Effect of adsorbents on diets with corn contaminated by mycotoxins on the productive performance and health of broilers

A total of 1,296 broiler chicken were housed, distributed in a completely randomized design with 6 treatments and 6 repetitions. The treatments consisted of a control diet formulated with corn considered adequate and without the addition of adsorbent, a diet formulated with corn naturally contaminated with mycotoxin (CCM) and four diets formulated with CCM and added with different commercial adsorbents. At the end of the first week and at 21 days of age of the birds, it was observed that the control diet resulted in greater (p < 0.05) live weight and weight gain in relation to the inclusion of CCM. The relative weight of the liver was lower for the control group compared to the groups receiving a diet with CCM and CCM + ads D. The relative weight of the proventriculus and cloacal bursa was lower for the control group compared to those who received a diet with CCM + ads B and CCM+ ads C. There was no significant effect (p > 0.05) of the diets on the analysis of serum biochemistry and the occurrence of fatty and hydropic degeneration in the liver of broilers. The use of adsorbents can mitigate the harmful effects of mycotoxins, however, these products have specific binding capacity to the type of mycotoxin present in food.(AU)

Mitigation of nivalenol using alcoholic fermentation and magnetic field application.

This study aimed at evaluating mitigation of nivalenol (NIV) in alcoholic fermentation with magnetic field application (MF). Mitigation was related to both the glutathione (GSH) redox molecule and the enzyme peroxidase (PO), which were synthesized by Saccharomyces cerevisiae US-05. Conditions under evaluation were NIV (0.2 µg mL-1), MF application (35 mT) and simultaneous use of mycotoxin and MF. The GSH content and the PO activity were increased when the culture contained NIV and the alcohol profile was altered after 48 h of fermentation. At the end of the alcoholic fermentation, NIV was mitigated by 56.5%. Therefore, this process is a promising method to reduce contamination by NIV, although the mycotoxin affects the chemical characteristics of the final product.

Mycotoxins in cereals and cereal-based products: Incidence and probabilistic dietary risk assessment for the Brazilian population.

A probabilistic dietary risk assessment on mycotoxins was conducted using the Monte Carlo Risk Assessment software, with consumption data from the 2008/2009 Brazilian Household Budget Survey for individuals who were at least 10 years old and occurrence data for 646 samples of rice, maize, wheat, and their products, collected in the Federal District and in the state of Rio Grande do Sul, Brazil. Processing factors were estimated and applied to concentration data. Chronic exposure was estimated for fumonisins (free and bound/hidden), deoxynivalenol (DON) (including the acetylated forms) and zearalenone (ZON) (including alfa-zearalenol) and acute exposure was estimated for DON. For the general population, the chronic exposure exceeded the safe exposure levels at the 95P for DON and at the 99P for fumonisins. Additionally, safe level exceedance occurred at the 97.5P for fumonisins and at the 95P for DON for teenagers, as well as at the 99P for fumonisins for women of child-bearing-age. No exceedances were found for chronic exposure to ZON and acute exposure to DON. Maize couscous contributed most of the total fumonisins (91%) and ZON intakes (~40%) and bread to total intake of DON (~30%). Further studies should be conducted with updated Brazilian consumption data, which should include information for individuals aged less than 10 years old.

Mycotoxins from Fusarium proliferatum: new inhibitors of papain-like cysteine proteases.

Papain-like cysteine proteases (PLCPs) in plants are essential to prevent phytopathogen invasion. In order to search for cysteine protease inhibitors and to investigate compounds that could be associated to pineapple Fusarium disease, a chemistry investigation was performed on Fusarium proliferatum isolated from Ananas comosus (pineapple) and cultivated in Czapek medium. From F. proliferatum extracts, nine secondary metabolites were isolated and characterized by nuclear magnetic resonance spectroscopy and mass spectrometry experiments: beauvericin (1), fusaric acid (2), N-ethyl-3-phenylacetamide (3), N-acetyltryptamine (4), cyclo(L-Val-L-Pro) cyclodipeptide (5), cyclo(L-Leu-L-Pro) cyclodipeptide (6), cyclo(L-Leu-L-Pro) diketopiperazine (7), 2,4-dihydroxypyrimidine (8), and 1H-indole-3-carbaldehyde (9). Compounds 1, 3, and 6 showed significant inhibition of papain, with IC50 values of 25.3 ± 1.9, 39.4 ± 2.5, and 7.4 ± 0.5 µM, respectively. Compound 1 also showed significant inhibition against human cathepsins V and B with IC50 of 46.0 ± 3.0 and 6.8 ± 0.7 µM, respectively. The inhibition of papain by mycotoxins (fusaric acid and beauvericin) may indicate a mechanism of Fusarium in the roles of infection process.

Effects of Fusarium graminearum and Fusarium poae on disease parameters, grain quality and mycotoxin contamination in barley (part II).

BACKGROUND: Barley is one of the most sown crops in the world, with multiple uses such as human consumption, animal feed and for the malting industry. This crop is affected by different diseases, such as Fusarium Head Blight (FHB), that causes losses in yield and quality. In the last years F. graminearum and F. poae were two of the most frequently isolated species in barley grains, so the aim of this study was to evaluate the interaction between these Fusarium species and the effects on disease parameters, grain quality and mycotoxin contamination on five barley genotypes under field conditions. RESULTS: Statistical differences between Fusarium treatments for some parameters depending mainly on the year/genotype were found. The results showed that the germination process was affected by both Fusarium species. As to grain quality and the different hordein fractions, it was observed that F. graminearum affects preferentially D and C-hordeins. Different concentrations of nivalenol, deoxynivalenol and their acetylated derivatives (3-acetyl deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol (15-ADON)) were detected. CONCLUSIONS: In the present work, no evidence of synergism between F. graminearum and F. poae were found regarding disease parameters and mycotoxin contamination. However, at least in the years with favorable climatic conditions to FHB development and depending on the barley genotype, a continuous monitoring is deemed necessary to prevent the negative impact on protein composition and germinative parameters © 2020 Society of Chemical Industry.

Comparative Sequence Analysis of TRI1 of Fusarium.

Trichothecene mycotoxins are a class of secondary metabolites produced by multiple genera of fungi, including certain plant pathogenic Fusarium species. Functional variation in the TRI1 gene produces a novel Type A trichothecene called NX-2 in strains of F. graminearum. Using a bioinformatics approach, a systematic analysis of 52 translated TRI1 sequences of Fusarium species, including five F. graminearum NX-2 producers and four F. graminearum non-NX-2 producers, was conducted to explain the functional difference of TRI1p of FGNX-2. An assessment of several signature motifs of fungal P450s revealed amino acid substitutions in addition to the post-translational N-X-S/T sequons motif, which is indicative of N-linked glycosylation of this TRI1-encoded protein characteristic of NX-2 producers. There was evidence of selection bias, where TRI1 gene sequences were found to be under positive selection and, therefore, under functional constraints. The cumulative amino acid changes in the TRI1p sequences were reflected in the phylogenetic analyses which revealed species-specific clustering with a distinct separation of FGNX-2 from FG-non-NX-2 producers with high bootstrap support. Together, our findings provide insight into the amino acid sequence features responsible for the functional diversification of this TRI1p.

Changes in masked forms of deoxynivalenol and their co-occurrence with culmorin in cereal-based products: A systematic review and meta-analysis.

This study was aimed to evaluate the fate of D3G, 3-ADON, and 15-ADON during various processing steps (milling, fermentation, baking and cooking with water) of different cereal-based products, as well as the co-occurrence of culmorin (CUL) and its derivatives (15-Hydroxy-CUL and 5-Hydroxy-CUL. Some databases such as Science Direct, PubMed, Scopus, and Embase were screened to collect the relevant published papers between January 1983 to October 2018, and 23 articles with 319 data were included. The baking resulted in reductions in the concentration of all types of investigated masked mycotoxins, i.e., 15-ADON (-25%) > 3-ADON (-15%) > D3G (-6%). Also, rank order of CUL and its derivatives based on occurrence was CUL (70%) > 15-Hydroxy-CUL (47%) > 5-Hydroxy-CUL (15%) and their rank based on their concentration was 5-Hydroxy-CUL (99.21 µg/kg) > CUL (48.84 µg/kg) > 15-Hydroxy-CUL (9.39 µg/kg) > Hydroxy -CUL (0.06 µg/kg) > 12-Hydroxy-CUL (0.05 µg/kg) > 14-Hydroxy-CUL (0.01 µg/kg).

Presence of mycotoxins in ready-to-eat food and subsequent risk assessment.

A study on a set of ready-to-eat meals (n = 328) based on cereals, legumes, vegetables, fish and meat was carried out to determine the natural presence of twenty-seven mycotoxins by both liquid chromatography and gas chromatography coupled mass spectrometry in tandem (MS/MS) after QuEChERS extraction. The occurrence of mycotoxins was headed by cereal samples with 35% of samples contaminated by at least one mycotoxin followed by vegetables (32%), legumes (15%) and lastly, 9% of fish and meat samples were contaminated. DON was the most detected mycotoxin in vegetables, meat, fish and cereals with an incidence of 13% 18% 19% and 60%, respectively, and the highest mean levels were found in fish (1.19 µg/kg) and vegetable (1.53 µg/kg), respectively. The highest levels means were for HT-2 toxin ranging from 4.03 to 7.79 µg/kg, in cereal and legume samples respectively. In this last, HT-2 toxin was also the most prevalent (54%). In meat samples, OTA resulted with highest value with 8.09 µg/kg. Likewise, PCA analysis revealed a high correlation between the mycotoxins and the food groups analyzed. The findings indicate that there is no toxicological concern associated with exposure to mycotoxins for consumers as all levels were in accordance with the legislation.

Characterization of Argentinian Endemic Aspergillus flavus Isolates and Their Potential Use as Biocontrol Agents for Mycotoxins in Maize.

Maize (Zea mays L.) is a highly valuable crop in Argentina, frequently contaminated with the mycotoxins produced by Aspergillus flavus. Biocontrol products formulated with atoxigenic (nontoxic) strains of this fungal species are well known as an effective method to reduce this contamination. In the present study, 83 A. flavus isolates from two maize regions of Argentina were characterized and evaluated for their ability to produce or lack of producing mycotoxins in order to select atoxigenic strains to be used as potential biocontrol agents (BCA). All of the isolates were tested for aflatoxin and cyclopiazonic acid (CPA) production in maize kernels and a liquid culture medium. Genetic diversity of the nonaflatoxigenic isolates was evaluated by analysis of vegetative compatibility groups (VCG) and confirmation of deletions in the aflatoxin biosynthesis cluster. Eight atoxigenic isolates were compared for their ability to reduce aflatoxin and CPA contamination in maize kernels in coinoculation tests. The A. flavus population was composed of 32% aflatoxin and CPA producers and 52% CPA producers, and 16% was determined as atoxigenic. All of the aflatoxin producer isolates also produced CPA. Aflatoxin and CPA production was significantly higher in maize kernels than in liquid medium. The 57 nonaflatoxigenic strains formed six VCG, with AM1 and AM5 being the dominant groups, with a frequency of 58 and 35%, respectively. In coinoculation experiments, all of the atoxigenic strains reduced aflatoxin from 54 to 83% and CPA from 60 to 97%. Members of group AM1 showed a greater aflatoxin reduction than members of AM5 (72 versus 66%) but no differences were detected in CPA production. Here, we described for the first time atoxigenic isolates of A. flavus that show promise to be used as BCA in maize crops in Argentina. This innovating biological control approach should be considered, developed further, and used by the maize industry to preserve the quality properties and food safety of maize kernels in Argentina.

Modified mycotoxins: An updated review on their formation, detection, occurrence, and toxic effects.

Modified mycotoxins are metabolites that normally remain undetected during the testing for parent mycotoxin. These modified forms of mycotoxins can be produced by fungi or generated as part of the defense mechanism of the infected plant. In some cases, they are formed during food processing. The various processing steps greatly affect mycotoxin levels present in the final product (free and modified), although the results are still controversial regarding the increase or reduction of these levels, being strongly related to the type of process and the composition of the food in question. Evidence exists that some modified mycotoxins can be converted into the parent mycotoxin during digestion in humans and animals, potentially leading to adverse health effects. Some of these formed compounds can be even more toxic, in case they have higher bioaccessibility and bioavailability than the parent mycotoxin. The modified mycotoxins can occur simultaneously with the free mycotoxin, and, in some cases, the concentration of modified mycotoxins may exceed the level of free mycotoxin in processed foods. Even though toxicological data are scarce, the possibility of modified mycotoxin conversion to its free form may result in a potential risk to human and animal health. This review aims to update information on the formation, detection, occurrence, and toxic effects caused by modified mycotoxin.

Quality and safety of maize (Zea mays L.) from Rondônia state storage units, Northern Brazil.

The quality and safety of maize (Zea mays L.) from different grain storage units (GSUs), located in the main producing region of Rondônia State, Northern Brazil, were evaluated. Maize grains (n = 76) stored in four GSUs were collected from July to November 2014 and evaluated for grain damages, humidity, fungi and fumonisins (FBs) content. The climate conditions data were also obtained from plant growing to storage periods. Regarding the moisture content and water activity (aw), these varied from 10.0% to 16.1% and 0.5 to 0.8, respectively. As expected, fungi spores were present in 94.8% of the samples, prevailing Fusarium genera, with a fungi colony maximum of 2.2 × 104 CFU g-1. Regarding FBs, 60.5% of the samples were contaminated, below Brazilian and United States maximum limits, but 9.2% had levels higher than the European legislation.

Ozonation of whole wheat flour and wet milling effluent: Degradation of deoxynivalenol (DON) and rheological properties.

The objective of this study was to evaluate the reduction on the levels of the mycotoxin deoxynivalenol (DON) in whole wheat flour (WWF) with different moisture levels, on the wet milling effluent through ozone (O3) processing, as well as the impact of ozonation on the rheological properties of flour. The results have shown that the reduction of DON was improved with increasing moisture and exposure time of WWF and wet milling effluent to ozone. The maximum reduction was about 80%, proving that ozonation is an effective and promising technology in reducing mycotoxins in different products. However, the process altered the rheological profile of WWF. Therefore, further studies are needed to better understand the process.

Natural occurrence of tenuazonic acid and Phoma sorghina in Brazilian sorghum grains at different maturity stages.

A survey of 100 samples of sorghum grains was carried out to determine Phoma spp. and tenuazonic acid (TA) contamination using molecular tools and LC-MS/MS. Sorghum samples were obtained at the following four grain maturity stages: milk (S1), soft dough (S2), hard dough (S3), and physiological maturity (S4). The results revealed a good correlation between Phoma and TA occurrence during grain development. The samples showed Phoma contamination with frequencies ranging from 2.4% (S1) to 87.4% (S4), and the molecular identification revealed P. sorghina as the only Phoma specie isolated. Tenuazonic acid was found in sorghum grains at all maturity stages. In S2, S3 and S4, 100% of the samples showed TA contamination with levels ranging from 20 to 1234µg/kg. Low levels of TA were detected in 36% of the samples collected at S1 stage. This is the first report of tenuazonic acid in Brazilian sorghum grains.

Phytotoxic Lipophilic Metabolites Produced by Grapevine Strains of Lasiodiplodia Species in Brazil.

Phytotoxic metabolites produced in liquid culture by six species of Lasiodiplodia isolated in Brazil and causing Botryosphaeria dieback of grapevine were chemically identified. As ascertained by LC/MS, L. brasiliense, L. crassispora, L. jatrophicola, and L. pseudotheobromae produced jasmonic acid, and L. brasiliense synthesized, besides jasmonic acid, also (3R,4S)-4-hydroxymellein. L. euphorbicola and L. hormozganensis produced some low molecular weight lipophilic toxins. Specifically, L. euphorbicola produced (-)-mellein, (3R,4R)-(-)- and (3R,4S)-(-)-4-hydroxymellein, and tyrosol, and L. hormozganensis synthesized tyrosol and p-hydroxybenzoic acid. This is the first report on the production of the above cited metabolites from L. euphorbicola and L. hormozganensis. The phytotoxic activity of the metabolites produced is also discussed and related to the symptoms these pathogens cause in the grapevine host plants.

Toxin distribution and sphingoid base imbalances in Fusarium verticillioides-infected and fumonisin B1-watered maize seedlings.

Fusarium verticillioides is a major maize pathogen and there are susceptible and resistant cultivars to this fungal infection. Recent studies suggest that its main mycotoxin fumonisin B1 (FB1) may be involved in phytopathogenicity, but the underlying mechanisms are mostly still unknown. This work was aimed at assessing whether FB1 disseminates inside the plants, as well as identifying possible correlations between the maize resistant/susceptible phenotype and the unbalances of the FB1-structurally-related sphingoid base sphinganine (Sa) and phytosphingosine (Pso) due to toxin accumulation. Resistant (RH) and susceptible hybrid (SH) maize seedlings grown from seeds inoculated with a FB1-producer F. verticillioides and from uninoculated ones irrigated with FB1 (20 ppm), were harvested at 7, 14 and 21 days after planting (dap), and the FB1, Sa and Pso levels were quantified in roots and aerial parts. The toxin was detected in roots and aerial parts for inoculated and FB1-irrigated plants of both hybrids. However, FB1 levels were overall higher in SH seedlings regardless of the treatment (infection or watering). Sa levels increased substantially in RH lines, peaking at 54-fold in infected roots at 14 dap. In contrast, the main change observed in SH seedlings was an increase of Pso in infected roots at 7 dap. Here, it was found that FB1 disseminates inside seedlings in the absence of FB1-producer fungal infections, perhaps indicating this might condition the fungus-plant interaction before the first contact. Furthermore, the results strongly suggest the existence of at least two ceramide synthase isoforms in maize with different substrate specificities, whose differential expression after FB1 exposure could be closely related to the susceptibility/resistance to F. verticillioides.

Liquid chromatography-tandem mass spectrometry characterization of five new leucinostatins produced by Paecilomyces lilacinus CG-189.

The fungus Paecilomyces lilacinus produces leucinostatins­peptaibiotics that exert a range of biological activities including antimalarial, antiviral, antitumor and phytotoxicity. In this paper, we developed an analytical method employing LC-MS/MS in the precursor ion and product ion scan modes to elucidate five new leucinostatins. Direct Infusion (DI-MS) helped to identify the most abundant leucinostatins: F, D B2, S, A and K. MS/MS analysis using a triple quadrupole operating at different scan modes is a versatile tool to study natural products, especially peptaibiotics. Although DI-MS full-scan analysis is rapid and sensitive, it cannot distinguish between peptide isomers. On the other hand, LC-MS/MS operated in the precursor ion and product ion modes is time consuming, but allows identifying the structure of isomers or isobar in crude extracts.

Characterization of Alternaria strains from Argentinean blueberry, tomato, walnut and wheat.

Alternaria species have the ability to produce a variety of secondary metabolite, which plays important roles in food safety. Argentina is the second largest exporter of fresh and processed food products to Europe, however, few studies on Alternaria mycotoxins and other bioactive secondary metabolites have been carried out on Argentinean cereals, fruit and vegetables. Knowing the full chemical potential and the distribution of Alternaria spp. on crops, it is necessary to establish a toxicological risk assessment for food products for human consumption. In the present study, 87 Alternaria strains from different substrates (tomato, wheat, blueberries and walnuts) were characterized according to morphology and metabolite production. Aggressive dereplication (accurate mass, isotopic patterns and lists of all described compounds from Alternaria) was used for high-throughput evaluation of the chemical potential. Four strains belonged to the Alternaria infectoria sp.-grp., 6 to the Alternaria arborescens sp.-grp., 6 showed a sporulation pattern similar to that of "M" according to Simmons, 1 to that of Alternaria vaccinii, and the remaining 70 constituted a diverse group belonging to morphological groups "G" and "H". The cluster analysis yielded 16 almost identical dendrograms and grouped the Alternaria strains into four clusters and 11 singletons and outlier groups. The chemical analysis showed that AOH and AME were the most common metabolites produced, followed by TEN, ALXs and TeA. The A. infectoria sp.-grp. had no metabolites in common with the rest of the strains. Several secondary metabolites isolated from large-spored Alternaria species or other fungal genera were detected, such as dehydrocurvularin, pyrenochaetic acid and alternarienonic acid. The strains isolated from tomato produced lower amounts of metabolites than strains from blueberries, walnut and wheat, although individual strains from tomato produced the highest amount of some metabolites. The A. infectoria sp.-grp. was unique to cereals, whereas strains classified as belonging to the A. arborescens sp.-grp or having sporulation pattern "M" were only isolated from tomatoes. Otherwise, no clear association between substrate and identity could be found. The analyses in the study show that at least 75% of the Argentinean strains are able to produce potential mycotoxins.

Bioactive secondary metabolites from Phomopsis sp., an endophytic fungus from Senna spectabilis.

Chemical investigation of an acetonitrile fraction from the endophytic fungus Phomopsis sp. led to the isolation of the new natural product 2-hydroxy-alternariol (7) together with the known compounds cytochalasins J (1) and H (2), 5'-epialtenuene (3) and the mycotoxins alternariol monomethyl ether (AME, 4), alternariol (AOH, 5) and cytosporone C (6). The structure of the new compound was elucidated by using 1-D and 2-D NMR (nuclear magnetic resonance) and high resolution mass spectrometry. The cytochalasins J (1) and H (2) and AOH (5) exhibited potent inhibition of the total ROS (reactive oxygen species) produced by stimulated human neutrophils and acted as potent potential anti-inflammatory agents. Moreover, cytochalasin H (2) demonstrated antifungal and acetylcholinesterase enzyme (AChE) inhibition in vitro.

Purification and biochemical characterization of three myotoxins from Bothrops mattogrossensis snake venom with toxicity against Leishmania and tumor cells.

Bothrops mattogrossensis snake is widely distributed throughout eastern South America and is responsible for snakebites in this region. This paper reports the purification and biochemical characterization of three new phospholipases A2 (PLA2s), one of which is presumably an enzymatically active Asp49 and two are very likely enzymatically inactive Lys49 PLA2 homologues. The purification was obtained after two chromatographic steps on ion exchange and reverse phase column. The 2D SDS-PAGE analysis revealed that the proteins have pI values around 10, are each made of a single chain, and have molecular masses near 13 kDa, which was confirmed by MALDI-TOF mass spectrometry. The N-terminal similarity analysis of the sequences showed that the proteins are highly homologous with other Lys49 and Asp49 PLA2s from Bothrops species. The PLA2s isolated were named BmatTX-I (Lys49 PLA2-like), BmatTX-II (Lys49 PLA2-like), and BmatTX-III (Asp49 PLA2). The PLA2s induced cytokine release from mouse neutrophils and showed cytotoxicity towards JURKAT (leukemia T) and SK-BR-3 (breast adenocarcinoma) cell lines and promastigote forms of Leishmania amazonensis. The structural and functional elucidation of snake venoms components may contribute to a better understanding of the mechanism of action of these proteins during envenomation and their potential pharmacological and therapeutic applications.

Cooccurrence of mycotoxins in maize and poultry feeds from Brazil by liquid chromatography/tandem mass spectrometry.

The objective of this study was to quantitatively evaluate mycotoxins in samples of maize and poultry feed produced in Brazil. A multimycotoxin method based on HPLC-MS/MS was applied to investigate the occurrence of toxical fungal metabolites in 119 samples collected from poultry feed factory integrated poultry farms: maize grain (74), poultry feed (36), and feed factory residue (9). Twenty of 101 fungal metabolites investigated were detected and quantified in the samples: aflatoxins B1, B2, G1, and G2, fumonisins B1, B2, and B3, hydrolyzed fumonisin B1, zearalenone, agroclavine, chanoclavine, deoxynivalenol, and nivalenol, and enniatin A, A1, B, B1, beauvericin, kojic acid, and moniliformin. Most samples were contaminated with more than one mycotoxin. All samples were contaminated with fumonisins, with medians values of 1,840 µ g/kg, 239 µ g/kg, and 23,676 µ g/kg for maize, feed, and factory residue samples, respectively. Surprisingly, beauvericin was detected in more than 90% of samples. The median contaminations of aflatoxin and trichothecenes were low, near LOD values. The factory residue presented highest contamination levels for all mycotoxins. This is the first study dealing with agroclavine, chanoclavine, enniatin A, A1, B, B1, beauvericin, and kojic acid contamination of maize and poultry feeds from Brazil.