Mycotoxin Testing Research Articles

マイコトキシン試験法：一斉分析からメタボロミクスへ

マイコトキシン試験法：一斉分析からメタボロミクスへ

Flow cytometry in rapid mycotoxin testing.

Flow cytometry in rapid mycotoxin testing.

Prevalence of Mycotoxins in Late-Season Corn Ear Molds in North Dakota

Widespread corn ear mold development in North Dakota and in other states was associated with cool, wet weather after physiological maturity in 2009. In response to concerns about possible grain mold and mycotoxin contamination in North Dakota, a survey of corn ear diseases was conducted in 2009 prior to harvest. Corn ears were collected from 94 field locations from 24 counties and were examined macroscopically and microscopically for mold identification. Mycotoxin testing was done with a screen for 17 tricothecenes using gas chromatography/mass spectrometry techniques. Cladosporium was the most commonly identified fungus with some Fusarium contaminated samples being observed. Other fungi identified include Alternaria, Penicillium, Aureobsidium, Rhizopus, Stemphylium and an unidentified non-pigmented fungus. Five mycotoxins were identified in 25 out of the 94 samples; deoxynivalenol, T-2, HT-2, zearalenone and nivalenol. Of the mycotoxin-positive samples, 88% had less than 2 ppm of any mycotoxin. Despite the growing season’s weather being favorable for corn ear mold development late in the season, observed mycotoxin levels were surprisingly low and most grain was deemed safe for use in food and feed channels. Surveys were planned in subsequent years, but late season molds did not develop in the 2010-2015 growing seasons.

Mycotoxin Testing with Aran Gas: Aflatoxin B1 Mycotoxin Destruction using Allotropic Oxygen O4, O5, and O6

Mycotoxin Testing with Aran Gas Aflatoxin B Mycotoxin Destruction using Allotropic Oxygen O O and O

Avian Hepatitis E Virus Infection in Organic Layers.

Between 2012 and 2014, 141 chickens from 10 organic layer flocks with a history of severe drop in egg production (up to 40%) and slight increased mortality (up to 1% per week) were submitted to the Avian Health and Food Safety Laboratory (Puyallup, WA). At necropsy, the most common finding was pinpoint white foci on the liver and regressed ova without any other remarkable lesions. Histologically, there was multifocal mild-to-severe acute necrotizing hepatitis present. No significant bacteria were recovered from liver samples, and tests for mycotoxins were negative. Twenty-six serum samples from four affected flocks tested were positive for avian hepatitis E virus (HEV) immunoglobulin Y antibodies. Avian HEV RNA was detected in 10 livers of chickens from two different affected flocks. The avian HEV was characterized by sequencing and determined to belong to genotype 2. The diagnosis of a clinical manifest HEV was based solely on the demonstration of specific viral RNA and the absence of other causative agents in samples from flocks, as the clinical sings and pathologic lesions were atypical.

Effectiveness of hand sorting, flotation/washing, dehulling and combinations thereof on the decontamination of mycotoxin-contaminated white maize

Maize is one of the major staple foods of Sub-Saharan Africa and is consumed as whole or dehulled grain. In this region, where the environmental conditions favour fungal growth and mycotoxin production, the majority of the population are subsistence consumers who, unfortunately, have little or no access to mycotoxin testing of their food. In an attempt to develop feasible reduction strategies in dietary mycotoxin exposure of the population, a three-factorial design experiment was conducted to examine and compare the efficacy of hand sorting, flotation, dehulling and combinations thereof in removing naturally occurring aflatoxins, fumonisins, nivalenol, deoxynivalenol and alternariol in shelled white maize. Regression analysis was used to determine the significant (p < 0.05) process variables on the removal of mycotoxins from the maize. Results from this experiment indicated that hand sorting had the greatest effect on mycotoxin removal, while flotation yielded the least effect. In particular hand sorting left < 6% of aflatoxin B1 and < 5% of fumonisin B1. Based on these results, hand sorting of maize grains is being recommended as a last line of defence against mycotoxin exposure among subsistence consumers.

Framework for Managing Mycotoxin Risks in the Food Industry

We propose a methodological framework for managing mycotoxin risks in the food processing industry. Mycotoxin contamination is a well-known threat to public health that has economic significance for the food processing industry; it is imperative to address mycotoxin risks holistically, at all points in the procurement, processing, and distribution pipeline, by tracking the relevant data, adopting best practices, and providing suitable adaptive controls. The proposed framework includes (i) an information and data repository, (ii) a collaborative infrastructure with analysis and simulation tools, (iii) standardized testing and acceptance sampling procedures, and (iv) processes that link the risk assessments and testing results to the sourcing, production, and product release steps. The implementation of suitable acceptance sampling protocols for mycotoxin testing is considered in some detail.

Survey of all mycobiota associated with rhizosphere and rhizoplane of different cultivated plants in new reclaimed soil, upper Egypt, and examination of the most common fungal isolates to produce mycotoxins

This survey was designed to study the diversity and occurrence of rhizosphere and rhizoplane fungi in the protectorate of Assiut in Egypt, followed by testing the ability of the most common isolated fungal strains to produce mycotoxins. Not many mycological studies have been carried out to describe the fungal flora of this area, which will be of great significance for the endemic mycobiota. Rhizosphere and rhizoplane samples were collected from the protectorate of Assiut, which represents one of the largest distinctive regions of newly reclaimed soil at the Assiut Governorate. The identification of the isolated fungi during our investigation was carried out using the morphological and microscopic features according to many references and confirmed by the Assiut University Mycology Center (AUMC). The most common four fungal species were examined for their capability to produce mycotoxins; in addition, chemical confirmatory tests for mycotoxins were examined.

Cell-based models for mycotoxin screening and toxicity evaluation: an update

This review presents the applications of cell-based models in mycotoxin research, with a focus on models for mycotoxin screening and cytotoxicity evaluation. Various cell-based models, cell and cell culture condition related factors, toxicity endpoints and culture systems as well as predictive value of cell-based bioassays are reviewed. Advantages, drawbacks and technical problems regarding set up and validation of consistent, robust, reproducible and high-throughput cell-based models are discussed. Various cell-based models have been developed and used as screening tests for mycotoxins but the data obtained are difficult to compare. However, the results highlight the potential of cell-based models as promising in vitro platforms for the initial screening and cytotoxicity evaluation of mycotoxins and as a significant analytical approach in mycotoxin research before any animal or human clinical studies. To develop cell-based models as powerful high-throughput laboratory platforms for the analysis of large numbers of samples, there are mainly two fundamental requirements that should be met, i.e. the availability of easy-to-use and, if possible, automated cell platforms and the possibility to obtain reproducible results that are comparable between laboratories. The transition from a research model to a test model still needs optimisation, standardisation, and validation of analytical protocols. The validation of a cell-based bioassay is a complex process, as several critical points, such as the choice of the cellular model, the assay procedures, and the appropriate use and interpretation of the results, must be strictly defined to ensure more consistency in the results. The development of cell-based models exploring the third dimension together with automation and miniaturisation will bring cellular platforms to a level appropriate for cost-effective and large-scale analysis in the field of mycotoxin research.

Looking for the best compromise in rapid food mycotoxin tests: speed, sensitivity, precision and accuracy

Mycotoxins provide additional challenges to food businesses in terms of successful management of food-safety management systems. These reflect, in part, an unusually high dependency on the activities of others in the supply chain to ensure that levels of contamination remain within set limits. Consequently analyses for mycotoxins by food businesses are primarily commissioned for one or a combination of two reasons: to determine compliance with regulatory or commercial standards or; as part of an exercise to verify the efficacy of the businesses foodsafety management systems. Given the regulatory/commercial implications, the standard of evidence needed to demonstrate (non)compliance will be the greater than that needed for simple verification. Consequently, decisions relating to matters of regulatory or commercial arbitration need to be based on agreed and well defined methods of analysis, which are normally laboratory-based. These data are also often sufficient to be used to verify foodsafety management systems. However, supply conditions may predicate the need for increased levels of verification and rapid mycotoxin test-kits have the potential to both meet this need and satisfy the requirements of statistical process control. Nevertheless, it is important to note that deployment of such test-kits cannot be considered to be a ‘turnkey’ exercise and that, as in the case of laboratory-based assays, care must be taken in the validation and subsequent verification of their use for a given material being used within a food business. In particular, this means demonstrating under local conditions that results from the use of these test-kits are comparable to those that would be obtained using official or reference methods.

Bt maize: a tool for improving food safety of grains at harvest

A new EU (European Union) regulation came into force in 2007 with Regulation (EC) No. 1126/2007 which established maximum levels for fumonisins B 1 and B 2 (4000 ppb), deoxynivalenol (1750 ppb) and zearalenone (350 ppb) in maize and maize products. In order to evaluate French maize food safety, studies were carried out by the national Biological Risk Monitoring (BRM) Network. In this study, field trials involving 84 plots were conducted with Bt maize (MON 810) and its isogenic non-Bt counterpart in 2005 and 2006 in South-western France. Mycotoxin levels were determined in grain at harvest. Fumonisins B 1 and B 2 , deoxynivalenol, and zearalenone were analyzed by LC-MS-MS and the results treated statistically using non parametric tests for mycotoxins and analysis of variance test for weather variables. As the climate was homogenous inside the experimental area, the transgenic event introduced into the maize was the only key parameter which differed between Bt and non- Bt maize plots. Our results showed that all mycotoxin families were not impacted in the same way. The efficacy of Bt maize reduced mycotoxins more than 90% for fumonisins and more than 50% for zearalenone although deoxynivalenol was lightly increased. Therefore a competition between the different Fusarium spp. which produced fumonisins or trichothecenes is hypothesized. According to Regulation (EC) No. 1126/2007, 93% of the maize of Bt maize plots were able to be commercialized compared to only 45% for non- Bt maize plots. The results of this work showed that Bt maize improved food safety and constituted an useful tool to reduce significantly mycotoxin levels in harvested and stored grains. Keywords : Bt (MON810) maize, Fumonisins B 1 and B 2 , Deoxynivalenol (DON), Zearalenone, EC regulation 1126/2007 thresholds

Lower mycotoxin levels in Bt maize grain

Mycotoxins produced by Fusarium spp. during plant cultivation induce severe diseases in animal and humans. In 2007 a European Union regulation set maximum concentrations of mycotoxins in maize and derivatives of 4000 ppb for fumonisins B1 and B2, 1750 ppb for deoxynivalenol, and 350 ppb for zearalenone. To assess the safety of French maize food, investigations are currently being carried out by the national Biological Risk Monitoring network. Here, 84 plots were cropped with the Bt maize MON 810 and its isogenic non-Bt counterpart in 2005 and 2006 in Southwestern France. Mycotoxin levels were measured in grain at harvest. Fumonisins B1 and B2, deoxynivalenol, and zearalenone were analysed by liquid chromatography-mass spectrometry. The data were analysed statistically using non-parametric tests for mycotoxins and analysis of variance for weather variables. As the climate was homogenous inside the experimental area, the transgenic event introduced into the maize was the only key parameter which differed between Bt and non-Bt maize plots. Our results show that Bt maize decreased concentrations of fumonisins by 90% and zearalenone by 50%, whereas the concentration of deoxynivalenol was slightly increased. Those findings suggest a competition among Fusarium species that produce fumonisins or trichothecenes. According to the European regulation, 93% of the Bt maize crops can be sold, compared with only 45% for non-Bt maize plots. Our results thus show that Bt maize improved food safety by greatly reducing mycotoxin levels in field crops in Southwestern France.

MICROBIAL CONTAMINATION OF SELECT DIETARY SUPPLEMENTS

ABSTRACT One hundred thirty‐eight dietary supplement samples comprised of alfalfa, Circu‐Care, coriander, cumin, echinacea, garlic, ginger, ginkgo, horse chestnut extract, juniper berries, licorice, psyllium, saw palmetto, St. John's wort, valerian, white willow bark, and various vitamins and minerals were obtained from local supermarkets and dietary supplement companies and analyzed for fungal contamination and the presence of aerobic mesophilic bacteria. Results indicated that the highest mold and yeast counts of 5.6 × 106 colony forming units (cfu) per gram product were found in alfalfa and the lowest (1.0 × 102 cfu/g) were present in ginger supplements. Potentially toxigenic molds were found in alfalfa, coriander, echinacea, garlic, ginkgo, juniper, licorice, psyllium and St. John's wort supplements. The most common fungi were aspergilli, followed by eurotia, penicillia and yeasts. Alternaria alternata, Fusarium, Cladosporium, Rhizopus and Phoma spp. were isolated less frequently. No molds or yeasts were found in synthetic vitamins, minerals, Circu‐Care and valerian. Aerobic mesophilic bacteria were isolated from all commodities tested. The highest aerobic plate count numbers (5.2 × 106–3.8 × 107 cfu/g) were recovered from alfalfa, whereas the lowest (&lt;100–5.5 × 102 cfu/g) were found in vitamins and minerals.PRACTICAL APPLICATIONSWith an ever‐increasing population utilizing dietary supplements in order to improve and sustain health and vitality, it is essential that these products are safe for human consumption. A very critical indicator of safety is the bacteriological and mycological quality of these commodities.The results of this investigation constitute an indicator of mycological/bacteriological contamination of a variety of dietary supplements. Similar testing has not been reported for several of the studied commodities. Therefore, our findings can serve as a basis for future mycological and mycotoxin testing of dietary supplements and eventually for developing guidelines in order to achieve and maintain safe microbial levels in these products.

The use of mycotoxin methodology in practice: a need for harmonization

Background In the EU, sampling and analysis for the official control of the levels of mycotoxins in foodstuffs should be performed in accordance with the methods and criteria set out in Commission Regulation 401/2006. For each mycotoxin, the values of recovery, repeatability and reproducibility of the analytical method selected by each laboratory must fall within the range of acceptability as prescribed in the Regulation. Aims Carry out a survey on current practices concerning the use and application of mycotoxin test methods for what are considered to be the most current commercially significant mycotoxins. Materials and Methods Nineteen control, commercial and research laboratories from 12 countries (United Kingdom, Italy, Belgium, Spain, Germany, The Netherlands, Bulgaria, Hungary, Greece, Turkey, New Zealand and China) participated in a survey of current practices concerning the use and application of methods for the determination of the principal mycotoxins found in foods and subject to regulatory control: [aflatoxins (AFs: AFB1, AFB2, AFG1, AFG2), aflatoxin M1 (AFM1) fumonisins (FBs: FB1, FB2), ochratoxin A (OTA), deoxynivalenol (DON), patulin (PAT), zearalenone (ZEA), and T-2 and HT-2 toxins]. Results and Discussion Fourteen of the laboratories surveyed were accredited to ISO 17025:2005 and the accreditation paralleled participation in proficiency testing schemes such as FAPAS®. Most of the laboratories declared that they received laboratory samples weighing between 0.004–1 kg. The number and types of food matrices analysed for each mycotoxin or group of mycotoxins varied consistently between mycotoxins, laboratories and countries. In general the highest number of food matrices capable of being assessed for a particular mycotoxin was for OTA followed - in decreasing order - by AFs, DON, ZEA, T-2/HT-2 toxins, FBs, AFM1 and PAT. Analysis for OTA, AFs, PAT, ZEA, DON, FBs, T-2/HT-2 toxins and AFM1, were performed in 95%, 84%, 74%, 74%, 63%, 58%, 58% and 53% of the laboratories, respectively. Most laboratories stated that they used HPLC coupled with either a fluorometer, ultraviolet or mass spectrometric (MS) detectors for detection and quantification of mycotoxins. Only one laboratory used GC/MS for analysis of T-2 and HT-2 toxins whereas two laboratories used TLC based methods for the determination of all mycotoxins except fumonisins. The use of LC-MS methodology by eight laboratories is remarkable because LC/MS is not an official method for mycotoxins within a CEN or AOAC context. Some mycotoxins are not amenable to all detection techniques reported above. ELISA kits were used in three laboratories for the analysis of AFs, OTA, ZEA, DON, FBs and/or T-2/HT-2 toxins. Several other test kits were used in one laboratory for the determination of OTA (six different test kits) and DON (eight different test kits). Six different definitions of limit of detection (LOD) and nine different definitions of limit of quantification (LOQ) have been reported by participating laboratories with the signal/noise ratio being the most popular (used by 40% of laboratories). In some cases the values of LOD, LOQ and measurement uncertainty for the same mycotoxin varied from laboratory to laboratory. In particular, a large variability of measurement uncertainty was noted that was probably due to non-harmonized interpretation of the term. Conclusion This survey suggests that the primary issues needing to be harmonized are: accreditation needed, appropriate size of laboratory sample, guidelines on the most convenient analytical method for each combination of mycotoxins/matrix, use of method validated through a collaborative study, participation in proficiency testing, use of reference/certified materials/standard solutions, use of the same definition/calculation for LOD, LOQ, recovery and measurement uncertainty.

Commercial use of rapid mycotoxin test kits: significance and potential harmonisation issues

A key requirement of any food safety management system is the process of demonstrating its continuous effectiveness (verification). Increasingly, rapid test methods are being used by food businesses as a tool to assist them in this process. This includes management of hazards presented by mycotoxins. These compounds provide a significant challenge to both food processors and regulatory agencies, since they are usually present in the raw material on receipt and subsequent process steps have only limited efficiency in effecting their removal. Within the commercial environment it is critical for those purchasing raw materials to satisfy themselves that any delivery meets regulatory and specification requirements. Decisions to accept or reject deliveries usually need to be taken within short time periods (minutes rather than hours). This requires the use of rapid and - at the same time - reliable analytical methodology; furthermore, the methods need to be of minimal complexity. Within the European Union (EU), analytical methods to determine mycotoxin levels for regulatory purposes must fulfil to certain performance criteria. This principle also holds true for rapid test kits. Current activities within the EU-funded project MoniQA have identified a number of areas in relation to the use of rapid test kits which need to be addressed. These include the criteria which determine robustness of such kits, operator competence and the issues relating to the fate of the rejected lots. Given the diverse stakeholder (e.g. regulator, food business and consumer) interest in the data generated from rapid test kits, a harmonised rationale for their use and also the actions taken upon the results obtained, is desirable.

Sampling Foods for Mycotoxins

It is difficult to obtain precise and accurate estimates of the true mycotoxin concentration of a bulk lot when using a mycotoxin-sampling plan that measures the concentration in only a small portion of the bulk lot. A mycotoxin-sampling plan is defined by a mycotoxin test procedure and a defined accept/reject limit. A mycotoxin test procedure is a complicated process and generally consists of several steps: (1) a sample of a given size is taken from the lot, (2) the sample is ground (comminuted) in a mill to reduce its particle size, (3) a subsample is removed from the comminuted sample, and (4) the mycotoxin is extracted from the comminuted subsample and quantified. Even when using accepted test procedures, there is uncertainty associated with each step of the mycotoxin test procedure. Because of this variability, the true mycotoxin concentration in the lot cannot be determined with 100% certainty by measuring the mycotoxin concentration in a sample taken from the lot. The variability for each step of the mycotoxin test procedure, as measured by the variance statistic, is shown to increase with mycotoxin concentration. Sampling is usually the largest source of variability associated with the mycotoxin test procedure. Sampling variability is large because a small percentage of kernels are contaminated and the level of contamination on a single seed can be very large. Methods to reduce sampling, sample preparation and analytical variability are discussed.

Recent advances on the use of adsorbent materials for detoxification of Fusarium mycotoxins

The extensive use of adsorbents in the livestock industry has led to the introduction of a wide range of new products on the market, most of them claiming high in vitro mycotoxin adsorption capacity. However, adsorbents that may appear effective in vitro do not necessarily retain their efficacy when tested in vivo. Studies performed in our laboratory during the past few years aiming to evaluate the efficacy of various adsorbent materials in binding Fusarium mycotoxins are reported. Adsorption experiments were performed in in vitro screening tests for Fusarium mycotoxins at different pHs; by in vivo tests using the increase of the sphinganine to sphingosine ratio in rat urine and tissues as a biomarker of fumonisin exposure; and by a dynamic, computer–controlled, gastrointestinal model simulating the gastrointestinal tract of healthy pigs. Most of the commercially available mycotoxin–binders failed in sequestering in vitro Fusarium mycotoxins. Only for a small number of adsorbent materials was the ability to bind more than one mycotoxin demonstrated. Cholestyramine was proven to be an effective binder for fumonisins and zearalenone in vitro, which was confirmed for zearalenone in experiments using a dynamic gastrointestinal model and for fumonisins in in vivo experiments. No adsorbent materials, with the exception of activated carbon, showed relevant ability in binding deoxynivalenol and nivalenol. The in vitro efficacy of activated carbon toward fumonisins was not confirmed in vivo by the biomarker assay. The dynamic gastrointestinal model was a reliable tool to study the effectiveness of adsorbent materials in reducing the bioaccessibility of Fusarium mycotoxins, as an alternative to the more difficult and time–consuming studies with domestic livestock.

Development of a generic PCR detection of deoxynivalenol- and nivalenol-chemotypes of Fusarium graminearum

Based on the intergenic sequences of Tri5–Tri6 genes involved in the mycotoxin pathways of Fusarium species, a generic PCR assay was developed to detect a 300 bp fragment of deoxynivalenol (DON)-chemotypes and a 360 bp sequence of nivalenol (NIV)- chemotypes of Fusarium graminearum. Mycotoxin chemotypes identified by the PCR assays were confirmed by the chemical analyses of HPLC or GC/MS. Further analysis of 364 F. graminearum isolates from 12 provinces of China showed that 310 were DON-chemotypes and 54 were NIV-chemotypes. Sequence analyses revealed that DON-chemotypes display more variations than NIV-chemotypes. This PCR assay could be used to detect mycotoxin-producing Fusarium-species and may thus help to develop strategies to avoid or reduce mycotoxin contamination of cereals. Also this assay may provide useful alternatives to antibody-based mycotoxin tests.

Soybean pod blight and root rot caused by lineages of the Fusarium graminearum and the production of mycotoxins

Surveys of soybean (Glycine max) seed grown in South Brazil revealed infection with Fusarium graminearum. To determine if members of this complex were pathogenic to soybean, six strains derived from soybean were added to soil at a rate of 10³ macroconidia/ ml or individual pods were inoculated with 10(4) macroconidia/ml. Seedlings grown in infested soil developed small necrotic lesions in the crown and upper roots. Pods inoculated with conidia developed large (&gt;1 cm), dark brown, necrotic lesions. Younger pods inoculated with the fungus blighted and dropped from the plant. Strains of the F. graminearum complex recovered from lesions on the crown, roots and pods of soybean plants were identified as lineage 1, 2 or 8 by obtaining the DNA sequence from the EF1-alpha gene and comparing it to strains of the known lineage. Two strains of F. graminearum lineage 7 from the U.S. caused similar symptoms of the disease on soybean. Mycotoxin tests on soybean and wheat (Triticum aestivum) indicate that most Brazilian strains produce nivalenol as the major trichothecene mycotoxin rather than deoxynivalenol. In addition, strains from lineages 2 and 8 produce the novel trichothecene, 3-acetylnivalenol.

Production of toxic metabolites in Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei: justification of mycotoxin testing in food grade enzyme preparations derived from the three fungi

Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei are three important production organisms used in industrial fermentations. Several of the fungal secondary metabolites produced by selected strains of these three fungi are capable of eliciting toxicity in animals. Among those toxic substances are the well-known mycotoxins 3-nitropropionic acid and ochratoxin A. However, many others, such as kojic acid, may not be true mycotoxins. The production, extraction, chemical structure, and the toxicity (expressed as LD 50) of these substances are reviewed. Production of toxic secondary metabolites in A. niger, A. oryzae, and T. reesei is strain-specific and environment-dependent. Considering all of the safety measures taken in the industrial production process, these three fungal species are safe to use. The recently revised JECFA specification for mycotoxins in food enzyme preparations is also discussed. The extent of mycotoxin tests in food enzyme preparations should be judged on a case-by-case basis, through a careful evaluation based on knowledge of taxonomy, biochemistry, and genetics. In many cases, the testing scope at the level of genus should be sufficient. In other cases, the scope can even be further narrowed based on scientific knowledge and assessment.