developed and utilized in numerous epidemiological studies to assess aflatoxin exposure and its associated health effects in these high-risk population groups. Recent evidence suggests that aflatoxin may be an underlying factor contributing to stunted child growth and may lower cell-mediated immunity, potentially increasing susceptibility to diseases. However, establishing a causal relationship between aflatoxin exposure and these adverse health outcomes remains a challenge, and the biological mechanisms for these effects have not been fully elucidated, warranting further research. Additionally, there is limited information on the health effects of co-exposure to aflatoxin with other mycotoxins. Recent advancements in biomarkers offer opportunities for crucial future research in this field. Aflatoxin contamination in crops is a global threat that not only compromises the safety of food and feed but also impacts the agricultural economy and crop-dependent small-scale industries.
Conditions that allow the aflatoxin contamination
Aspergillus, is a fungus found especially in areas with hot and humid climates. Climate change is expected to have an impact on the presence of aflatoxins in food in Europe. As aflatoxins are known to be genotoxic and carcinogenic, exposure through food should be kept as low as possible. Mycotoxin production depends on the food source, enzymes, and various environmental factors. However, the conditions that are favorable for aflatoxigenic fungi are not always conducive to the production of aflatoxins. There are several factors which are as;
Physical factors
Various physical factors play a crucial role in aflatoxin contamination, with pH, light, moisture, temperature, water, relative humidity, and atmospheric gases being significant contributors. Aflatoxin-producing molds or fungi exhibit adaptability to a wide pH range (1.7–9.3), with the optimum range being 3–7. Lower pH (3 > pH > 1) restricts fungal growth, while slightly higher pH (6 > pH > 3) promotes both fungal and aflatoxin production. An initial pH of 5 promotes Aflatoxin B (AFB) production, whereas a higher pH of 7 promotes Aflatoxin G (AFG) production. The composition of the growth medium also influences pH.
Light conditions significantly impact fungal growth and aflatoxin production. Darkness enhances aflatoxin production, while exposure to sunlight inhibits it. Moisture content is a critical factor, favoring aflatoxin contamination under high moisture conditions due to the conducive environment for fungal growth. Relative humidity at 85% is optimal for aflatoxin production, and higher levels, such as 95%, further increase aflatoxin production. However, water levels do not have a reported effect on aflatoxin contamination. Aspergillus flavus, the primary aflatoxin producer, exhibits robust survival capabilities over a broad temperature range from 12 °C to 48 °C, with the optimal temperature for growth falling between 28 °C and 37 °C. Aflatoxins can be produced across a wide temperature range, but the optimal temperature for aflatoxin production is 25-35 °C. Higher temperatures tend to favor AFB production over AFG, while at lower temperatures, both AFB and AFG production is equal. The availability of oxygen (O2) and carbon dioxide (CO2) also influences aflatoxin production. Higher levels of CO2 and lower levels of O2 inhibit aflatoxin production and fungal growth.
Nutritional factors
Aflatoxin production is significantly influenced by the substrate and various nutritional factors, including carbon, amino acids, nitrogen, lipids, and trace elements. Substrates rich in carbohydrates support higher production compared to oil, as carbohydrates readily provide the carbon essential for robust fungal growth. Glucose, ribose, sucrose, xylose, and glycerol are excellent carbohydrate substrates, while peptone, lactose, and sorbose fail to stimulate aflatoxin production. Nitrogen, present in the form of nitrite and nitrate, enhances aflatoxin production by A. flavus through various mechanisms.
Lipids play a crucial role in aflatoxin production, with biosynthesis initiated by lipophilic epoxy fatty acids. Fungal growth and aflatoxin production are induced by ergosterol oxidation. Consequently, lipids serve as both a substrate to obtain an acyl-CoA starter and a signaling molecule. Aflatoxin production and accumulation are higher in full-fat substrates compared to low-fat substrates. Additionally, vitamins, amino acids, and metal ions collectively promote aflatoxin production. Amino acids such as glycine, glutamate, alanine, and bivalent metals like zinc and magnesium contribute to promoting aflatoxin production.
Biological factors
Biological factors encompass fungal species, weeds, and insect-induced injuries. Weeds primarily function as competitors, inducing plant stress that correlates with aflatoxin production. The quantity of aflatoxin generated is predominantly influenced by the types of fungi present. Insect-induced wounds in plants cause stress and create a conducive environment for aflatoxigenic fungi contamination. The variation in aflatoxin production is also contingent on the fungal strains involved. A. flavus tends to produce fewer aflatoxins compared to A. parasiticus. Notably, A. flavus emerges as the principal species responsible for aflatoxin production and crop contamination. Its prevalence in soil, coupled with its saprobe characteristics, enables it to thrive on various organic nutrient substrates, including compost piles, plant debris, cotton, deceased insects, stored grains, field crops, animal remains, and animal fodder. Pre-harvest contaminations of field crops are frequent due to the natural presence of A. flavus in soil, while post-harvest contamination occurs during storage, where A. flavus can spoil stored food grains. A. flavus lacks host specificity, contaminating both monocot and dicot seeds.
Contamination
Aspergillus flavus extensively contaminates a diverse array of food commodities, encompassing cereals (such as maize, sorghum, pearl millet, rice, and wheat), oilseeds (peanut, soybean, sunflower, and cotton), spices (including chilies, black pepper, turmeric, coriander, and ginger), nuts (almond, Brazil nut, pistachio, walnut, and coconut), yams, and various milk products. Displaying a green appearance, the A. flavus fungus demonstrates the ability to proliferate even under challenging conditions. Injuries inflicted by insects and nematodes create openings for these fungi to infiltrate. Mycotoxins, including aflatoxins, can emerge during the growth stages of specific crops. The occurrence of particular mycotoxins is influenced by a combination of climate, sources of fungal inoculum (or suitability of the fungal substrate), potential insect vectors, and the plant’s response (or its susceptibility to fungal infestation). Insect-induced damage exposes vulnerable tissues to colonization by toxigenic fungi, leading to subsequent aflatoxin production. The intrusion of fungi into crops not only compromises the self-defence mechanisms of crop plants but also results in the contamination of crop seeds, leading to aflatoxin production. Fungal infections adversely impact the growth and yield of crops, ultimately causing a loss of market value.
Methods of Aflatoxin Detection
The most widely employed method is the Enzyme-Linked Immunosorbent Assay (ELISA), followed by various chromatographic techniques such as High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-Mass Spectroscopy (LCMS), and Thin Layer Chromatography (TLC). Methods that yield rapid results are particularly valuable for handling large sample sizes. Polymerase Chain Reaction (PCR), Fluorescence/Near-Infrared Spectroscopy (FS/NIRS), and Hyper Spectral Imaging (HSI) fall into this category. The PCR technique is primarily utilized for molecular-level detection of Aflatoxins-producing fungi, specifically A. flavus. Genes identified during the molecular screening of A. flavus, responsible for Aflatoxin biosynthesis, serve as target genes for Aflatoxin detection using multiplex PCR.
The visible/Near-Infrared technique (VNIR) is also employed for toxin contamination detection, although recent studies have focused on enhancing the image quality by combining HSI with chemometric data analysis. This combination has resulted in improved identification of AFB1 on maize kernel surfaces. Advancements in analytical techniques have facilitated the simultaneous detection of aflatoxins with other toxic compounds. Time-Resolved Fluorescence Immuno-Chromatographic Assay (TRFICA) and anti-idiotypic nanobody-Phage Display-mediated Immune-Polymerase Chain Reaction (PD-IPCR) are employed for the concurrent detection of aflatoxins and zearalenone, providing detection limits of 0.05 ng/mL and 0.03 ng/mL, respectively, for aflatoxins. Another recent method detects aflatoxins and fumonisins (a group of mycotoxins derived from Fusarium spp.) through Color-encoded Lateral Flow Immuno-Assay (CLFIA). Additionally, the use of biosensors for AFB1 detection in crop products, as opposed to chromatographic or spectrophotometric methods, has gained popularity. Biosensors offer easy and rapid detection, reduced expenses, minimal sample pretreatment, portability, and on-the-spot identification of aflatoxins using an electrochemical enzyme-linked oligonucleotide array.
Measures to Control Aflatoxin Contamination
The extent and intensity of contamination are contingent upon factors such as temperature, humidity, and conditions prevalent in both soil and storage environments. The most efficacious strategy for curtailing mycotoxin contamination lies in prevention, focusing on the exclusion or reduction of toxigenic mold proliferation and toxin production in vulnerable food crops. Practical implementation involves minimizing fungal infections during crop growth through prompt drying and proper storage of harvested crops, employing effective anti-mold preservatives, and adhering to correct postharvest processing, transport, and distribution practices.
Aflatoxin contamination in crops poses a severe threat to production, the food market, health, and economics. Diverse approaches, encompassing physical, chemical, and biological methods, have been devised to mitigate aflatoxin contamination in crops. Effectively preventing mycotoxin contamination during storage predominantly hinges on meticulous moisture control of the crop. This involves eliminating insect activity, which can create favorable microclimates for toxigenic fungal growth, preventing moisture migration, condensation, or water leaks, and curbing rodent activity to prevent increased moisture levels from urination. In essence, conditions that impede fungal growth will invariably curtail or eliminate mycotoxin production.
Harvesting during dry weather generally results in less alarming mycotoxin contamination. Conversely, challenges arise when harvesting takes place in highly humid weather. In numerous developing countries, the amalgamation of insufficient drying equipment and humid atmospheric conditions leads to unacceptable levels of aflatoxin in harvested groundnuts, tree nuts, and other food commodities.
Measures to prevent aflatoxin contamination.
Physical methods
Various physical methods, including steam under pressure, dry roasting, and other cooking techniques, have proven effective in managing or diminishing aflatoxin contamination in numerous crops. A significant reduction in aflatoxin levels, ranging from 40% to 73%, has been documented by subjecting seed samples to heat at 180 °C. Roasting groundnut and corn seeds with 30% moisture at 100 °C for 2 h resulted in an impressive 85% reduction in aflatoxin content. Moreover, roasting at 150 °C for 15 min led to a notable decrease of 70% and 79% in the concentrations of AFB1 and AFG1, respectively. The application of various radiations, such as UV and Infrared radiation, also demonstrated an effective reduction in aflatoxin concentration when applied to seeds. Sunlight emerges as a crucial factor in the detoxification of AFB1 in multiple crops. Exposure to sunlight for a duration of 10–12 h led to a reduction in AFB1 content in artificially infected maize (80%) and groundnut (17%). Additionally, gamma radiation exposure has been identified as another method to diminish aflatoxin levels.
Chemical methods
Numerous chemicals, including acids, alkalis, oxidizing agents, aldehydes, and various gases, have demonstrated efficacy in suppressing aflatoxigenic fungal growth and aflatoxin production when applied in appropriate concentrations. Among gases, ozone stands out as the most effective in promoting aflatoxin degradation in legumes and cereals through an electrophilic attack on carbon bonds within the furan ring. However, the high cost associated with ozone treatment limits its practicality for post-harvest crop treatment. Certain chemicals, such as sodium bisulfite, calcium hydroxide, formaldehyde, sodium hypochlorite, sodium borate, and sorbents, have shown significant potential in reducing aflatoxin levels across various food commodities.
Moreover, some food additives, when combined with specific physical factors like temperature and moisture, inhibit fungal growth and aflatoxin production. Notably, certain food preservatives like propionic acid, crystal violet, p-amino benzoic acid, benzoic acid, boric acid, and sodium acetate have demonstrated inhibitory effects on A. flavus growth and aflatoxin production. Even under conditions favoring A. flavus growth, several propionic acid salts, such as calcium and sodium propionates, have exhibited the ability to diminish aflatoxin formation in maize. Azole fungicides, particularly prochloraz, have been employed as effective tools to control fungal growth and aflatoxin production, surpassing the efficacy of tebuconazole.
Biological methods
Biological agents, including bacteria, yeasts, molds, and algae, exhibit diverse capabilities for aflatoxin degradation in the environmental context. The detoxification of aflatoxins through biological agents involves two processes: absorption and enzymatic decadence. Microorganisms can directly absorb aflatoxins by binding to their cell wall constituents through effective internalization or aggregation. Even dead microorganisms possess the ability to absorb aflatoxins, offering potential applications in the development of probiotic-based bio-filters for fluid decontamination.
Additionally, aflatoxin degradation can occur through intra or extracellular enzymes, with the resulting end products primarily being water and CO2. In a study assessing the efficacy of different microbial strains, Flavobacterium aurantiacum B-184 emerged as the most effective for aflatoxin degradation. Various bacterial strains, including Bacillus velezensis DY3108, demonstrated notable effectiveness against AFB1 degradation, with the cell-free supernatant of the latter exhibiting a robust AFB1 degradation activity of 91.5%. Furthermore, the inoculation of antagonistic strains of Pseudomonas, Trichoderma, Ralstonia, Lactobacilli, Burkholderia, and Bacillus spp. led to a noteworthy reduction in A. flavus growth in pre-harvest crops. Surprisingly, the use of non-toxigenic strains of A. flavus and other molds emerged as an effective control agent, inhibiting aflatoxin contamination.
Further Reading
Aflatoxin Contamination in Corn
Aflatoxin Contamination of Pistachio nuts
Contaminant Detection: Tools for Ensuring Food Safety
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