Preliminary survey of the occurrence of mycotoxins in cereals and estimated exposure in a northwestern region of Mexico.

Mycotoxins have several toxicological implications. In the present study, we evaluate the presence of aflatoxin B1 (AFB1), ochratoxin A (OTA), and fumonisin (FB1) in paddy rice, polished rice, and maize from the fields and markets in Nayarit State (Mexico). The results indicated the presence of AFB1 in 21.21% of paddy rice samples and 11.11% of market maize samples. OTA was present in only 3.03% (one sample) of paddy rice samples. FB1 was detected in 87.50% and 88.88% of maize samples from field and market, respectively. The estimated human exposure was calculated for FB1 using the probable daily intake (PDI), which suggested that FB1 could contribute to the development of diseases through the consumption of contaminated maize. Positive samples indicated that some rice and maize samples were not suitable for human consumption. Further efforts are needed to continue monitoring mycotoxins and update national legislation on mycotoxins accordingly.

Exploring polyamine metabolism of Alternaria alternata to target new substances to control the fungal infection.

Polyamines are essential for all living organisms as they are involved in several vital cell functions. The biosynthetic pathway of polyamines and its regulation is well established and, in this sense, the ornithine descarboxylase (ODC) enzyme acts as one of the controlling factors of the entire pathway. In this work we assessed the inhibition of the ODC with D, l-α-difluoromethylornithine (DFMO) on Alternaria alternata and we observed that fungal growth and mycotoxin production were reduced. This inhibition was not completely restored by the addition of exogenous putrescine. Actually, increasing concentrations of putrescine on the growth media negatively affected mycotoxin production, which was corroborated by the downregulation of pksJ and altR, both genes involved in mycotoxin biosynthesis. We also studied the polyamine metabolism of A. alternata with the goal of finding new targets that compromise its growth and its mycotoxin production capacity. In this sense, we tested two different polyamine analogs, AMXT-2455 and AMXT-3016, and we observed that they partially controlled A. alternata's viability in vitro and in vivo using tomato plants. Finding strategies to design new fungicide substances is becoming a matter of interest as resistance problems are emerging.

Targeting Fusarium graminearum control via polyamine enzyme inhibitors and polyamine analogs.

Fusarium graminearum not only reduces yield and seed quality but also constitutes a risk to public or animal health owing to its ability to contaminate grains with mycotoxins. Resistance problems are emerging and control strategies based on new targets are needed. Polyamines have a key role in growth, development and differentiation. In this work, the possibility of using polyamine metabolism as a target to control F. graminearum has been assessed. It was found that putrescine induces mycotoxin production, correlating with an over expression of TRI5 and TRI6 genes. In addition, a homolog of the Saccharomyces cerevisiae TPO4 involved in putrescine excretion was up-regulated as putrescine concentration increased while DUR3 and SAM3 homologues, involved in putrescine uptake, were down-regulated. When 2.5 mM D, l-α-difluoromethylornithine (DFMO) was added to the medium, DON production decreased from 3.2 to 0.06 ng/mm(2) of colony and growth was lowered by up to 70 per cent. However, exogenous putrescine could overcome DFMO effects. Five polyamine transport inhibitors were also tested against F. graminearum. AMXT-1505 was able to completely inhibit in vitro growth and DON production. Additionally, AMXT-1505 blocked F. graminearum growth in inoculated wheat spikes reducing DON mycotoxin contamination from 76.87 µg/g to 0.62 µg/g.

Temperature-dependent production of various PlcR-controlled virulence factors in Bacillus weihenstephanensis strain KBAB4.

The Bacillus cereus sensu lato complex has recently been divided into several phylogenetic groups with clear differences in growth temperature range. However, only a few studies have investigated the actual pathogenic potential of the psychrotolerant strains of the B. cereus group at low temperature, and little information is available concerning gene expression at low temperature. We found that vegetative cells of the psychrotolerant B. weihenstephanensis strain KBAB4 were pathogenic against the model insect Galleria mellonella at 15°C but not at 30°C. A similar temperature-dependent difference also was observed for the supernatant, which was cytotoxic to Vero epithelial cell lines and to murine macrophage J774 cells at 15°C but not at 30°C. We therefore determined the effect of low temperature on the production of various proteins putatively involved in virulence using two-dimensional protein gel electrophoresis, and we showed that the production of the Hbl enterotoxin and of two proteases, NprB and NprP2, was greater at a growth temperature of 15°C than at 30°C. The quantification of the mRNA levels for these virulence genes by real-time quantitative PCR at both temperatures showed that there was also more mRNA present at 15°C than at 30°C. We also found that at 15°C, hbl mRNA levels were maximal in the mid- to late exponential growth phase. In conclusion, we found that the higher virulence of the B. cereus KBAB4 strain at low temperature was accompanied by higher levels of the production of various known PlcR-controlled virulence factors and by a higher transcriptional activity of the corresponding genes.

Modelling Aspergillus flavus growth and aflatoxins production in pistachio nuts.

Aflatoxins (AFs) are the main contaminants in pistachio nuts. AFs production in pistachio has been attributed to Aspergillus flavus. The aim of this study was to apply existing models to predict growth and AFs production by an A. flavus isolated from pistachios as a function of moisture content and storage temperature of pistachios in order to test their usefulness and complementarities. A full factorial design was used: the moisture content levels assayed were 10, 15, 20, 25 and 30% and incubation temperatures were 10, 15, 20, 25, 30, 37 and 42 °C. Both kinetic and probability models were built to predict growth of the strain under the assayed conditions. Among the assayed models, cardinal ones gave a good quality fit for radial growth rate data. Moreover, the progressive approach, which was developed based on a reduced number of experimental points led to an improved prediction in the validation step. This is quite significant as may allow for improved experimental designs, less costly than full factorial ones. Probability model proved to be concordant in 91% of the calibration set observations. Even though the validation set included conditions around the growth/no-growth interface, there was a 100% agreement in the predictions from the data set (n = 16, cut off = 0.5) after 60 days. Similarly, the probability for AF presence was rightly predicted in 89% of the cases. According to our results EC maximum aflatoxin levels would be surpassed in a period as short as 1 month if pistachio nuts reach 20 °C, unless %mc is ≤10%.

Mycotoxins occurrence and fungal populations in different types of silages for dairy cows in Spain.

Silages constitute a major component of the feed ration for dairy cows, being a potential source of mycotoxins due to the possible contamination by filamentous fungi capable of producing these toxic compounds. In this study, samples of different kinds of silages collected from farms located in four regions of Spain, were analysed to evaluate the occurrence of aflatoxins (AFs) and Fusarium mycotoxins. Lactic acid bacteria and fungal populations as well as pH and water activity were also studied. Penicillium, Geotrichum and Monascus were the main fungi identified in all the silages examined. The incidence of AFs was low (10 % of positive samples). Fusarium mycotoxins were detected in 40 % of the samples and fumonisins (FBs) were the most commonly detected. Maize silage was the most heavily contaminated type of silage. Levels of mycotoxins detected in positive samples did not exceed the EU guidance values. The lack of relationship between Fusarium counts and its mycotoxin concentrations suggested that mycotoxin production possibly occurred pre-ensiling or immediately post-ensiling. Outcomes showed that mould growth and mycotoxin contamination in silages should be regularly monitored in order to minimize the exposure of dairy cows to contaminated feed.

Effect of Capsicum carotenoids on growth and aflatoxins production by Aspergillus flavus isolated from paprika and chilli.

The aim of this study was to determine the effect of a carotenoid mixture (Capsantal FS-30-NT), containing capsanthin and capsorubin, on growth and aflatoxins (AF) production of AF-producing Aspergillus flavus isolates. Each isolate, previously isolated from paprika and chilli, was inoculated on Czapek Yeast extract Agar (CYA) medium supplemented with different amounts of capsantal (0-1%) and incubated at 10, 15 and 25 °C during 21 days. Growth rates and lag phases were obtained, and AF production was determined at 7, 14 and 21 days. None of the isolates grew at 10 °C and one isolate (UdLTA 3.193) hardly grew at 15 °C. Capsantal addition had no effect over lag phases and growth rates at 15 °C. At 25 °C capsantal reduced growth rates and increased lag phases. However, the effect of capsantal on AF production was inconclusive, because it depended on temperature or time, and most of the times it was not significant. Low temperature has been a crucial factor in AF production, regardless of the capsantal concentration tested. Industrial storage temperature for paprika and chilli use to be approximately 10 °C, so if this temperature is maintained mould growth and AF production should be prevented.

First report on mould and mycotoxin contamination of pistachios sampled in Algeria.

Mycotoxin contamination of pistachios represents a serious food safety hazard. The aim of this study was to evaluate fungal contamination and aflatoxin (AF) and ochratoxin A (OTA) occurrence in pistachio sampled in Algeria and to study the mycotoxigenic capacities of the isolates. A total of 31 pistachio samples were collected from retail outlets from different regions of Algeria. The most frequently found fungi were Penicillium spp. (38%), Aspergillus section Nigri (30%) and A. flavus (22%). A total of 56.5% of A. flavus isolates were able to produce AFB(1) and AFB(2). No A. section Nigri uniseriate isolate was OTA producer, whereas OTA production capacity was detected in 33.3% of the A. section Nigri biseriate. At least one of the potentially ochratoxigenic species was found in 64.5% of samples. Despite the high number of pistachio samples containing AFs and OTA-producing isolates, only two samples contained AFs (always below the EU maximum tolerable level) and only one sample showed OTA contamination. This is the first report on the occurrence of toxigenic moulds and mycotoxins in pistachios from Algerian market.

Review. Ochratoxin A: presence in human plasma and intake estimation.

Ochratoxin A (OTA) is a fungal toxic secondary metabolite that can be found in several foodstuffs and thereby ingested by humans. One way to assess exposure of humans to OTA is the determination of the levels of this mycotoxin in blood plasma from a certain population. Such studies have been done in many countries, both in healthy people and nephropathy patients. Relationships with individual characteristics were investigated in several cases. Thus, most studies found no correlation with age, either with gender. However, the few studies that found correlation between OTA plasma levels and gender showed that men presented the highest values. When sampling was done over more than one season, the highest OTA plasma levels were found mostly in summer. Differences within regions of a country were related to dietary habits of each area. OTA levels of group populations showed variations from year to year, whereas intraindividual repetitions showed no specific trend. Daily intake of the toxin can be estimated from OTA plasma concentrations by the Klaassen equation. OTA toxicokinetics are considered in this review. Calculated daily intake of OTA by different studies did not overpass the proposed tolerable daily intakes of OTA.

Biomonitoring of Fusarium spp. mycotoxins: perspectives for an individual exposure assessment tool.

Fusarium species are probably the most prevalent toxin-producing fungi of the northern temperate regions and are commonly found on cereals grown in the temperate regions of America, Europe and Asia. Among the toxins formed by Fusarium we find trichothecenes of the A-type or B-type, zearalenone, fumonisins or nivalenol. The current exposure assessment consists of the qualitative and/or quantitative evaluation based on the knowledge of the mycotoxin occurrence in the food and the dietary habits of the population. This process permits quantifying the mycotoxin dietary intake through deterministic or probabilistic methods. Although these methods are suitable to assess the exposure of populations to contaminants and to identify risk groups, they are not recommended to evaluate the individual exposition, due to a low accuracy and sensitivity. On the contrary, the use of biochemical indicators has been proposed as a suitable method to assess individual exposure to contaminants. In this work, several techniques to biomonitor the exposure to fumonisins, deoxynivalenol, zearalenone or T-2 toxin have been reviewed.

Usefulness of the analytical control of aflatoxins in feedstuffs for dairy cows for the prevention of aflatoxin M1 in milk.

Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1) and can be excreted in milk of cows after consuming aflatoxin (AF)-contaminated feed. The aim of this research was to assess the levels of total AFs in samples of feedingstuff for dairy cows (n = 193) and the levels of AFM1 in raw bulk tank milk samples (n = 375), in order to estimate the ratio of "AFB1 feed input" versus "AFM1 milk output" in four specific regions of Spain. Moreover, the correlation between the raw materials used as ingredients of the total mixed ration (TMR) and the presence of AFs was studied. About one-third (34.7%) of the feed samples were positive for total AFs in a range of 0.05-6.45 µg/kg, and 12.4% were positive for AFB1. AFM1 was detected in 18.9% of bulk milk samples, with concentrations ranging from 0.009 to 1.36 µg/kg. While none of the feed samples exceeded the European Union (EU) maximum content for AFB1 in feedingstuff for dairy animals of 5 µg/kg, three bulk milk samples exceeded the EU maximum level for AFM1 in milk of 50 ng/kg. The transfer ratio AFB1/AFM1, which was derived from AFB1 levels in feed, AFM1 levels in bulk tank milk, feed intake, and milk yield data, was 0.6-6%, which corresponded well with the range of published carry-over data for aflatoxins. Statistical analyses showed that the main sources of AFB1 in TMR were maize silage, bagasse, soya bean husk, maize, alfalfa hay, cotton seed and compound feed, thus special attention should be paid in controlling these raw materials when used in TMR preparation. Although the analysis of AFs in feed did not correlate with the presence of AFM1 in milk, monitoring feedstuffs is a useful tool in order to try and minimise AF-contamination of milk.

The dlt operon of Bacillus cereus is required for resistance to cationic antimicrobial peptides and for virulence in insects.

The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus, which is both an opportunistic human pathogen and an insect pathogen. The Delta dlt(Bc) mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodoptera frugiperda. Delta dlt(Bc) was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. mellonella and Spodoptera littoralis. We detected the dlt operon in three gram-negative genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in Delta dlt(Bc)). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans.

Mycelial growth and ochratoxin A production by Aspergillus section Nigri on simulated grape medium in modified atmospheres.

AIMS: To evaluate the impact of modified atmosphere packaging on in vitro growth of Aspergillus carbonarius and Aspergillus niger, and possible effects on ochratoxin A (OTA) biosynthesis. METHODS AND RESULTS: Ochratoxigenic isolates belonging to the species A. carbonarius and A. niger were grown on a synthetic grapejuice medium (SNM) and packaged in combinations of controlled O(2) (1% and 5%) and CO(2) levels (0% and 15%), and in air as a control. Colony diameters were recorded every 3 days up to 21 days, and OTA was analysed after 7, 14 and 21 days. The greatest reductions in mycelial growth rate were observed at 1% O(2) followed by 1% O(2)/15% CO(2), whereas 5% O(2) stimulated the growth of all isolates. OTA production by A. carbonarius and A. niger isolates was minimized at 1% O(2)/15% CO(2) and 1% O(2), respectively, after 7 days of incubation. Maximal OTA accumulation after 7 days was observed for all isolates in the control pack and at 5% O(2). CONCLUSIONS: Of the atmospheres tested, only 1% O(2) combined with 15% CO(2) consistently reduced fungal growth and OTA synthesis by A. carbonarius and A. niger. SIGNIFICANCE AND IMPACT OF THE STUDY: Storage under modified atmospheres is unlikely to be suitable as the sole method for OTA minimization and grape preservation; other inhibitory factors are necessary.

Predicting the growth/no-growth boundary and ochratoxin A production by Aspergillus carbonarius in pistachio nuts.

Black aspergilli are the main fungal contaminants in pistachio nuts. Ochratoxin A (OTA) production has been repeatedly reported in Aspergillus section Nigri; OTA has been occasionally detected in pistachio nuts in high concentrations. The aim of this study was to develop suitable validated models to predict the growth and OTA production boundaries by an Aspergillus carbonarius isolated from pistachios as a function of moisture content and storage temperature of pistachios. A full factorial design was used: the moisture content levels assayed were 12.5%, 17.9%, 24.0%, 29.5% and 34.8% and the incubation temperatures were 10, 15, 20, 25, 30, 37 and 42 degrees C. A probability model was built to predict the growth of the strain under the assayed conditions, which proved to be concordant in 73-91% of the cases with the observed probabilities in the validation test specifically chosen for limiting growth conditions. Similarly, the probability for the presence of OTA was correctly predicted in 90% of the cases. OTA accumulation was mainly a function of the temperature of storage, with a sharp increase at <15-20 degrees C; this value was very different from 30 to 35 degrees C, which was optimum for growth. Increasing OTA levels were found with increasing moisture content in the pistachio nuts. The impact of these results in the implementation of HACCP plans in Western Asian countries, is discussed. Probability models were applied in this work to mycotoxin accumulation for the first time, however, further research is required in the kinetics of mycotoxins accumulation to develop proper empirical models.

A review of the mycotoxin adsorbing agents, with an emphasis on their multi-binding capacity, for animal feed decontamination.

Contamination of animal feed with mycotoxins still occurs very often, despite great efforts in preventing it. Animal feeds are contaminated, at low levels, with several mycotoxins, particularly with those produced by Aspergillus and Fusarium genera (Aflatoxin B1, Ochratoxin A, Zearalenone, Deoxynivalenol and Fumonisina B1). In animal feed, to date, only Aflatoxin B1 is limited through EU regulation. Consequently, mycotoxins cause serious disorders and diseases in farm animals. In 2009, the European Union (386/2009/EC) approved the use of mycotoxin-detoxifying agents, as feed additives, to prevent mycotoxicoses in farm animals. The present review gives an overview of the problem of multi-mycotoxin contamination of feed, and aims to classify mycotoxin adsorbing agents (minerals, organic, and synthetic) for feed decontamination, focusing on adsorbents with the ability to bind to multiple mycotoxins, which should have a more effective application in farms but they are still little studied in scientific literature.

Use of a MS-electronic nose for prediction of early fungal spoilage of bakery products.

A MS-based electronic nose was used to detect fungal spoilage (measured as ergosterol concentration) in samples of bakery products. Bakery products were inoculated with different Eurotium, Aspergillus and Penicillium species, incubated in sealed vials and their headspace sampled after 2, 4 and 7 days. Once the headspace was sampled, ergosterol content was determined in each sample. Different electronic nose signals were recorded depending on incubation time. Both the e-nose signals and ergosterol levels were used to build models for prediction of ergosterol content using e-nose measurements. Accuracy on prediction of those models was between 87 and 96%, except for samples inoculated with Penicillium corylophilum where the best predictions only reached 46%.

Effects of water activity and temperature on germination and growth profiles of ochratoxigenic Penicillium verrucosum isolates on barley meal extract agar.

The aim of this work was to study the combined effects of water activity (aw, 0.75-0.99), temperature (10-30 degrees C) and isolate on spore germination and mycelial growth of ochratoxigenic Penicillium verrucosum isolates on barley meal extract agar (BMEA). Lag phases prior to germination (h), germination rates (h(-1)) and growth rates (mm day(-1)) were obtained at each set of conditions and for all the isolates. Analysis of variance showed a significant influence of the abiotic factors assayed on fungal development. Minimum aw levels found for germination and mycelial growth were 0.80 and 0.85, respectively, while maximum germination and growth rates were obtained at 0.95-0.99 aw level. Although maximum germination was obtained at 20 degrees C, significant differences were not found between growth rates at 20 and 30 degrees C. Data were modelled by MLR regressions and response surface models were obtained. Spore germination and mycelial growth of ochratoxigenic P. verrucosum isolates are significantly affected by water activity and temperature. Moreover, different isolates may have slightly different response to environmental factors. Predictive models obtained may allow a rough prediction of germination/growth as a function of the storage temperature and moisture content of barley grains.

Mapping of Aspergillus Section Nigri in Southern Europe and Israel based on geostatistical analysis.

Geostatistical analysis was applied to the incidence of Aspergillus Section Nigri and A. carbonarius in Southern Europe and Israel for the 3-year period 2001-2003 to facilitate identification of regions of high risk from contamination with these fungi and production of ochratoxin. The highest incidence of black aspergilli was normally observed at harvesting. At this grape growth stage, spatial variability of black aspergilli was significantly related to latitude and longitude, showing a positive West-East and North-South gradient. Predictive maps of infected berries incidence were drawn and showed the same trend in the 3 years, but incidence was highest in 2003, followed by 2001 and 2002. The highest incidence was always observed in Israel, Greece and Southern France, associated with the highest incidence of A. carbonarius. Southern Spain and Southern Italy also had relevant incidence of black aspergilli. The thermo-wetness maps for the 3 years showed a trend similar to the incidence of black aspergilli. The coldest and wettest year was 2002, while 2003 was the hottest and driest, particularly during August, with Israel being the hottest and driest country, followed by Greece and Southern Italy. This indicates that meteorological conditions can contribute to explain spatial distribution variation of black aspergilli within the Mediterranean basin.

Effect of 1-methylcyclopropene on the development of black mold disease and its potential effect on alternariol and alternariol monomethyl ether biosynthesis on tomatoes infected with Alternaria alternata.

Ethylene is a naturally produced plant regulator involved in several plant functions, such as regulation of fruit ripening. Inhibition of ethylene perception by using 1-methylcyclopropene (1-MCP) slows down the ripening of the fruit maintaining its quality and freshness. The use of 1-MCP is a commercial strategy commonly used in the food industry to extend the postharvest life of several fruits, including tomatoes. To assess how 1-MCP affected infection by Alternaria alternata on tomatoes, three different cultivars were artificially inoculated with 5µL of an A. alternata conidial suspension (10(5)conidia/mL). Tomatoes were treated with 0.6µL/L of 1-MCP for 24h. Spiked but untreated tomatoes were considered controls. Then, fruit were stored 6days at 10°C and one more week at 20°C to simulate shelf-life. Fungal growth development and mycotoxin production (alternariol, AOH and alternariol monomethyl ether, AME) were assessed both on the first and on the second week. After the first 6days at 10°C, in just one variety the black mold disease was higher in the 1-MCP treated samples. However, after two weeks of storage, in all cases, tomatoes treated with 1-MCP showed more significant fungal growth disease. Regarding mycotoxin production, no large differences were observed among different treatments, which was corroborated with gene expression analysis of pksJ, a gene related to AOH and AME biosynthesis.

The effect of enhanced carotenoid content of transgenic maize grain on fungal colonization and mycotoxin content.

Novel strategies that address vitamin A deficiency have been developed, such as high-carotenoid maize, a biofortified transgenic maize line rich in carotenoids generated by genetic transformation. The South African white maize inbred (M37W), which is devoid of carotenoids, was engineered to accumulate high levels of ß-carotene (provitamin A), lutein, and zeaxanthin. Maize seeds contaminated with fumonisins and other mycotoxins pose a serious threat to both humans and livestock. During three consecutive harvests, the fungal incidence and the fumonisin and aflatoxin content of maize seeds grown in an experimental field in Catalonia (Northeastern Spain) were evaluated. Fungal infection was similar in high-carotenoid maize and its isogenic line, with Fusarium verticillioides being the most prevalent fungus in all the harvests. Neither Aspergillus spp. nor aflatoxin contamination was found. Fumonisin levels were lower in high carotenoid than in its isogenic line, but this reduction was statistically significant in only 2 of the 3 years of study. Our results suggest that high carotenoid content reduces fumonisin levels in maize grains.