Chitosan-glucose derivative as effective wall material for probiotic yeasts microencapsulation.

A chitosan-glucose derivative (ChG) with lower antimicrobial activity against whey native probiotic yeast K. marxianus VM004 was synthesized by the Maillard reaction. The ChG derivative was characterized by FT-IR, 1H NMR, and SLS to determine the structure, deacetylation degree (DD), and molecular weight (Mw). In addition, we evaluated the antioxidant, cytotoxic, and antimicrobial activities of ChG. ChG was then used for microencapsulation of K. marxianus VM004 by spray drying. The microcapsules were characterized by evaluating their encapsulation yield, encapsulation efficiency, morphology, tolerance to the gastrointestinal tract, and viability during storage. The results indicated that a non-cytotoxic product with lower MW and DD and higher antioxidant activity than native chitosan was obtained by the Maillard reaction. The yeast ChG microcapsules exhibited an encapsulation efficiency >57 %, improved resistance to gastrointestinal conditions, and enhanced stability during storage. These results demonstrate that ChG may be a promising wall material for the microencapsulation of probiotic yeasts.

Fist report on fumagillin production by Aspergillus fumigatus sensu stricto gliotoxigenic strains recovered from raw cow milk and clinical samples in Argentina / Primer reporte en Argentina sobre la producción de fumagilina por cepas deAspergillus fumigatus sensu stricto productoras de gliotoxina, aisladas de lechecruda y de casos clínicos

Abstract In Argentina there are no reports on Aspergillus fumigatus fumagillin-producingstrains. In this study we describe the isolation and mycotoxin production capacity of ten A.fumigatus strains isolated from farm and clinical samples. Farm strains were isolated frommilk samples taken from dairy cows in Córdoba province, some of which were associated withsubclinical mastitis. A culture medium was defined to optimize fumagillin production and adetection method was developed by HPLC chromatography. It is known that in addition to thehost immune status, strain virulence is a fundamental characteristic that will determine itspathogenicity and, in this sense, fumagillin is considered to be among the virulence factors. Inthe present work, all the strains tested for the production of fumagillin were able to synthesizeit, highlighting that the strain A. fumigatus RC2243, from a milk sample from a cow with clinicalmastitis, was the most productive. The existence of fumagillin-producing strains represents apotential risk of mycotoxins being transferred to raw milk, constituting a public health risk.

Resumen En Argentina no existen reportes sobre cepas de Aspergillus fumigatus productoras de fumagilina. En este trabajo se describe el aislamiento y la producción de dicha micotoxina clínicaspor 10 cepas, provenientes del medioambiente rural y aisladas de muestras clínicas. Las cepasde origen rural fueron aisladas de vacas lecheras en tambos de la provincia de Córdoba, yalgunas de esas cepas se asociaron a casos de mastitis subclínica. Se definió la composición deun medio de cultivo para optimizar la producción de fumagilina y se desarrolló un método decromatografía HPLC para su determinación. Es conocido que, además del estado inmunitario delhuésped, la virulencia de la cepa es una de las características fundamentales que determinansu potencial patogénico y, en este sentido, la fumagilina es considerada un factor de virulencia. En el presente trabajo todas las cepas estudiadas fueron capaces de sintetizarla y la cepa A.fumigatus RC2243, proveniente de leche de una vaca con mastitis subclínica, se destacó comola cepa más productora. La existencia de cepas productoras de fumagillina representa un riesgopotencial por el pasaje de dicha micotoxina a la leche, lo cual constituye un problema para lasalud pública.

Fist report on fumagillin production by Aspergillus fumigatus sensu stricto gliotoxigenic strains recovered from raw cow milk and clinical samples in Argentina.

In Argentina there are no reports on Aspergillus fumigatus fumagillin-producing strains. In this study we describe the isolation and mycotoxin production capacity of ten A. fumigatus strains isolated from farm and clinical samples. Farm strains were isolated from milk samples taken from dairy cows in Córdoba province, some of which were associated with subclinical mastitis. A culture medium was defined to optimize fumagillin production and a detection method was developed by HPLC chromatography. It is known that in addition to the host immune status, strain virulence is a fundamental characteristic that will determine its pathogenicity and, in this sense, fumagillin is considered to be among the virulence factors. In the present work, all the strains tested for the production of fumagillin were able to synthesize it, highlighting that the strain A. fumigatus RC2243, from a milk sample from a cow with clinical mastitis, was the most productive. The existence of fumagillin-producing strains represents a potential risk of mycotoxins being transferred to raw milk, constituting a public health risk.

Fusarium species and moniliformin occurrence in sorghum grains used as ingredient for animal feed in Argentina.

BACKGROUND: A survey on Fusarium species and moniliformin (MON) occurrence in sorghum grains collected from one of the main sorghum-producing areas of Argentina was conducted. Also, growth of F. thapsinum, one of the main sorghum pathogens, and MON production under different water activity (aw ) conditions on a sorghum-based medium were determined. RESULTS: Infection of sorghum grains by Fusarium species ranged from 82.5 to 99%; closely related species F. verticillioides, F. thapsinum and F. andiyazi were the most frequently recovered, followed by F. proliferatum and F. subglutinans. By sequencing a portion of the translation elongation factor-1α (TEF-1α) gene and by maximum parsimony analysis, F. verticillioides and closely related species were identified as F. thapsinum, F. andiyazi and F. verticillioides. Species within the F. graminearum species complex (FGSC) were isolated in high frequency. Maximum growth rates of 12 F. thapsinum strains were obtained at 0.995 aw . All evaluated strains were able to produce MON at all aw values tested, but MON production was higher at 0.995-0.982 aw . MON was detected in 41% of the samples at levels ranging from 363.2 to 914.2 µg kg-1 . CONCLUSION: This study provides new data on the occurrence of Fusarium species in sorghum grains destined for animal consumption in Argentina. The production of MON at different aw values showed that the toxin can be produced under field conditions. The risk to livestock exposed to daily low levels of MON associated with the toxin occurrence in the sorghum grains analyzed is unknown. © 2018 Society of Chemical Industry.

Effect of prepubertal nutrition on cellular apoptosis and proliferation in at term placenta of Anglo-Nubian goats.

The aim of this study was to evaluate the effect of feed restriction with or without monensin supplementation, followed by a re-feeding period on cellular apoptosis and proliferation in at term placenta of Anglo-Nubian goats. To evaluate the induction of apoptosis through the intrinsic pathway, proteins Bax and Bcl-2 were determinated. The apoptosis was related with the cell proliferation indices through Ki67 determination. The treatments were applied for 250 days and were (a) ad libitum feeding (control; n = 5); (b) restricted feeding at 70% of control (restricted; n = 7); and (c) restricted with monensin supplementation (monensin; n = 7). After treatments, all the animals were fed to support their requirements. After parturition, 27 placentas were gathered. The placental cellular structure was studied by high-resolution light microscopy and transmission electron microscopy; the cellular proliferation was determined by Ki67 index, and Bax and Bcl-2 proteins were localized by immunohistochemical analysis. Differences in cell proliferation through the Ki67 index were found in monensin group placentas. Monensin supplementation stimulated the placental cell proliferation reversing the effect of feed restriction during the peripuberal period. A significant increase of Bcl-2 in placentas of restricted group was found, and it would provide a protective effect on the placental structure. A lack of the Bcl-2 protective effect was observed in control and monensin group placentas, probably meaning that the observed apoptosis would be induced through the intrinsic signalling pathway. A balance between apoptosis and cell proliferation is necessary to maintain tissue homoeostasis during caprine placental development.

Molecular characterization of Aspergillus fumigatus isolated from raw cow milk in Argentina: Molecular typing of A. fumigatus from raw cow milk.

Aspergillus fumigatus, the major etiological agent of human and animal aspergillosis, is a gliotoxinogenic species into section Fumigati commonly found in contaminated animal environments. In dairy herds, exposed areas of lactating cows, as mammalian glandule, can be easily contaminated by them. This study was aimed to identify A. fumigatus sensu lato strains (identified based on morphology) isolated from raw cow milk at species level, by morphological and molecular techniques, and to estimate their genetic variability. Forty-five A. fumigatus strains showed similar RAPD profiles (generated with PELF and URP1F primers) to each other and to A. fumigatus sensu stricto reference strains; also, they were almost identical to clinical human and feed-borne A. fumigatus strains included in the assay, since their similarity coefficient ranged from 0.7 to 1.00. Therefore, all strains were characterized as belonging to A. fumigatus sensu stricto species. This result was supported by sequencing the benA gene of selected strains and by maximum parsimony analysis. In addition, RAPD fingerprinting demonstrated intra-specific genetic variability into the A. fumigatus sensu stricto cluster. The results found in this study strengthen the fact that A. fumigatus sensu stricto is the predominant species in the Aspergillus section Fumigati found in animal environments such as dairy herd environments, while other species such as A. novofumigatus, A. fumigatiaffinis, A. udagawae and A. lentulus may be rarely isolated. Since no differences between animal and human strains were observed they can become pathogenic also for farm handlers'. Moreover, the presence of A. fumigatus sensu stricto in raw cow milk is probably a very important risk factor since milk and its by-products are generally indented for human consumption, then gliotoxin could be transferred to them.

Validation of a liquid chromatography/tandem mass spectrometry method for the detection of aflatoxin B1 residues in broiler liver.

Aflatoxin B1 is a carcinogenic and mutagenic mycotoxin produced mainly by Aspergillus flavus and Aspergillus parasiticus. It is the predominant mycotoxin found in raw materials used for the manufacture of broiler feeds. The aim of the present study was to develop a new and optimized method for the detection and quantification of aflatoxin B1 (AFB1) residues in broiler liver using solid phase extraction (SPE) clean-up and liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS) detection. The method was validated for linearity, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ). The validation parameters indicated satisfactory linearity (r2>0.99), accuracy and precision (4.57% intra-day RSD; 14.65% inter-day RSD) a very high recovery (99±13%) and high sensitivity achieved for AFB1 in animal samples (LOD=0.017 and LOQ=0.050ng/g). The method was effective for the detection and quantification of AFB1 residues in broiler liver and could also be potentially used for detecting AFB1 in other edible animal tissues after natural or experimental AFB1 exposure with high sensitivity and precision.

Encapsulated whey-native yeast Kluyveromyces marxianus as a feed additive for animal production.

Whey is the main byproduct of the cheese industry. While the composition is variable, it retains up to 55% of milk nutrients. The beneficial features of whey indicates a promising source of new potentially probiotic strains for the development of food additives destined for animal production. The aim of this study was to identify Kluyveromyces spp. isolated from whey, to study some probiotic properties and to select the best strain to be encapsulated using derivatised chitosan. Kluyveromyces marxianus strains (VM003, VM004 and VM005) were isolated from whey and identified by phenotypic and molecular techniques. These three yeast strains were able to survive under gastrointestinal conditions. Moreover, they exhibited weak auto-aggregation and co-aggregation with pathogenic bacteria (Salmonella sp., Serratia sp., Escherichia coli and Salmonella typhimurium). In general the K. marxianus strains had a strong antimicrobial activity against pathogenic bacteria. The potential probiotic K. marxianus VM004 strain was selected for derivatised-chitosan encapsulation. Material treated with native chitosan exhibited a strong antimicrobial activity of K. marxianus, showing a total growth inhibition at 10 min exposure. However, derivatised-chitosan encapsulation showed a reduced antimicrobial activity. This is the first study to show some probiotic properties of whey-native K. marxianus, in vitro. An encapsulation strategy was applied using derivatised chitosan.

Characterization and genetic variability of feed-borne and clinical animal/human Aspergillus fumigatus strains using molecular markers.

Aspergillus fumigatus, the major etiological agent of human and animal aspergillosis, is a toxigenic fungus largely regarded as a single species by macroscopic and microscopic features. However, molecular studies have demonstrated that several morphologically identified A. fumigatus strains might be genetically distinct. This work was aimed to apply PCR-restriction length fragment polymorphisms (PCR-RFLP) and random amplification of polymorphic DNA (RAPD) molecular markers to characterize a set of feed-borne and clinical A. fumigatus sensu lato strains isolated from Argentina and Brazil and to determine and compare their genetic variability. All A. fumigatus strains had the same band profile and those typical of A. fumigatus sensu stricto positive controls by PCR-RFLP. Moreover, all Argentinian and Brazilian strains typified by RAPD showed similar band patterns to each other and to A. fumigatus sensu stricto reference strains regardless of their isolation source (animal feeds or human/animal clinical cases) and geographic origin. Genetic similarity coefficients ranged from 0.61 to 1.00, but almost all isolates showed 78% of genetic similarly suggesting that genetic variability was found at intraspecific level. Finally, benA sequencing confirmed its identification as A. fumigatus sensu stricto species. These results suggest that A. fumigatus sensu stricto is a predominant species into Aspergillus section Fumigati found in animal environments as well as in human/animal clinical cases, while other species may be rarely isolated. The strains involved in human and animal aspergillosis could come from the environment where this fungus is frequently found. Rural workers and animals would be constantly exposed.

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.

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.

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.

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.

Characterization of Aspergillus species based on fatty acid profiles.

Cellular fatty acid (FA) composition was utilized as a taxonomic tool to discriminate between different Aspergillus species. Several of the tested species had the same FA composition and different relative FA concentrations. The most important FAs were palmitic acid (C16:0), estearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2), which represented 95% of Aspergillus FAs. Multivariate data analysis demonstrated that FA analysis is a useful tool for differentiating species belonging to genus Aspergillus. All the species analyzed showed significantly FA acid profiles (p < 0.001). Furthermore, it will be possible to distinguish among Aspergillus spp. in the Flavi Section. FA composition can serve as a useful tool for the identification of filamentous fungi.

Characterization of Aspergillus species based on fatty acid profiles

Cellular fatty acid (FA) composition was utilized as a taxonomic tool to discriminate between different Aspergillus species. Several of the tested species had the same FA composition and different relative FA concentrations. The most important FAs were palmitic acid (C16:0), estearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2), which represented 95 percent of Aspergillus FAs. Multivariate data analysis demonstrated that FA analysis is a useful tool for differentiating species belonging to genus Aspergillus. All the species analyzed showed significantly FA acid profiles (p < 0.001). Furthermore, it will be possible to distinguish among Aspergillus spp. in the Flavi Section. FA composition can serve as a useful tool for the identification of filamentous fungi.

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.