Up to 80% of food items of plant origin worldwide are estimated to be contaminated with mycotoxins, toxic secondary metabolites of fungi, at levels above the limit of detection (LOD) [1
]. Mycotoxins threaten the health and productivity of humans and domesticated animals through dietary exposure at both acute and sub-acute contamination levels in the diet [2
]. Many countries regulate the levels of mycotoxins allowed in imported goods, and mycotoxins are becoming an important non-tariff trade barrier [4
]. Climate change will alter the distribution of mycotoxin producing fungi, including both Aspergillus
spp. For example, hot, dry conditions that exacerbate plant stress will increase the contamination of food and feed with carcinogenic aflatoxins [5
]. Cumulative exposure to mycotoxins can be reduced by careful management of these natural toxins across the food and feed production chains. Mitigation strategies developed in Europe, China and other parts of the world have helped to reduce the absolute number of highly contaminated batches of raw food and feedstuffs in Europe [1
Recently, two large four-year EU-projects with Chinese participation MyToolBox [6
] and MycoKey [7
], focused on reducing mycotoxin contamination, have been successfully completed. The goal of the MyToolBox project [8
] was to develop an integrated, user-friendly e-platform for managing mycotoxins by stakeholders along global food and feed chains [9
]. The e-platform contains information on management practices for mitigating mycotoxin contamination in both pre- and post-harvest settings and for safe-use options, with an on-line decision support tool based on predictive models to assist participants, as needed. The project applied a multi-disciplinary approach to the problem and engaged partners who were academics, industry scientists, IT specialists, policy makers and potential end-users. This diversity was reflected in the consortium that implemented the project, which consisted of 23 organizations based in 10 European countries and China [7
China is a global leader in the production of maize, peanuts and wheat for human food and animal feed. These crops are susceptible to colonization by mycotoxin-producing fungi and to mycotoxin contamination, sometimes at quite high levels. A ten-year survey of feed samples showed that more than 20% of samples from the Far East (China, Japan and South Korea), exceeded the maximum EU guidance levels for deoxynivalenol and zearalenone [10
]. Hence, raw materials in the Chinese market contaminated with mycotoxins at levels that exceed EU regulatory limits are expected. The occurrence of aflatoxins and in particular aflatoxin B1 in the peanut supply chain poses a clear risk for European [11
] and Chinese consumers. Based on data from the Rapid Alert System for Food and Feed (RASFF), mycotoxin contamination exceeding the maximum allowable limit is the most common reason for rejection of food products at the outer European Economic Area (EEA) borders. For example, over the last five years RASFF reported more than 300 alerts of aflatoxin levels exceeding legal limits in peanuts imported from China [12
]. For the major mycotoxins, detailed limits for food and feed are in place in China [13
]. In 2017, China released the National Food Safety Standard for Maximum Levels of aflatoxin B1, aflatoxin M1, deoxynivalenol, patulin, ochratoxin A and zearalenone in foods (GB 2761-2017) as an update of the standard GB2761-2011 [14
]. Worth noticing, is that these limits correspond with the maximum levels established in the EU for most of the commodities listed. Exchange of expertise between the EU and China enables the sharing of experiences, expansion of scientific interaction networks, and synergistic solutions to mycotoxin contamination problems of interest to all parties.
A significant portion of the MyToolBox project focused on personal interactions between EU scientists and their Chinese counterparts at three Chinese universities and research institutes, and broader collaborations that went beyond the formal relationships. A major MyToolBox goal was to strengthen international cooperation in research and innovation for mycotoxins and to invigorate ties between China and Europe in the area of mycotoxin reduction.
In Europe, MyToolBox pre-harvest studies focused on alternative plant protection products and soil treatments in northwestern Europe to mitigate Fusarium
]. Farming systems, including Aspergillus
-resistant maize hybrids, were tested in southeastern Europe. Joint EU–China biocontrol projects focusing on the identification of new biocontrol agents (e.g., atoxigenic Aspergillus flavus
) were developed and the results implemented to reduce aflatoxin contamination in southeast Europe (maize) and in China (peanuts). MyToolBox post-harvest studies evaluated silo management practices for wheat storage in Italy and peanut storage in China and developed CO2
respiration models that enabled the design and production of improved sensors for silo monitoring [16
]. Evaluation of innovative milling techniques and the effects of thermal processing on wheat and wheat-based products, provided evidence for a reduction in deoxynivalenol contamination in the final products [19
]. Testing for efficient safe-use options included the use of genetically altered enzymes (FUMzyme®
) to minimize mycotoxin levels in Distillers Dried Grains with Solubles (DDGS), a protein rich by-product produced during bioethanol fermentation. The same type of enzyme, in addition to specific toxin binders, were used as feed additives in China to reduce exposure of swine to fumonisins (FUMzyme®
) and of dairy cattle to aflatoxins (Bentonite) [20
]. As a result, the EU–China partnership within MyToolBox contributed to the standard setting process for authorization of mycotoxin-detoxifying feed additives in China, with current EU guidelines for the registration of detoxifying feed additives serving as starting points for writing similar Chinese legislation. The EU–China partnership enabled the collection and analysis of data on mycotoxin contamination in winter wheat from various parts of Europe and China with a validated analytical method. Based on these collection data, contamination prediction models were customized for different climatic regions [21
]. The customized prediction models are available through the e-tool [9
] and can assist farmers in identifying potential risks to their crops.
While scientific publications are valuable for defining the status of, and progress in, a specific field of research, they are less useful for projecting where a field is going. To help identify future directions, MyToolBox participants, including the Chinese Academy of National Food and Strategic Reserves Administration in cooperation with Romer Labs, organized a Stakeholder Workshop on Strategies for Effective Mycotoxin Management in China. The workshop was held in Beijing from 16–17 April 2019. The program had six major sections: Biocontrol, Forecasting, Sampling and Analysis, Silo Management, Detoxification, and Safe Use Options, each with Chinese and European presenters in formal meeting presentations and stakeholders in forward-looking discussion sessions. The discussion sessions were run as Nominal Group style roundtable discussions at the end of each day following the presentations [22
]. This style of group discussion has been used previously to set goals for research in mycotoxin control [23
] and sorghum and millet improvement in Africa [25
]. In Beijing, six simultaneous discussion sections were formed—one for each of the three subject areas in which presentations were made on each of the two days. The goal was to identify areas of future research and collaboration, i.e., how to build on the significant progress reported in the presentations in terms of work needed in China and in terms of EU–China interactions.
Climate change is resulting in more extreme weather events in many parts of the world, with increased heat and drought two examples of ways that these changes stress crop plants. Changes in both the EU and China increased the geographic range where aflatoxin contamination can occur and intensification of the problem in areas where contamination already occurs. Within the MyToolBox project, an atoxigenic Aspergillus flavus
strain, Af01, which is native to Serbia, was used as a biocontrol agent in irrigated and non-irrigated maize fields in Serbia during the 2016 and 2017 cropping seasons. This biocontrol treatment reduced aflatoxin contamination, on average, by 73% (range 51–83%). Thus, aflatoxin contamination of maize in Serbia can be reduced through biological control with a native atoxigenic A. flavus
To comply with the Nagoya protocol [28
] on biodiversity and to limit the spread of introduced strains of Aspergillus
, Chinese workers are following a parallel process to develop local atoxigenic strains of A. flavus
for use in China on maize and peanuts. There is high demand in China for a sustainable pre-harvest method that reduces or eliminates aflatoxin contamination. Follow-up issues include: (i) how to apply the biocontrol agents in a commercial setting, (ii) identifying microorganisms responsible for degradation of mycotoxins, and (iii) creating a standard process for continuous development of new biocontrol agents in China. Stakeholders were very interested in future EU–China collaborations to develop and implement biocontrol agents for various regions in China, and to maintain and update control methods, possibly by using the MyToolBox e-platform. Biocontrol research currently is riding a wave of scientific popularity with the success of AflaSafe [29
] for the reduction of aflatoxins in maize and peanuts in sub-Saharan Africa [30
]. This product can be easily adapted to or developed for particular in-country situations [31
Forecasting has become a much more prominent issue with multiple forecasting models now available for multiple toxins in multiple crops [21
]. Although some models incorporate only weather variables, most also include farm-specific agronomic information and geographic location. Some of the models are empirical, i.e., defined by relationships between input and output data, but others are mechanistic, i.e., are based on the biology of the toxin-producing fungi growing under particular conditions. The existing models are not perfect, but several can give 80% or more accuracy in locations to which they have been adapted [21
]. MyToolBox has various forecasting models available in the dynamic part of the e-tool. These models were improved and validated with real data obtained during the project.
The Chinese stakeholders were very interested in the mycotoxin forecasting models, their development, their implementation, and especially in using machine learning in combination with big data. The need to transform both analytical data and field survey data to usable information for farmers and researchers remains to be addressed. Data sharing for supply chain analysis, tailored model development for Chinese end-users, and the cost-effectiveness of forecasting models/DSSs are important areas of interest for future EU–China collaborations.
3.3. Sampling and Analysis
Sampling and analysis protocols are key to estimating risks and enforcing food safety and phytosanitary requirements. The irregular distribution of mycotoxins within a sample can make obtaining a representative sample difficult. In the European Union, sampling and analysis for official sampling and control of (multiple) mycotoxins is regulated by Regulation (EC) No. 401/2006 [40
] and 2014/519 [41
], setting requirements for the method’s precision, repeatability and reproducibility (amongst others)for different foods. In recent years significant advances have been made to increase the speed of the analysis, lower the limits of detection and increase the number of compounds that can be measured. A MyToolBox goal was to identify cost-effective methods for required sampling and analysis, e.g., for routine screening or enforcing legislation [42
]. The most commonly used analytical methods for the determination of various mycotoxins simultaneously are based on liquid chromatography coupled with mass-spectrometry (LC-MS/MS) [43
]. In case of non-official controls for the onsite detection of a limited number of mycotoxins, enzyme-linked immunosorbent assays (ELISA), e.g., for aflatoxin B1 in maize or deoxynivalenol in wheat, or lateral flow devices (LFD) for deoxynivalenol in wheat, might provide quicker or more cost-effective alternatives [42
]. In Europe, ELISA-based analysis for screening was slightly more effective than LC-MS/MS or LFD-based methods when analyzing wheat batches for deoxynivalenol, with differences in accuracy for deoxynivalenol contamination in wheat found to be minimal [42
]. Surprisingly, on-site detection with LFDs turned out to be the least cost-effective method [43
Sampling and analysis were of great interest to the Chinese stakeholders, and this group had more stakeholders present than any other. All aspects of sampling and analysis had the stakeholders’ attention—starting from ensuring high quality samples were collected by properly trained personnel, to transport to the analytical facilities, and subsequent high quality analyses of the materials collected. Many stakeholders were interested in collaborating with MyToolBox and its successors through the exchange of harmonized analytical methods, increased availability of analytical standards, and the use of rapid screening methods to make initial evaluations of mycotoxin contamination on-site. Finally, there was interest in collaboration on methods for tracking and tracing samples with the associated batches of raw materials, food or feed from which the samples were taken.
3.4. Silo Management
Chinese stakeholders expressed interest in effective post-harvest management of staple foodstuffs especially rice, maize, wheat and peanuts to minimize both losses and export rejections due to mycotoxin contamination. Regional differences in the quality of the harvested product and silo design and management require tailor-made solutions rather than generic approaches to solving problems. At present, short-term sensing systems for the key abiotic factors such as relative humidity (RH), temperature or CO2 are used in Chinese storage facilities, and development of permanent sensors and models to use the data these sensors report has been an important step forward. A real time Decision Support System (DSS) was developed in the MyToolBox project which can be used in conjunction with biological models for marginal and optimum abiotic conditions for the initiation of fungal growth and potential contamination of stored wheat with zearalenone or ochratoxin A or of stored maize or peanuts with aflatoxins. Once changes in CO2 levels in real time inside a silo could be visualized it was possible to identify regions within a silo where fungal/insect respiration is occurring and where stored materials can be contaminated by mycotoxins. MyToolBox partners from China and UK have worked together closely on-site at Chinese storage facilities to improve post-harvest management by implementing effective remedial actions, e.g., aeration or removal of potentially contaminated material from the silo, based on trial versions of the DSS.
Management of silos and other post-harvest issues are critical for managing losses of food after it has been produced. Moving materials from farm gate to storage and preparation of harvested crops prior to storage can significantly affect the time the crop can be stored and the quality of the material that is taken from storage. In principle, the most important thing to do is to reduce moisture content below a fixed threshold (varies by crop). China has a range of post-harvest practices and storage facilities that are rapidly being modernized and that require sophisticated trained managers. The form of these storage structures can be uniquely Chinese and practical management solutions may be unique as well, even though the problems being addressed are similar to those encountered elsewhere.
With increased surveillance and lower limits of allowable contamination, the amount of material that could potentially need detoxification is increasing.
Feed additives for mycotoxin detoxification are substances which, when incorporated in animal feed, either bind mycotoxins so they are no longer bioavailable or act as bio-transforming agents converting the toxins into less biologically active products. The scientific literature covering mycotoxin detoxification in animal feed is large and numerous substances have been promoted either as physical or biological adsorbents, or as microbiological/enzymatic transformation agents. Particularly exhaustive is the 2009 report commissioned by European Food Safety Authority (EFSA) to review mycotoxin-detoxifying agents used as feed additives, their mode of action, efficacy and feed/food safety [44
Feeding trials are crucial to evaluate the efficacy of mycotoxin detoxifying feed additives in target species. In the last decade, the focus of these in vivo studies has shifted from the sole assessment of performance data to the evaluation of specific mycotoxin biomarkers in biological matrices of exposed animals. The biomarker data enable demonstration of increased excretion/reduced deposition of mycotoxins, which is an important criterion for registration of mycotoxin-detoxifying feed additives in the EU.
The efficacy of feed additives to reduce the absorption of dietary aflatoxin B1 in dairy cows and absorption of dietary fumonisin B1 in pigs were investigated through in vivo experiments conducted at the Feed Research Institute of the Chinese Academy of Agricultural Sciences CAAS (CAAS-FRI). Both studies were undertaken in China and used EU mycotoxin detoxification additives for aflatoxins in dairy cows and for fumonisins in pigs. MyToolBox partners successfully tested the efficacy of these feed additives under local conditions in China according to EC Regulation No. 429/2008 for the evaluation of feed additives. This test was an unprecedented step forward in establishing common scientific (and ethical) bases for feeding trials jointly recognized by the EU and China [20
Globalization implies increased import–export exchanges combined with different, and decreasing, allowable contamination levels. Thus, post-harvest practices that minimize mycotoxin contamination remain important considerations, a point that also was reflected in the round table discussions. In addition to effective chemical–physical treatments, e.g., cleaning, sorting, roasting, binding agents or ammoniation/chlorination/ozonation, there was growing interest in enzymatic treatments targeted towards particular toxins. Lack of familiarity with many of these processes led to questions about the properties and results of these treatments. Future Chinese cooperation with MyToolBox and its successors should include developing common operational protocols and identifying active agents, reference materials, and analytical standards to monitor safety of the treated end products.
3.6. Safe Use Option
When the topics for the round table discussion sessions were established there was no clear differentiation between the topics of “detoxification” and “safe use options”. Detoxification focuses on animal feed additives, while the focus of safe use options is on detoxification technologies outside of animal feed, e.g., enzymes used in biofuel production. Both topics were included in this session which led to overlap in topic identification and goal setting between the two topics. In the MyToolBox context the use of binders falls in the detoxification category. The use of mycotoxin binders that prevent absorption of toxins in the gut of animals that consume contaminated feed is an important “detoxification” option. The “safe use option” in the MyToolBox context defines the application of mycotoxin degrading enzymes, which were developed and registered for the use in animal feed, in processes such as bioethanol and biogas production. During bioethanol production, mycotoxin concentration increases by a factor of three on a dry weight basis in DDGS compared with the starting grain [45
]. The use of mycotoxin detoxifying enzymes in the fermentation process increases valorization of DDGS as a feed ingredient, because grain batches with mycotoxin levels that exceed limit and guidance values can have these values greatly reduced even while using the contaminated grain is used as a substrate for biofuel production. Alternatively, highly contaminated grain can be used as a feedstock for bioethanol production to generate biogas as an energy source.
When strategies to produce raw materials with low levels of mycotoxin contamination have failed, as have post-harvest strategies, such as proper storage, cleaning and sorting or adding binders, the remaining materials are unfit for use as food or feed. Instead of destruction, the contaminated material could be used for bioethanol production, but high levels of contaminated material may reduce process efficiency and result in DDGS with too much mycotoxin to be used as animal feed. MyToolBox partners adapted mycotoxin degrading enzymes to the bioethanol process to increase fermentation efficiency and produce DDGS with mycotoxin levels below legal limits for use in animal feed.