Aflatoxin-degrading Bacillus sp. strains degrade zearalenone and produce proteases, amylases and cellulases of agro-industrial interest.

Zearalenone, an oestogenic mycotoxin produced by Fusarium sp., occurs naturally in agricultural commodities. Economic losses and health concerns associated to mycotoxins has attracted research interest towards exploring novel approaches to detoxify mycotoxin-contaminated food and feed. The aim of the present work was to study the ability of 11 aflatoxin-degrading Bacillus strains to degrade ZEA. In addition, a qualitative assessment of protease, amylase and cellulase activity of the studied Bacillus strains was made. All strains were able to degrade 58-96.9% ZEA after 72 h. Toxicity towards Artemia salina was significantly reduced (P < 0.0001). Degradation extracts fluorescence decreased 50% indicating a probable cleavage of the lactone ring. Strains RC1A, RC3A and RC6A showed a remarkable enzymatic activity, showing potential to be used as feed additives.

Probiotic bacteria and yeasts adsorb aflatoxin M1 in milk and degrade it to less toxic AFM1-metabolites.

The presence of contaminants such as aflatoxins (AFs) in dairy products constitutes a serious risk to the health of consumers, especially children who are most sensitive to the adverse effects of AFs. The presence of Aflatoxin M1 (AFM1) in milk is a public health problem since dairy products are massively consumed worldwide. The aim of the present work was to select microorganisms capable of reducing AFM1 entry into the food chain through adsorption/degradation strategies. Moreover, the toxicity of AFM1 degradation products was evaluated. All tested strains had the capacity to adsorb 19%-61% AFM1 in milk. These strains also had the ability to degrade AFM1 into metabolites less toxic than the original toxin. Moreover, this is the first study to report harmless and probiotic Pediococcus pentosaceus and Kluveromyces marxianus have the ability to adsorb and degrade AFM1 to less toxic metabolites in milk.

Presence of aiiA homologue genes encoding for N-Acyl homoserine lactone-degrading enzyme in aflatoxin B1-decontaminating Bacillus strains with potential use as feed additives.

Microbial degradation of aflatoxins (AFs) is an alternative to the use of mycotoxin binders. The lactone ring is a possible target for microbial enzymes and its cleavage reduces AFs toxicity. The aim of this study was to isolate and identify Bacillus strains able to degrade AFB1 to less toxic metabolites and to identify aiiA genes encoding for N-acyl-homoserine lactone (AHL) lactonase to possibly correlate detoxification with the production of this enzyme. Eleven soilborne Bacillus strains were isolated and identified by MALDI-TOF MS. Ten cultures and eight cell free culture supernatants (CFCS) were able to significantly (P < 0.05) degrade 27.78-79.78% AFB1. Cell lysates were also able to degrade AFB1 (P < 0.05). Exposure to 70 and 80 °C did not affect enzyme activity. Aflatoxin B1 toxicity towards Artemia salina was reduced after degradation by each of the Bacillus strains. B. subtilis RC1B, B. cereus RC1C and B. mojavensis RC3B, amplified a fragment of 753 pb corresponding to the aiiA gene encoding for AHL lactonase. AFB1 degradation by the strains tested was due to the extracellular and intracellular enzymes. If demonstrated to be safe, these could be used to detoxify AFB1 in contaminated food or feed.

Use of yeast (Pichia kudriavzevii) as a novel feed additive to ameliorate the effects of aflatoxin B1 on broiler chicken performance.

The aim of this study was to evaluate the efficacy of autochthonous Pichia kudriavzevii as a novel bioadsorbent for aflatoxin B1 (AFB1). The selection of this yeast was based on the AFB1 adsorption capacity previously demonstrated in vitro (Magnoli et al. 2016). One-day-old Cobb broilers (n = 160) were randomly assigned to four dietary treatments (T1: basal diet (B); T2: B + 0.1% yeast; T3: B + AFB1, 100 µg/kg; T4: B + 0.1% yeast + AFB1, 100 µg/kg). Performance parameters (average daily weight gain body, average daily consumption, feed conversion ratio, carcass weight, and dead weight), biochemical parameters (albumin, globulin, and albumin/globulin), liver pathological changes, and AFB1 residual levels in the liver and excreta were evaluated. Significant differences (P < 0.05) in performance parameters were observed among treatments and controls: T3 group showed the lowest average daily body weight gain value while in T4 group, the value of this parameter increased significantly (P < 0.05). T3 and T4 groups showed the lowest and highest values for average daily feed consumption, respectively. The feed conversion ratio (FC) showed no significant differences among treatments. T3 group showed the lowest dead weight and carcass weight compared with T1 group. The biochemical parameters showed no significant differences among treatments. T3 group showed macroscopic and microscopic liver changes compared to the control. Aflatoxin B1 levels (µg/g) were detected in broiler livers and showed significant differences among treatments (P < 0.05). In conclusion, native P. kudriavzevii incorporation (0.1%) in broiler diets containing AFB1 was shown to be effective in ameliorating the adverse effects of AFB1 on production.

Gliotoxin production by Aspergillus fumigatus strains from animal environment. Micro-analytical sample treatment combined with a LC-MS/MS method for gliotoxin determination.

In this study, gliotoxin production by Aspergillus fumigatus strains from animal environment is studied. Moreover, a rapid, easy and environment-friendly micro-analytical sample treatment procedure coupled with LC-MS/MS was applied for the determination of gliotoxin from A. fumigatus cultures. The ability of gliotoxin production by 143 strains was assayed in yeast extract sucrose agar, and 1 ml of chloroform was used for toxin extraction without further clean-up. Mean recoveries at two spiking levels (2500 and 7000 ng/g; n = 6) were 100.3 ± 6.6 % relative SD (RSD) and 92.4 ± 3.8 % RSD. Repeatability and within-laboratory reproducibility for different concentration levels of gliotoxin (25 to 1000 ng/ml; n = 12) ranged from 0.3 to 5.4 % RSD and from 3.9 to 12.7 % RSD, respectively. The detection limit of the analytical method was 3.5 ng/g. The ability for gliotoxin production by A. fumigatus revealed that 61.5 % of the strains were able to produce the toxin at levels ranging from LOQ to 3430.5 ng/g. However, all the tested samples had similar percentages of producing strains (81.8 to 86.6 %). The micro-analytical sample treatment coupled with LC-MS/MS detection is a precise and useful methodology for determining gliotoxin from fungal extracts of A. fumigatus and allows working both fast and safely and also reducing the effect on the environment. This toxin plays a critical role in the pathobiology of A. fumigatus, and its presence in animal environments could affect animal health and productivity; in addition, there are risks of contamination for rural workers during handling and storage of animal feedstuffs.

Genotoxicity and cytotoxicity evaluation of probiotic Saccharomyces cerevisiae RC016: a 60-day subchronic oral toxicity study in rats.

AIMS: To acquire data on the safety-in-use of the probiotic Saccharomyces cerevisiae RC016 and test its ability to reduce genotoxicity caused by dietary aflatoxins (AFs). METHODS AND RESULTS: The probiotic was orally administered to Wistar rats. Six groups (n = 6) were arranged: feed and probiotic controls, two levels of AFs-contaminated feed and two treatments including both the probiotic and the toxin. Genotoxiciy and cytotoxicity were evaluated with the bone marrow micronuclei assay and the comet assay and internal organs were macroscopically and microscopically examined. The tested S. cerevisiae strain did not cause genotoxicity or cytotoxicity in vivo, and it was able to attenuate AFs-caused genotoxicity. Saccharomyces cerevisiae RC016 did not cause any impairment on the rats' health and it showed no negative impact on the weight gain. Moreover, RC016 improved zootechnical parameters in AFs-treated animals. The beneficial effects were likely to be caused by adsorption of AFs to the yeast cell wall in the intestine and the consequent reduction in the toxin's bioavailability. CONCLUSIONS: The dietary administration of RC016 does not induce genotoxicity or cytotoxicity to rats. SIGNIFICANCE AND IMPACT OF THE STUDY: Incorporation of RC016 in the formulation of feed additives increases animal productivity. Similar effects may even occur in human food applications.

Mycobiota and mycotoxins in malted barley and brewer's spent grain from Argentinean breweries.

AIMS: To evaluate mycobiota and aflatoxins B(1) (AFB(1)), B(2) (AFB(2)), G(1) (AFG(1)), G(2) (AFG(2)) and fumonisin B(1) (FB(1)) contamination in different malted barley types and brands and brewer's grain collected from a major Argentinean brewery. METHODS AND RESULTS: Total fungal counts were performed using the plate count method. Aflatoxin B(1), AFB(2), AFG(1), AFG(2) and Zearalenone (ZEA) analyses were performed by thin-layer chromatography (TLC). Fumonisin B(1) was determined by HPLC. Eighty-three percentage of the malted barley (100% M1, 50% M2 and 100% M3) and 61% of brewer's grain samples had a count >1 × 10(4) CFU g(-1). Yeasts were isolated from all malt and brewer's grain samples. Genera containing some of the most important mycotoxin producer species--Fusarium ssp., Aspergillus ssp., Penicillium ssp. and Alternaria ssp.--were isolated from the analysed samples, along with other environmental saprophytic fungi such as Geotrichum ssp., Mucorales and Cladosporium ssp. All samples were contaminated with 104-145 µg kg(-1) FB(1). Eighteen per cent of brewer's grain samples were contaminated with 19-44.52 µg kg(-1) AFB(1). Aflatoxin B(2), AFG(1), AFG(2) and ZEA were not detected in any of the analysed samples. CONCLUSIONS: Fungal and mycotoxin contamination in malt and brewer's grain is an actual risk for animal and human health. SIGNIFICANCE AND IMPACT OF THE STUDY: This study may be useful for assessing the risk of mycotoxins in Argentinean beers and especially in animal feeds.

Mycological survey and ochratoxin A natural contamination of swine feedstuffs in Rio de Janeiro State, Brazil.

Mycotoxin contamination of animal feeds represents a hazard to human and animal health due to potential transmission to meat and milk. Barley by-products are alternative feeding supplies for animal production. The aims of this assay were to study the mycobiota of feedstuffs and finished swine feed, to determine the ability of Aspergillus and Penicillium isolates to produce ochratoxin A (OTA) and to evaluate OTA occurrence in these substrates. Corn, brewers' grains and finished swine feed samples were collected from different factories. Fungal counts were higher than 2.8x10(4)CFU g(-1). Fusarium, Aspergillus and Penicillium genera were isolated at high levels. A 23.7% of the isolates produced 9-116 microg kg(-1) of OTA in vitro. Corn samples (44%) were contaminated with 42-224 microg kg(-1) of OTA. Finished feed (31%) and brewers' grains samples (13%) were contaminated with 36-120 microg kg(-1) and 28-139 microg kg(-1) of OTA, respectively. This is the first scientific report on contamination by OTA-producer molds and OTA in swine feedstuffs from Brazil. The presence of OTA in raw materials and finished feed requires periodic monitoring to prevent mycotoxicoses in animal production, reduce economic losses and minimize hazards to human health.

Determination of mycobiota and mycotoxins in pig feed in central Argentina.

AIMS: To evaluate the mycobiota and natural levels of aflatoxins, fumonisins and zearalenone present in compound feed and home-corn grains intended for fattening pigs. METHODS AND RESULTS: Total fungi, Fusarium and Aspergillus species occurrence were examined. Aflatoxins and zearalenone were detected by thin-layer chromatography and fumonisins by high-pressure liquid chromatography. Fungal counts were generally higher than 1 x 10(5) colony forming units (CFU) ml(-1). Aspergillus flavus, Aspergillus parasiticus and Fusarium verticillioides were the most prevalent species. FB(1) and FB(2) were detected in all feed and corn samples. Aflatoxin B(1) was detected in 33.33% of initial and growing feed and in 44.44% of final feed samples. It was not detected in corn samples. All feed and corn samples were negative for AFB(2), AFG(1), AFG(2) and ZEA presence during all growing stages tested. CONCLUSIONS: Fungal counts at all growing periods exceeded the levels proposed as feed hygienic quality limits. Aflatoxin levels in all feeds and fumonisin levels in many samples were higher than the established regulations. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of mycotoxins indicates the existence of contamination. This fact requires periodic monitoring to prevent the occurrence of mycotoxicosis in animal production, to reduce the economic losses and to minimize hazards to human health.

Fungi and selected mycotoxins from pre- and postfermented corn silage.

AIM: To determine fungal genera, Aspergillus and Fusarium species and aflatoxin B(1) (AFB(1)), zearalenone (ZEA), deoxynivalenol (DON), fumonisin B(1) (FB(1)) contamination from pre- and postfermented corn silage produced in the most important region of Argentina where silage practice is developed. METHODS AND RESULTS: Sampling of corn silos was performed manually through silos in transects at three levels: upper, middle and low sections. AFB(1) and FB(1) were quantified by high-performance liquid chromatography, zearalenone by enzyme-linked immunosorbent assay and DON by gas chromatography. Over 90% of the samples showed counts higher than 1 x 10(4) CFU g(-1). Aspergillus flavus and Fusarium verticillioides were the prevalent species. Some tested samples were contaminated with AFB(1), ZEA, DON and FB(1). CONCLUSIONS: This study demonstrates the presence of fungi and AFB(1), ZEA, DON and FB(1) contamination in corn silage in Argentina. SIGNIFICANCE AND IMPACT OF THE STUDY: This manuscript makes a contribution to the knowledge of mycotoxins in Argentinean silage in particular because the environmental conditions in this country differ from those of most reports. The comparison of pre- and postfermentation silage is also outstanding. Therefore, information on fungi and mycotoxins present in silage--an increasingly popular commodity--is useful to estimate potential risk for animal and human health.