Binding of Aflatoxin B1 to Lactic Acid Bacteria and Saccharomyces cerevisiae in vitro: A Useful Model to Determine the Most Efficient Microorganism

Mycotoxins are toxic fungal metabolites found as contaminants in many agricultural products. Feeds contaminated with mycotoxins have a health risk to animals and, as a consequence, may cause big economical losses due to the low efficacy of animal husbandry (Richard, 2007). In addition, directly or indirectly (animal by-products) contaminated foods may also have a health risk to humans (CAST, 2003; Hussein & Brasel, 2001; Wild, 2007). Aflatoxins (AFs), a group of potent mycotoxins with mutagnic, carcinogenic, teratogenic, hepatotoxic and immunosupresive properties, are of particular importance because of their major occurrence and adverse effects on animal and human health, generalized as ?aflatoxicosis? (CAST, 2003; Hussein & Brasel, 2001; Magnoli et al., 2011). The AFs are produced by genus Aspergillus, mainly A. flavus, A. parasiticus and A. nomius, that grow on a variety of raw material during growth, harvest, storage and transportation of for example, the cereal used in the preparation of food and feed commodities (Ito et al., 2001; Kurtzman et al., 1987; Payne, 1998; Pereyra et al., 2010). The investigation of strategies to prevent the presence of AFs in foods, as well as, to eliminate, inactivate or reduce the bio-availability of these mycotoxins in contaminated products include physical, chemical, and biological methods (Bueno et al., 2001; CAST, 2003; Kabak et al., 2006). Limitations such as the loss of nutritional and sensory qualities of the product, the expensive equipment required for these techniques and the impossibility to guarantee the desired results, have allowed us to consider the hipothesis that foods and feeds can always be potentially contaminated with aflatoxins. For instance, in the poultry industry aflatoxin B1 (AFB1) is almost an unavoidable feed contaminant and levels from 0-200 ng/g have been reported (Dalcero et al., 1997). On the other hand, it is known that lactic acid bacteria (LAB) and some yeast, principally Saccharomyces cerevisiae, are capable to bind AFs in liquid media, apparently to cell wall components, polysaccharides and peptidoglycans of LAB (Haskard et al., 2001; Latinen et al., 2004) and glucomannans of yeast (Karaman et al., 2005; Raju & Devegowda 2000) and therefore could be used as potential mycotoxin decontaminating (Armando et al 2011; El-Nezami et al., 1998; Haskard et al., 2000, 2001; Hernandez-Mendoza et al., 2009; Lee et al., 2003; Peltonen et al., 2001; Shetty et al., 2007). The inclusion of appropriate microorganisms in the contaminated diet could prevent the absorption of mycotoxins during their passage in the gastrointestinal tract and eliminated in the faeces (Bueno et al., 2007; El-Nezami et al., 2000; Gratz et al., 2004; Gratz et al., 2007). Moreover, Kankaanpää et al. (2000) showed that the binding of AFB1 to the surface of LAB reduced their adhesive properties, and the accumulation of aflatoxins in the intestine may therefore be reduced via the increased excretion of an aflatoxin-bacteria complex. These considerations encouraged the recent emphasis on biological methods, but mainly focused on preventing AFs absorption in the gastrointestinal tract of the consumers, including these microorganisms in the diet and so prevent the aflatoxicosis effects. The first step in this direction is the selection of the most efficient microorganism for AFB1 removing and while many researchers have assayed LAB and yeast with AFB1 binding abilities (Ciegler et al., 1966; El-Nezami et al., 1998; Gourama & Bullerman, 1995; Haskard et al., 2001; Line et al., 1994; Oatley et al., 2000) no clear mechanism for this effect has been provided. Thus, this selection frequently is performed using a single concentration of AFB1, but we demonstrated that the microorganism efficiency may change when the mycotoxin concentration is modified (Bueno et al., 2007; Pizzolitto, 2011), therefore the microorganism selected could not be the most competent. In this context, we investigated the nature of the interaction between different microorganisms and AFB1 molecule, which led us to develop a model to explain the binding of AFB1 by LAB and Saccharomyces cerevisiae strains. This model allows an estimation of two important parameters related to a microorganism´s capacity for dietary decontamination: the number of binding sites for AFB1 in the surface microorganism (M) and the equilibrium constant of the process involved (Keq), both of them are useful in the selection of the most suitable microorganism in a wide range of AFB1 concentration (Bueno et al., 2007). In adittion, studies of viability of the microorganisms in the salivary and gastrointestinal tract, cell adhesion, autoaggregation, coaggregation and antimicrobial activity against pathogen strains, were also evaluated as a way to research potential beneficial properties on the host (Armando et al., 2011). Thus, in this chapter we describe the development and application of an in vitro methodology to evaluate the aflatoxin B1 binding ability, gastrointestinal tolerance and potential beneficial properties of Saccharomyces cerevisiae strains, useful to select the more appropriated microorganism to be assayed in expensive, complicated but necessary in vivo studies.Fil: Pizzolitto, Romina Paola. Universidad Nacional de Río Cuarto; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Bueno, Dante Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Armando, María Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; ArgentinaFil: Cavaglieri, Lilia Reneé. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Dalcero, Ana Maria. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Salvano, Mario Armando. Universidad Nacional de Río Cuarto; Argentin

Fungi and mycotoxins from pre and post storage brewer’s grain intended for bovine intensive-rearing

The aim of the study was to determine the mycobiota and natural levels of mycotoxins such as aflatoxin B1 (AFB1), ochratoxin A (OTA), fumonisin B1 (FB1) and deoxinivalenol (DON) present in brewers grains pre and post stored intended for bovine intensive-rearing. Post stored (80%) samples had counts higher than 1 x 104 colony-forming units (CFU/g). Cladosporium spp., and Aspergillus spp. were isolated at high frequencies. Aspergillus flavus was the prevalent isolated species. Pre stored (70%) and post stored (100%) samples showed AFB1 levels over the recommended limits (20 µg/Kg) and OTA levels were below the recommended limits (50 µg/Kg). While pre and post stored samples did not show FB1 and DON natural contamination levels. The presence of mycotoxins in this substrate indicates the existence of contamination. Regular monitoring of feeds is required in order to prevent chronic and acute toxic syndromes related to this kind of contamination.Fil: Keller, Luiz Antonio Moura. Universidade Federal Rural do Rio de Janeiro; Brasil. Conselho Nacional de Desenvolvimento Científico e Tecnológico; BrasilFil: Pereyra, Carina Maricel. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cavaglieri, Lilia Reneé. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Dalcero, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; ArgentinaFil: Rosa, C. A. R. Conselho Nacional de Desenvolvimento Científico e Tecnológico; Brasil. Universidade Federal Rural do Rio de Janeiro; Brasi

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 B1 production, micoflora, and potential aflatoxin B1 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 B1 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 × 105 to 4.4 × 109 CFU g−1. Aspergillus spp. prevailed over other fungal genera. Aspergillus flavus was the prevalent species followed by A. fumigatus. Aflatoxin B1 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 B1  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

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

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. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.Fil: Keller, Luiz Antonio Moura. Universidade Federal Rural do Rio de Janeiro; BrasilFil: Keller, Kelly Moura. Universidade Federal Rural do Rio de Janeiro; BrasilFil: Monge, Maria del Pilar. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigación en Micología y Micotoxicología. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación en Micología y Micotoxicología; ArgentinaFil: Pereyra, Carina Maricel. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alonso, Veronica Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; ArgentinaFil: Cavaglieri, Lilia Reneé. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Chiacchiera, Stella Maris. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigación en Micología y Micotoxicología. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación en Micología y Micotoxicología; ArgentinaFil: R Rosa, C. A.. Universidade Federal Rural do Rio de Janeiro; Brasi

Access to Research Veterinary Medicine International Volume

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

Saccharomyces cerevisiae como agente probiótico e possível adsorvente de aflatoxina B1 em condições simuladas do trato intestinal de peixes

The aim of this study was to evaluate in vitro the probiotic potential and absorption of Saccharomyces cerevisiae for the aflatoxin B1 in simulated fish intestinal tract conditions. Three yeast strains were used, two from brewery: S. cerevisiae RC1 and S. cerevisiae RC3 and one from a fish farming environment: S. cerevisiae A8L2. The selected yeasts were subjected to the following in vitro tests: homologous inhibition, self-aggregation, co-aggregation, antibacterial activity, gastrointestinal conditions tolerance and adsorption of AFB1. All S. cerevisiae strains showed good capability of self-aggregation and co-aggregation with pathogenic bacteria. All yeast strains were able to survive the gastrointestinal conditions. In acidic conditions, the factors (strain vs. time) had interaction (P=0.0317), resulting in significant variation among the strains tested in the time periods analyzed. It was observed that there was also interaction (P=0.0062) in intestinal conditions, with an increased number of cells in the 12-hour period for all strains tested. In the adsorption test, the A8L2 strain was statistically more effective (P<0.005) for both AFB1 concentrations evaluated in this study (10 and 25ng/mL). Thus, it was observed that the strains of S. cerevisiae have potential probiotic and adsorbent of AFB1.Objetivou-se, com esta pesquisa, avaliar in vitro o potencial probiótico e adsorvente de Saccharomyces cerevisiae para aflatoxina B 1 em condições simuladas do trato intestinal de peixes. Foram utilizadas três cepas de leveduras, sendo duas provenientes de cervejaria: S. cerevisiae RC1 e S. cerevisiae RC3, e uma de ambiente de piscicultura: S. cerevisiae A8L2. As leveduras selecionadas foram submetidas aos seguintes testes in vitro: inibição homóloga, autoagregação, coagregação, atividade antibacteriana, viabilidade às condições gastrointestinais e adsorção de AFB 1 . Todas as estirpes de S. cerevisiae mostraram boa capacidade de autoagregação e coagregação com bactérias patogênicas. Todas as estirpes de levedura foram capazes de sobreviver às condições gastrointestinais. Em condições ácidas, os fatores (cepa x tempo) tiveram interação (P=0,0317), resultando em variações significativas entre as cepas testadas nos períodos de tempo analisados. Observou-se que também houve interação (P=0,0062) em condições intestinais, havendo um aumento do número de células no período de 12h para todas as cepas avaliadas. No ensaio de adsorção, a estirpe A8L2 foi a mais eficaz estatisticamente (P<0,005), para as duas concentrações de AFB 1 avaliadas neste estudo (10 e 25ng. mL -1 ). Dessa forma, conclui-se que as cepas de Saccharomyces cerevisiae possuem potencial probiótico e adsorvente de AFB 1.Fil: Pinheiro, R. E. E.. Universidade Federal Do Piaui.; BrasilFil: Rodrigues de Campos, Ana Maria. Universidade Federal Do Piaui.; BrasilFil: Lima, Carlos Eduardo. Universidade Federal Do Piaui.; BrasilFil: Santos, J. T. O.. Universidade Federal Do Piaui.; BrasilFil: Pereyra, Carina Maricel. Universidad Nacional de Río Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; ArgentinaFil: Torres, Adriana Mabel. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigación en Micología y Micotoxicología. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación en Micología y Micotoxicología; ArgentinaFil: Cavaglieri, Lilia Reneé. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; ArgentinaFil: Lopes, J. B.. Universidade Federal Do Piaui.; BrasilFil: Muratori, M. C. S.. Universidade Federal Do Piaui.; Brasi

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.Fil: Pizzolitto, Romina P. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico, Químicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Armando, María R. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico, Químicas y Naturales. Departamento de Microbiología e Inmunología; ArgentinaFil: Combina, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cavaglieri, Lilia Reneé. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico, Químicas y Naturales. Departamento de Microbiología e Inmunología; ArgentinaFil: Dalcero, Ana María. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico, Químicas y Naturales. Departamento de Microbiología e Inmunología; ArgentinaFil: Salvano, Mario A. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico, Químicas y Naturales. Departamento de Biología Molecular; Argentin

Characterization of Aspergillus species based on fatty acid profiles

Submitted by Sandra Infurna ([email protected]) on 2018-07-10T12:22:53Z No. of bitstreams: 1 marioj_gatti_etal_IOC_2008.pdf: 340819 bytes, checksum: 0b1e15fe67438cea2db176e1aef202e5 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-07-10T12:40:21Z (GMT) No. of bitstreams: 1 marioj_gatti_etal_IOC_2008.pdf: 340819 bytes, checksum: 0b1e15fe67438cea2db176e1aef202e5 (MD5)Made available in DSpace on 2018-07-10T12:40:21Z (GMT). No. of bitstreams: 1 marioj_gatti_etal_IOC_2008.pdf: 340819 bytes, checksum: 0b1e15fe67438cea2db176e1aef202e5 (MD5) Previous issue date: 2008Universidade Federal Rural do Rio de Janeiro. Departamento de Microbiologia e Imunologia Veterinária. Seropédica, RJ, Brasil.Universidade Federal Rural do Rio de Janeiro. Departamento de Tecnologia de Alimentos. Seropédica, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Avaliação e Promoção da Saúde Ambiental. Rio de Janeiro, RJ, Brasil.Universidade Federal Rural do Rio de Janeiro. Departamento de Microbiologia e Imunologia Veterinária. Seropédica, RJ, Brasil.Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Rio Cuarto, Córodba, Argentina.Universidade Federal Rural do Rio de Janeiro. Departamento de Microbiologia e Imunologia Veterinária. Seropédica, RJ, Brasil.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