A Systematic Review of the Efficacy of Interventions to Control Aflatoxins in the Dairy Production Chain—Feed Production and Animal Feeding Interventions

The study presents a systematic review of published scientific articles investigating the effects of interventions aiming at aflatoxin reduction at the feed production and animal feeding phases of the milk value chain in order to identify the recent scientific trends and summarize the main findings available in the literature. The review strategy was designed based on the guidance of the systematic review and knowledge synthesis methodology that is applicable in the field of food safety. The Web of Science and EBSCOhost online databases were searched with predefined algorithms. After title and abstract relevance screening and relevance confirmation with full-text screening, 67 studies remained for data extraction, which were included in the review. The most important identified groups of interventions based on their mode of action and place in the technological process are as follows: low-moisture production using preservatives, acidity regulators, adsorbents and various microbiological additives. The results of the listed publications are summarized and compared for all the identified intervention groups. The paper aimed to help feed producers, farmers and relevant stakeholders to get an overview of the most suitable aflatoxin mitigation options, which is extremely important in the near future as climate change will likely be accompanied by elevated mycotoxin levels.

The primary data source was peer-reviewed scientific articles, risk assessments, as well as primary research. The intention was to provide results with data from the findings of the publications (can be seen in the Supplementary Materials), therefore, reviews containing no data were not considered.

Review Team
The core review team consisted of ten individuals with relevant (agriculture, food safety, microbiology and veterinary public health) and methodological (knowledge synthesis) expertise. The method-related activities were implemented and executed by the core team members who met regularly throughout the review procedure. Prior to implementing the review, the review protocol, the proposed approach and the inclusion and exclusion criteria during the screening and selection of relevant articles were shared with members of the group for feedback.

Review Question, Scope and Eligibility Criteria
The key review question was: What is the effect of targeted feed production, animal feeding or animal husbandry interventions on aflatoxin levels of corn, feed, milk and milk products from feed processing to milk production? It was framed by using the PICO (population, intervention, comparison, outcome) process which is widely used in systematic human health reviews and had been adapted to our purposes.
The population of interest included corn produced for animal (cattle and other ruminants) feeding purposes. Interventions comparing the effect with other interventions or with situations where no interventions were implemented were sought. The main examined outcome was the aflatoxin level in corn-based feed, aflatoxin levels in animals and milk, as well as animal health, status and zootechnical parameters. This paper focused on the interventions applicable at the feed production and animal feeding intervention phases of the milk processing chain.

Search Strategy
Studies that evaluated the effect of agricultural interventions on aflatoxin levels of corn and in animals were identified by searching the Web of Science (https://www.webofscience. com (accessed on 2 January 2022)) and EBSCOhost (https://www.ebsco.com (accessed on 2 January 2022)) online databases. The keyword selections comprised combinations of terms related to the targeted population of interest and intervention types, hence, the following general form was used: [Keywords regarding the fungal infections which affect the aflatoxin levels of corn (e.g., aflatoxin OR Aspergillus); all separated by OR] AND [types of the population of interest (e.g., milk OR corn); all separated by OR] AND [types of intervention (e.g., preservation OR rodent control); all separated by OR]. Searches were run in the publication titles, abstracts and keywords and were restricted to only those studies which were published in English from 2013 to November 2019. More details on the search algorithms are reported in Appendix A. A limitation of the review process was that publications that cover the topic but are written in a language other than English are not indexed by EBSCOhost or Web of Science; the studies that did not meet the inclusion/exclusion criteria or did not fall into the defined timeframe were excluded from the review.

Title and Abstract Relevance Screening (AS)
Abstract screening was performed based on relevance regarding the research question in the Zotero web application (https://www.zotero.org (accessed on 2 January 2022)). Abstract selection was performed by two independent reviewers. In case of conflicting opinions regarding the exclusion or inclusion of a publication based on the content of the abstract, the decision was made by an independent supervisor. As the two domains investigated (storage and feed; farm) had many articles in common, the duplicates between the domains were also excluded. The inclusion and exclusion criteria for the title and abstract relevance screening are summarized in Appendix B.

Relevance Confirmation during Full Text Screening
The papers selected during the title and abstract relevance screening were accessed as full-text articles and relevance confirmation was performed with the help of a predefined form (Appendix C) by single reviewers. During this phase, the papers which did not investigate the effect of interventions on aflatoxins in maize, the papers not in English and the papers that did not contain data on the magnitude of the effect of interventions on aflatoxins available for extraction were excluded. The papers passing this stage were assessed in detail and data were extracted from them in a subsequent step. The keywords were aimed at searching for cattle as the target population; however, if relevant information was found with results for other ruminants, they were not excluded during relevance screening.

Data Extraction
As the main objective of the study was to summarize the effect of various interventions on aflatoxins, data providing evidence on intervention effectiveness were extracted from the selected papers. The extraction was performed by a single reviewer with the help of a data extraction form (Appendix D). The form included data fields on paper identification (authors, title, publication details), on the main characteristics of the study (point in the food chain, intervention category), on intervention details (intervention description, target population/sample, outcome measured, description of the outcome and the data extracted from the outcomes) and on the study quality indicators (study design, number (magnitude) of samples, level of data reported, dose-response gradient, region of the study). Study quality indicators were used to prioritize outcomes in cases when conflicting outcomes were present throughout the search corpus.

Results of the Review Process
A process flow diagram of the knowledge synthesis process for the review is shown in Figure 1.
The 67 papers which were subject for detailed assessment and data extraction contained 126 different interventions. The key characteristics of the 126 relevant interventions are shown in Table 1.

Summary of the Key Findings Regarding Feed Production and Animal Feeding Interventions
The risk reduction interventions regarding aflatoxins during feed production and animal feeding encompass a multitude of various methods. The most important identified groups of interventions based on their mode of action and place in the technological process are the following: low-moisture production using preservatives, acidity regulators, adsorbents and various microbiological additives. A summary of the key findings is presented here. The detailed extracted data and information related to the intervention groups and individual interventions are presented in the Supplementary Materials.

Low-Moisture Production
Aflatoxins are heat-stable molecules and cannot be eliminated using different heat treatments, therefore aflatoxin formation prevention is essential. The rapid drying of agricultural products to reduce their moisture content is an important method, which prevents the formation of favourable conditions for the growth of fungi. Chiewchan et al. (2015) [21] reviewed the possibilities of application of different drying methods to control mould growth and aflatoxin production. The first method was drying sheep feed composed of crushed maize, wheat bran and peanut meal as a thin layer using a hot air oven at 80 • C for 6 h. This technology resulted in a 57.6% reduction of aflatoxins. The second method, drying under sunlight at ambient temperature for 2 days, resulted in an 83.7% reduction of aflatoxins. However, in other studies, drying whole maize kernels at 40-70 • C presented no eradication efficacy regarding aflatoxins.

Preservatives
Testing of naturally occurring antimicrobials for the preservation of food and feed products has been receiving increasing attention due to the growing concern of microbial resistance towards conventional preservatives. The safety of chemical preservatives generated a strong debate since they are considered responsible for many carcinogenic and teratogenic attributes as well as residual toxicity.
Koc and Kara (2014) [22] investigated the antifungal potential of thyme, laurel and rosemary essential oils against A. flavus and A. parasiticus in order to use them as an alternative to preservatives such as potassium sorbate.
Each essential oil-especially at higher concentrations-showed antifungal activities against A. parasiticus and A. flavus. The storage time had no significant effect on the antifungal activity. The most promising essential oil was thyme oil, which featured the highest inhibition of mould growth at all concentrations, followed by the preservative, potassium sorbate, then rosemary and laurel oils.
In another study, conducted by Garcia et al. (2012) [23], the antifungal capacity of the Equisetum arvense extract was tested against A. flavus. The inhibitory effect of the extract was only observed in the inoculated treatments regardless of the incubation time, with a 45% population reduction.
Ashgar et al. (2018) [24] investigated the antifungal activity of and reduction of aflatoxin production by iron (Fe), copper (Cu) and silver (Ag) nanoparticles (NPs) extracted from green tea and black tea leaves. Ag NPs showed the highest antifungal activity and aflatoxin reduction in comparison to Fe NPs and Cu NPs.
The study also examined the effect of NPs on AFB1 adsorption activity in different conditions. The adsorption activities of the metal NPs followed the order of Fe NPs > Cu NPs > Ag NPs, but they were not significantly different from each other at p ≤ 0.05. The study concluded that metal NPs may be utilized as a possible aflatoxin adsorbent in human food and animal feed such as rice, wheat, maize, red chili peppers and poultry feed.
The effectiveness of four additives was tested by Shi et al. (2017) [25] in distillers wet grains (DWG) and condensed distillers solubles (CDS), namely sodium bisulfite, sodium hypochlorite, citric acid and ammonium persulfate. According to the results, sodium bisulfite was not highly efficient in degrading aflatoxins neither in DWG (24% reduction) nor in CDS (35%). Among the four additives tested, sodium hypochlorite was the most effective (42% reduction in DWG and 56% in CDS), but it bleached the substrate and left an off odour, therefore the authors concluded that sodium hypochlorite is not suitable for aflatoxin degradation in food and feed products. Citric acid and ammonium persulfate reduced aflatoxin levels by 31-51% and the effect of citric acid could be further enhanced by increasing the addition level and prolonging the heating time.

Acidity Regulators
Organic acids-similarly to some nutrients like selenium [26]-are natural preservatives and antioxidants and are present in feed as common constituents or could be added artificially to enhance their flavour. They are also suitable for aiding the degradation of AFB1 in food. Aiko et al. (2016) [27] investigated the effect of three different organic acids on the degradation of AFB1 at high temperatures. The results showed that among acetic acid, citric acid and lactic acid, the latter was the most efficient in degrading AFB1, and the efficacy increased with concentration, heating temperature and duration. The most effective degradation of AFB1 was observed at the 1 mol L −1 concentration of lactic acid when heated for 60 min to 80 • C.
Singh and Mandal (2014) [28] studied the efficacy of fumaric and citric acids in preventing the biosynthesis of aflatoxins in poultry feed. The results showed that at the 11% moisture level, none of the studied aflatoxins were recorded in any of the treatments, but with the increase in moisture in feed, the production of aflatoxins also increased. It was concluded that storage of feed for 1 month with 13% moisture content is only safe if the pro-duction of aflatoxins is inhibited by adding fumaric acid or citric acid at the concentration of 0.2% and 0.45%, respectively.
Propionic acid is a very effective and cheap mould inhibitor; however, it has an unpleasant odour and corrosive nature, which hinder its use in food and feed products. This problem can be solved using its salts, sodium and calcium propionates, which have no offensive odour and are not corrosive but have a fungistatic effect [29]. Alam et al. (2014) [30] studied the effects of calcium propionate, water activity (a w ), and incubation time on the total fungal count and aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) production in broiler feed. All the factors (preservative, a w and storage time) alone and in combination significantly reduced the total fungal count and aflatoxin production in the feed. All the aflatoxins increased over the storage time; however, the increase was moderate in the preserved feed that contained a lower amount of available water. This study proved that calcium propionate along with decreased water activity can serve as an effective tool for controlling mould incidence and aflatoxin production.

Adsorbents
Decontamination of feed by adding adsorbents to the diet that can bind aflatoxin molecules is a common way of controlling aflatoxin contamination and thereby protecting animal and human health from adverse effects of aflatoxins. Clay-based feed additives are most frequently used for this purpose; charcoal, glucomannan and plant-based products are also used or investigated. The most important questions regarding the adsorbents used as feed additives are their effectiveness and their physiological effects, therefore the examined parameters in the studies presented can be grouped into the following categories ( Table 2): in vitro examinations of aflatoxin adsorption/binding capacity, antifungal activity, ruminal fermentation parameters and in vivo examinations of aflatoxin degradation/reduction in animals (aflatoxin levels in blood, urine, faeces, milk), carryover/transfer rate, reduction of aflatoxin excretion, as well as animal health status/zootechnical parameters like blood (plasma) parameters, performance (e.g., milk yield, feed intake, milk composition), general health status and immune status.
This section provides an insight regarding the abovementioned adsorbent types, their combinations and toxin-binding premixes (containing not only technological additives, but other beneficial substances as well) by giving a brief description of the outcomes of the studies in this field, summarized in Table 2. The chapter is divided by considering the adsorbent type and the study type-comparative or single-substance investigation.
In conclusion, almost all the studies presented that the investigated adsorbents or adsorbent-based premixes are efficient regarding aflatoxin adsorption or reduction of aflatoxin levels, transfer rate and excretion of aflatoxins. Most of the results were statistically significant regarding the examined parameters. The exceptions were as follows:      Legend: ++-significant; +-not significant or not indicated in the study; 0-no change; empty cell-not examined. Different results for different food additives examined in the same study are separated with "/". 1 Parameters such as aflatoxin levels in blood, urine, faeces, milk. 2 Generally calculated from the AFM1 concentration and milk yield. 3 Including liver and kidney functions (if measured).
In the study of Pate et al. (2018) [36], there was no change in aflatoxin excretion after adding aluminosilicate clay to the diet, which was explained by increased milk yield and feed efficiency parameters by the authors.
Kissel et al. (2012) [42] found no effects regarding aflatoxin reduction in the case of glucomannan and aluminosilicate blend and modified glucomannan treatment.
Weatherly et al. (2018) [44] found no change in the transfer rate and excretion parameters when adding yeast fractions and bentonite to the diet; however, aflatoxin B1 levels in faeces decreased in the study in a quadratic manner.
In the study of Ogunade et al. (2016) [55], no differences were shown regarding aflatoxin levels, transfer rate and excretion. The feed additives used were Saccharomyces cerevisiae fermentation products containing a low or high dose of a chlorophyll-based additive or a low dose of a chlorophyll-based additive and sodium bentonite clay. However, when AFB1 was withdrawn from the diet, AFM1 concentrations decreased rapidly in the treated groups.
There is an unambiguous relationship regarding the interactions between aflatoxins and adsorbents. Some studies investigated the nature of these relationships from various aspects. In the studies of Maki [50] showed that the adsorbent at high AFB1 concentrations was not effective. Weatherly et al. (2018) [44] found a quadratic decrease in AFB1 reduction by the adsorbent treatment in faeces.
Regarding animal health status and zootechnical parameters, it can be concluded that no negative effects of the adsorbent treatments were shown in any of the studies for any of the examined parameters. The results were mainly neutral-meaning that the feed additives did not have any adverse effects on the animals. Nine studies showed positive effects regarding any of the parameters belonging to this group (Table 2) [37,39,43,44,50,51,[53][54][55]; there was significant improvement in the following parameters: blood (plasma) parameters and performance in the case of glucomannan treatment in the study of Akhtar et al. (2016) [39]; general health status for all the three examined feed additives in the study of Naveed et al. (2018) [53]; immune status in the case of a Saccharomyces cerevisiae fermentation product containing a dose of a chlorophyll-based additive in the study of Ogunade et al. (2016) [55].

Microbes and Enzymes
Biodegradation of aflatoxins by microorganisms and other biological organisms is an increasingly studied area as it provides an alternative for the control and elimination that is safe and has the potential not only to remove the aflatoxins, but also to extinguish its adverse health effects. Probiotic strains may also have beneficial effects on general animal health. Yeast preparations are commonly used in feed additive premixes for mycotoxin decontamination; besides, lactobacilli are well-studied for this purpose. Lactic acid bacteria (LAB) and different yeast strains are also widely used to initiate and improve silage fermentation. The decline in pH correlates with the lactic acid concentration produced by LAB, which have antimicrobial properties; besides that, yeasts may also have an adverse effect on moulds with the production of killer toxins.
In this chapter, publications found in the experimental period regarding aflatoxindecreasing potential of microbes and enzymes are classified based on the type of organisms: yeasts, lactic acid bacteria, other microbes and enzymes of Basidiomycota.
The studies usually contain experiments regarding aflatoxin adsorption/binding (in vitro), antifungal activity (in vitro), aflatoxin degradation in feed or in animals (e.g., serum levels, carryover (in vivo)), animal health status (e.g., body weight gain, feed intake (in vivo)) and zootechnical parameters (e.g., dry matter, crude protein, in vitro digestibility).
In some cases, environmental effects (pH, temperature) and dose dependency (number of colony-forming units, aflatoxin concentration), etc., were also studied.
Below, a brief description of the experiments and outcomes of the studies are presented together with the most relevant data (quantified results).
Generally, all the listed publications report positive (mainly significant) results regarding aflatoxin control by microbes and/or enzymes. Besides the known and practically used microbes such as Saccharomyces strains, there is an abundance of promising research aiming at new candidates that are isolated from normal animal microbiota, thereby increasing the probability of colonization [56][57][58][59], while other studies focus on affordable mass production options [60]. Antimicrobial/antifungal effects of LAB were shown in the studies of Dogi et al. (2013 and [61,62], and Drobná et al. (2017) [57], while the synergistic effects of different LAB strains on aflatoxin degradation were published in the study of Zielinska and Fabiszewska (2018) [63]. Strains isolated from novel sources, such as Korean kimchi [64], Tunisian artificial butter [65,66] and feedstuff [67] are also presented. Besides novel yeast [56,68] and bacterial strains [57][58][59][60]65,67,69], the use of Basidiomycota extracellular enzymes has also been investigated with promising results regarding aflatoxin degradation [70][71][72]. Results regarding Aspergillus/aflatoxin inhibition/degradation and decreasing the adverse effects caused by aflatoxins are summarized in Table 3. Findings for other related studied topics that have been investigated in numerous publications are summarized hereunder.

Use of Cell-Free Supernatants
There were altogether five publications with experiments using supernatants of cultures. Three of them compared the efficiency regarding aflatoxin inhibition/degradation of supernatants versus intracellular extracts, cell pellets or viable cells [59,67,69]. In all the cases, the supernatants showed significantly better results than the others. The comparative studies were usually conducted with experimental cultures (not commonly used in practice for aflatoxin degradation at the time of publication) such as microbial consortium TMDC [69], Bacillus shackletonii [67] and Escherichia coli [59]. In the studies of Drobná et al. (2017) and Rather et al. (2014) [57,64], supernatants of Lactobacillus species were used, with significant results in aflatoxin reduction. This indicates that feed additives containing supernatants of bacteria may be the most effective for aflatoxin degradation.    The culture supernatant of the tested isolate was more effective than viable cells and cell extracts Legend: ++-significant; +-not significant or not indicated in the study; 0-no change; --negative effect; empty cell-not examined. Results of the same publication are indicated with a thick frame. 1 Parameters such as carryover rate, aflatoxin excretion, aflatoxin levels in blood, urine, faeces, milk.

Cell Viability
Yeast cell wall is often used in toxin-binding feed additive premixes; nonetheless, whole cells of microbes are used for aflatoxin-degrading experiments in many studies.  [76] compared different types of yeast preparations and concluded that cell wall and autolysed yeast showed high AFM1-binding capability; however, viable cells were not included in the comparison [73,74,76]. Regarding bacteria, a strong strain dependency can be seen from the results of the studies, for example, Ma et al. (2017) [78] concluded that dead Lactobacillus plantarum PT5B was more effective than viable cells; however, L. plantarum MON03 showed better results when using live cell preparations in the study of Jebali et al. (2015) [65].

Effect of pH and Temperature
The effect of pH on aflatoxin inhibition/degradation was studied in six publications. Strains belonging to the Lactobacillus genus were shown to function optimally at acidic pH (2.5-6, with the optimal value of 4) [57,61,65,78], which is favourable to tolerate gastric conditions. However, in the case of Bacillus shackletonii [67] and Escherichia coli [59], pH 8 and 8.5 proved to be effective, respectively. In these two cases, the optimal temperatures for aflatoxin-degrading enzymes proved to be effective (70 and 55 • C, respectively) than in the case of other microbes.

Dose Dependency
The number of initial cells reported varied in terms of the units of measurement and place of administration (depending on the experiment type), but it can be generally said that more microbes produced better results regarding aflatoxin decontamination [56,78].
For the initial aflatoxin concentration, in the case of studies examining extracellular enzymes of Basidiomycota, high initial aflatoxin concentrations inhibited aflatoxin degradation [71,72]. In the study of Intanoo et al. (2018) [58], inhibition at high aflatoxin concentrations was shown for bacteria; however, yeast strains functioned well at high aflatoxin concentrations as well. A positive correlation was shown for yeasts in the study of Magnoli et al. (2016) [56] as well.

Conclusions
Based on the systematic search of scientific literature, the main findings regarding intervention options for effective reduction and control of aflatoxins were identified and summarized. A detailed briefing containing data of the main results of the studies can be found in the Supplementary Materials. The identified studies in the animal feeding topic form the four main topics: low-moisture production, preservatives, acidity regulators, adsorbents and various microbiological additives.
As aflatoxins are heat-stable molecules, prevention steps are essential. Some findings indicated promising results for drying methods; however, other studies showed controversial results, meaning that the circumstances and conditions must be more precisely studied. As conventional preservatives may lead to antimicrobial resistance, naturally occurring preservatives are being more and more investigated, with positive results for essential oils and nanoparticles. Acidity regulators, as well as natural preservatives, antioxidants and flavour enhancers, were also capable of reducing aflatoxin levels in the studies found.
Using adsorbent-based feed additives is a common way of controlling aflatoxin contamination in practice. Several publications investigated the aflatoxin degradation capabilities of different clay types by themselves, in comparison with other clays, used together with other clays or other types of adsorbents or used as ingredients in feed premixes (Table 2). In conclusion, almost all the publications showed efficiency (in most cases with statistical significance) regarding aflatoxin adsorption or reduction of aflatoxin levels, transfer rate and excretion of aflatoxins. The most important concerns regarding adding adsorbents to an animal's diet are the potential changes in the animal's health status and zootechnical parameters as adsorbents may bind useful ingredients of the diet as well. According to the results of the studies found by systematic search, it can be concluded that no negative effects could be shown in any of the studies for any of the examined parameters. The results were mainly neutral-meaning that the feed additives did not have any adverse effects on the animals, and in some cases, on the contrary, were positive for animal health status parameters.
Using biological organisms such as microorganisms as feed additives is a well-studied area according to the results of the systematic review. Not only does it provide an option for safe prevention of aflatoxin formation and removal of aflatoxins, but it might also extinguish its adverse health effects and have beneficial effects on the general animal health. In the studies found, these were the mainly investigated areas. In general, all the listed publications reported positive (mainly significant) results regarding aflatoxin control by microbes and/or their enzymes; however, the optimal conditions of use (e.g., effect of pH and temperature, dosage) and form of usage (dead or viable cells) are strongly dependent on the type of organism. However, cell-free supernatants produced the best results in all the cases.
Ensuring product compliance is always the responsibility of the producer. Because of this and also animal welfare issues, it is of utmost importance for farmers to be able to choose the best, most suitable and fit-for-purpose animal feeding options to protect the health of the animals and thereby protect human health by placing safe milk and meat products on the market. Furthermore, in the case of aflatoxins, human health cannot be guaranteed by only maintaining the levels under the legal limits as it is a carcinogenic compound, of which any consumed amount is to be avoided. This systematic review helps feed and feed additive producers and authorities and might also help farmers or advisors of farmers, veterinarians, farmer associations to get an overview of the most suitable aflatoxin mitigation options, which will be extremely important in the near future as climate change will likely be accompanied by elevated mycotoxin levels.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this manuscript.

Conflicts of Interest:
The authors declare no conflict of interest. Table A1. Point in the food chain: storage and feed-producing facility.

Search Date 28 March 2019
Databases EBSCOhost Intervention phase Storage and feed-producing facility Search string TI (aflatoxin OR "aflatoxin B*" OR "aflatoxin M*" OR AFM* OR AFB* OR Aspergillus) AND TI Feed AND TI (maize OR "zea mays" OR corn) AND TI (storage OR silo-bag OR rotation OR aeration OR (modif* atmosphere) OR pest control OR insect control OR rodent control OR preservation OR "aflatoxin reduc*" OR torrefaction OR irradiat* OR ammonia* OR acidificat* OR microorganism transformation OR enzymatic transformation OR (solvent extract*) OR roughage* OR forage* OR silage OR ensilage* OR silage additive* OR "by-products" OR cgf OR corn gluten feed OR ddgs OR distillers dried grain* with soluble* OR whey OR buttermilk OR permeate OR concentrate* OR biotransform* OR degrad* OR binding OR adsorbent* OR absorbent* OR clay or HSCAS OR "sodium calcium aluminosilicate" OR charcoal OR bentonite OR zeolite OR clinoptilolite OR silicate* OR chlorofillin OR "lactic acid bacteri*" OR ferment*) Number of records 0 Table A2. Point in the food chain: storage and feed-producing facility.

Databases EBSCOhost
Intervention phase Storage and feed-producing facility Search string AB (aflatoxin OR "aflatoxin B*" OR "aflatoxin M*" OR AFM* OR AFB* OR Aspergillus) AND AB Feed AND AB (maize OR "zea mays" OR corn) AND (storage OR silo-bag OR rotation OR aeration OR (modif* atmosphere) OR pest control OR insect control OR rodent control OR preservation OR "aflatoxin reduc*" OR torrefaction OR irradiat* OR ammonia* OR acidificat* OR microorganism transformation OR enzymatic transformation OR (solvent extract*) OR roughage* OR forage* OR silage OR ensilage* OR silage additive* OR "by-products" OR cgf OR corn gluten feed OR ddgs OR distillers dried grain* with soluble* OR whey OR buttermilk OR permeate OR concentrate* OR biotransform* OR degrad* OR binding OR adsorbent* OR absorbent* OR clay or HSCAS OR "sodium calcium aluminosilicate" OR charcoal OR bentonite OR zeolite OR clinoptilolite OR silicate* OR chlorofillin OR "lactic acid bacteri*" OR ferment*) Field AB (abstract or author-supplied abstract) Search mode      (((TS = (aflatoxin OR "aflatoxin B*" OR "aflatoxin M*" OR AFM* OR AFB* OR Aspergillus)) AND TS = (Feed)) AND TS = (maize OR "zea mays" OR corn)) AND TS = (binding OR adsorbent* OR absorbent* OR clay or HSCAS OR "sodium calcium aluminosilicate" OR charcoal OR bentonite OR zeolite OR clinoptilolite OR silicate* OR chlorofillin OR "lactic acid bacteri*" OR ferment*) Field TS (topic)   Specify the methods used to decide whether a study met the inclusion criteria of the review, including how many reviewers screened each record and each report retrieved, whether they worked independently, and if applicable, details of automation tools used in the process.

2-3
Data collection process 9 Specify the methods used to collect data from reports, including how many reviewers collected data from each report, whether they worked independently, any processes for obtaining or confirming data from study investigators, and if applicable, details of automation tools used in the process. List and define all outcomes for which data were sought. Specify whether all results that were compatible with each outcome domain in each study were sought (e.g., for all measures, time points, analyses), and if not, the methods used to decide which results to collect.

4, 28 (Appendix D)
3, Tables 2 and 3 10b List and define all other variables for which data were sought (e.g., participant and intervention characteristics, funding sources). Describe any assumptions made about any missing or unclear information.

4, 28 (Appendix D)
Study risk of bias assessment 11 Specify the methods used to assess risk of bias in the included studies, including details of the tool(s) used, how many reviewers assessed each study and whether they worked independently, and if applicable, details of automation tools used in the process.

Effect measures 12
Specify for each outcome the effect measure(s) (e.g., risk ratio, mean difference) used in the synthesis or presentation of results.

2, not applicable
Synthesis methods

13a
Describe the processes used to decide which studies were eligible for each synthesis (e.g., tabulating the study intervention characteristics and comparing against the planned groups for each synthesis (item #5)).
not applicable 13b Describe any methods required to prepare the data for presentation or synthesis, such as handling of missing summary statistics, or data conversions.
not applicable 13c Describe any methods used to tabulate or visually display results of individual studies and syntheses. Tables 2 and 3,  Supplementary  Materials   13d Describe any methods used to synthesize results and provide a rationale for the choice(s). If meta-analysis was performed, describe the model(s), method(s) to identify the presence and extent of statistical heterogeneity, and software package(s) used.

4-5,
not applicable 13e Describe any methods used to explore possible causes of heterogeneity among study results (e.g., subgroup analysis, meta-regression).
not applicable 13f Describe any sensitivity analyses conducted to assess robustness of the synthesized results. not applicable Reporting bias assessment 14 Describe any methods used to assess risk of bias due to missing results in a synthesis (arising from reporting biases). not applicable Certainty assessment 15 Describe any methods used to assess certainty (or confidence) in the body of evidence for an outcome. not applicable

Study selection 16a
Describe the results of the search and selection process, from the number of records identified in the search to the number of studies included in the review, ideally using a flow diagram.

16b
Cite studies that might appear to meet the inclusion criteria, but which were excluded, and explain why they were excluded. Describe and explain any amendments to information provided at registration or in the protocol. not applicable Support 25 Describe sources of financial or non-financial support for the review, and the role of the funders or sponsors in the review. 22

3-4
Competing interests 26 Declare any competing interests of review authors. 22 Availability of data, code and other materials 27 Report which of the following are publicly available and where they can be found: template data collection forms; data extracted from included studies; data used for all analyses; analytic code; any other materials used in the review.