Lactic Acid Bacteria as Potential Agents for Biocontrol of Aflatoxigenic and Ochratoxigenic Fungi.

Aflatoxins (AF) and ochratoxin A (OTA) are fungal metabolites that have carcinogenic, teratogenic, embryotoxic, genotoxic, neurotoxic, and immunosuppressive effects in humans and animals. The increased consumption of plant-based foods and environmental conditions associated with climate change have intensified the risk of mycotoxin intoxication. This study aimed to investigate the abilities of eleven selected LAB strains to reduce/inhibit the growth of Aspergillus flavus, Aspergillus parasiticus, Aspergillus carbonarius, Aspergillus niger, Aspergillus welwitschiae, Aspergillus steynii, Aspergillus westerdijkiae, and Penicillium verrucosum and AF and OTA production under different temperature regiments. Data were treated by ANOVA, and machine learning (ML) models able to predict the growth inhibition percentage were built, and their performance was compared. All factors LAB strain, fungal species, and temperature significantly affected fungal growth and mycotoxin production. The fungal growth inhibition range was 0-100%. Overall, the most sensitive fungi to LAB treatments were P. verrucosum and A. steynii, while the least sensitive were A. niger and A. welwitschiae. The LAB strains with the highest antifungal activity were Pediococcus pentosaceus (strains S11sMM and M9MM5b). The reduction range for AF was 19.0% (aflatoxin B1)-60.8% (aflatoxin B2) and for OTA, 7.3-100%, depending on the bacterial and fungal strains and temperatures. The LAB strains with the highest anti-AF activity were the three strains of P. pentosaceus and Leuconostoc mesenteroides ssp. dextranicum (T2MM3), and those with the highest anti-OTA activity were Leuconostoc paracasei ssp. paracasei (3T3R1) and L. mesenteroides ssp. dextranicum (T2MM3). The best ML methods in predicting fungal growth inhibition were multilayer perceptron neural networks, followed by random forest. Due to anti-fungal and anti-mycotoxin capacity, the LABs strains used in this study could be good candidates as biocontrol agents against aflatoxigenic and ochratoxigenic fungi and AFL and OTA accumulation.

Machine learning approach for predicting Fusarium culmorum and F. proliferatum growth and mycotoxin production in treatments with ethylene-vinyl alcohol copolymer films containing pure components of essential oils.

Fusarium culmorum and F. proliferatum can grow and produce, respectively, zearalenone (ZEA) and fumonisins (FUM) in different points of the food chain. Application of antifungal chemicals to control these fungi and mycotoxins increases the risk of toxic residues in foods and feeds, and induces fungal resistances. In this study, a new and multidisciplinary approach based on the use of bioactive ethylene-vinyl alcohol copolymer (EVOH) films containing pure components of essential oils (EOCs) and machine learning (ML) methods is evaluated. Bioactive EVOH-EOC films were made incorporating cinnamaldehyde (CINHO), citral (CIT), isoeugenol (IEG) or linalool (LIN). Several ML methods (neural networks, random forests and extreme gradient boosted trees) and multiple linear regression (MLR) were applied and compared for modeling fungal growth and toxin production under different water activity (aw) (0.96 and 0.99) and temperature (20 and 28 °C) regimes. The effective doses to reduce fungal growth rate (GR) by 50, 90 and 100% (ED50, ED90, and ED100) of EOCs in EVOH films were in the ranges 200 to >3330, 450 to >3330, and 660 to >3330 µg/fungal culture (25 g of partly milled maize kernels in Petri dish), respectively, depending on the EOC, aw and temperature. The type of EVOH-EOC film and EOC doses significantly affected GR in both species and ZEA and FUM production. Temperature also affected GR and aw only affected GR and FUM production of F. proliferatum. EVOH-CIT was the most effective film against both species and ZEA and FUM production. Usually, when the EOC levels increased, GR and mycotoxin levels in the medium decreased although some treatments in combination with certain aw and temperature values induced ZEA production. Random forest models predicted the GR of F. culmorum and F. proliferatum and ZEA and FUM production better than neural networks or extreme gradient boosted trees. The MLR mode provided the worst performance. This is the first approach on the ML potential in the study of the impact that bioactive EVOH films containing EOCs and environmental conditions have on F. culmorum and F. proliferatum growth and on ZEA and FUM production. The results suggest that these innovative packaging systems in combination with ML methods can be promising tools in the prediction and control of the risks associated with these toxigenic fungi and mycotoxins in food.

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.

Risk management of ochratoxigenic fungi and ochratoxin A in maize grains by bioactive EVOH films containing individual components of some essential oils.

Aspergillus steynii and Aspergillus tubingensis are possibly the main ochratoxin A (OTA) producing species in Aspergillus section Circumdati and section Nigri, respectively. OTA is a potent nephrotoxic, teratogenic, embryotoxic, genotoxic, neurotoxic, carcinogenic and immunosuppressive compound being cereals the first source of OTA in the diet. In this study bioactive ethylene-vinyl alcohol copolymer (EVOH) films containing cinnamaldehyde (CINHO), linalool (LIN), isoeugenol (IEG) or citral (CIT) which are major components of some plant essential oils (EOs) were produced and tested against A. steynii and A. tubingensis growth and OTA production in partly milled maize grains. Due to the favourable safety profile, these bioactive compounds are considered in the category "GRAS". The study was carried out under different water activity (0.96 and 0.99 aw), and temperature (24 and 32 °C) conditions. ANOVA showed that class of film, fungal species, aw and temperature and their interactions significantly affected growth rates (GR), ED50 and ED90 and the doses for total fungal growth inhibition and OTA production. The most effective EVOH films against both species were those containing CINHO. ED50, ED90 and doses for total growth and OTA inhibition were 165-405, 297-614, 333-666 µg of EVOH-CINHO/plate (25 g of maize grains), respectively, depending on environmental conditions. The least efficient were EVOH-LIN films. ED50, ED90 and doses for total growth and OTA inhibition were 2800->3330, >3330 and >3330 µg of EVOH-LIN/plate (25 g of maize grains), respectively. The effectiveness of the bioactive films increased with increasing doses. Overall, A. tubingensis was less sensitive to treatments than A. steynii. Depending on the species, aw and temperature affected GR and OTA production in a different way. In A. steynii cultures, optimal growth occurred at 0.96 aw and 32 °C while optimal OTA production happened at 0.99 aw and 32 °C. In A. tubingensis cultures optimal growth happened at 0.99 aw and 32 °C, although the best conditions for OTA production were 0.99 aw and 24 °C. Thus, these species can be very competitive in warm climates and storage conditions. The EVOH-CINHO films followed by EVOH-IEG and EVOH-CIT films, designed in this study and applied in vapour phase, can be potent antifungal agents against A. steynii and A. tubingensis and strong inhibitors of OTA biosynthesis in maize grains at very low doses. This is the first study on the impact that interacting environmental conditions and bioactive films containing individual components of EOs have on the growth of these ochratoxigenic fungi and on OTA production in maize grains.

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.

Impact of bioactive packaging systems based on EVOH films and essential oils in the control of aflatoxigenic fungi and aflatoxin production in maize.

Aspergillus flavus and A. parasiticus are the most common fungal species associated with aflatoxin (AF) contamination of cereals, especially maize, and other agricultural commodities. AFB1, the most frequent and toxic metabolite, is a powerful hepatotoxic, teratogenic and mutagenic compound. Effective strategies to control these fungal species and AFs in food and feed are required. Active packaging film containing essential oils (EO) is one of the most innovative food packaging concepts. In this study, ethylene-vinyl alcohol (EVOH) copolymer films incorporating EO from Origanum vulgare (ORE), Cinnamomum zeylanicum (CIN) or their major active constituents, carvacrol (CAR) and cinnamaldehyde (CINHO), respectively, were developed and assayed to control growth of A. flavus and A. parasiticus and AF production in maize grains under different aw and temperature regimens. EO doses assayed in cultures were in the range 0.25-4.0mg/Petri dish. The factors aw, temperature, type of EVOH-EO film and fungal species significantly influenced the ED50 values of all assayed films. Growth rate (GR) of both species was usually higher at 0.99 than at 0.96 aw and at 37°C than at 25°C. However, the contrary was found with regard to AF production. The order of efficacy of EVOH-EO films to control growth of both species and AF production was EVOH-CINHO>EVOH-CAR>EVOH-ORE>EVOH-CIN. The effective dose (ED50) (mg EO/plate) for EVOH-CINHO and EVOH-CIN films against A. flavus were in the ranges of 0.125 and 2.475-3.500 and against A. parasiticus in the ranges of 0.121-0.133 and 2.275-3.625, respectively. Under the assayed conditions, the ED90 for EVOH-CINHO film were 0.22-0.23mg/plate for both species. It was the most effective bioactive film to control fungal growth (vapour phase) and AF production, regardless of aw and temperature. This is the first study about the impact that interacting environmental conditions and bioactive EVOH-CINHO, EVOH-ORE, EVOH-CIN EVOH-CAR films have on the growth of aflatoxigenic fungi and on AF production in maize grains.

Neurotoxic effects of ochratoxin A on the subventricular zone of adult mouse brain.

Ochratoxin A (OTA), a mycotoxin that was discovered as a secondary metabolite of the fungal species Aspergillus and Penicillium, is a common contaminant in food and animal feed. This mycotoxin has been described as teratogenic, carcinogenic, genotoxic, immunotoxic and has been proven a potent neurotoxin. Other authors have previously reported the effects of OTA in different structures of the central nervous system as well as in some neurogenic regions. However, the impact of OTA exposure in the subventricular zone (SVZ) has not been assessed yet. To elucidate whether OTA affects neural precursors of the mouse SVZ we investigated, in vitro and in vivo, the effects of OTA exposure on the SVZ and on the neural precursors obtained from this neurogenic niche. In this work, we prove the cumulative effect of OTA exposure on proliferation, differentiation and depletion of neural stem cells cultured from the SVZ. In addition, we corroborated these results in vivo by immunohistochemistry and electron microscopy. As a result, we found a significant alteration in the proliferation process, which was evidenced by a decrease in the number of 5-bromo-2-deoxyuridine-positive cells and glial cells, as well as, a significant decrease in the number of neuroblasts in the SVZ. To summarize, in this study we demonstrate how OTA could be a threat to the developing and the adult SVZ through its impact in cell viability, proliferation and differentiation in a dose-dependent manner.

Effect of carbendazim and physicochemical factors on the growth and ochratoxin A production of Aspergillus carbonarius isolated from grapes.

Carbendazim is a systemic fungicide that is commonly used on several crops (tobacco, fruit, vegetables, cereals, etc.). This fungicide is used to control fungal infections in vineyards. It is indicated against Botrytis cinerea, Uncinula necator, Plasmopara viticola and other fungi and can be used either alone or coupled with other fungicides. However, there is a lack of in-depth studies to evaluate its effectiveness against growth of Aspergillus carbonarius isolated from grapes and OTA production. A medium based on red grape juice was used in this study. Preliminary studies were performed at 0.98 a(w) and 25 degrees C using carbendazim concentrations over a wide range (1-2000 ng/ml medium) to control both growth of a strain of A. carbonarius isolated from grape and its ability to produce OTA. As the lag phase increased considerably at levels > 1000 ng/ml of medium, detailed studies were carried out in the range 50-450 ng/ml of medium at 0.98-0.94 a(w) and 20-28 degrees C. Statistical analysis (multifactor ANOVA) of the data revealed that the three factors assayed and the interactions a(w)-carbendazim concentration and a(w)-temperature had significant effects on lag phase duration. The highest lag-times were observed at 0.94 a(w,) 20 degrees C, and with 450 ng carbendazim/ml. The three factors also had significant effects of the growth rate and there was an interaction between a(w) and temperature. The growth rate of A. carbonarius in these cultures is favoured by high water availability and relatively high temperatures. However, addition of carbendazim at the assayed levels did not significantly influenced fungal growth rate. Accumulation of OTA was studied as a function of four factors (the three previously considered, and time). All factors had significant effects on the accumulation of OTA. There were also two significant interactions (a(w)-temperature and temperature-time). On the basis of the results obtained, carbendazim does not increase the lag phase of A. carbonarius except at relatively low a(w) and temperatures. It does not substantially decrease fungal growth rate once growth is apparent but it appears to cause an increase in OTA accumulation in the medium at the doses assayed. Carbendazim, which is widely used against fungal infections in grape, can positively influence OTA production by A. carbonarius in field, which can increase OTA content in grape juices and wines.

An overview of ochratoxin A in beer and wine.

Ochratoxin A (OTA) is a mycotoxin produced mainly by several fungal species of the genera Aspergillus and Penicillium. This mycotoxin has been shown to be nephrotoxic, hepatotoxic, teratogenic and carcinogenic to animals and has been classified as a possible carcinogen to humans. OTA occurs in a variety of foods, including beer and wine. Reports on OTA occurrence in beer indicate that this is a worldwide problem due to the widespread consumption of this beverage. At present, the European Union (EU) has not set a maximum allowable limit (MAL) for this mycotoxin in beer, although there is a limit in barley and malt. Studies carried out in different countries agree in the high proportion of samples contaminated with OTA although levels are, usually, below 0.2 ng/ml. OTA occurrence has been related to the contamination of malt barley with ochratoxigenic species, particularly Penicillium verrucosum. OTA produced in grains is carried to wort and, although fermentation decreases the concentration, the toxin is not eliminated. Reducing the fungal contamination of malt barley is the most promising strategy for reducing OTA in beer. With regard to wine, surveys on the presence of OTA have been conducted worldwide. The proportion of wines in which OTA is detected is very high (above 50%) in some countries (especially in the Mediterranean basin) although only a few wines contained concentrations exceeding the MAL laid down by the EU (2.0 ng/ml). A gradient of concentration is usually recognized; OTA levels decrease in the order red, rose, and white wine but also with increasing latitude of the producing countries. OTA presence in wines is due to the black aspergilli, mainly A. carbonarius, which can grow on grapes in the vineyards and produce the toxin. At grape crushing, the juice can be contaminated with the toxin which is carried over into wine, where it persists due to its stability. Pre- and post-harvest treatments are being investigated to diminish contamination of wines as much as possible.

Comparison of different sample treatments for the analysis of ochratoxin A in must, wine and beer by liquid chromatography.

Ochratoxin A (OTA) is a mycotoxin produced by some species of Aspergillus and Penicillium verrucosum. It has been found in foods and feed all over the world. There is a great concern about OTA because it is nephrotoxic and probably, carcinogenic to humans. Most of analytical methods developed for OTA in wine, beer and other products are based on LC with fluorescence detection (LC-FLD). In the present work, various procedures for extraction and/or clean-up for determination of OTA in musts, wine and beer by LC-FLD were compared: (1) dilution with polyethylen glycol 8000 and NaHCO3 solution and clean-up an on immunoaffinity column (IAC); (2) extraction with chloroform and IAC clean-up; solid-phase extraction (SPE) on (3) reversed-phase (RP) C18; (4) RP phenylsilane and (5) Oasis HLB cartridges. SPE on phenylsilane and Oasis HLB have not been reported for OTA analysis in beverages. The same LC-FLD conditions and concentration ratio were used. The former procedure was simple, rapid and provided flat baselines, free from most impurity peaks, high OTA recoveries and quite repeatable results. RP C18 using methanol-acetic acid (99.5:0.5) as elution solvent provided good recoveries and precision, thus becoming a cheaper but interesting alternative at 0.1-1 ng/ml spiking levels. Oasis HLB cartridges were usually better than phenylsilane. Possible binding of OTA to proteins or other components was tested by acid treatment before extraction but no significant differences with controls appeared.