Emerging Issues on Tropane Alkaloid Contamination of Food in Europe.

The occurrence of tropane alkaloids (TAs), toxic plant metabolites, in food in Europe was studied to identify those TAs in food most relevant for human health. Information was extracted from the literature and the 2016 study from the European Food Safety Authority. Calystegines were identified as being inherent TAs in foods common in Europe, such as Solanum tuberosum (potato), S. melongena (eggplant, aubergine), Capsicum annuum (bell pepper) and Brassica oleracea (broccoli, Brussels sprouts). In addition, some low-molecular-weight tropanes and Convolvulaceae-type TAs were found inherent to bell pepper. On the other hand, atropine, scopolamine, convolvine, pseudotropine and tropine were identified as emerging TAs resulting from the presence of associated weeds in food. The most relevant food products in this respect are unprocessed and processed cereal-based foods for infants, young children or adults, dry (herbal) teas and canned or frozen vegetables. Overall, the occurrence data on both inherent as well as on associated TAs in foods are still scarce, highlighting the need for monitoring data. It also indicates the urge for food safety authorities to work with farmers, plant breeders and food business operators to prevent the spreading of invasive weeds and to increase awareness.

The aflatoxin situation in Africa: Systematic literature review.

Contamination of African staple foods is a major issue for human and animal health, nutrition, and trade. This review aimed to collect and synthesize the available evidence on geographical spread, scale of contamination, disease burden, economic impact, and mitigation measures for aflatoxins in Africa by way of a systematic literature review. This knowledge can enhance management strategies for the major challenges to combat aflatoxins. The search was conducted by applying a predefined search strategy, using bibliographic databases and websites, covering the period 2010 to 2018. Results showed that maize, peanuts, and animal feeds were the most studied commodities. For maize, all studies indicated mean AFB1 to exceed the European Union legal limit. From studies on contamination levels and biomarkers, it is clear that overall exposure is high, leading to a substantial increase in long-term disease burden. In addition, concentrations in food occasionally can reach very high levels, causing acute aflatoxicoses. The trade-related impact of aflatoxin contamination was mainly evaluated from the standpoint of aflatoxin regulation affecting products imported from Africa. There was a limited number of studies on health-related economic impacts, pointing out a gap in peer-reviewed literature. A number of mitigation measures have been developed, but proof of cost-effectiveness or even costs alone of the practices is often lacking. We recommend more emphasis to be put in peer-reviewed studies on evidence-based cost-effective mitigation strategies for aflatoxins, on the scale and spread of the problem and its impacts on public health and economics for use in evidence-based policies.

The MyToolbox EU-China Partnership-Progress and Future Directions in Mycotoxin Research and Management.

Affordable and practical tools for farmers and food processors along the chain are required to efficiently reduce the risk of mycotoxin contamination of crops, feeds and foods. Developing new tools and enhancing existing ones was the mission of MyToolBox-a four-year EU-project that included important Chinese partners and joint research efforts. To identify future directions in mycotoxin research and management in China and their role in China-EU relations, a unique stakeholder workshop including group discussions was organized in Beijing. Six related topics: biocontrol, forecasting, sampling and analysis, silo management, detoxification, and the development of safe use options for contaminated materials were covered. The discussions clearly identified a critical need for smart, integrated strategies to address mycotoxin issues to attain safer food and feed, and to minimize losses and export rejections. Managing data on when, where and the size of mycotoxin contamination events and identifying the institution(s) to manage them are complex issues in China. Studies of microbes and novel, genetically-altered enzymes to limit pre-harvest contamination and to manage post-harvest product detoxification and alternate uses of contaminated materials are in the early stages in China. Further efforts are needed to increase the visibility of mycotoxin problems beyond the scientific and research communities.

Simultaneous quantification of ergot and tropane alkaloids in bread in the Netherlands by LC-MS/MS.

Atropine and scopolamine are tropane alkaloids (TAs), which are regulated for cereal-based foods for children in the EU. For ergot alkaloids (EAs) in cereals and cereal-based food harmonised legislation is not yet established. A fast and straightforward method, which employs extraction by acidified water/methanol followed by ultra-filtration prior to analysis by LC-MS/MS, was validated in bread for 20 EAs and six TAs. LOQs for individual alkaloids ranged from 0.3 to 1.2 µg kg-1, while recoveries ranged from 65% to 94% and repeatability from 3.4% to 17%. A survey was conducted in the Netherlands on 40 retail samples of bread (wheat, rye, wheat-rye, multi-grain) collected in 2014 and 2018. TAs, including atropine and scopolamine, were not detected. Eighteen different EAs were detected and total levels varied between

Are visual processes causally involved in "perceptual simulation" effects in the sentence-picture verification task?

Many studies have shown that sentences implying an object to have a certain shape produce a robust reaction time advantage for shape-matching pictures in the sentence-picture verification task. Typically, this finding has been interpreted as evidence for perceptual simulation, i.e., that access to implicit shape information involves the activation of modality-specific visual processes. It follows from this proposal that disrupting visual processing during sentence comprehension should interfere with perceptual simulation and obliterate the match effect. Here we directly test this hypothesis. Participants listened to sentences while seeing either visual noise that was previously shown to strongly interfere with basic visual processing or a blank screen. Experiments 1 and 2 replicated the match effect but crucially visual noise did not modulate it. When an interference technique was used that targeted high-level semantic processing (Experiment 3) however the match effect vanished. Visual noise specifically targeting high-level visual processes (Experiment 4) only had a minimal effect on the match effect. We conclude that the shape match effect in the sentence-picture verification paradigm is unlikely to rely on perceptual simulation.

Straightforward analytical method to determine opium alkaloids in poppy seeds and bakery products.

A straightforward method to determine the content of six opium alkaloids (morphine, codeine, thebaine, noscapine, papaverine and narceine) in poppy seeds and bakery products was developed and validated down to a limit of quantification (LOQ) of 0.1mg/kg. The method was based on extraction with acetonitrile/water/formic acid, ten-fold dilution and analysis by LC-MS/MS using a pH 10 carbonate buffer. The method was applied for the analysis of 41 samples collected in 2015 in the Netherlands and Germany. All samples contained morphine ranging from 0.2 to 240mg/kg. The levels of codeine and thebaine ranged from below LOQ to 348mg/kg and from below LOQ to 106mg/kg, respectively. Sixty percent of the samples exceeded the guidance reference value of 4mg/kg of morphine set by BfR in Germany, whereas 25% of the samples did not comply with the limits set for morphine, codeine, thebaine and noscapine by Hungarian legislation.

Fate of pyrrolizidine alkaloids during processing of milk of cows treated with ragwort.

To investigate the fate of pyrrolizidine alkaloids (PAs) during milk processing, milk of cows treated via rumen fistula with a mixture of 84% (w/w) ragwort (Jacobaea vulgaris, syn. Senecio jacobaea) and 16% narrow-leaved ragwort (Senecio inaequidens) was processed using laboratory scale heating systems with industrial settings. Pasteurised and sterilised (UHT) milk were produced, as well as set-type yoghurt and cheese. Samples were analysed for 29 PAs using LC-MS/MS, of which 11 PAs were detected above LOQ in the samples (0.1 µg l-1). Alterations in the PA concentration and composition between the standardised milk and the corresponding end-product(s) were evaluated. The heat treatments applied for pasteurisation and UHT sterilisation to prepare semi-skimmed consumption milk did not affect the PA levels in the end-products. In yoghurt, after fermentation of standardised milk (6 h, pH 4.4), 73% of total PAs were recovered. The PA concentration, specifically dehydrojacoline, was decreased, although not quantifiable, during cheese production. A further decrease of 38% during 6 weeks of ripening was observed. The results show that the PA concentration of natural contaminated cow's milk is not affected by heat treatment applied for pasteurised and sterilised milk, but that microbial fermentation of the milk leads to a lowered PA concentration in yoghurt and cheese. This is probably due to microbiological degradation, since PAs are fairly stable under acidic conditions.

Survey of moniliformin in wheat- and corn-based products using a straightforward analytical method.

A straightforward analytical method was developed and validated to determine the mycotoxin moniliformin in cereal-based foods. Moniliformin is extracted with water and quantified with liquid chromatography tandem mass spectrometry, and its presence confirmed with liquid chromatography-Orbitrap-high-resolution mass spectrometry. The method was validated for flour, bread, pasta and maize samples in terms of linearity, matrix effect, recovery, repeatability and limit of quantification. Quantification was conducted by matrix-matched calibration. Positive samples were confirmed by standard addition. Recovery ranged from 77 to 114% and repeatability from 1 to 14%. The limit of quantification, defined as the lowest concentration tested at which the validation criteria of recovery and repeatability were fulfilled, was 10 µg/kg. The method was applied to 102 cereal-based food samples collected in the Netherlands and Germany. Moniliformin was not detected in bread samples. One of 22 flour samples contained moniliformin at 10.6 µg/kg. Moniliformin occurred in seven out of 25 pasta samples at levels around 10 µg/kg. Moniliformin (MON) was present in eight out of 23 maize products at levels ranging from 12 to 207 µg/kg.

Fate of enniatins and deoxynivalenol during pasta cooking.

The fate of deoxynivalenol and enniatins was studied during cooking of commercially available dry pasta in the Netherlands in 2014. Five samples containing relatively high levels of deoxynivalenol and/or enniatins were selected for the cooking experiment. Cooking was performed in duplicate on different days, under standardised conditions, simulating house-hold preparation. Samples were extracted with a mixture of acetonitrile/water followed by salt-induced partitioning. The extracts were analysed by LC-MS/MS. The method limits of detection were 8µg/kg for deoxynivalenol, 10µg/kg for enniatin A1 and 5µg/kg for enniatins A, B and B1. During the cooking of the five dry pasta samples, 60% of the deoxynivalenol and 83-100% of the enniatins were retained in the cooked pasta. It is recommended to study food processing fate of mycotoxins through naturally contaminated materials (incurred materials).

Tropane and ergot alkaloids in grain-based products for infants and young children in the Netherlands in 2011-2014.

An LC-MS/MS multi-method was developed to simultaneously quantify ergot alkaloids (EAs) and tropane alkaloids (TAs) in 113 cereal-based food for infants and young children. To assess yearly variation, samples were collected in 2011, 2012 and 2014. EAs were detected in 54% and TAs in 22% of the samples. Mean EA levels in the three sampling years were 10.6, 6.2 and 8.6 µg kg(-1), respectively (maximum: 115.4 µg kg(-1)), indicating that exposure to EAs would not have exceeded the health-based guidance values set by EFSA in 2012. Mean TA levels were 3.9, 2.4 and 0.4 µg kg(-1), respectively (maximum: 80.8 µg kg(-1)). The acute reference dose for TAs, derived by EFSA in 2013, would have been exceeded by young children when consuming some of the products sampled in 2011-2012. TA levels had decreased drastically in 2014, possibly due to measures taken by producers as response to the EFSA Opinion.

Are effects of common ragwort in the Ames test caused by pyrrolizidine alkaloids?

It has previously been demonstrated by others that acetone extracts of Senecio jacobaea (syn. Jacobaea vulgaris, common or tansy ragwort) test positive in the Salmonella/microsome mutagenicity test (Ames test). Pyrrolizidine alkaloids (PAs) are thought to be responsible for these mutagenic effects. However, it was also observed that the major PA present in common ragwort, jacobine, produced a negative response (with and without the addition of rat liver S9) in Salmonella test strains TA98, TA100, TA1535 and TA1537. To investigate which compounds in the plant extracts were responsible for the positive outcome, the present study investigated the contents and mutagenic effects of methanol and acetone extracts prepared from dried ground S. jacobaea and Senecio inaequidens (narrow-leafed ragwort). Subsequently, a fractionation approach was set up in combination with LC-MS/MS analysis of the fractions. It was shown that the positive Ames test outcomes of S. jacobaea extracts are unlikely to be caused by PAs, but rather by the flavonoid quercetin. This study also demonstrates the importance of identifying compounds responsible for positive test results in bioassays.

A straightforward method to determine flavouring substances in food by GC-MS.

A straightforward GC-MS method was developed to determine the occurrence of fourteen flavouring compounds in food. It was successfully validated for four generic types of food (liquids, semi-solids, dry solids and fatty solids) in terms of limit of quantification, linearity, selectivity, matrix effects, recovery (53-120%) and repeatability (3-22%). The method was applied to a survey of 61 Dutch food products. The survey was designed to cover all the food commodities for which the EU Regulation 1334/2008 set maximum permitted levels. All samples were compliant with EU legislation. However, the levels of coumarin (0.6-63 mg/kg) may result in an exposure that, in case of children, would exceed the tolerable daily intake (TDI) of 0.1mg/kg bw/day. In addition to coumarin, estragole, methyl-eugenol, (R)-(+)-pulegone and thujone were EU-regulated substances detected in thirty-one of the products. The non-EU regulated alkenylbenzenes, trans-anethole and myristicin, were commonly present in beverages and in herbs-containing products.

Inter-laboratory comparison study for pyrrolizidine alkaloids in animal feed using spiked and incurred material.

Pyrrolizidine alkaloids (PAs) are hepatotoxic metabolites produced by plants. PAs in animal feed can cause acute or chronic intoxications in animals and can be transferred to milk. An inter-laboratory comparison study among 12 laboratories, using their own methods of analysis, was conducted for the detection and quantification of PAs in animal feed. The participants were asked to quantify PAs in a blank test sample, a blank test sample to be spiked with a provided spiking mixture of seven PA standards, and a test sample contaminated with common groundsel (Senecio vulgaris). Ten of the participating laboratories used an LC-MS/MS method, one used an LC-ToF-MS method, and one used a GC-MS method. None of the laboratories reported false-negative samples, while two laboratories reported false-positive results in the blank sample. z-scores were calculated for each laboratory for seven PAs in test samples B and C. z-scores varied considerably between laboratories for the concentrations of the free bases and less for the N-oxides, probably due to the lower levels of the free bases as compared with the N-oxides in the contaminated feed. Questionable or unsatisfactory results for the z-scores were obtained for 8% of the cases for the spiked sample and for 12% of the incurred sample. Three laboratories scored consequently positive or negative results. No preferred method for quantification of PAs in feed could be identified within the methods used for this study due to the relatively small number of participants. It was concluded that this inter-laboratory study shows that the methods used for PA detection need further development for accurate estimation of PAs in contaminated feed.

Development and validation of an LC-MS/MS method for the detection of phomopsin A in lupin and lupin-containing retail food samples from the Netherlands.

Phomopsins (PHO) are mycotoxins produced by the fungus Diaporthe toxica (also referred to as Phomopsis leptostromiformis). Lupin is the most important host crop for this fungus and PHO are suspected as cause of lupinosis, a deadly liver disease, in sheep. Lupin is currently in use to replace genetically modified soy in many food products available on the European market. However, a validated method for analysis of PHO is not available until now. In this work, a dilute-and-shoot LC-MS/MS-based method was developed for the quantitative determination and identification of phomopsin A (PHO-A) in lupin and lupin-containing food. The method involved extraction by a mixture of acetonitrile/water/acetic acid (80/20/1 v/v), dilution of the sample in water, and direct injection of the crude extract after centrifugation. The method was validated at 5 and 25 µg PHO-A kg(-1) product. The average recovery and RSD obtained were 79% and 9%, respectively. The LOQ (the lowest level for which adequate recovery and RSD were demonstrated) was 5 µg PHO-A kg(-1). Identification of PHO-A was based on retention time and two transitions (789 > 226 and 789 > 323). Using the average of solvent standards from the sequence as a reference, retention times were all within ± 0.03 min and ion ratios were within ± 12%, which is compliant with European Union requirements. The LOD (S/N = 3 for the least sensitive transition) was 1 µg PHO-A kg(-1) product. Forty-two samples of lupin and lupin-containing food products were collected in 2011-2012 from grocery stores and internet shops in the Netherlands and analysed. In none of the samples was PHO-A detected.

Fungal Infection and Presence of Deoxynivalenol and Zearalenone in Cereals Grown in The Netherlands.

In 1991 and 1993 cereals were sampled during harvest in The Netherlands. The samples were tested for the presence of molds and the samples of 1993 were additionally tested for the mycotoxins deoxynivalenol and zearalenone. The molds were identified to genus level and those belonging to the genus Fusarium to species level. The total fungal infection of cereals in 1991 did not differ from 1993, with a median value of 5.0 log CFU g-1 in both years. The incidences of the genera Aspergillus , Penicillium , the group of Mucor and Rhizopus , Cladosporium , and Fusarium differed considerably between the two years, possibly caused by the different weather conditions. The numbers of samples infected with Fusarium were much higher in 1993 (83%) than in 1991 (34%). In 1991, no Fusarium was detected in samples from the southern part of The Netherlands, as opposed to 1993, when Fusarium was found in all regions sampled. The most dominant Fusarium species in 1991 were Fusarium culmorum and Fusarium avenaceum . In 1993, Fusarium poae , Fusarium culmorum , and Fusarium crookwellense dominated. All these Fusarium species are known mycotoxin producers. Three percent of the cereal samples of 1993 contained deoxynivalenol and 1% contained zearalenone in levels of over 500 µg kg-1 and 200 µg kg-1, respectively. This study has shown that the incidences of various fungal genera and Fusarium species in cereals in The Netherlands can vary from year to year. Considerable numbers of toxigenic Fusarium molds can occur and Fusarium mycotoxins may be present.