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.

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.

Bakery by-products based feeds borne-Saccharomyces cerevisiae strains with probiotic and antimycotoxin effects plus antibiotic resistance properties for use in animal production.

The aim of this study was to select S. cerevisiae strains able to exert probiotic and antimycotoxin effects plus antibiotics resistance properties for use in animal production. S. cerevisiae LL74 and S. cerevisiae LL83 were isolated from bakery by-products intended for use in animal feed and examined for phenotypic characteristics and nutritional profile. Resistance to antibiotic, tolerance to gastrointestinal conditions, autoaggregation and coaggregation assay, antagonism to animal pathogens and aflatoxin B1 binding were studied. S. cerevisiae LL74 and S. cerevisiae LL83 showed resistance to all the antibiotics assayed (ampicillin, streptomycin, neomycin, norfloxacin, penicillin G, sulfonamide and trimethoprim). The analysis showed that exposure time to acid pH had a significant impact onto the viable cell counts onto both yeast strains. Presence of bile 0.5% increased significantly the growth of the both yeast strains. Moreover, they were able to tolerate the simulated gastrointestinal conditions assayed. In general, the coaggregation was positive whereas the autoaggregation capacity was not observed. Both strains were able to adsorb AFB1. In conclusion, selected S. cerevisiae LL74 and S. cerevisiae LL83 have potential application to be used as a biological method in animal feed as antibiotic therapy replacement in, reducing the adverse effects of AFB1 and giving probiotic properties.

Avaliação in vitro da adsorção de aflatoxina B1 por produtos comerciais utilizados na alimentação animal / In vitro evaluation of aflatoxin B1 adsorption by commercial products used in animal feed

The aim of this study was to evaluate the aflatoxin B1 (AFB1) adsorption capacity, in vitro, by commercial products used in animal feed. Many studies are being conducted for the decontamination of aflatoxins in feed. The commercial products destined to fish feed that are available as probiotics and are formed by strains of bacteria and yeasts used in most mycotoxins adsorption assays. Three commercial products were studied: A, consisting of Bacillus subtilis, Bifidobacterium bifidum, Enterococcus faecium and Lactobacillus acidophilus; B, consisting of dry yeast of Saccharomyces cerevisiae from brewery; and C, consisting of Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus. Five suspensions of the maximum dose recommended by the manufacturer of each product (0; 25; 50; 75 and 100%) were tested against AFB1 (1000 ng.mL-1) in microtubes to determine the adsorption capacity. To simulate the pH of the stomach and intestine of the Nile tilapia (Oreochromis niloticus), phosphate buffered saline solutions (PBS) at pH 1.5 and 7.5, respectively, were formulated. Microtubes were introduced into a centrifuge with mechanical agitation at 37ºC for 1 h and then centrifuged for 10 min at 14.000 rpm; the supernatants were quantified by high-performance liquid chromatography. The commercial products in the maximum concentration were capable of adsorbing AFB1 in amounts from 45.01 to 129.59; from 123.90 to 215.59; and from 209.98 to 370.73 (ng.mL-1), respectively. It was concluded that all commercial products, which are added to animal feed, adsorbed AFB1 under simulated gastrointestinal pH conditions and are potential candidates for AFB1 adsorption for future in vivo studies.

Objetivou-se avaliar a capacidade de adsorção in vitro de aflatoxina B1 (AFB1) por produtos comerciais utilizados na alimentação animal. Muitas pesquisas estão sendo realizadas para a descontaminação de AFB1 em alimentos. Os produtos comerciais utilizados frequentemente na alimentação de peixes, disponíveis na forma de probióticos, são formados por cepas de bactérias e leveduras utilizadas na maioria dos ensaios de adsorção de micotoxinas. Foram utilizados três produtos comerciais: A, composto por Bacillus subtilis, Bifidobacterium bifidum, Enterococcus faecium e Lactobacillus acidophilus; B, por leveduras secas de Saccharomyces cerevisiae provenientes de cervejaria; e C, por Bacillus subtilis, Bacillus licheniformis e Bacillus pumilus. Cinco suspensões da dose máxima recomendada pelo fabricante de cada produto (0; 25; 50; 75 e 100%) foram testadas contra AFB1 (1000 ng.mL-1) em microtubos para determinação da capacidade de adsorção. Para simular o pH do estômago e do intestino de tilápias do Nilo (Oreochromis niloticus) foram formuladas soluções tampão fosfato salino (PBS), com pH 1,5 e 7,5; respectivamente. Os microtubos foram introduzidos em uma centrífuga com agitação mecânica, a 37ºC por 1 h e depois centrifugados por 10 min a 14.000 rpm; os sobrenadantes foram quantificados por cromatografia líquida de alta eficiência. Os produtos comerciais, nas concentrações máximas, foram capazes de adsorver AFB1 em quantidades de 45,01 a 129,59; 123,90 a 215,59 e 209,98 a 370,73 ng.mL-1, respectivamente. Concluiu-se que todos os produtos comerciais analisados adsorvem AFB1 em condições simuladas de pH gastrointestinal e são candidatos potenciais para adsorção de AFB1 para futuros ensaios in vivo.

Avaliação in vitro da adsorção de aflatoxina B1 por produtos comerciais utilizados na alimentação animal / In vitro evaluation of aflatoxin B1 adsorption by commercial products used in animal feed

Objetivou-se avaliar a capacidade de adsorção in vitro de aflatoxina B1 (AFB1) por produtos comerciais utilizados na alimentação animal. Muitas pesquisas estão sendo realizadas para a descontaminação de AFB1 em alimentos. Os produtos comerciais utilizados frequentemente na alimentação de peixes, disponíveis na forma de probióticos, são formados por cepas de bactérias e leveduras utilizadas na maioria dos ensaios de adsorção de micotoxinas. Foram utilizados três produtos comerciais: A, composto por Bacillus subtilis, Bifidobacterium bifidum, Enterococcus faecium e Lactobacillus acidophilus; B, por leveduras secas de Saccharomyces cerevisiae provenientes de cervejaria; e C, por Bacillus subtilis, Bacillus licheniformis e Bacillus pumilus. Cinco suspensões da dose máxima recomendada pelo fabricante de cada produto (0; 25; 50; 75 e 100%) foram testadas contra AFB1 (1000 ng.mL-1) em microtubos para determinação da capacidade de adsorção. Para simular o pH do estômago e do intestino de tilápias do Nilo (Oreochromis niloticus) foram formuladas soluções tampão fosfato salino (PBS), com pH 1,5 e 7,5; respectivamente. Os microtubos foram introduzidos em uma centrífuga com agitação mecânica, a 37ºC por 1 h e depois centrifugados por 10 min a 14.000 rpm; os sobrenadantes foram quantificados por cromatografia líquida de alta eficiência. Os produtos comerciais, nas concentrações máximas, foram capazes de adsorver AFB1 em quantidades de 45,01 a 129,59; 123,90 a 215,59 e 209,98 a 370,73 ng.mL-1, respectivamente. Concluiu-se que todos os produtos comerciais analisados adsorvem AFB1 em condições simuladas de pH gastrointestinal e são candidatos potenciais para adsorção de AFB1 para futuros ensaios in vivo.(AU)

The aim of this study was to evaluate the aflatoxin B1 (AFB1) adsorption capacity, in vitro, by commercial products used in animal feed. Many studies are being conducted for the decontamination of aflatoxins in feed. The commercial products destined to fish feed that are available as probiotics and are formed by strains of bacteria and yeasts used in most mycotoxins adsorption assays. Three commercial products were studied: A, consisting of Bacillus subtilis, Bifidobacterium bifidum, Enterococcus faecium and Lactobacillus acidophilus; B, consisting of dry yeast of Saccharomyces cerevisiae from brewery; and C, consisting of Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus. Five suspensions of the maximum dose recommended by the manufacturer of each product (0; 25; 50; 75 and 100%) were tested against AFB1 (1000 ng.mL-1) in microtubes to determine the adsorption capacity. To simulate the pH of the stomach and intestine of the Nile tilapia (Oreochromis niloticus), phosphate buffered saline solutions (PBS) at pH 1.5 and 7.5, respectively, were formulated. Microtubes were introduced into a centrifuge with mechanical agitation at 37ºC for 1 h and then centrifuged for 10 min at 14.000 rpm; the supernatants were quantified by high-performance liquid chromatography. The commercial products in the maximum concentration were capable of adsorbing AFB1 in amounts from 45.01 to 129.59; from 123.90 to 215.59; and from 209.98 to 370.73 (ng.mL-1), respectively. It was concluded that all commercial products, which are added to animal feed, adsorbed AFB1 under simulated gastrointestinal pH conditions and are potential candidates for AFB1 adsorption for future in vivo studies.(AU)

Potentiation of the effect of a commercial animal feed additive mixed with different probiotic yeast strains on the adsorption of aflatoxin B1.

This study potentiates the adsorbent effect for aflatoxin B1 (AFB1) of a commercial additive (CA) of animal feed, containing inactive lysate of three Saccharomyces cerevisiae strains, active enzymes, adsorbents and a selenium-amino acid complex, when the additive was mixed separately with three S. cerevisiae strains. Levels of AFB1 of 20 and 50 ng g(-1) were used to determine the binding capacity of different concentrations of CA alone and in the presence of yeast strains, as well as toxin desorption, under gastrointestinal conditions. The viability of yeasts in the presence of CA was evaluated. The results show that the CA did not affect the viability of the yeast strains assayed. CA alone showed a low percentage adsorption. At 20 and at 50 ng g(-1), CA was highly efficient in adsorbing AFB1 when combined with RC016 and RC012 strains respectively. Desorption of AFB1 by CA alone and in combination with the yeasts increased with increasing levels of CA. The results demonstrate the improvement of CA in AFB1 adsorption once it is mixed with live yeasts.

Efficacy of corn silage inoculants on the fermentation quality under farm conditions and their influence on Aspergillus parasitucus, A. flavus and A. fumigatus determined by q-PCR.

Laboratory-scale silos were prepared to evaluate the efficacy of two different lactic acid bacteria (LAB) on the fermentation quality and mycobiota of corn silage. Their influence on Aspergillus species' variability by using the q-PCR technique was studied. Silage inoculated with Lactobacillus rhamnosus RC007 or L. plantarum RC009 were compared with uninoculated silage. Silos were opened after 1, 7, 45, 90 and 120 days after ensiling. At the end of the ensiling period, silos were left open for 7 days to evaluate aerobic stability. Rapid lactic acid production and decline in pH values were seen in the early stages of fermentation in silage inoculated with L. rhamnosus RC007. After aerobic exposure, a significant decline in lactic acid content was observed in untreated and L. plantarum RC009-inoculated silages. Counts for yeasted and toxigenic fungus remained lower, after aerobic exposure, in L. rhamnosus RC007-inoculated silage, in comparison with L. plantarum RC009 and uninoculated silages. Comparing the influence exerted by both BAL, it was observed that L. rhamnosus RC007 was more efficient at inhibiting the three fungal species tested whose DNA concentrations, determined by q-PCR, oscillated near the initial value (pre-ensiling maize). The ability of L. rhamnosus RC007 to produce lactic acid rapidly and the decline in pH values in the early stages of the fermentation along with the reduction of yeast and mycotoxicogenic fungus after aerobic exposure shows its potential as a bio-control inoculant agent in animal feed.

Inhibitory effect of sodium metabisulphite and chlorine on growth of Aspergillus spp. and Penicillium spp. strains isolated from marine shrimp / Efeito inibitório do metabissulfito de sódio e do cloro sobre o crescimento de cepas de Aspergillus spp. e Penicilium spp. isoladas de camarão marinho

The sodium metabisulphite (SMB) is used in shrimp farming to prevent melanosis and the 5.0 ppm chlorine (CL) concentration used in the shrimp processing is efficient as a bactericide, but there is no evidence of the effectiveness of these chemical compounds as fungicides. Therefore, the aim of this study was to evaluate the in vitro effect of sodium metabisulphite (SMB) and chlorine (CL) on the growth of Aspergillus and Penicillium species isolated from marine shrimp in different stages of processing. The samples were collected from a frozen shrimp processing industry, located in Piauí State, Brazil. The total fungi and occurrence of Aspergillus and Penicillium species were evaluated. For in vitro sensibility test using the diffusion disk in agar method, five concentrations of SMB (0%, 1%, 3%, 5% and 10%) and six of CL (0, 1, 2, 3, 4 and 5 µg mL-1) were used. The fungal counts in the different processing stages ranged from 1.74 to 3.38 CFU g-1. Twenty-nine Aspergillus strains were isolated, prevailing A. versicolor (59.3%) and twenty-two of Penicillium, prevailing P. citrinum (74%). One strain of A. flavus was AFB1 producer. All the isolated strains of P. citrinum produced citrinin. All tested species were in vitro sensitive to 3% of SMB, except the A. flavus. The 10% concentration of SMB inhibited the in vitro growth of all strains. The CL concentrations tested did not inhibit the studied species growth and SMB concentrations above 3.0% inhibited in vitro the growth of the tested strains.

O metabissulfito de sódio (SMB) é usado na carcinicultura para evitar a melanose e a concentração de 5,0ppm de cloro (CL), utilizada no beneficiamento do camarão, é eficiente como bactericida, porém não há comprovação da eficácia destes compostos químicos como fungicida. Desse modo, o objetivo deste estudo foi avaliar o efeito in vitro do metabissulfito de sódio (SMB) e cloro (CL) sobre o crescimento de espécies de Aspergillus e Penicillium isolados de camarão marinho em diferentes estágios de processamento. As amostras foram coletadas de uma indústria de processamento de camarão congelado, localizada no Estado do Piauí, Brasil. Fungos totais e ocorrência das espécies de Aspergillus e Penicillium foram avaliados. Para o teste in vitro de sensibilidade pelo método disco-difusão em ágar, foram utilizadas cinco concentrações de SMB (0%, 1%, 3%, 5% e 10%) e seis de CL (0, 1, 2, 3, 4 e 5µg mL-1). As contagens fúngicas nos diferentes estágios de processamento variaram de 1,74 a 3,38UFC g-1. Foram isoladas 29 cepas de Aspergillus, prevalecendo o A. versicolor (59,3%) e 22 de Penicillium, prevalecendo o P. citrinum (74%). Uma cepa de A. flavus era produtora de AFB1. Todas as cepas de P. citrinum isoladas produziram citrinina. Todas as espécies testadas foram sensíveis in vitro a 3% do SMB, com exceção do A. flavus. A concentração de 10% do SMB inibiu in vitro o crescimento de todas as cepas. As concentrações de CL testadas não inibiram o crescimento das espécies isoladas e concentrações de SMB acima de 3,0% inibiram in vitro o crescimento das linhagens testadas.

Qualidade microbiológica de castanhas de caju (Anacardium occidentale, L) industrializadas e processadas artesanalmente / Study on the microbiological quality of industrialized and handmade cashew nuts (Anacardium occidentale L)

O objetivo deste estudo foi avaliar a qualidade microbiológica de castanhas industrializadas e das castanhas artesanalmente processadas, comercializadas por ambulantes em Teresina (PI). Foram coletadas 40 amostras de castanhas, sendo 21 amostras de castanhas industrializadas de três marcas (“A”, “B” e “C”) e 19 amostras de castanhas processadas artesanalmente (“D”), nas quais foram realizadas a determinação de coliformes a 35 °C e a 45 °C (NMP/g), a pesquisa de Salmonella spp. e a contagem de fungos. As amostras da marca “D” apresentaram maiores valores de coliformes a 35 °C (1,16 × 101 NMP/g); para coliformes a 45 °C, foram detectados valores de 7,0 NMP/g, e de 1,22 × 102 UFC/g para fungos e leveduras. Nas amostras da marca “A”, os valores para coliformes a 35 °C e 45 °C foram de 4,0 NMP/g e, para fungos e leveduras,de 1,0 × 102 UFC/g. Foram isoladas 43 cepas fúngicas. Do gênero Aspergillus, houve maior prevalência da espécie Aspergillus niger agregados (64,7%), e as espécies P. corylophillum (33,3%) e P. citrinum (29,2%) do gênero Penicillium. As amostras de castanhas industrializadas e processadas artesanalmente apresentaram condições higiênico-sanitárias satisfatórias e de acordo com a legislação vigente.

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.

Fungi and mycotoxins in feed intended for sows at different reproductive stages in Argentina.

The aim of this study was to evaluate fungi and contamination levels of aflatoxin B(1), ochratoxin A, fumonisin B(1), and zearalenone in raw materials and finished feed intended for sows at different reproductive stages. Total fungi, Aspergillus, Penicillium, and Fusarium species occurrence, were examined. Aspergillus flavus, A. niger aggregate spp., and F. verticillioides were the prevalent species. Fungal counts exceeded the levels proposed as feed hygienic quality limits (1 x 10(4) colony forming units) at all reproductive stages. Aflatoxin B(1), ochratoxin A, fumonisin B(1), and zearalenone were detected by high-pressure liquid chromatography. Aflatoxin levels in 80% samples of finished sow feeds were over the permitted levels of 0.02 mug g(-1) (mean 228.2 +/- 95 mug Kg(-1)). Fumonisin B(1) was detected in all tested raw materials at levels that varied from 50.3 to 1137.64 mug Kg(-1) and finished feed samples at levels that ranged from 99.8 to 512.4 mug Kg(-1). Aflatoxin B(1), zearalenone, and ochratoxin A were not detected in raw materials. All finished feeds were negative for zearalenone contamination whereas all nonpregnant gilt samples were contaminated with low OTA levels (mean 0.259 +/- 0.123). This fact requires periodic monitoring to prevent the occurrence of mycotoxicosis in animal production, to reduce the economic losses, and to minimize hazards to human health.