The presence of mycotoxins associated with cereals and their derived products constitutes a serious health threat, and their presence has become a matter of control and regulation in many countries.
Mycotoxins are chemical compounds that are naturally produced by certain types of fungi (mainly Fusarium spp., Aspergillus spp., and Penicillium spp.). These compounds are toxic, both for people and animals who consume them, and, as a result, they constitute a public health risk.
The fungi that produce mycotoxins can potentially contaminate cereals at any point during the production process, from sowing to flowering, ripening, and harvest through storage and transport. Most mycotoxins are chemically stable and tend to withstand the processing of products, even thermal treatments at high temperatures in some cases. Integral management of the production process can reduce the presence of mycotoxins, and, for this reason, it is necessary to take measures at each point in the production process to perform analyses and routinely confirm their efficacy to ensure cereals and cereal-based products meet the regulatory requirements that are in force.
Cereals Present in Food
Cereals and their derived products are part of the basic foundation of the human diet. They are also one of the main food sources for animals intended for human consumption.
According to the Food and Agriculture Organization of the United Nations (FAO) report “Crop Prospects and Food Situation” published in September 2019 (9), worldwide cereal production during the 2019–2020 growing season reached 2.708 million metric tons (MMT), 16% of which was produced by the United States. In addition, world cereal stocks are forecasted to represent 847 MMT by the end of the season. The global cereals trade is projected to total 415 MMT for 2019–2020. Argentina, Australia, Canada, the European Union, the Black Sea region, and the United States are the six major wheat exporters.
Studies of mycotoxin prevalence in cereal products around the world vary according to different factors, such as origin, climate, and test methods used. For this reason, it is difficult to obtain accurate data. However, according to studies performed by the European Food Safety Authority (EFSA), the prevalence of mycotoxins in cereals is between 60 and 80% (5).
The combination of stored cereal stocks and worldwide trade poses a risk for the contamination of cereals by fungal mycotoxin producers and increases their likelihood of entering the global food supply (12). Knowledge of consumption patterns and prevalence data allows us to understand the scope and importance of a possible contamination event caused by mycotoxins (and the potential for mycotoxicosis).
Health and Economic Consequences
Specific mycotoxin effects vary according to their chemical makeup. In general terms, we can say that a possible contamination event has important implications on three different levels: human health, animal health, and technological challenges.
Human Health. Mycotoxicosis (poisoning) can result from consumption of products contaminated with mycotoxins. Consumption of mycotoxins can result in two different types of mycotoxicosis: acute and chronic.
Acute Mycotoxicosis. Acute mycotoxicosis signals the presence of high levels or dosages of mycotoxins. This type of exposure usually takes place in developing countries, where control resources are very limited.
Chronic Mycotoxicosis. Chronic mycotoxicosis usually takes place when an individual is exposed to small amounts of mycotoxins through frequent consumption of contaminated food(s). This type of poisoning is worrisome in the long-term because some mycotoxins are carcinogenic and genotoxic (i.e., there is the potential for genetic mutations during embryonic development). Long-term exposure can also affect the kidneys, liver, and immune system in humans.
Animal Health. As in the case of mycotoxicosis in humans, acute mycotoxicosis can take place in animals. Symptoms vary depending on the dose and type of mycotoxin that causes the mycotoxicosis. The main effects are gastric symptoms, such vomiting and diarrhea. More severe neurological symptoms, such as paralysis, paresis, convulsions, and even death, also can occur in acute mycotoxicosis events. The symptoms of chronic mycotoxicosis are associated with a reduction in food intake due to a refusal of food, delayed growth, less homogenous flocks, lower production of milk, eggs, or meat, and even fertility problems. The economic impact in both cases can be very high.
Technological Challenges. Mycotoxins can also affect certain microorganisms that are used in food production processes, such as the production of beer, in which microorganisms are used for fermentation. Likewise, mycotoxins negatively affect the quality of the grain, thereby reducing the quality of end products.
Types of Mycotoxins
There are a number of economically and technologically important mycotoxins. Although there is a greater tendency for contamination at different points in the production process depending on the specific cereal and mycotoxin, we can reasonably divide contamination points into two groups: field and storage and transportation.
Contamination in the Field. The main fungi that contaminate cereals in the field are Fusarium spp. This fungus infects the branches, steams, or leaves of plants. Environmental moisture and temperature conditions are critical during the contamination and propagation process. Field contamination is typically heterogeneous and not evenly distributed across an entire field or crop.
An infection in the crop also can cause symptomatology in the plants. This allows for an initial visual screening that may raise suspicions about the presence of mycotoxins. Nevertheless, visual screening is not an ideal diagnostic tool, because asymptomatic infections may take place. In asymptomatic cases, there is no evidence of alterations in the plant; however, mycotoxins can be found during subsequent analyses. The main mycotoxins produced by Fusarium spp. include
- T-2 and HT-2 toxins
- Deoxynivalenol (DON)
- Nivalenol (NIV)
- Zearalenone (ZEN)
Contamination during Storage and Transportation of Cereals. Aspergillus spp. and Penicillium spp. are the two main fungi that are likely to contaminate cereals during storage and transport. Temperature and moisture are the two critical factors that favor the proliferation of microorganisms and the production of mycotoxins after harvest. These two factors drive mycotoxin contamination that occurs when storage, transport, and/or drying conditions are not well controlled. The main mycotoxins produced by these fungi include
- Aflatoxins and ochratoxins (Aspergillus spp.)
- Ochratoxins and citrinin (Penicillium spp.)
Importance of Integral Management
The contamination of cereals due to the presence of fungi can occur at any point in the production process, from planting to the transportation of grain. For this reason, it is very important to practice integral management of the entire production and distribution process to prevent, control, and reduce the presence of mycotoxins in cereals (2,3,4,10).
The Good Agricultural Practices published by the Codex Alimentarius Commission (3) includes suggestions for best practices during production, harvest, and distribution of cereal products.
With regard to crops, the suggestions made are focused on good agricultural practices, crop rotation, selection of resistant varieties, control of moisture in grain, treatment, and analyses to confirm the absence of fungi and mycotoxins during the different phases of the harvest process.
After harvest, it is very important to perform adequate screening of cereals, separating contaminated and suspect grains from sound grains. Another concern after harvest is the drying process. It is not recommended that grain with a high moisture content be stored for an extended period. The grain should be dried as soon as possible to reach moisture levels around 14.5%.
The suggestions made for the storage and transport of grain are very similar (1). With regard to this second point, particularly if it involves great distances, transportation might be considered as “storage on the move.” Storage and transportation facilities must be kept clean and dry. It is extremely important to reinforce cleaning practices and equipment to avoid cross contamination in cases where previous product was contaminated. In addition, the presence of fungal spores in these facilities could contaminate the stored product; thus, good sanitation and upkeep of facilities are encouraged.
As mentioned earlier, moisture and temperature are two critical issues in control of mycotoxins, and as a result transportation and storage facilities must guarantee the protection of cereals against rain or any other source of water. Facilities must also be well ventilated to promote reduced temperatures of bulk cereals or store product in cool conditions. These precautions will limit the growth of fungi.
Pests, such as spider mites, insects, or rodents, in addition to being a distinct sanitation risk in their own right, may be carriers of fungal spores and, thereby, contaminate products (11). For this reason, they must be kept out of storage and transportation facilities. Treatments with approved pesticides can be used when considered necessary. Finally, antifungal or preservative agents such propionic acid can contribute to the elimination of fungi and, consequently, reduce the production of mycotoxins.
Testing is necessary before storage and transport of cereals to determine the concentration of mycotoxins in a given product lot. Through testing, it is also possible to check the efficacy of the preventive measures implemented, as well as verify compliance with legal limits established for the product in the country that is its destination.
According to a study conducted by FAO in 2004 (8), 89 countries had established legal limits for the presence of mycotoxins in foods. Only 13% of the global population lives in countries in which there is no information available regarding the legislation of these limits. Currently, more than 100 countries have enacted legislation.
Each country formulates laws and legislation based on different considerations, including, among others, the characterization of the risks within the country itself, the availability of analytical methods, and/or the current legislation in other countries with whom they have business dealings. For this reason, we find variations in the limits established in different countries, making international commerce more complex (7,15,16).
It is important to perform mycotoxin analyses to ensure that companies comply with local regulations in the country of destination, taking into account whether the regulations define the analytical or sampling requirements that must be fulfilled. Examples of established mycotoxin limits in the United States (15,16) and the European Union (7) are provided in Table I.
Mycotoxin analyses are basic tools for managing the risks associated with mycotoxins. When implementing these analyses, it is very important to perform appropriate sampling of the batch to be analyzed. Fungi tend to reproduce at isolated points in a batch, especially in the case of stored products. This causes the distribution of mycotoxins within a batch to be heterogeneous, which increases the probability of obtaining false negatives or rejecting batches that are in compliance with the limits established due to inadequate sampling.
The sample to be analyzed must be representative of the entire lot. For this reason, primary samples or subsamples must be taken at different points in the lot. With regard to bulk products, different samples at different depths need to be taken. The incremental number will then be blended to generate to a standardized sample. After completing standardization of the sample, a portion will be taken as the lab sample for analysis.
Manuals and regulations that explain how to perform official sampling processes are available. In the United States, the USDA Federal Grain Inspection Service (FGIS) has published two manuals that define appropriate sampling protocols (13,14). These manuals provide instructions that explain how to design and collect adequate samples, as well as the required size of each sample. European Commission Regulation 401/2006 (6) also provides instructions on official sampling protocols and analytical methods for control of mycotoxins in food products.
Several techniques are available for use in detecting and/or quantifying mycotoxins. The most common techniques are enzyme-linked immunosorbent assay (ELISA), lateral flow assay, high-performance liquid chromatography (HPLC), liquid chromatography–mass spectrometer (LC MS/MS), and others.
ELISA. ELISA techniques are based on the antigen–antibody reaction. ELISA techniques are selected when screening or routine tests are performed. These are quick methods and do not require a great degree of specialized skill by the technical staff. The quantity or reactant is small, and the equipment is not expensive. Highly sensitive, semi-quantitative or qualitative ELISA techniques (low detection levels) and specific kits are widely available.
ELISA kits do have some shortcomings, however, in that only one mycotoxin can be detected. Costs are higher in cases where multiple mycotoxins need to be analyzed. There is also the potential for cross-reactions with other mycotoxins or compounds, which can lead to false positive results. In the case of positive results, follow-up testing generally is necessary.
Lateral Flow Assay. As with ELISA techniques, lateral flow assay is an immunoassay based on the antibody–antigen reaction. One advantage of lateral flow assay is that this method is simpler than ELISA techniques, and the required sample preparation is easy.
One disadvantage of lateral flow assay is that it is a qualitative method, and the detection limits are not always adequate for objective analysis. However, whether detection limits are adequate will depend on the limits established by the legislation for the specific country for which the product is destined. As with ELISA, cross-reactions with other substances can lead to false positive results.
These considerations notwithstanding, lateral flow assay is the method of choice for performing on-the-spot checks in the field or warehouse where decisions need to be made quickly.
HPLC. HPLC is a molecular separation technique that allows for the accurate detection of multiple substances. Compounds are separated in a chromatography column and then detected and quantified by a fluorescence detector. As a result, this is a technique with high specificity and sensitivity.
HPLC is a quantitative technique that allows for the detection of very low contamination levels with high precision. Some of the analytical methods that utilize HPLC allow for the extraction of mycotoxins (e.g., B1, B2, G1, and G2 aflatoxins or T-2 and HT-2 mycotoxins), with individual data and quantification provided for each toxin. However, the sweeps are quite limited because they are focused only on mycotoxins of a similar nature.
One disadvantage of HPLC is that it requires complex equipment and skilled staff to develop the necessary techniques. Sample preparation is also more complex. A purification or clean up stage generally is needed to facilitate the detection of mycotoxins.
LC MS/MS. Liquid chromatography linked with a mass spectrometer (LC MS/MS) is an HPLC variation in which the detector used is a mass spectrometer. Detection and quantification are based on the molecular weight and ions produced by the analyte of interest. It is a very sensitive and specific technique that allows for very low detection levels. Multianalyte sweeps using LC MS/MS can identify and quantify multiple mycotoxins unequivocally in a single run.
Although this technique is more expensive due to the equipment and staff requirements, it is increasingly being used due to its high reliability and the ability to quantify multiple mycotoxins in a single analysis.
Other Techniques. To reduce delivery times and facilitate more rapid analysis, new applied technologies are enabling the development of techniques utilizing detection by enzymatic biosensors, immunological biosensors, infrared spectrometry, and others.
Mycotoxins pose an extremely serious threat to public health and can cause major losses both at the human and economic levels. Cereals are one of the primary food sources that are susceptible to contamination due to the presence of mycotoxin-producing fungi. Contamination with mycotoxins can take place at any time in the production cycle, from sowing in the field through grain storage and transport. As a result, it is necessary to carry out integral risk management, from the field to the processing facility, and to take measures during each stage, including transport.
Mycotoxin analyses allow companies to measure the efficacy of the measures taken to reduce the presence of mycotoxins and to check for compliance with the legal limits established for the product in countries for which it is destined. Performing routine analyses will reduce the risk of contamination and product recalls by reducing and removing raw materials that test positive for the presence of mycotoxins.
Pablo de Vicente is the manager of AENOR Laboratory. He holds a bachelor’s degree in veterinary medicine from the Universidad Complutense de Madrid (UCM), Spain, and a technician in food quality control degree. Pablo has been working for more than 19 years in the field of food safety and quality. He began as a veterinary inspector in different slaughterhouses in Madrid and later went to work for private companies in food testing and consulting laboratories. In 2007 Pablo joined AENOR, participating in the development of the new food testing laboratory project, and in 2015 he was appointed manager of AENOR Laboratory. LinkedIn: www.linkedin.com/in/pablodvl.