Cytotoxicity in Vero cells and cytokines analyses in Balb/c mice as safety assessments of the probiotic mixture Saccharomyces cerevisiae RC016 and Lactobacillus rhamnosus RC007 for use as a feed additive.

The objective was to carry out cytotoxicity assays in Vero cells and cytokines analyses in Balb/c mice as safety assessments to evaluate the probiotic mixture (M) Saccharomyces cerevisiae RC016 (Sc) and Lactobacillus rhamnosus RC007 (Lr) for use as feed additive. Vero cells (104 cells per well) were exposed to Sc (2·08 × 107 , 2·08 × 106 ; 2·08 × 105 cells per ml), Lr (8·33 × 107 ; 8·33 × 106 ; 8·33 × 105 cells per ml) and their M (1 : 1). Sc concentrations did not affect the Vero cells viability; in contrast, they were lower when exposed to Lr (P Ë‚ 0·0001). Vero cells showed increasing viability with M decreasing concentrations (91% viability with M2). Control BALB/c mice received only phosphate buffer saline and the others received the M. The IL-10, IL-6 and TNFα concentrations from intestinal fluid were analysed and no significant differences were observed among treatments. The same occurred with the ratio between IL-10/TNF-α. Beneficial effects of probiotics are associated with the regulation of the excessive inflammatory response; it is desirable they can modulate the cytokines production only under pathological conditions. Here, M administration to healthy mice did not induce negative side effects and expands the knowledge about beneficial effects of using probiotic microorganisms in mixture for feed additives development.

Aflatoxin B1 adsorption/desorption dynamics in the presence of Lactobacillus rhamnosus RC007 in a gastrointestinal tract-simulated model.

AIMS: (i) To determine the aflatoxin B1 (AFB1 ) adsorption and desorption dynamics in the presence of Lactobacillus rhamnosus RC007 under simulated transit of AFB1 at each gastrointestinal tract (GIT-saliva, stomach and intestine) stage consecutively and then, separately, (ii) to study the ability of L. rhamnosus RC007 to biotransform AFB1 as a strategy that complements the adsorption process. METHODS AND RESULTS: The AFB1 adsorption and desorption assay simulating the GIT passage of AFB1 (93·89 ng g-1 ) in the presence of L. rhamnosus RC007 (108 CFU per ml) was conducted. Moreover, lactic acid production was determined. Results demonstrated that predominant environmental conditions in salivary solution induced a low AFB1 adsorption, while the transit through the gastric solution and intestinal solution allowed high percentages of adsorption and did not generate significant AFB1 desorption. CONCLUSIONS: The AFB1 adsorption and desorption dynamics in the presence of L. rhamnosus RC007 was favoured by gastric and intestinal environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge of the adsorption dynamics of AFB1 with a micro-organism of interest will allow predicting its behaviour at each stage of the GIT.

Novel yeast isolated from broilers' feedstuff, gut and faeces as aflatoxin B1 adsorbents.

AIMS: To isolate and characterize native yeast strains from broilers' environment as feedstuff, faeces and gut, and to evaluate their binding capacity for aflatoxin B1 (AFB1 ). METHODS AND RESULTS: A total of nine yeast strains were isolated: three from feedstuff identified as Pichia kudriavzevii (2) and Clavispora lusitaniae (1), two from gut identified as Candida tropicalis and four from faeces identified as Cl. lusitaniae (3) and Cyberlindnera fabianii (1). AFB1 binding percentages varied among yeast strains and with AFB1 concentrations. To carry out adsorption studies, one strain from each genus and each origin was selected as follows: Cl. lusitaniae and P. kudriavzevii from feedstuff, Cl. lusitaniae and Cy. fabianii from faeces and Ca. tropicalis from gut. The most appropriate concentrations for cells and toxin were 107 cells per ml and 100 ng ml-1 of AFB1 respectively. All the tested yeast strains showed similar adsorption capacities independently of the origin. The adsorption isotherm studies in all yeasts assayed showed behaviour of L type or Langmuir and a varied affinity for the toxin. The stability of the AFB1 -yeast complex demonstrated the irreversibility of the binding process. CONCLUSION: Yeast strains tested in this study constitute potential AFB1 adsorbents and they possess the advantage to be native from the avian environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study makes a contribution to using native yeasts from broilers' environment for controlling chronic aflatoxicosis in avian production.

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.

The corn influence on the adsorption levels of aflatoxin B1 and zearalenone by yeast cell wall.

AIMS: To in vitro evaluate the influence of the corn on the adsorption levels of aflatoxin B1 (AFB1) and zearalenone (ZEA) by yeast cell walls (YCWs). METHODS AND RESULTS: Two commercial YCWs were studied. The YCWs contain different percentages of polysaccharides. YCW1 and 2 contain 5.9 and 21% of mannans and 17.4 and 23% of ß-glucans, respectively. Each YCW was resuspended in pH 2 and pH 6 buffer solutions. Corn was used to study the matrix influence. An aliquot of 500 µl YCW suspension was added to each microtube containing 500 µl of 0.1, 0.25, 0.5, 1, 2.5 and 5 µg ml(-1) AFB(1) or 0.5, 5, 10, 20 and 50 µg ml(-1) ZEA. Microtubes were kept with mechanical agitation at 37 °C for 30 min and then centrifuged for 10 min at 16,873 g and; the supernatants were quantified by high-pressure liquid chromatography. The amount of bound toxin was plotted as a function of the amount of added toxin according to mathematical expressions proposed by three theoretical models. Both YCWs were capable of adsorbing AFB(1) and ZEA in amounts from 0.061 to 0.40 and from 0.10 and 0.26 g g(-1), respectively. In the presence of the matrix, both adsorbents were not able to adsorb AFB(1) . However, they could adsorb ZEA at levels from 0.03 to 0.23 g g(-1). CONCLUSIONS: Both YCWs adsorbed ZEA in the presence of corn and also under simulated gastrointestinal pH conditions. These results suggest that the studied YCWs are potential candidates for ZEA adsorption. SIGNIFICANCE AND IMPACT OF THE STUDY: Several in vitro assays have informed the ability of different substrates including yeast walls to adsorb AFB(1) and ZEA; none of them have evaluated their ability to adsorb AFB(1) and ZEA in the presence of the corn. The corn matrix can influence the adsorption phenomena of these mycotoxins.

Fungi and mycotoxins in silage: an overview.

The present revision shows the early and current knowledge in the field of silage fungi and mycotoxins explaining the relevance of fungi and mycotoxins in silage. The problem does not end in animal disease or production losses as mycotoxins in feed can lead to the presence of their metabolic products in dairy products, which will be eventually affecting human health, mainly infants. Silage is green forage preserved by lactic fermentation under anaerobic conditions. This ecosystem maintains its quality and nutritional value depending on interactions among physical, chemical and biological agents. Forages used for ensilage are naturally in contact with yeasts and filamentous fungi, and the contamination often occurs in the field and can also occur during harvesting, transport, storage. Moreover, postharvest poor management can lead to a rapid spoilage. Studies on fungal contamination of dairy cattle feed have shown how corn silage influences the contamination degree of feed supplied to livestock. Increasing knowledge in this area will help elucidate the influence that this microbiota exerts on production and/or degradation of mycotoxins present in silage. Some of these fungi, although opportunist pathogens, are relevant epidemiologically and represent a high risk of contamination to farm workers who handle them improperly.

Statistical optimization of culture conditions for biomass production of probiotic gut-borne Saccharomyces cerevisiae strain able to reduce fumonisin B1.

AIM: To evaluate the ability of probiotic Saccharomyces cerevisiae RC016 strain to reduce fumonisin B(1) (FB(1)) in vitro and to optimize the culture conditions for the growth of the yeast employing surface response methodology. METHODS AND RESULTS: Using Plackett-Burman screening designs (PBSD) and central composite designs (CCD), an optimized culture medium containing (g l(-1)) fermentable sugars provided by sugar cane molasses (CMs), yeast extract (YE) and (NH(4))(2) HPO(4) (DAP) was formulated. The S. cerevisiae RC016 strain showed the greatest binding at all assayed FB1 concentration. The CMs, YE, DAP concentrations and incubation time influenced significantly the biomass of S. cerevisiae RC016. CONCLUSION: A combination of CMs 17%; YE 4·61 g l(-1) and incubation time 60 h was optimum for maximum biomass of S. cerevisiae RC016. SIGNIFICANCE AND IMPACT OF THE STUDY: The importance of this work lies in the search for live strains with both probiotic and fumonisin B1 decontamination properties that could be sustainably produced in a medium just containing cheap carbon, nitrogen and phosphorus sources and would be included in a novel product to animal feed.

Fungal contamination and determination of fumonisins and aflatoxins in commercial feeds intended for ornamental birds in Rio de Janeiro, Brazil.

The purposes of this study were to determine the distribution of total mycobiota, to determine the occurrence of Aspergillus spp., Penicillium spp. and Fusarium spp. and to detect and quantify fumonisin B1 and aflatoxin B1 in birds' feedstuffs. Sixty samples from different commercial feeds were collected. Analysis of the total mycobiota was performed and total fungal counts were expressed as CFU g(-1). The isolation frequency (%) and relative density (%) of fungal genera and species were determined. Mycotoxins determination was carried out using commercial ELISA kits. The 48% of standard, 31% of premium and only 9% of super premium feed samples were found above of recommended limit (1 × 10(4) CFU g(-1)). Aspergillus (82%), Cladosporium (50%) and Penicillium (42%) were the most frequently isolated genera. Aspergillus niger aggregate (35%), Aspergillus fumigatus (28%) and Aspergillus flavus (18%) had the highest relative densities. Contamination with fumonisins was detected in 95% of total samples with levels from 0·92 to 6·68 µg g(-1), and the aflatoxins contamination was found in 40% of total samples with levels between 1·2 and 9·02 µg kg(-1). Feed samples contaminated with fumonisins and aflatoxins are potentially toxic to birds.

Typing clinical and animal environment Aspergillus fumigatus gliotoxin producer strains isolated from Brazil by PCR-RFLP markers.

UNLABELLED: Aspergillus fumigatus, a well-known human and animal pathogen causing aspergillosis, has been historically identified by morphological and microscopic features. However, recent studies have shown that species identification on the basis of morphology alone is problematic. The aim of this work was to confirm the taxonomic state at specie level of a set of clinical (human and animal) and animal environment A. fumigatus strains identified by morphological criteria applying a PCR-RFLP assay by an in silico and in situ analysis with three restriction enzymes. The A. fumigatus gliotoxin-producing ability was also determined. Previous to the in situ PCR-RFLP analysis, an in silico assay with BccI, MspI and Sau3AI restriction enzymes was carried out. After that, these enzymes were used for in situ assay. All A. fumigatus strains isolated from corn silage, human aspergillosis and bovine mastitis and high per cent of the strains isolated from cereals, animal feedstuff and sorghum silage were able to produce high gliotoxin levels. Also, all these strains identified by morphological criteria as A. fumigatus, regardless of its isolation source, had band patterns according to A. fumigatus sensu stricto by PCR-RFLP markers. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspergillus fumigatus is a well-known human and animal pathogen causing aspergillosis. In this study, clinical (human and animal) and animal environment strains were able to produce high gliotoxin levels and had band profiles according to A. fumigatus sensu stricto by PCR-RFLP markers. The results obtained here suggest that strains involved in human and animal aspergillosis could come from the animal environment in which A. fumigatus is frequently found. 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.

Adsorption of ochratoxin A and zearalenone by potential probiotic Saccharomyces cerevisiae strains and its relation with cell wall thickness.

AIMS: To examine Saccharomyces cerevisae strains with previously reported beneficial properties and aflatoxin B1 binding capacity, for their ability to remove ochratoxin A (OTA) and zearalenone (ZEA) and to study the relation between cell wall thickness and detoxificant ability of yeast strains. METHODS AND RESULTS: A mycotoxin binding assay at different toxin concentrations and the effect of gastrointestinal conditions on mycotoxin binding were evaluated. Ultrastructural studies of yeast cells were carried out with transmission electronic microscopy. All tested strains were capable of removing OTA and ZEA. Saccharomyces cerevisiae RC012 and RC016 showed the highest OTA removal percentage, whereas RC009 and RC012 strains showed the highest ZEA removal percentages. The cell diameter/cell wall thickness relation showed a correlation between cell wall amount and mycotoxin removal ability. After exposure to gastrointestinal conditions, a significant increase in mycotoxin binding was observed. CONCLUSIONS: All tested Saccharomyces cerevisiae strains were able to remove OTA and ZEA, and physical adsorption would be the main mechanism involved in ochratoxin A and ZEA removal. Gastrointestinal conditions would enhance adsorption and not decrease mycotoxin-adsorbent interactions. SIGNIFICANCE AND IMPACT OF THE STUDY: Live strains with mycotoxin binding ability and beneficial properties are potential probiotics that could be included in animal feed. Previous and present results suggest that the RC008 and RC016 strains are very promising candidates for functional feed product development.

Gliotoxin contamination in and pre- and postfermented corn, sorghum and wet brewer's grains silage in Sao Paulo and Rio de Janeiro State, Brazil.

AIMS: The aim of this study was to determine total fungal counts and the relative density of Aspergillus fumigatus and related species in silage samples intended for bovines before and after fermentation as well as to monitor the natural occurrence of gliotoxin in silage samples (pre- and postfermentation). METHODS AND METHODS: The survey was performed in farms located in São Paulo and Rio de Janeiro States in Brazil. In addition, the ability of A. fumigatus strains and related species strains to produce gliotoxin was also evaluated. A total of 300 samples were taken, immediately after opening of the silo (3-5 months) and during the ensiling period. Fungal counts were done by the surface-spread method. Gliotoxin production ability of isolates and natural contamination were determined by HPLC. RESULTS: All postfermented samples had a total number of moulds exceeding 1 × 10(4) CFU g(-1), with Aspergillus sp. as the most prevalent genus. Frequency of strains, among A. fumigatus and related species, was able to produce gliotoxin was similar in pre- and postfermented samples, except for sorghum, which showed differences between both kinds of samples. The highest toxin levels were produced by strains isolated from postfermented samples. More than 50% of the samples showed gliotoxin contamination levels that exceeded concentrations known to induce immunosuppressive and apoptotic effects in cells. CONCLUSIONS: The present data suggest that care should be taken because gliotoxin contamination in feedstuffs could affect productivity and also present a health risk for herds. SIGNIFICANCE AND IMPACT OF THE STUDY: Gliotoxin was found at quite important concentrations levels in pre- and postfermented substrates and its presence could therefore probably affect the productivity and health of herds. Current conservation and management practices do not avoid contamination with A. fumigatus on silage. Therefore, farm workers should be adequately protected during its handling.

Combined effects of aflatoxin B1 and corticosterone treatment on selected performance indices and liver histopathology in Japanese quail.

Animal feed may be contaminated with different mycotoxins, with aflatoxin B(1) (AFB(1)) being a very common and toxic compound. Considering that birds normally have to cope with different stressful situations at the same time, the present study aims to evaluate the effects of feed contamination with AFB(1) in combination with corticosterone treatment in drinking water (a model to induce physiological stress in birds) on selected performance indices: BW, feed conversion, egg production, and macroscopic and microscopic liver alterations. At 5 wk of age, quails were randomly assigned to 1 of 6 dietary treatment groups that resulted from the combination of the presence or absence of corticosterone in drinking water (5 mg/L) with the presence or absence of AFB(1) contamination (0, 100, or 500 µg/kg). The animals remained in these treatments from 5 to 11 wk of age. There were 6 replicates per treatment, each containing 2 males and 2 females. Contamination with 100 µg of AFB(1) per kilogram of feed induced no changes in BW, feed conversion, and egg production parameters. Quail fed with 500 µg of AFB(1) per kilogram of feed showed significant decreases in BW and feed consumption compared with their control counterparts. Corticosterone in combination with 500 µg of AFB(1) per kilogram of feed intensified the negative effects observed on BW and feed consumption and also had negative effects on feed conversion rate and egg production parameters, suggesting that the adverse effects of contamination with AFB(1) are intensified in situations of chronic stress. Quail treated with 500 µg of AFB(1) per kilogram showed hepatocytes with degree 1 and 2 lesions, and all quail treated with 500 µg of AFB(1) per kilogram of feed in combination with corticosterone showed degree 2 liver lesions (i.e., hepatocytes with fatty macro and microvacuoles and necrosis). This result is also consistent with the hypothesis that chronic stress exacerbates the effect of AFB(1) contamination. In conclusion, this study suggests that the negative effects of AFB(1) contamination are increased when overlapped with chronic stressful stimulation.

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.

Sodium bentonite and monensin under chronic aflatoxicosis in broiler chickens.

Clay feed additives have been increasingly incorporated into animal diets to prevent aflatoxicosis. Due to the nonselective nature of the binding interaction, many important components of the diets could also be made unavailable because of these feed additives. The anticoccidial monensin (MON) could also be sequestered by these clays. The use of sodium bentonite (Na-B) from a mine in the province of Mendoza, Argentina, was investigated as a sequestering agent to prevent the effects of 100 µg/kg of dietary aflatoxin B(1) (AFB(1)). In vitro studies demonstrated that the above Na-B was a good candidate to prevent aflatoxicosis. They also showed that MON competes with AFB(1) for the adsorption sites on the clay surface and effectively displaces the toxin when it is in low concentration. Even though the levels of MON in diets, approximately 55 mg/kg, are high enough to not be significantly changed as a consequence of the adsorption, they can further affect the ability of the clays to bind low levels of AFB(1). An in vivo experiment carried out with poultry showed that 100 µg/kg of AFB(1) does not significantly change productive or biochemical parameters. However, liver histopathology not only confirmed the ability of this particular Na-B to prevent aflatoxicosis but also the decrease of this capacity in the presence of 55 mg/kg of MON. This is the first report stressing this fact and further research should be performed to check if this behavior is a characteristic of the assayed Na-B or of this type of clay. On the other hand, the presence of MON should also be taken into account when assaying the potential AFB(1) binding ability of a given bentonite.

Effect of low levels of aflatoxin B1 on performance, biochemical parameters, and aflatoxin B1 in broiler liver tissues in the presence of monensin and sodium bentonite.

Aflatoxins (AF) are a major problem in broiler production and are significant economic and public health burdens worldwide. A commercial sodium bentonite (Na-B) adsorbent was used to prevent the effect of AF [50 µg of aflatoxin B1 (AFB1)/kg of feed] in broiler productivity, biochemical parameters, macroscopic and microscopic liver changes, and AFB1 liver residues. The influence of Na-B (0.3%) and monensin (MON, 100 mg/kg), alone or in combination, was investigated in depth. The dietary treatments were as follows: treatment (T) 1: basal diet (B); T2: B + MON; T3: B + Na-B; T4: B + Na-B + MON; T5: B + AFB1; T6: B + AFB1 + Na-B + MON; T7: B + AFB1 + MON; T8: B + AFB1 + Na-B. Birds were fed dietary treatments for 28 d (d 18 to 46). No significant differences (P < 0.05) were observed among treatments with respect to broiler performance, biochemical parameters, or relative liver weights. With the exception of T8, all livers showed histopathological alterations, with accumulation of fat vacuoles. The normal appearance of livers from T8 showed the protective effect of Na-B against aflatoxicosis. The residual AFB1 levels in livers from T5 to T8 ranged from 0.2 to 1.0 ng/g and were higher in livers from T6 (P < 0.05). Results of this study indicate a competition between AFB1 and MON for adsorption sites on Na-B when feed contains low levels of the toxin, indicating a nonselective adsorption capacity of this particular Na-B. In addition, significant levels of AFB1 in livers indicate that this determination is an important technique not only for diagnosis of aflatoxicosis in broilers, but also for quality control of avian products.

Aspergillus fumigatus toxicity and gliotoxin levels in feedstuff for domestic animals and pets in Argentina.

AIMS: To evaluate gliotoxin production by Aspergillus fumigatus strains isolated from feedstuff intended for domestic animals and pets, and to determine the amount of gliotoxin in these substrates. METHODS AND RESULTS: A total of 150 feedstuff samples were collected. They were composed of 30 samples each of five different feed types (pigs, poultry, cattle, horse and pets). Aspergillus fumigatus gliotoxin production ability and gliotoxin presence in feedstuff was determined by HPLC. Aspergillus fumigatus strains were isolated from all of the tested samples. Strains from cattle, horses and pet food were able to produce gliotoxin. Corn silage samples intended for cattle did not show gliotoxin contamination. All the other tested samples had gliotoxin levels ranging from 29 to 209 microg g(-1). Horse and poultry feed samples had the greatest contamination frequency. CONCLUSIONS: Feed samples contaminated with gliotoxin are potentially toxic to animals. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of gliotoxin could affect animal productivity and health. Moreover, there are risks of contamination to farm workers handling improperly stored animal feed. Aspergillus fumigatus strains isolated from different sources should be investigated to determine prevention and control strategies.

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.

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.

Beneficial effects of Saccharomyces cerevisiae RC016 in weaned piglets: in vivo and ex vivo analysis.

Probiotics represents an alternative to replace antibiotics as growth promoters in animal feed and are able to control enteric bacterial diseases and to improve gut immunity. Saccharomyces cerevisiae RC016 showed previously inhibition/coagregation of pathogens) and mycotoxins adsorbent ability (aflatoxin B1, ochratoxin A and zearalenone). The aim of this work was to evaluate beneficial properties of S. cerevisiae RC016 in a non-inflammatory in vivo model in weaned piglets and in an intestinal inflammation ex vivo model induced by the mycotoxin deoxynivalenol (DON). Secretory immunoglobulin A (s-IgA) levels, intestinal cytokines, goblet cells and production parameters were evaluated in a pig model. For the in vivo assays, twelve pigs were weaned at 21 days and assigned to two groups: Control (n=6) and Yeast (n=6). Animals received yeast strain for three weeks. After 22 days the small intestine was recovered for determination of goblet cells and s-IgA. For the ex vivo assay, jejunal explants were obtained from 5 weeks old crossbred piglets and treated as follow: (1) control; (2) treated for 3 h with 10 µM DON used as an inflammatory stressor; (3) incubated with 107 cfu/ml yeast strain; (4) pre-incubated 1 h with 107 cfu/ml yeast strain and then treated for 3 h with 10 µM DON. CCL20, interleukin (IL)-1ß, IL-8 and IL-22 gene expression was determined by qPCR. Oral administration of S. cerevisiae RC016 increased s-IgA, the number of goblet cells in small intestine and all the growth parameters measured. In the ex vivo model, the cytokine profile studied showed a potential anti-inflammatory effect of the administration of the yeast. In conclusion, S. cerevisiae RC016 is a promising candidate for feed additives formulation to improve animal growth and gut immune system. This yeast strain could be able to improve the gut health through counteracting the weaning-associated intestinal inflammation in piglets.