Determination of regulated and emerging mycotoxins in organic and conventional gluten-free flours by LC-MS/MS

Gluten-free cereal products have grown in popularity in recent years as they are perceived as “healthier” alternatives and can be safely consumed by celiac patients, and people with gluten intolerance or wheat allergies. Molds that produce mycotoxins contaminate cereal crops, posing a threat to global food security. Maximum levels have been set for certain mycotoxins in cereal flours; however, little is known about the levels of emerging mycotoxins in these flours. The aim of this study was to develop an efficient, sensitive, and selective method for the detection of four emerging (beauvericin and enniatins A1, B, and B1) and three regulated (aflatoxin B1, zearalenone, and deoxynivalenol) mycotoxins in gluten-free flours. Ultrasound-assisted matrix solid-phase dispersion was used in the extraction of these mycotoxins from flour samples. The validated method was utilized for the LC-MS/MS analysis of conventional and organic wholegrain oat and rice flours. Six of the seven target mycotoxins were detected in these samples. Multi-mycotoxin contamination was found in all flour types, particularly in conventional wholegrain oat flour. Despite the low detection frequency in rice flour, one sample was found to contain zearalenone at a concentration of 83.2 µg/kg, which was higher than the level set by the European Commission for cereal flours. The emerging mycotoxins had the highest detection frequencies; enniatin B was present in 53% of the samples at a maximum concentration of 56 µg/kg, followed by enniatin B1 and beauvericin, which were detected in 46% of the samples, and at levels reaching 21 µg/kg and 10 µg/kg, respectively. These results highlight the need to improve the current knowledge and regulations on the presence of mycotoxins, particularly emerging ones, in gluten-free flours and cereal-based product

Mycotoxins: The Hidden Danger in Foods

Mycotoxins are secondary metabolites synthesized by a variety of fungal species such as Aspergillus, Penicillium, Fusarium, and Alternaria. These secondary metabolites are toxic and have a significant impact if they enter the production and food chain. Mycotoxins have attracted worldwide attention because of their impact on human health, huge economic losses, and domestic and foreign trade. Although more than 400 mycotoxins have been identified, most studies have focused on aflatoxins (AF), ochratoxin A (OTA), Fusarium toxins, fumonisin (FUM), zearalenone (ZEA), trichothecenes (TCT), and deoxynivalenol/nivalenol due to food safety and economic losses. This chapter will be addressing the type of mycotoxins, its importance in food industry, preventive measures, and implementation of hazard analysis critical control point (HACCP) to control mycotoxin

The global overview of the occurrence of mycotoxins in cereals: a three-year survey

peer-reviewedMycotoxins are secondary metabolites from molds that can contaminate the food and cause serious health problems in consumers. Aflatoxins, deoxynivalenol, fumonisins, zearalenone, T-2 toxins, ochratoxin A, are mycotoxins with acute carcinogenic, mutagenic, teratogenic, hepatotoxic, estrogenic effects. Cereals are very important and strategic grains that are very susceptible to mycotoxin contamination. In this study, the most recent studies about mycotoxins' occurrence in cereals (wheat, corn, rice, oats, barley, rye, sorghum) from 2018 to 2020 were reviewed. It can be concluded that the majority of the studies in the last three years have been done on corn, wheat, and rice, respectively. According to the results, the hazard of aflatoxin B1 in wheat, corn, and rice is serious as it was higher than the EU limit in most of the studies. Because of climate change globally, the fungal population and mycotoxin patterns in different regions and crops are changing. Therefore, the development of practical control and management strategies is essential to ensure crop safety

Validação de métodos para determinação de zearalenona e fumonisinas totais por fluorimetria em amostras reduzidas de milho.

Dois métodos para determinação de micotoxinas em grãos de milho foram internamente validados com base nos critérios: limite de detecção (LD), limite de qualificação (LQ), linearidade, recuperação, repetitividade e estabilidade. Zearalenona e fumonisinas foram extraídas a partir de quantidades reduzidas de amostra, passadas através de colunas de imunoafinidade específicas, eluídas e quantificadas em fluorímetro. Para a zearalenona, LD e LQ foram 0,02 mg kg-1 e 0,07 mg kg-1, respectivamente, a recuperação das amostras fortificadas de milho foi maior do que 78%, o coeficiente de variação (CV) foi de 4,9% e uma correlação linear foi encontrada entre as leituras e os níveis de fortificação, com coeficiente de correlação r =0,997. Para fumonisinas totais, os resultados foram LD = 0,6 mg kg-1, LQ = 1,9 mg kg-1, recuperação = 88%, CV = 17% e r = 0,977. A estabilidade da análise de fumonisinas totais foi avaliada e constatada por um período de tempo de pelo menos três meses, em um nível de significância de 5%. Os resultados são satisfatórios, tendo em vista a nova regulamentação brasileira sobre o assunto. A redução em 80% na massa das amostras e outros ajustes feitos na rotina da análise em relação ao método de referência foram aprovados para fins de pesquisa, levando a uma economia substancial de reagentes e a uma redução substancial na geração de resíduos perigosos contendo metanol.bitstream/item/94175/1/bol-86.pd

Development and Validation of Analytical Methods for Mycotoxins in Food, Medicinal Herbs and Feed

This thesis concerns with the development and the validation of analytical methods for the determination of the mycotoxins aflatoxin B1, zearalenone and patulin, which occur frequently in food and feed. The toxic syndromes produced by them when ingested are known as mycotoxicoses. One of the first reports in history of mycotoxicoses is ergotism, caused by the fungus Claviceps purpurea. Nowadays ergotism is of minor importance; however the problem of mycotoxicoses and long term sub-acute exposure has not faded. Therefore, regulations have been established in many countries, and reliable testing methodology is needed to implement and enforce the regulatory limits. So far, several hundred different mycotoxins have been discovered, exhibiting different structural diversity, with various chemical and physicochemical properties, but only a few present significant food safety challenges. Among these are aflatoxins and ochratoxin A (produced by Aspergillus sp.), fumonisins, trichothecenes such as T-2, HT-2 toxins, deoxynivalenol and zearalenone (produced by Fusarium sp.), patulin (produced by Penicillium sp.) and ergot alkaloids (produced by Claviceps sp.) the most frequent occurring mycotoxins with the highest potential to adverse effects in humans and animals. The work of this thesis can be clustered into three parts as follows: (I) Method comparison and collaborative trial for the determination of aflatoxin B1 in medicinal herbs. This study was initiated upon the request of the European Pharmacopoeia since the regulatory limits for aflatoxin B1 in medicinal herbs were discussed in that moment. The methodology used has been adopted from existing methods for the determination of aflatoxin B1 in food. The food is extracted with an organic solvent followed by immunoaffinity clean-up and reversed-phase high performance liquid chromatography with fluorescence detection. The aim was to select the most suitable method parameters in order to obtain a method that allows the precise determination of aflatoxin B1 in a variety of medicinal herbs. Therefore acetone-water and methanol-water were tested as extraction solvents. Further, the influence of different post-column derivatisation options with electrochemically generated bromine, photochemical reaction and chemical bromination was compared. In addition, two different calculation modes peak height versus peak area were investigated concerning the precision on the evaluation of the rather small peaks that are obtained for aflatoxin B1 at low contamination levels. The different method parameters were applied in the collaborative study to three matrices: senna pods, ginger root and devil’s claw root. As a result, the method with all tested variations was found to be fit-for-purpose for the determination of aflatoxin B1 in medicinal herbs at levels of 1 µg/kg and above. It could be concluded that the tested derivatisation methods had no influence on the analytical result in a range of 1 - 3 µg/kg for aflatoxin B1 in medicinal herbs. This is an interesting conclusion as control laboratories often have a preference for one or the other derivatisation method depending on their experience with one or the other system and its availability. 'A second method was adopted by single-laboratory validation for the determination of aflatoxin B1 in tiger nuts. The interest on tiger nuts rose on the fact of recent entries in the Rapid Alert System for Food and Feed regarding contamination with aflatoxin B1 in tiger nuts. This system allows the European Commission, EU member states and other associated countries to share information and take immediate action when potentially dangerous food or feed is detected on the market or at the border. Additionally a small survey of aflatoxin B1 content in chufa, which is a tiger nuts based soft drink in Spain, was conducted with the adopted method. The detection limit and the quantification limit were 0.02 µg/kg and 0.06 µg/kg respectively. The mean recovery at a level of 2 µg/kg was 88 % (n = 6) and the coefficient of variation 9 %. (II) Development and validation of an analytical method for the determination of zearalenone in infant food as well as in animal feed. The main challenge was to allow the determination of zearalenone at rather low concentrations in infant food, while being also able to deal with complex and more challenging matrices such as compound animal feed, due to their abundant interferences compounds. Previous work performed the determination of zearalenone in cereal grains and at higher levels. The method was validated in an international interlaboratory trial in which laboratories from EU member states, China, Turkey and Uruguay participated. Method performance parameters for both baby food and animal feed were calculated based on results for spiked samples blind duplicates at two levels and based on results for naturally contaminated samples blind duplicates at three levels. Test portions of the samples were spiked at levels of 20 µg/kg and 30 µg/kg zearalenone in baby food and at levels of 100 µg/kg and 150 µg/kg zearalenone in animal feed. As a result the method showed acceptable within-laboratory and between-laboratory precision for each matrix, as required by European legislation. Therefore, the newly developed method allows the enforcement of EU legislative limits for zearalenone in foods for infants at 20 µg/kg. (III) Development and validation of an analytical method for the determination of patulin in juices and purees for infants. The main challenge was to stress the method to determine patulin reliably at levels of 10 µg/kg in products intended for infants and young children. Previously developed methods for patulin were either collaboratively tested at higher levels, indicating that the lower limit for reliable quantification of patulin in such products was around 25 µg/kg or higher, or no validation data except single-laboratory validation were available. Method development focussed on improved and simplified extraction and clean-up procedures. A single liquid-liquid extraction in the presence of sodium sulfate as water binding agent showed sufficient extraction recovery rates for patulin in combination with a solid-phase extraction method, which trapped interfering substances and allowed the purification of patulin extracts without any pre-conditioning of the SPE cartridge. As a result, purees can be extracted without previous enzymatic treatment, as it is required by other methods that use multiple liquid-liquid extractions. Patulin was well separated from the main interfering compound 5-hydroxymethylfurfural during chromatography when using RP-HPLC columns that allow the separation of rather polar substances with mobile phases of more 99% of water. Additionally to this method A and due to the large number of laboratories that intended to participate in the validation process, the participants were split into two groups and a second method B was validated. This method B is a slightly modified version with the same principle as the one previously published by MacDonald et al. in 2000. The main modifications related to the aliquotation. Patulin is extracted three times from the juice or the de-pectinated puree with neat ethyl acetate. The combined ethyl acetate phases were re-extracted with sodium carbonate solution and evaporated. The residue was then re-dissolved in 0.1 % acetic acid solution and separated by HPLC as in method A. Both methods were tested for the determination of patulin in apple juice and fruit puree at the proposed European regulatory limit of 10 µg/kg. The methods were validated in an international interlaboratory trial in which laboratories from EU Member States, Japan and Brazil participated. Method performance parameters for both apple juice and fruit puree were calculated based on results for spiked samples blind duplicates at two levels and based on results for naturally contaminated samples blind duplicates at three levels for both methods. Test portions of the samples were spiked at levels of 10 µg/kg and 25 µg/kg patulin for both apple juice and fruit puree. In conclusion, the new developed method A showed acceptable within-laboratory and between-laboratory precision for both juice and puree at all levels, while method B only fulfilled partially the performance parameters as required by current EU legislation. Therefore, the newly developed method allows the enforcement of EU legislative limits for patulin in foods for infants at 10 µg/kg. Finally the development and in-house validation of a method for determination of patulin using ultra high pressure liquid chromatography in combination with a mass selective detector, resulting in a better chromatographic and analyte selective separation within a shorter time is described. This is of interest especially for projects in which larger amounts of results need to be generated. A survey with more than 200 samples of baby foods, apple purees and tomato purees from the EU food market was performed with this method. It indicated that during the period of 2006 almost all products were free of patulin and all products were compliant with current EU legislation

The fate of mycotoxins in non-alcoholic lactic acid maize meal fermentation.

Thesis (M.Med.Sc.)-University of Natal, Durban, 2003.This study was aimed at investigating the potential of lactic acid fermentation in reducing myco toxin concentration in maize meal products. Maize meal was spiked separately with aflatoxin Bi, fumonism Bi, and zearalenone, and fermented for four days. During this period the concentration of each toxin and the pH of the fermented maize meal were monitored. There was a significant (p= 0.000) decrease in the concentration of all the mycotoxins, with a percentage reduction of 55-69 by the third day and 68-75 by the fourth day, respectively. Commercial amahewu samples were also screened for the presence of these three mycotoxins, and the results indicated that the samples were not contaminated with detectable levels of these toxins. An attempt was made to characterise the metabolic derivatives (by-products) of each mycotoxin following lactic acid maize meal fermentation. To achieve this maize meal samples were separately spiked with each of mycotoxin, fermented for four days and screened for specific mycotoxin derivatives (by-products) using GC/MS, HPLC and relevant standards (i.e. partially hydrolysed fumonisin Bi, aflatoxin B2a, a- and Pzearalenol). None of the targeted derivatives could be detected in the fermented maize meal samples. The potential cytotoxicity of the mycotoxin-spiked fermented samples was investigated using an SNO cell line. The fermented toxin-spiked maize meal samples with a starter culture were comparatively less toxic (29 - 36%) to SNO oesophageal cells than samples spiked with toxin without a starter culture (24 - 30%). However, this observed difference was not statistically significant (p = 0.295 - 0.681). Furthermore, cells that were only inoculated with the cell culture medium had significantly (p = 0.000) high percentage cell viability. This study indicates that it is possible to significantly reduce the concentration of mycotoxins using lactic acid maize fermentation to trace levels. However, such a reduction will not significantly alter the possible chronic toxic effects of such toxins in the diet, particularly a maize based diet containing poor quality protein. The trace amounts of these toxins in fermented and unfermented maize meal should continue to be a cause for concern

Mycotoxins and Food Safety

Foodborne illnesses are a global public health concern with implications worldwide. Mycotoxins are naturally occurring toxins produced by microfungi that are capable of causing disease and death in. living organisms. They are recognized as a major economic problem due to their impact on human health, animal productivity, and.domestic and.international.trade. This book provides updated information about foodborne mycotoxins, their toxicities, new determination methods, prevention strategies, and regulations around the world

Phytoalexins from crucifers : probing detoxification pathways in Sclerotinia sclerotiorum

This thesis investigates two aspects of phytoalexin metabolism by the phytopathogenic fungus Sclerotinia sclerotiorum (Lib) de Bary: (i) determination of detoxification pathways of structurally different molecules; (ii) design and synthesis of potential inhibitors of enzyme(s) involved in detoxification steps.First, the transformations of important cruciferous phytoalexins by the economically important stem rot fungus, S. sclerotiorum, were investigated. During these studies a number of new metabolic products were isolated, their chemical structures were determined using spectroscopic techniques, and further confirmed by synthesis. The metabolic products did not show detectable antifungal activity against S. sclerotiorum which indicated that these metabolic transformations were detoxification processes. Overall, the results of these transformations suggested that S. sclerotiorum produces various enzymes that can detoxify cruciferous phytoalexins via different pathways. While the detoxifications of strongly and moderately antifungal phytoalexins such as brassilexin, sinalexin, and 1-methoxybrassinin were fast and led to glucosylated products, the transformations of the weakly antifungal phytoalexins brassicanal A, spirobrassinin and 1-methoxyspirobrassinin were very slow and yielded non-glucosylated compounds.Next, the design of potentially selective inhibitors of the brassinin detoxification enzyme, BGT, was sought. Two sets of potential inhibitors of BGT were designed: (i) a group was based on the structure of brassinin, where the indole ring of brassinin was replaced with benzofuran, thianaphthene, 7-azaindole and pyrazolo[1,5-a]pyridine and/or the position of side chain was changed from C-3 to C-2; and (ii) another group based on the structure of camalexin where the thiazole ring of camalexin was replaced with a phenyl group. The syntheses and chemical characterization of these potential detoxification inhibitors, along with their antifungal activity, as well as screening using fungal cultures and cell-free extracts of S. sclerotiorum, were examined. The results of these screening indicated that 3-phenylindoles, 3-phenylbenzofuran, 5-fluorocamalexin, methyl (indol-2-yl)methyl-dithiocarbamate, methyl (benzofuran-3-yl)methyldithiocarbamate and methyl (benzo-furan-2-yl)methyldithiocarbamate could slow down the rate of detoxification of brassinin in fungal cultures and also in cell-free extracts of S. sclerotiorum. Among the designed compounds, 3-phenylindole appeared to be the best inhibitor both in fungal cultures and in cell-free extracts. Metabolism studies of all the designed compounds using fungal cultures of S. sclerotiorum indicated that they were metabolized by S. sclerotiorum to glucosyl derivatives, although at much slower rates.It is concluded that some inhibitors that can slow down the rate of metabolism of brassinin could be good leading structures to design more active inhibitors of BGT

Trace analysis of environmentally important species

A flow injection hydride generation atomic absorption (AAS) method has been developed for the analysis of arsenic species. The technique has been optimised for the analysis of arsenite, arsenate, monomethylarsonate(MMA) and dimethylarsinate(DMA) with detection limits of 9, 35, 24 and 24 ppb respectively being achieved. The method described offers the advantage of the reproducible use of small volumes and the ability to achieve rapid sample throughput. The optimised hydride generation AAS method was then investigated as a detector for HPLC. The resulting hyphenated technique allows the separation and detection of the individual arsenic species at ppm levels. As lower detection limits are required for the analysis of arsenic species in real samples an on-line preconcentration technique has been developed, resulting in improved detection limits and the removal of matrix interferences. Finally a matrix solid phase dispersion technique was developed for the extraction of arsenic species from fish which did not result in the loss of information on speciation. A sensitive and reliable method was developed for the determination of aflatoxins Bj, B2, Gj, and G2, ochratoxin A and zearalenone in animal feed ingredients. A multi-toxin extraction and clean-up procedure was used, with dichloromethane: 1 M hydrochloric acid (10:1) being used for the extraction and gel permeation chromatography being used for the clean-up. The liquid chromatographic method developed for the separation of the six mycotoxins involved gradient elution with reverse-phase Cjg column and fluorescence detection. Recoveries, repeatability and reproducibility have been determined on maize, palm and wheat. The detection limits varied depending on the type of feed

An investigation of mycotoxin induced damage and remediation strategies in porcine intestinal cells

Mycotoxins are naturally occurring secondary metabolites, produced by fungal species, and can be toxic to both humans and animals when consumed. Deoxynivalenol (DON) is one of the most commonly occurring mycotoxins and is found to be a common contaminant of cereal grains that are consumed by humans and animals. Consumption of DON contaminated feed can result in vomiting, refusal of feed and reduced weight gain. Zearalenone (ZEN) is an oestrogenic mycotoxin that has been shown to have a negative effect on the reproductive function of animals. The structure of ZEN resembles that of naturally occurring oestrogens, which allows it to bind to oestrogenic receptors, resulting in hormonal disturbances. It has been shown that pigs are most susceptible to both DON and ZEN toxicity through their feeds. The European Food Safety Authority (EFSA) has suggested that the maximum level DON in pig feed should not exceed 0.9 ppm and ZEN in feed for sows and fattening pigs should not exceed 0.25 ppm. DON and ZEN are commonly found to co-occur as both are produced by the Fusarium species. Their common co-occurrence makes it a critical issue in the agriculture industry. Organic selenium yeasts are frequently used as an animal feed supplement as it has a positive impact on animal health. Mycotoxin binders that reduce the amount of mycotoxin absorbed by animals are also used as supplements to animal feeds. In this thesis, the effect of DON and ZEN, individually and combined, on the cell viability, DNA damage and apoptosis of porcine intestinal epithelial (IPEC-J2) cells was studied. Additionally, the potential ameliorative effects of organic selenium yeast and polyunsaturated fatty acids from a mycotoxin binder (Mycosorb A+) against mycotoxin-induced damage was investigated. This research illustrates the damaging effects of the co-occurring mycotoxins and a potential mitigation strategy against such damage