Claudia Falavigna

In Vitro Digestion Assay for Determination of Hidden Fumonisins in Maize

Hidden fumonisins have received great attention in the last years as they have been frequently found in maize products in addition to the free forms. Several papers have shown that interaction with macromolecular components such as protein and starch is at the base of the phenomenon: although the nature of the interaction (covalent or not) is still not clarified, the occurrence of hidden forms is generally revealed by the application of an alkaline hydrolysis procedure. In this study, an in vitro digestion model has been applied to raw maize to evaluate the possible release of hidden fumonisins under gastrointestinal conditions. Upon digestion of the food matrix, an increased amount of total detectable fumonisins was observed in comparison with the analysis on the nondigested matrix, an amount even higher than that calculated through the application of the hydrolysis procedure. Besides the analytical issues, our data have serious implications, since consumers may be exposed to a systematic higher risk than that estimated by conventional techniques.

Role of maize hybrids and their chemical composition in Fusarium infection and fumonisin production

This study was designed to investigate the role of hybrids in maize Fusarium section Liseola interaction and fumonisin production, with particular emphasis on the occurrence and accumulation of hidden fumonisins in maize (masking phenomenon). In this 2 year study, naturally infected field crops were chosen with 10 maize hybrids, six of them grown in both years. Maize samples collected in 2010 showed a higher incidence of fungal infection as well as higher fumonisin contamination than those obtained in 2009 but a very similar incidence of F. section Liseola. Fumonisin masking was confirmed in raw maize, with a lower amount of hidden forms as compared to free fumonisins detected in the year with higher contamination. The chemical composition of the different hybrids was determined and correlated with the contamination data: the results obtained highlight the main role of fatty acids, with a higher fumonisin contamination in hybrids showing a higher linoleic acid content and a higher masking action in hybrids with higher oleic to linoleic ratio. These results represent a good basis to explain maize hybrid susceptibility to F. section Liseola infection, fumonisin contamination, and masking not related to a specific commercial hybrid but extendable to all hybrids.

Masked fumonisins in processed food: co-occurrence of hidden and bound forms and their stability under digestive conditions

Fumonisins can occur in foodstuffs as free forms (prent, partially hydrolysed, or totally hydrolysed), as covalently bound fumonisins (bound fumonisins) and as non-covalently bound fumonisins (hidden fumonisins). The formation of several covalently bound fumonisin B1 conjugates upon reaction with sugars and/or amino acids has been reported in the literature so far, but occurrence data are still very poor. Since very little is known about the relevance of both hidden and bound fumonisins in processed products, the present study aimed to evaluate the occurrence of both masked forms in maize-based food products and to study their stability under digestive conditions. The behaviour of covalently bound fumonisin B1 conjugates upon in vitro digestion was evaluated, demonstrating their stability under these conditions. In addition, the co-occurrence of hidden and bound fumonisins in maize-based food products was investigated by application of both alkaline hydrolysis and simulated in vitro digestion assay. The e...

Fatty acid esters of fumonisins: first evidence of their presence in maize

Fumonisin derivatives obtained by esterification of fumonisin B1 (FB1) with palmitic, oleic and linoleic fatty acids have been recently described, but never reported in raw maize so far. In this study, the presence of oleoyl-EFB1 (EFB1OA) and linoleoyl-EFB1 (EFB1LA) in raw maize is reported by means of a suitable LC-ESI-MS/MS method. In addition, the production of EFB1 derivatives by three Fusarium verticillioides strains is described on malt extract–based media and on corn meal–based growth media. EFB1OA and EFB1LA were produced by all considered strains in corn meal medium, with EFB1LA > EFB1OA. On the contrary, EFB1OA and EFB1LA were never observed in Fusarium cultures grown on a malt extract medium, suggesting that the esterification of FB1 can occur only in a complex matrix such as maize.

Fumonisins B, A and C profile and masking in Fusarium verticillioides strains on fumonisin-inducing and maize-based media.

The production of fumonisin B, A and C and hidden and partially hydrolysed fumonisin occurrence was investigated in 3 strains of Fusarium verticillioides isolated from maize, cultured for 21-45days on malt extract medium at 25°C and 0.955-0.990 water activity (a(w)). Fumonisin A-B and C series were produced by all the strains in all conditions studied, with B-fumonisin≫C-fumonisin>A-fumonisin following a similar trend. The dynamic of fumonisin production was significantly influenced by factors considered and their interaction, with a(w)=0.990 as favourable condition in ITEM 10026 and ITEM 10027. All fumonisins were maximised at 30days incubation in ITEM 10027 and ITEM 1744 and at 45days incubation in ITEM 10026. Partially hydrolysed fumonisins were detected only for the B-group. Hidden fumonisins were never observed in Fusarium cultures grown on malt extract medium but were detected in the additional trial on maize-based medium, suggesting that the masking phenomenon can occur only in a complex matrix.

LC/ESI-MS/MS analysis outlines the different fumonisin patterns produced by F. verticillioides in culture media and in maize kernels.

Fumonisins are a family of food-borne mycotoxins with a wide spectrum of toxicological activities, produced by Fusarium verticillioides. Twenty-eight fumonisin analogues have been characterised so far, which can be separated into four main groups, identified as fumonisin A, B, C and P, being fumonisin B the most widely occurring in maize and corn-based food. In this work, major and minor fumonisin analogues produced by F. verticillioides have been determined by the development of a suitable tandem mass spectrometry procedure for target compound identification and quantification. The method has been applied to the determination of the major fumonisins in culture media of F. verticillioides and in mouldy maize. In addition to the main fumonisins produced by F. verticillioides, also secondary compounds such as FB4, FB5, FAs and FCs have been detected in both fungal liquid cultures and contaminated maize samples. The use of this method for quantification of major and minor fumonisins may be useful for an exhaustive evaluation of their occurrence and toxicological relevance in food; moreover, it may be applied for a better definition of the fumonisin biosynthetic pathways in different growing media as well as in maize.

Cornmeal and starch influence the dynamic of fumonisin B, A and C production and masking in Fusarium verticillioides and F. proliferatum.

Fusarium verticillioides and F. proliferatum can infect maize ears and produce fumonisins. The fumonisin B (FB) series is the most prolifically produced, followed by fumonisin C (FC), A (FA) and P (FP); moreover hidden forms of fumonisins have been detected in maize and derivatives. There is a lack of information about which maize component may affect fumonisin pattern production. Therefore, in this work we studied the role of cornmeal and corn starch, as the sole source of nutrition, in the production dynamic of all fumonisin series, hidden forms included, in different strains of F. verticillioides and F. proliferatum incubated at 25°C for 7-45days. Both Fusarium species produced high amounts of FB, following the chemotype FB1>FB2>FB3; FC and FA were produced in lesser amounts, showing the chemotypes: FA2+FA3>FA1 and FC1>FC2+FC3>FC4, respectively; while no FP were detected. F. verticillioides was more prolific than F. proliferatum in fumonisin production (ten times more on average) in all the tested conditions. Fumonisin production was higher in cornmeal than in starch based medium in both Fusarium species; FA and FC were detected only in the former medium. An important role of amylopectin as an inducing factor for fumonisin biosynthesis was suggested, as were acid pH conditions. Fumonisin hidden forms may occur in cornmeal medium, whereas they were never found at significant levels in corn starch medium.

Hydrolysed fumonisin B1 and N-(deoxy-D-fructos-1-yl)-fumonisin B1: stability and catabolic fate under simulated human gastrointestinal conditions

Abstract Food processing may induce thermal degradation of fumonisins in corn via Maillard-type reactions, or alkaline hydrolysis via loss of the two tricarballylic acid moieties. In the former case, N-(1-deoxy-D-fructos-1-yl)-fumonisin B1 (NDF) can be formed, while the latter derivative is called hydrolysed fumonisin B1 (HFB1). The aim of this study was to deepen the knowledge about the gastrointestinal stability of HFB1 and NDF in humans. Due to the lack of standard, NDF was chemically synthesised and cleaned up in high purity to be used for further experiments. While NDF is already partially cleaved (about 41%) during simulated digestion, it remained rather stable towards human colon microflora. In contrast to this, HFB1 is partially metabolised by the colon microflora to unknown compounds after 24 h of fermentation, as seen by a loss of about 22%. Concluding, the cleavage of NDF during digestion as well as the likely metabolisation of HFB1 emphasise the need for animal trials to ascertain their toxicity in vivo.

Oleoyl and linoleoyl esters of fumonisin B1 are differently produced by Fusarium verticillioides on maize and rice based media

Fatty acid esters of fumonisins, namely oleoyl- and linoleoyl esters of fumonisin B1 (EFB1OA and EFB1LA, respectively), are modified forms of fumonisins whose formation and occurrence have been reported so far in naturally infected maize and in artificially inoculated rice. There is a lack of knowledge about the mechanism of formation, mainly in relation to the role played by the substrate. Therefore, in this work we studied the dynamics of accumulation of the toxin and its esters, together with their precursor, in maize and rice based media inoculated with different strains of F. verticillioides and incubated at 25 °C for 7-45 days. The production pattern of FB1 and its modified forms was significantly influenced by growth media, reaching a higher concentration in cornmeal compared to rice based medium. Similarly, cornmeal was more supportive for the conversion of FB1 by considering the esterification rate, with a prevalence of linoleoyl esters compared to oleoyl esters resembling the OA/LA rate in both media. The conversion of FB1 into fatty acid esters was also shown as strain-related. Results, thus, strongly support the hypothesis that fatty acid esters of FB1 are produced by the fungus itself at a late stage of growth, or at a certain point of FB1 accumulation in the medium, using fatty acids from the substrate.

Chapter Three - Mycotoxins from Alternaria: Toxicological Implications

Abstract Alternaria toxins are mycotoxins produced by Alternaria species. These fungi cause serious diseases in many crops such as cereals, oil seeds, and fruits. More than 70 Alternaria toxins have been reported and only some of them have been physicochemically characterized. Among the most common in food commodities, alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT), and tenuazonic acid were mainly studied. Available in vitro data indicate that AOH, AME, and ALT are hydroxylated, mostly to catechol metabolites, and form glucuronide and sulfate conjugates. AOH and AME are genotoxic in bacteria and mammalian cells in vitro . Although there are no in vivo genotoxicity or carcinogenicity data available for Alternaria toxins, some indications of precancerous changes have been reported in esophageal mucosa of mice. In this review, the state of the art about the toxic effects of the major Alternaria toxins will be reported, and new data about their stability under in vitro human simulated gastrointestinal conditions will be described.