Comparative Analysis of Machine Learning Methods to Predict Growth of F. sporotrichioides and Production of T-2 and HT-2 Toxins in Treatments with Ethylene-Vinyl Alcohol Films Containing Pure Components of Essential Oils.

The efficacy of ethylene-vinyl alcohol copolymer films (EVOH) incorporating the essential oil components cinnamaldehyde (CINHO), citral (CIT), isoeugenol (IEG), or linalool (LIN) to control growth rate (GR) and production of T-2 and HT-2 toxins by Fusarium sporotrichioides cultured on oat grains under different temperature (28, 20, and 15 °C) and water activity (aw) (0.99 and 0.96) regimes was assayed. GR in controls/treatments usually increased with increasing temperature, regardless of aw, but no significant differences concerning aw were found. Toxin production decreased with increasing temperature. The effectiveness of films to control fungal GR and toxin production was as follows: EVOH-CIT > EVOH-CINHO > EVOH-IEG > EVOH-LIN. With few exceptions, effective doses of EVOH-CIT, EVOH-CINHO, and EVOH-IEG films to reduce/inhibit GR by 50%, 90%, and 100% (ED50, ED90, and ED100) ranged from 515 to 3330 µg/culture in Petri dish (25 g oat grains) depending on film type, aw, and temperature. ED90 and ED100 of EVOH-LIN were >3330 µg/fungal culture. The potential of several machine learning (ML) methods to predict F. sporotrichioides GR and T-2 and HT-2 toxin production under the assayed conditions was comparatively analyzed. XGBoost and random forest attained the best performance, support vector machine and neural network ranked third or fourth depending on the output, while multiple linear regression proved to be the worst.

Potential Health Risk Associated with Mycotoxins in Oat Grains Consumed in Spain.

Spain is a relevant producer of oats (Avena sativa), but to date there has been no study on the occurrence/co-occurrence of mycotoxins in oats marketed in Spain. The present study is addressed to overcome this lack of knowledge. One hundred oat kernel samples were acquired across different Spanish geographic regions during the years 2015-2019 and analyzed for mycotoxin content using an ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method and matrix-matched calibration. The focus was on the regulated mycotoxins although other relevant mycotoxins were considered. The percentage of incidence (levels ≥ limit of detection), mean and range (ng/g) of mycotoxins were as follows: zearalenone (66%, mean 39.1, range 28.1-153), HT-2 toxin (47%, mean 37.1, range 4.98-439), deoxynivalenol, (34%, mean 81.4, range 19.1-736), fumonisin B1 (29%, mean 157.5, range 63.2-217.4), and T-2 toxin, (24%, mean 49.9, range 12.3-321). Fumonisin B2, 3-acetyldeoxynivalenol, aflatoxins B1, B2, and G2, and ochratoxin A were also detected at low levels, but aflatoxin G1 was not. The maximum limits established by the European Commission for unprocessed oats were not exceeded, except for zearalenone (in one sample), and the sum of aflatoxins (in two samples). Mycotoxin co-occurrence at quantifiable levels in the same sample (two to five combinations) was found in 31% of samples. The most common mixtures were those of HT-2 + T-2 toxins alone or together with deoxynivalenol and/or zearalenone.

Electrochemical identification of toxigenic fungal species using solid-state voltammetry strategies.

An electrochemical methodology for the characterization of mycotoxin-producing fungal species from the genera Aspergillus and Fusarium using solid-state voltammetry is described. Upon attachment of fungal colony microsamples to glassy carbon electrodes in contact with aqueous acetate buffer, characteristic voltammetric signals mainly associated to the oxidation of polyphenolic metabolites are recorded. The possibility of fungi-localized electrochemical processes was assessed by means of electron microscopy and field emission scanning electrochemical microscopy coupled to the application of oxidative potential inputs. Using pattern recognition methods, the determined voltammetric profiles were able to discriminate between mycotoxin-producing fungi from different sections and to identify selected toxigenic species of the Aspergillus and Fusarium genera isolated from grapes and cereals.

Selected plant essential oils and their main active components, a promising approach to inhibit aflatoxigenic fungi and aflatoxin production in food.

Recent research has showed that Aspergillus flavus and Aspergillus parasiticus are aflatoxigenic species that can become very competitive in the framework of climate change. Aflatoxins show carcinogenic, mutagenic, immunotoxic and teratogenic effects on human and animals. Effective and sustainable measures to inhibit these species and aflatoxins in food are required. Origanum vulgare and Cinnamomum zeylanicum essential oils (EOs) and their major active constituents, carvacrol and cinnamaldehyde, respectively, were assayed for inhibiting these species and aflatoxin production in maize extract medium under different environmental conditions. Doses of 10-1000 mg l-1 were assayed and the effective doses for 50 (ED50) and 90% (ED90) growth inhibition were determined. The ED50 of cinnamaldehyde, carvacrol, oregano EO, and cinnamon EO against A. flavus were in the ranges 49-52.6, 98-145, 152-505, 295-560 mg l-1 and against A. parasiticus in the ranges 46-55.5, 101-175, 260-425 and 490-675 mg l-1, respectively, depending on environmental conditions. In A. flavus treatments ED90 were in the ranges 89.7-90.5, 770-860 and 820->1000 mg l-1 for cinnamaldehyde, carvacrol and cinnamon EO, and in A. parasiticus treatments in the ranges 89-91, 855->1000 and 900->1000 mg l-1, respectively. ED90 values for oregano EO against both species were >1000 mg l-1. Growth rates of both species were higher at 37 than at 25°C and at 0.99 than at 0.96 aw. Aflatoxin production was higher at 25 than at 37°C. Stimulation of aflatoxin production was observed at low doses except for cinnamaldehyde treatments. The effectiveness of EOs and their main constituents to inhibit fungal growth and aflatoxin production in contact assays was lower than in vapour phase assays using bioactive EVOH-EO films previously reported.

Assessment of azole fungicides as a tool to control growth of Aspergillus flavus and aflatoxin B1 and B2 production in maize.

Aspergillus flavus is a highly aflatoxin (AF)-producing species infecting maize and other crops. It is dominant in tropical regions, but it is also considered an emerging problem associated with climate change in Europe. The aim of this study was to assess the efficacy of azole fungicides (prochloraz, tebuconazole and a 2:1 (w/w) mixture of prochloraz plus tebuconazole) to control the growth of A. flavus and AF production in yeast-extract-sucrose (YES) agar and in maize kernels under different water activities (aw) and temperatures. Aflatoxins B1 and B2 were determined by LC with fluorescence detection and post-column derivatisation of AFB1. In YES medium and maize grains inoculated with conidia of A. flavus, the growth rate (GR) of the fungus and AFB1 and AFB2 production were significantly influenced by temperature and treatment. In YES medium and maize kernels, optimal temperatures for GR and AF production were 37 and 25°C, respectively. In maize kernels, spore germination was not detected at the combination 37ºC/0.95 aw; however, under these conditions germination was found in YES medium. All fungicides were more effective at 0.99 than 0.95 aw, and at 37 than 25ºC. Fungicides effectiveness was prochloraz > prochloraz plus tebuconazole (2:1) > tebuconazole. AFs were not detected in cultures containing the highest fungicide doses, and only very low AF levels were found in cultures containing 0.1 mg l-1 prochloraz or 5.0 mg l-1 tebuconazole. Azoles proved to be highly efficient in reducing A. flavus growth and AF production, although stimulation of AF production was found under particular conditions and low-dosage treatments. Maize kernels were a more favourable substrate for AF biosynthesis than YES medium. This paper is the first comparative study on the effects of different azole formulations against A. flavus and AF production in a semi-synthetic medium and in maize grain under different environmental conditions.