Nicotine in the button mushroom Agaricus bisporus, endogenous biosynthesis?

In early 2009 nicotine was unexpectedly detected in dried mushroom samples. As its origin has not yet been elucidated, this study addressed possible endogenous synthesis of nicotine. Therefore, Agaricus bisporus fruiting bodies were grown in a representative and controlled (nicotine-free) setup. Fruiting bodies (fresh versus stored, intact versus processed (sliced/cooked)) from different harvest days and flushes were analysed with a validated, sensitive dilute-and-shoot UHPLC-MS/MS methodology for nicotine and its precursors putrescine and nicotinic acid. Neither storage nor processing initiated any endogenous nicotine biosynthesis (detection limit 1.6 ng g-1 fresh weight). In contrast, putrescine and nicotinic acid were detected in all samples, with increasing amounts in the different treatments. In silico analysis of the fully sequenced genome of A. bisporus confirmed its inability to produce nicotine. The data obtained do not provide evidence for natural, endogenous presence of nicotine in mushrooms, indicating an exogenous contamination source (e.g. contamination during hand-picking, sample preparation/analysis).

Cultured meat and challenges ahead: A review on nutritional, technofunctional and sensorial properties, safety and legislation.

Cultured meat is introduced as a valuable traditional meat equivalent. However, before marketable end products are available, several hurdles need to be overcome. Among others, these issues comprise obtaining an optimal nutritional profile and approaching the texture, the colour and the unique flavour and taste of conventional meat. Furthermore, the impact of processing on these matters is also still subject of future research. Moreover, more profound knowledge on food-safety aspects, like microbial contamination, prions, possible genetically engineered starting material, etc., and ways to reduce such risks will determine the future success of cultured meat products. Undoubtedly, correct terminology and adequate definitions also require further attention, as these form the starting point of legislative/regulatory aspects. This review provides a state-of-the-art overview on nutritional, technofunctional and sensorial properties, and food-safety and legislative/regulatory aspects on cultured meat production. Additionally, the various challenges and future steps of these aspects of cultured meat are highlighted.

Multi-Class UHPLC-MS/MS Method for Plant Toxins and Cyanotoxins in Food Supplements and Application for Belgian Market Samples.

The presence of plant toxins and/or cyanotoxins in food supplements implies consumer health risks. Therefore, a targeted ultra-high performance liquid chromatographic-tandem mass spectrometric method to detect/quantify 25 toxins simultaneously in food supplement formulations was developed and validated. Full validation for tablets/powders and secondary validation for a liquid and soft gel capsule indicated that most compounds were efficiently extracted (≥ 75%), while others were only partly extracted (18 - 61%). Trueness was fulfilled (70 - 120%), with some exceptions (mostly at the lowest validation level). Intralaboratory repeatability, intra- and interlaboratory reproducibility values of ≤ 20%, ≤ 25%, and ≤ 25% were obtained for most, respectively. Matrix effects were found to be significant for most compounds. Good sensitivity (µg/kg level) was observed for galegin(e), lycopsamine, lycorine, rubiadin, skimmiamine, and vascin(e), in contrast to helveticoside, lucidin, lucidin-3-primveroside, plumbagin(e), and thujone, which were detected at the mg/kg level. The other compounds were characterized by a sensitivity between 10 to 1000 µg/kg. The validated methodology was applied for 52 food supplements (tablets, capsules, liquids/syrup, etc.) purchased from the Belgian market. In more than 25% of the samples, one or more toxins were detected (concentrations determined using standard addition). Lycopsamine, microcystin LR, solamargine, thujone, and vasicin(e) were the most frequently detected toxins. A clear link between the toxins detected and the plant species on the food supplement ingredient list could not always be established. This generic "dilute-and-shoot" procedure can be used for further research on toxins in food supplements and by extension other plant/algae-based food/feed commodities (herbs, edible flowers, etc.).

Effects of ascorbic acid on patulin in aqueous solution and in cloudy apple juice.

Degradation of the mycotoxin patulin (PAT) and the generation of (less toxic) breakdown products, such as (E/Z)-ascladiol (ASC-E/Z) and desoxypatulinic acid (D-PAT), can occur due to chemical, physical and biological treatments. Our study focused on the chemical degradation of PAT in the presence of ascorbic acid (AA) both for pure PAT standard in acidified aqueous solution and for PAT-contaminated cloudy apple juice (CAJ) (obtained via addition of apple mash produced from apples inoculated with Penicillium expansum). Within this framework, different concentrations of AA were evaluated, as well as the presence/absence of oxygen and different storage temperatures. In order to do so, an in-house methodology allowing a good separation of PAT from its reaction and breakdown products was optimized first. The highest PAT reduction (60%) in CAJ with an initial PAT concentration of 100 µg/kg and 0.25% (w/v) AA was achieved after 6 days of incubation at 22 °C in the presence of oxygen. It was also found that the treatment by AA resulted in the generation of degradation products less toxic than PAT (such as (E/Z)-ASC). In conclusion, AA used to improve numerous product quality aspects (e.g. colour (less browning), nutritional value, etc.) and considered as a safe food additive (Food and Drug Administration (FDA) (1999)) has an effect on PAT degradation. It was shown that such degradation generated less toxic compounds in the presence of oxygen. In view of consumers' safety, fortification of apple juice (and possibly apple-based products) with AA could be helpful within an integrated system to ensure products with low levels of patulin. The optimum conditions for such an approach within a legal and practical point of view need to be further explored.

The role of roughage provision on the absorption and disposition of the mycotoxin deoxynivalenol and its acetylated derivatives in calves: from field observations to toxicokinetics.

A clinical case in Belgium demonstrated that feeding a feed concentrate containing considerable levels of deoxynivalenol (DON, 1.13 mg/kg feed) induced severe liver failure in 2- to 3-month-old beef calves. Symptoms disappeared by replacing the highly contaminated corn and by stimulating ruminal development via roughage administration. A multi-mycotoxin contamination was demonstrated in feed samples collected at 15 different veal farms in Belgium. DON was most prevalent, contaminating 80% of the roughage samples (mixed straw and maize silage; average concentration in positives: 637 ± 621 µg/kg, max. 1818 µg/kg), and all feed concentrate samples (411 ± 156 µg/kg, max. 693 µg/kg). In order to evaluate the impact of roughage provision and its associated ruminal development on the gastro-intestinal absorption and biodegradation of DON and its acetylated derivatives (3- and 15-ADON) in calves, a toxicokinetic study was performed with two ruminating and two non-ruminating male calves. Animals received in succession a bolus of DON (120 µg/kg bodyweight (BW)), 15-ADON (50 µg/kg BW), and 3-ADON (25 µg/kg) by intravenous (IV) injection or per os (PO) in a cross-over design. The absolute oral bioavailability of DON was much higher in non-ruminating calves (50.7 ± 33.0%) compared to ruminating calves (4.1 ± 4.5%). Immediately following exposure, 3- and 15-ADON were hydrolysed to DON in ruminating calves. DON and its acetylated metabolites were mainly metabolized to DON-3-glucuronide, however, also small amounts of DON-15-glucuronide were detected in urine. DON degradation to deepoxy-DON (DOM-1) was only observed to a relevant extent in ruminating calves. Consequently, toxicity of DON in calves is closely related to roughage provision and the associated stage of ruminal development.

Development, validation and application of an ultra high performance liquid chromatographic-tandem mass spectrometric method for the simultaneous detection and quantification of five different classes of veterinary antibiotics in swine manure.

In this study, a fast, simple and selective ultra high performance liquid chromatographic-tandem mass spectrometric (UHPLC-MS/MS) method for the simultaneous detection and quantification of colistin, sulfadiazine, trimethoprim, doxycycline, oxytetracycline and ceftiofur and for the detection of tylosin A in swine manure was developed and validated. First, a simple extraction procedure with acetonitrile and 6% trichloroacetic acid was carried out. Second, the supernatant was evaporated and the pellet was reconstituted in 1 ml of water/acetonitrile (80/20) and 0.1% formic acid. Extracts were filtered and analyzed by UHPLC-MS/MS on a Kinetex C18 column using gradient elution. The method developed was validated according to the criteria of Commission Decision 2002/657/EC. Recovery percentages varied between 94% and 106%, repeatability percentages were within the range of 1.7-9.2% and the intralaboratory reproducibility varied between 2.8% and 9.3% for all compounds, except for tylosin A for which more variation was observed resulting in a higher measurement uncertainty. The limit of detection and limit of quantification varied between 1.1 and 20.2 and between 3.5 and 67.3 µg/kg, respectively. This method was used to determine the presence and concentration of the seven antibiotic residues in swine manure sampled from ten different manure pits on farms where the selected antibiotics were used. A link was found between the antibiotics used and detected, except for ceftiofur which is injected at low doses and degraded readily in swine manure and was therefore not recovered in any of the samples. To the best of our knowledge, this is the first method available for the simultaneous extraction and quantification of colistin with other antibiotic classes. Additionally, colistin was never extracted from swine manure before. Another innovative aspect of this method is the simultaneous detection and quantification of five different classes of antibiotic residues in swine manure.

A multiresidue liquid chromatographic/tandem mass spectrometric method for the detection and quantitation of 15 nonsteroidal anti-inflammatory drugs (NSAIDs) in bovine meat and milk.

This study concerns a validated liquid chromatographic/tandem mass spectrometric (LC-MS/MS) multiresidue method for the simultaneous detection, identification, and quantitation of 15 nonsteroidal anti-inflammatory drugs (NSAIDs) in bovine meat and milk. The NSAIDs considered are carprofen, diclofenac, flufenamic acid, flunixin (5-hydroxyflunixin as marker metabolite in milk), ketoprofen, mefenamic acid, meloxicam, 4-methylaminoantipyrine (marker metabolite of metamizole in meat and milk), naproxen, niflumic acid, phenylbutazone (and metabolite oxyphenbutazone), ramifenazone, salicylic acid, and tolfenamic acid. These compounds were chosen as representatives of different chemical subclasses of NSAIDs. Flunixin-d3, diclofenac-d4, 4-aminoantipyrine-d3, and phenylbutazone-d10 were used as internal standards. Performance characteristics were validated according to the Commission Decision 2002/657/EC (Off J Eur Communities, L221: 8-36). Recovery percentages varied between 81 and 114% for bovine meat and between 79 and 118% for milk. Repeatability percentages were within the range of 1-12% for meat and between 1 and 17% for milk, whereas the intralaboratory reproducibility varied between 3 and 19% for meat and between 3 and 23% for milk. The decision limit and the detection capability for bovine meat were within the range of 0.5-579 µg kg(-1)and 0.6-642 µg kg(-1), respectively. Those for milk were within the range of 0.12-55 µg kg(-1) and 0.14-61 µg kg(-1), respectively. The methods developed were successfully applied for proficiency test samples and routine samples analyzed in the laboratory. The methodology concerns fast, user-friendly, and sensitive methods, which can be easily extended for other compounds and matrices. In general, such multiresidue methods contribute to the reduction of human exposure to these veterinary drug residues by consumption of contaminated bovine-derived products such as meat and milk.

Detection and identification of xerophilic fungi in Belgian chocolate confectionery factories.

Chocolate confectionery fillings are generally regarded as microbiologically stable. The stability of these fillings is largely due to the general practice of adding either alcohol or preservatives. Consumer demands are now stimulating producers to move away from adding alcohol or other preservatives to their confectionery fillings and instead to search for innovative formulations. Such changes in composition can influence the shelf life of the product and may lead to spoilage by xerophilic fungi. The aim of this study was to test whether the production environment of Belgian chocolate confectionery factories and common ingredients of chocolate confectioneries could be potential sources of contamination with xerophilic fungal species. In the factory environment, the general and strictly xerophilic fungal spore load was determined using an RCS Air Sampler device in combination with DG18 and MY50G medium, respectively. Four basic ingredients of chocolate confectionery fillings were also examined for fungal spore levels using a direct plating technique. Detected fungi were identified to species level by a combination of morphological characterization and sequence analysis. Results indicated a general fungal spore load in the range of 50-250 colony forming units per cubic meter of air (CFU/m(3) air) and a more strict xerophilic spore load below 50 CFU/m(3) air. These results indicate rather low levels of fungal spores present in the factory environment. The most prevalent fungi in the factory environment were identified as Penicillium spp., particularly Penicillium brevicompactum. Examination of the basic ingredients of confectionery fillings revealed nuts to be the most likely potential source of direct contamination. In nuts, the most prevalent fungal species identified were Eurotium, particularly Eurotium repens.

Development of an ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for the simultaneous determination of salicylic acid, jasmonic acid, and abscisic acid in rose leaves.

This paper describes a method to detect and quantitate the endogenous plant hormones (±)-2-cis-4-trans-abscisic acid, (-)-jasmonic acid, and salicylic acid by means of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in hybrid rose leaf matrices. Deuterium-labeled [(2)H6] (+)-2-cis-4-trans-abscisic acid, [(2)H6] (±)-jasmonic acid, and [(2)H4]-salicylic acid were used as internal standards. Rose samples (10 mg) were extracted with methanol/water/acetic acid (10:89:1) and subsequently purified on an Oasis MCX 1 cm(3) Vac SPE cartridge. Performance characteristics were validated according to Commission Decision 2002/657/EC. Recovery, repeatability, and within-laboratory reproducibility were acceptable for all phytohormones tested at three different concentrations. The decision limit and detection capability for (±)-2-cis-4-trans-abscisic acid, (-)-jasmonic acid, and salicylic acid were 0.0075 and 0.015 µg/g, 0.00015 and 0.00030 µg/g, and 0.0089 and 0.018 µg/g, respectively. Matrix effects (signal suppression or enhancement) appeared to be high for all substances considered, implying the need for quantitation based on matrix-matched calibration curves.

Influence of cultivar and harvest time on the amounts of isoalliin and methiin in leek (Allium ampeloprasum var. porrum).

Extracts of 31 leek cultivars were analyzed using liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to determine the distribution of the two most abundant S-alk(en)yl-l-cysteine sulfoxides (ACSOs) in leek, that is, isoalliin and methiin. The isoalliin concentration of the white shaft and green leaves of the 31 leek cultivars varied from 15 to 53 mg/g dry weight (dw) and from 9 to 45 mg/g dw, respectively, whereas the methiin concentration varied from 3 to 16 mg/g dw and from 1 to 10 mg/g dw, respectively. Leek cultivar and tissue had an effect on the ACSO amounts. Cultivars Artico and Apollo F1 rated highest for the mean isoalliin and methiin concentration, respectively. In general, the whole leek plant of the winter leek cultivars contained a significantly higher ACSO amount than the summer and autumn cultivars. To determine whether this difference was attributed to the cultivar background or time of harvest, ACSOs were also quantitated in nine leek hybrids at four different stages during the next growth season. The amounts of ACSO changed significantly during the growth season, indicating the importance of harvest at specific time moments, although there was still an effect of cultivar on the ACSO amounts.

Restriction analysis of an amplified rodA gene fragment to distinguish Aspergillus fumigatus var. ellipticus from Aspergillus fumigatus var. fumigatus.

A previous multidisciplinary study indicated that gliotoxin-producing Aspergillus fumigatus Fresen. isolates from silage commodities mostly belonged to its variant A. fumigatus var. ellipticus Raper & Fennell. Sequence analysis revealed the presence of a single nucleotide polymorphism at five positions in a fragment of the rodA gene (coding for a hydrophobin rodletA protein) between Aspergillus fumigatus var. fumigatus and Aspergillus fumigatus var. ellipticus. A method was developed to distinguish these two types of isolates based on restriction analysis of this rodA gene fragment using the HinfI restriction enzyme. In addition, in silico analysis of 113 rodA gene fragments retrieved from GenBank was performed and confirmed the suitability of this method. In conclusion, the method developed in this study allows easy distinction between A. fumigatus var. fumigatus and its variant ellipticus. In combination with the earlier developed PCR-restriction fragment length polymorphism method of Staab et al. (2009, J Clin Microbiol 47: 2079), this method is part of a sequencing-independent identification scheme that allows for rapid distinction between similar species/variants within Aspergillus section Fumigati, specifically A. fumigatus, A. fumigatus var. ellipticus, Aspergillus lentulus Balajee & K.A. Marr, Neosartorya pseudofischeri S.W. Peterson and Neosartorya udagawae Y. Horie, Miyaji & Nishim.

Ultrahigh-performance liquid chromatographic-tandem mass spectrometric multimycotoxin method for quantitating 26 mycotoxins in maize silage.

A multianalyte method was developed to identify and quantitate 26 mycotoxins simultaneously in maize silage by means of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The extraction and cleanup procedure consists of two extraction steps followed by purification on a Waters Oasis HLB column. The method developed was validated with the requirements of Commission Decision 2002/657/EC taken into account. The limit of detection and quantitation ranges were 5-348 and 11-695 ng/g, respectively. Apparent recovery varied between 61 and 116%, whereas repeatability and reproducibility were within the ranges of 3-45 and 5-49%, respectively. The method developed was successfully applied for maize silage samples taken at the cutting surface and 1 m behind that surface. Mainly Fusarium toxins (beauvericin, deoxynivalenol, enniatins, fumonisins, fusaric acid, and zearalenone) were detected, but postharvest toxins such as mycophenolic acid and roquefortine C were identified as well.

Mycotoxin production by pure fungal isolates analysed by means of an uhplc-ms/ms multi-mycotoxin method with possible pitfalls and solutions for patulin-producing isolates.

This study is the first report of applying an ultra high performance liquid chromatography/tandem mass spectrometric (UHPLC-MS/MS) multi-mycotoxin method to identify and quantify the mycotoxins produced by pure fungal isolates grown on Yeast Extract Sucrose (YES) agar. The method developed concerns a triple extraction procedure based on methanol, dichloromethane and ethyl acetate. The total extract was chromatographically separated on an UHPLC BEH C18 column and analyzed with a triple quadrupole mass spectrometer. Performance characteristics (specificity, linearity, possible matrix effects, recovery, repeatability, reproducibility and limit of detection) were evaluated by spiking experiments with blank agar plugs and the analytes. Verrucarol was used as internal standard. Recovery percentages varied between 56 and 125%, whereas the limit of detection ranged from 1 to 1,500 ng g(-1) with the exception of NIV, PAT and ZEA. The method was successfully applied for examining the in vitro mycotoxin production by Aspergillus fumigatus, A. flavus and A. niger. The mobile phases used for chromatographic separation were slightly modified when studying patulin-producing molds due to signal interference between this mycotoxin and an unknown metabolite. This modified method was successfully applied for Penicillium roqueforti, P. paneum and P. carneum grown on YES agar medium. Application of the multi-mycotoxin UHPLC-MS/MS method developed may be of great importance for studying the mycotoxin capacity of fungal isolates under varying growth conditions, in order to obtain a better insight into the conditions which induce or suppress mycotoxin production by pure fungal isolates or from a chemotaxonomic point of view.

Differential inlA and inlB expression and interaction with human intestinal and liver cells by Listeria monocytogenes strains of different origins.

In this study, a number of Listeria monocytogenes strains of different origins were evaluated for in vitro invasion capacity for various human cell types (monocytic THP-1, enterocytic Caco-2, and hepatocytic HepG2 cells) and for expression levels of specific virulence genes. For THP-1 cells, no differences between clinical and nonclinical L. monocytogenes strains in invasion capacity or in production of the proinflammatory cytokine interleukin-8 (IL-8) were observed, whereas for the Caco-2 and HepG2 cells, significant differences in invasion capacity were noticed. On average, the clinical strains showed a significantly lower invasion capacity than the nonclinical L. monocytogenes strains. Furthermore, it was shown that the clinical strains induce lower IL-8 levels in HepG2 cells than do the nonclinical strains. This observation led us to study the mRNA expression levels of inlA, inlB, and ami, important virulence genes mediating adhesion and invasion of eukaryotic cells, by real-time reverse transcription-PCR for 27 clinical and 37 nonclinical L. monocytogenes strains. Significant differences in inlA and inlB expression were observed, with clinical strains showing a lower expression level than nonclinical strains. These observations were in accordance with in vitro invasion of Caco-2 and HepG2 cells, respectively. The results of this study indicate that differential expression levels of inlA and inlB possibly play a role in the virulence capacities of L. monocytogenes strains. The lower capacity of clinical strains to invade HepG2 cells and to induce IL-8 is possibly a mechanism of immune evasion used by specific L. monocytogenes strains.