Hydrolytic enzymes production by Aspergillus section Nigri in presence of butylated hydroxyanisole and propyl paraben on peanut meal extract agar / Producción de enzimas hidrolíticas por Aspergillus sección Nigri en presencia de hidroxianisol butilado y propilparabeno en medio de cultivo de agar con extracto de cacahuete

Background. In the last years, food grade antioxidants are used safely as an alternative to traditional fungicides to control fungal growth in several food and agricultural products. Aims. In this work, the effect of butylated hydroxyanisole (BHA) and propyl paraben (PP) on two hydrolytic enzyme activity (β-d-glucosidase and α-d-galactosidase) by Aspergillus section Nigri species under different water activity conditions (aW; 0.98, 0.95 and 0.93) and incubation time intervals (24, 48, 72 and 96 h) was evaluated on peanut-based medium. Methods. The activity of two glycosidases, β-d-glucosidase and α-d-galactosidase, was assayed using as substrates 4-nitrophenyl-β-d-glucopyranosido and 4-nitrophenyl-α-d-galactopyranosido, respectively. The enzyme activity was determined by the increase in optical density at 405 nm caused by the liberation of p-nitrophenol by enzymatic hydrolysis of the substrate. Enzyme activity was expressed as micromoles of p-nitrophenol released per minute. Results. The major inhibition in β-d-glucosidase activity of A. carbonarius and A. niger was found with 20 mmol l−1 of BHA or PP at 0.98 and 0.95 aW, respectively, whereas for α-d-galactosidase activity a significant decrease in enzyme activity with respect to control was observed in A. carbonarius among 5 to 20 mmol l−1 of BHA or PP in all conditions assayed. Regarding A. niger, the highest percentages of enzyme inhibition activity were found with 20 mmol l−1 of BHA or PP at 0.95 aW and 96 h. Conclusions. The results of this work provide information about the capacity of BHA and PP to inhibit in vitro conditions two of the most important hydrolytic enzymes produced by A. carbonarius and A. niger species (AU)

Antecedentes. En los últimos años, para controlar el crecimiento fúngico, en lugar de los fungicidas tradicionales, tanto en la industria alimentaria como en los productos agrícolas se utilizan antioxidantes como aditivos alimentarios bien tolerados y sin riesgos de efectos adversos. Objetivos. En el presente estudio, en un medio de cultivo con cacahuete, se examinó el efecto de hidroxianisol butilado (BHA) y propilparabeno (PP) sobre la actividad de 2 enzimas hidrolíticas (β-d-glucosidasa y α-d-galactosidasa) producidas por especies de Aspergillus sección Nigri, en función de diferentes valores de actividad de agua del sustrato (aW; 0,98, 0,95 y 0,93) y tiempos de incubación (24, 48, 72 y 96 h). Métodos. La actividad de las 2 glucosidasas (β-d-glucosidasa y α-d-galactosidasa) se evaluó usando como sustrato 4-nitrofenil-β-d-glucopiranósido y 4-nitrofenil-α-d-galactopiranósido, respectivamente. La actividad enzimática se determinó mediante el aumento de la densidad óptica a 405 nm provocado por la liberación de p-nitrofenol, resultado de la hidrólisis enzimática del sustrato. La actividad enzimática se expresó como micromoles de p-nitrofenol liberado por minuto. Resultados. La mayor inhibición en la actividad de β-d-glucosidasa de Aspergillus carbonarius y Aspergillus niger se observó con 20 mmol l−1 de BHA o PP a 0,98 y 0,95 aW, respectivamente. Comparado con el control, en A. carbonarius se detectó una disminución significativa de la actividad de α-d-galactosidasa con 5-20 mmol l−1 de BHA o PP en todas las condiciones examinadas. Con respecto a A. niger, los porcentajes mas elevados de inhibición enzimática se observaron con 20 mmol l−1 de BHA o PP a 0,95 aW y un tiempo de incubación de 96 h. Conclusiones. Los resultados del presente estudio proporcionan información sobre la capacidad de BHA y PP para inhibir dos de las enzimas más importantes producidas por las especies A. carbonarius y A. niger (AU)

Hydrolytic enzymes production by Aspergillus section Nigri in presence of butylated hydroxyanisole and propyl paraben on peanut meal extract agar.

BACKGROUND: In the last years, food grade antioxidants are used safely as an alternative to traditional fungicides to control fungal growth in several food and agricultural products. AIMS: In this work, the effect of butylated hydroxyanisole (BHA) and propyl paraben (PP) on two hydrolytic enzyme activity (ß-d-glucosidase and α-d-galactosidase) by Aspergillus section Nigri species under different water activity conditions (aW; 0.98, 0.95 and 0.93) and incubation time intervals (24, 48, 72 and 96h) was evaluated on peanut-based medium. METHODS: The activity of two glycosidases, ß-d-glucosidase and α-d-galactosidase, was assayed using as substrates 4-nitrophenyl-ß-d-glucopyranosido and 4-nitrophenyl-α-d-galactopyranosido, respectively. The enzyme activity was determined by the increase in optical density at 405nm caused by the liberation of p-nitrophenol by enzymatic hydrolysis of the substrate. Enzyme activity was expressed as micromoles of p-nitrophenol released per minute. RESULTS: The major inhibition in ß-d-glucosidase activity of A. carbonarius and A. niger was found with 20mmoll(-1) of BHA or PP at 0.98 and 0.95 aW, respectively, whereas for α-d-galactosidase activity a significant decrease in enzyme activity with respect to control was observed in A. carbonarius among 5 to 20mmoll(-1) of BHA or PP in all conditions assayed. Regarding A. niger, the highest percentages of enzyme inhibition activity were found with 20mmoll(-1) of BHA or PP at 0.95 aW and 96h. CONCLUSIONS: The results of this work provide information about the capacity of BHA and PP to inhibit in vitro conditions two of the most important hydrolytic enzymes produced by A. carbonarius and A. niger species.

Inhibitory properties of Enterococcus spp. isolated from faeces of healthy dogs.

The aim of the present study was to evaluate the inhibitory effect by the cross-streak method of nine Enterococcus faecium strains isolated from faeces of healthy dogs and their treated and non-treated cell-free supernatant (CFS) by the well-diffusion test on the growth of potentially pathogenic bacteria isolated from clinical cases and aflatoxigenic Aspergillus section Flavi and the consequent aflatoxin B1 (AFB1) production. Results obtained from the cross-strake assay showed that E. faecium MF1, GJ18 and GJ40 presented the major inhibitory activity against all pathogenic strains assayed; E. faecium GJ40 produced the larger inhibitory zones (26-27 mm). Well-diffusion test results showed that the majority of the enterococci strains CFS had antimicrobial activity against the pathogenic microorganisms, especially on Gram negative indicators. Cell-free supernatant of E. faecium GJ40 was the one that produced the largest inhibition zones (14 to 21 mm) in the majority of the indicator microorganisms assayed. All supernatants treated with 10 N NaOH (pH6) showed no inhibitory effect on the indicator strain assayed. With respect to fungal inhibition, any of the CFS assayed significantly inhibited the Aspergillus strains growth. But, in general, all CFS reduced AFB1 production from 8 to 87%. The results demonstrate that enterococci isolated from healthy dog feaces produce substances with the capacity to inhibit some potential pathogenic bacteria growth and the capacity of inhibiting or reducing the AFB1 production in vitro.

Evaluation of Saccharomyces cerevisiae strains as probiotic agent with aflatoxin B1 adsorption ability for use in poultry feedstuffs.

In this study the aflatoxin B1 (AFB1) removal capacity, the tolerance to salivary and gastrointestinal conditions, autoaggregation and coaggregation with pathogenic bacteria of Saccharomyces cerevisiae strains isolated from broiler feces, were evaluated. Only four of twelve isolated strains were identified as Saccharomyces cerevisiae using molecular techniques. The results obtained in AFB1 binding studies indicated that the amount of AFB1 removed was both strain and mycotoxin-concentration dependent. Therefore, a theoretical model was applied in order to select the most efficient strain to remove AFB1 in a wide range of mycotoxin concentration. The results indicated that S. cerevisiae 08 and S. cerevisiae 01 strains were the most efficient microorganisms in the mycotoxin removal. Viability on simulated salivary and gastrointestinal conditions was investigated and S. cerevisiae 08 strain showed the best results, achieving 98% of total survival whereas S. cerevisiae 01 reached only 75%. Autoaggregation and coaggregation assays showed S. cerevisiae 08 as the most appropriate strain, mainly because it was the unique strain able to coaggregate with the four bacterial pathogens assayed. Consequently, S. cerevisiae 08 is the best candidate for future in vivo studies useful to prevent aflatoxicosis. Further quantitative in vitro and in vivo studies are required to evaluate the real impact of yeast-binding activity on the bioavailability of AFB1 in poultry. However, this study could be useful in selecting efficient strains in terms of AFB1 binding and provide an important contribution to research into microorganisms with potential probiotic effects on the host.

Analysis of fumonisin B1 removal by microorganisms in co-occurrence with aflatoxin B1 and the nature of the binding process.

The objectives of this investigation were to evaluate the ability of Saccharomyces cerevisiae CECT 1891 and Lactobacillus acidophilus 24 to remove fumonisin B(1) (FB(1)) from liquid medium; to determine the nature of the mechanism involved in FB(1)-microorganism interaction and to analyze whether the presence of aflatoxin B(1) (AFB(1)) interferes with the removal of FB(1) and vice versa. The results obtained indicated that: (i) both microorganisms were able to remove FB(1) from liquid medium; (ii) the removal was a fast and reversible process; (iii) cell viability was not necessary; (iv) the amount of FB(1) removed was both toxin- and microorganism concentration-dependent; (v) the process did not involve chemical modification of FB(1) molecules; and (vi) cell wall structural integrity of the microorganisms was required for FB(1) removal. Consequently, we propose that the mechanism involved in the removal of FB(1) is a physical adsorption (physisorption) of the toxin molecule to cell wall components of the microorganisms. It is highly probable that FB(1) and AFB(1) co-occur in contaminated foods, since the fungal genera Aspergillus and Fusarium frequently occur simultaneously. Therefore, we analyzed whether the presence of AFB(1) interferes with the removal of FB(1) by the microorganisms previously evaluated, and vice versa. Studies of co-occurrence of both mycotoxins clearly showed that they did not compete for binding sites on the microorganism cell wall and the presence of one toxin did not modify the efficiency of the organism in the removal of the other mycotoxin. These findings may be useful for optimization of mycotoxin binding and provide an important contribution to research on microorganisms with ability to remove these secondary metabolites.

Evaluation of aflatoxin B1 and ochratoxin A in interacting mixed cultures of Aspergillus sections Flavi and Nigri on peanut grains.

The influence of inoculum size on the colony-forming units, production of aflatoxin B1 (AFB1) and ochratoxin A (OTA) was determined when Aspergillus flavus and A. niger aggregate strains were cultured alone and in pairs on irradiated peanut grains at 28°C and 0.97 water activity (aW). The results showed a marked influence of inoculum factor on fungal counts, AFB1 and OTA production in single and paired cultures. Fungal counts of the A. niger aggregate strain in interacting cultures at 7, 14 and 21 days of incubation were significantly higher than those observed in the A. flavus strain, except in the mixed culture with 10(2) spores/ml of both strains. In all mixed culture assays, the AFB1 production was significantly reduced in comparison with the accumulation of mycotoxin in single cultures. A total inhibition in AFB1 production was observed in some interactions as 10(2) spores/ml of A. flavus and 10(3) spores/ml of A. niger aggregate strain at 7 and 14 days, among others. With regard to OTA production, a stimulation in the interacting cultures was observed at all inoculum sizes and incubation period. The highest levels of OTA accumulation were observed at 14 days for all interacting cultures. The maximum level was reach in the culture 10(3) spores/ml of A. niger aggregate and 10(4) spores/ml of A. flavus (p < 0.001). These results suggest that, under optimal environmental conditions in peanut grains, the interaction between A. flavus and A. niger aggregate strains could result in an inhibition of AFB1 and in a stimulation of OTA production.

Mycobiota and mycotoxins contamination in raw materials and finished feed intended for fattening pigs production in eastern Argentina.

The aim of the present study was to determine the mycobiota and natural levels of mycotoxins such as zearalenone, fumonisin B(1), aflatoxin B(1) and ochratoxin A present in raw materials and finished fattening pig feed. Samples were examined for total fungi and genera distribution. Zearalenone, FB(1), AFB(1) and OTA contamination were determined using high pressure liquid chromatography and thin layer chromatography. Milled maize and finished feed samples showed fungal contamination over than 1 × 10(4) CFU/g. All samples contained at least one of the main mycotoxigenic genera Aspergillus, Fusarium and Penicillium. A. flavus and F. verticillioides were the most prevalent species. Only some Aspergillus section Nigri strains from suckling pig to growing pig samples were able to produce OTA. A. flavus strains from milled maize, wheat bran, suckling pig to growing pig samples were able to produce AFB(1). All samples were positive for FB(1). Sucking pig, piglet, growing and boar feed samples showed ZEA natural contamination. AFB(1) and OTA contamination were not detected. There was a 100% correlation between FB(1) and ZEA contamination in sucking pig, piglet, growing and boar feed samples; 50% piglet samples and 67% suckling pig samples showed ZEA levels over the recommended limits. The present study has shown the occurrence of two mycotoxins, FB(1) and ZEA in feed intended for fattening pig consumption. In animal production, the simultaneous presence of toxicogenic fungi and low dietary levels of mycotoxins in field conditions can cause possible health impacts and lost performance in pigs from feeding spoiled feeds.

Surveillance of Aflatoxin and Microbiota Related to Brewer's Grain Destined for Swine Feed in Argentina.

Córdoba province in the center of Argentina is an important area of swine production. The use of industry by-product (brewer's grain) as feedstuff for swine is a regular practice and increases animal performance on these animals production. The occurrence of aflatoxin contamination is global, causing severe problems especially in developing countries. No reports on aflatoxin B(1) production, micoflora, and potential aflatoxin B(1) producing microorganism from brewer's grain are available. The aims of this study were (1) to isolate the microbiota species from brewer's grain, (2) to determine aflatoxin B(1) natural contamination levels, and (3) to determine the ability of Aspergillus section Flavi isolates to produce aflatoxins in vitro. Physical properties, total fungal counts, lactic acid bacteria, and fungal genera distribution were determined on this substrate. In 65% of the samples, fungal counts were higher than recommended by GMP, and lactic bacterium counts ranged from 1.9 × 10(5) to 4.4 × 10(9) CFU g(-1). Aspergillus spp. prevailed over other fungal genera. Aspergillus flavus was the prevalent species followed by A. fumigatus. Aflatoxin B(1) levels in the samples were higher than the recommended limits (20 ng g(-1)) for complementary feedstuffs. Several Aspergillus section Flavi strains were able to produce aflatoxin B(1) in vitro. Inadequate storage conditions promote the proliferation of mycotoxin-producing fungal species. Regular monitoring of feeds is required in order to prevent chronic and acute toxic syndromes related to this kind of contamination.

Comparative analysis of the mycobiota and mycotoxins contaminating corn trench silos and silo bags.

BACKGROUND: Silage is one of the most important feed sources for bovines. Mycotoxin contamination of feedstuffs is a worldwide concern. The aim of this study was to compare mycobiota and levels of aflatoxin B1 (AFB1), fumonisin B1 (FB1), deoxynivalenol (DON), zearalenone (ZEA) and patulin (PAT) in corn trench silos and silo bags. RESULTS: Dry matter was higher in trench silos. Counts varied from not detected to 108 CFU g⁻¹ in both trench silos and silo bags. Isolation frequencies of Aspergillus spp. and Fusarium spp. were higher in trench silos, whereas Penicillium spp. was higher in silo bags. Silo bags showed less diversity than trench silos. Strains isolated produced AFB1, FB1 and PAT. In trench silos, AFB1 was the only mycotoxin detected (1-160 µg kg⁻¹). In silo bags AFB1 levels varied from 5.8 to 47.4 µg kg⁻¹. DON was detected in two silo bag samples. CONCLUSION: When handling is adequate the reduction of mould and mycotoxin contamination in silo bags is considerable. This study will enable estimation of the mycotoxicological risk of different ensiling practices and determination of the most adequate method to minimize economic losses and reduce hazard to animal and human health.

Ochratoxin A in serum of swine from different Brazilian states.

The aims of the current study were to monitor the presence of ochratoxin A (OTA) in the serum of slaughtered swine and to investigate its distribution in 4 major geographical regions of Brazil. A total of 400 samples of serum were collected from 4 major states of Brazil (100 samples each). Ochratoxin A concentrations were determined by high-performance liquid chromatography. In Santa Catarina State, 60% of the samples had OTA concentrations ranging from 4.01 to 75.4 mg/l. In Mato Grosso State, 75% of the samples had OTA concentrations ranging from 0.17 to 46.79 mg/l. Bahia State samples had OTA concentrations ranging from 2.72 to 4.13 mg/l in 36% of the samples, whereas 68% of the samples from Rio de Janeiro State had OTA concentrations ranging from 0.16 to 115 mg/l. Only Santa Catarina State and Rio de Janeiro State had serum samples that exceeded 75 mg/l OTA in 20% and 2% of the samples, respectively. A direct relationship between the higher concentrations of OTA in serum from the States of Santa Catarina and Rio de Janeiro and the highest concentrations of OTA in food intended for animal consumption in the same 2 Brazilian states was found in the present study. Ochratoxin A distribution in foodstuffs is very heterogeneous, and an alternative method by which to monitor the presence of OTA in feed includes analyzing swine serum samples, which reflect the toxin content of the ingested feed. This strategy could prevent the occurrence of ochratoxicosis in animal production, reduce economic losses, and minimize hazards to human health.

Ecophysiology of Aspergillus section nigri species potential ochratoxin a producers.

After aflatoxins, ochratoxin A (OTA) is the most studied mycotoxin due to the toxicological significance in human and animal diets. OTA presence has been extensively reported worldwide in the last decade in several agricultural products. The main OTA producer in tropical and temperate climates is Aspergillus carbonarius followed by species belonging to A. niger aggregate. Currently, many scientists worldwide have studied the influence of water activity and temperature for growth and biosynthesis of OTA by these species on synthetic media. This article reviews ecophysiological studies of Aspergillus section Nigri strains on synthetic media and natural substrates. The results of these investigations suggest that significant amounts of OTA can be produced in only five days and that the use of different storage practices, such as a(W) and temperature levels below 0.930 and 15 °C, respectively, allow controlling fungal contamination and minimizing the OTA production in several products as peanuts, corn, dried grapes and derived products for human consumption.

An outbreak of acute aflatoxicosis on a chinchilla (Chinchilla lanigera) farm in Argentina.

Chinchillas (Chinehilla lanigera) are known to be very sensitive to aflatoxins, and often a large number of animals die if toxicosis occurs. An outbreak of acute aflatoxicosis on a chinchilla farm in Argentina is described in the present study. A commercial feed suspected of causing the death of 200 animals was sampled. Livers from 9 dead chinchillas were analyzed for their macroscopic and microscopic characteristics via necropsy and histopathology. Aflatoxins B(1), B(2), G(1), and G(2) were determined, by thin layer chromatography, to be in the feed. Macroscopic inspection of livers revealed general enlargement, pale-yellowish coloration, hypertrophy, rounded borders, and increased friability. Size and color were remarkably different from a healthy organ. Histopathologic analyses of hepatic parenchyma showed severe, diffuse cytoplasmic vacuolation of hepatocytes. Sudan III staining confirmed the presence of lipid within the vacuoles. The feed was positive for aflatoxin B(1) in quantities that exceeded the recommended levels. Histologic lesions were typical of aflatoxin intake. Monitoring feed for mycotoxins is crucial to prevent outbreaks of toxicosis, to improve management practices, and to diminish exposure risk of animals and humans to these harmful toxins.

Occurrence of ochratoxin A and ochratoxigenic mycoflora in corn and corn based foods and feeds in some South American countries.

Cereals and cereal- derived products constitute the base of human and animal feeding in South American countries. This review attempts to give an overview of the ochratoxin A (OTA) occurrence and potential sources of OTA contamination in those products. The environmental conditions as humidity and temperature in the colonization of the substrates by Aspergillus section Nigri isolated from corn kernels were also discussed. The available information on the ochratoxigenic mycoflora and OTA presence in corn, corn based food and feed is limited. Only few surveys have been carried out in Argentina, Ecuador and Brazil; which showed that Aspergillus niger aggregate and A. ochraceus species would be the main source of OTA. It's possible to emphasize that, the species A. carbonarius has not been isolated from these substrates and Penicillium verrucosum was isolated only from pig feeds of Argentinean samples in low percentage. Studies about the ecophysiology of ochratoxigenic fungi and OTA occurrence are in progress in Latin America to reduce the impact of this toxin in the food chain.

Mycobiota in poultry feeds and natural occurrence of aflatoxins, fumonisins and zearalenone in the Rio de Janeiro State, Brazil.

The intake of mycotoxin-contaminated feeds can lead to nutrient losses and may have adverse effects on animal health and on productivity. The aims of this study were (1) to determine the mycobiota present in poultry feed samples, and (2) to evaluate the natural occurrence of aflatoxin B(1), fumonisin B(1) and zearalenone. Fungal counts were similar between all culture media tested (10(3 )CFU g(-1)). The most frequent genus isolated was Penicillium spp. (41.26%) followed by Aspergillus spp. (33.33%) and Fusarium spp. (20.63%). High precision liquid chromatography was applied to quantify aflatoxin B(1) and fumonisin B(1). Thin layer chromatography was used to determine zearalenone levels. Aflatoxin B(1 )values ranged between 1.2 and 17.5 microg kg(-1). Fumonisin B(1) levels ranged between 1.5 and 5.5 microg g(-1). Zearalenone levels ranged between 0.1 and 7 microg g(-1). The present study shows the simultaneous occurrence of two carcinogenic mycotoxins, aflatoxin B(1) and fumonisin B(1), together with another Fusarium mycotoxin (zearalenone) in feed intended for poultry consumption. Many samples contained AFB(1 )levels near the permissible maximum and it could affect young animals. A synergistic toxic response is possible in animals under simultaneous exposure.

Occurrence of ochratoxin A-producing fungi in commercial corn kernels in Argentina.

Potentially ochratoxigenic Aspergillus and Penicillium species were identified and the natural occurrence of ochratoxin A (OTA) in corn kernels was evaluated. Likewise, the capacity to produce OTA by Aspergillus section Nigri and Circumdati was investigated. A total of 50 corn samples for human consumption was collected in the south of Córdoba Province. The surface-disinfected method for mycobiota determination was used. The OTA detection was performed by HPLC. OTA production was tested in strains belonging to section Nigri and Circumdati. Statistical analysis demonstrated that the specie A. flavus was isolated in higher frequency (p<0.01) from corn kernels in DRBC and DG18 media. The percentage of corn kernels contaminated by A. niger var. niger was similar in DRBC and DG18 media. The frequency of grains contaminated by A. flavus and A. niger var. awamori was higher than A. niger var. niger and A. japonicus var. japonicus (p<0.01) in DG18 media. The other potentially ochratoxigenic species, A. ochraceus, was isolated between 5% and 10% of the corn kernels in DG18 and DRBC media, respectively. The OTA producing species P. verrucosum was not isolated. All samples of corn were OTA negative (<1 ng g(-1)). Thirty strains (25%) of the black Aspergillus were OTA producers. From four strains of A. ochraceus isolated, only one produced OTA. Due to the storage variable conditions could not be adequate in this substrate, the presence of ochratoxigenic strains of section Nigri and OTA needs to be evaluated for a longer time to establish the toxicological risk for human beings. The contamination of stored corn kernels with A. flavus and Aspergillus section Nigri was significant.

Alternaria Mycotoxins in Sunflower Seeds: Incidence and Distribution of the Toxins in Oil and Meal.

A survey of 150 sunflower-seed samples was carried out to evaluate the contamination from infection with Alternaria alternata with alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA). A high percentage of the samples was contaminated with AOH (85%), AME, (47%), and TA (65%). The average levels detected were 187 µg/kg for AOH, 194 µg/kg for AME, and 6,692, µg/kg for TA. When sunflower seeds fermented by Alternaria alternata were processed under laboratory conditions to obtain the oil and meal, different distributions of Alternaria toxins between the oil and the meal were observed: whereas AOH, AME, and TA were detected in the meal, only AME and TA were detected in the oil, and the latter in a low percentage.