Ameliorative effects of Lactobacillus against Aflatoxin B1 / Efeitos benéficos do Lactobacillus contra a Aflatoxin B1

Abstract Lactobacilli are probiotics with Aflatoxin (AF) detoxification ability, found in fermented products, GIT of animals and environment. Purpose of this study was to investigate the ability of broiler isolates of Lactobacillus against Aflatoxin B1 (AFB1). For this purpose, 5 isolates of Lactobacillus from broiler gut were incubated with 100 ppb AFB1 in aqueous environment and effect of different parameters (cell fractions, time, temperature, pH) on detoxification was determined by HPLC. The ameliorative effect of Lactobacillus salivarius (LS) against AFB1 was studied in broiler. The results revealed that LS (CR. 4) showed the best results (in vitro) as compared to other isolates (L. salivarius (CR. 3, CR, 4), L. agilis (CE. 2.1, CE. 3.1) and L. crispatus (CE. 28). Cell debris of CR. 4 showed significantly higher detoxification (P<0.05). Maximum amount of AFB1 was detoxified at 30°C (97%), pH 4.0 (99%) and 6 h (99.97%). In vivo study showed that AFB1 decreased weight gain (1,269 ± 0.04 gm/ bird), feed consumed (2,161 ± 0.08 gm/ bird), serum total protein (2.42 ± 0.34 gm/ dl), serum albumin (0.5 ± 0.2 2 gm/dl) and antibody titer (4.2 ± 0.83). Liver function enzymes were found (alanine transaminase (ALT): 32 ± 10.7 U/L) and aspartate transaminase (AST): 314.8 ± 27 U/L) elevated in AFB1 fed broilers. Treatment with 1% LS not only decreased the toxic effects of AFB1 (group D) but also improved the overall health of broilers due to its probiotic effects (p<0.05) as compared to control negative (group A). The detoxification ability of LS was better than commercial binder (CB) (0.2% Protmyc). It was concluded that detoxification of AFB1 by Lactobacillus was strain, temperature, pH and time dependent. LS has detoxification ability against AFB1 in vivo.

Resumo Os lactobacilos são probióticos com capacidade de desintoxicação da Aflatoxina (AF), encontrados em produtos fermentados, TGI de animais e meio ambiente. O objetivo deste estudo foi investigar a capacidade de isolados de frango de corte de Lactobacillus contra a Aflatoxina B1 (AFB1). Para tanto, 5 isolados de Lactobacillus de intestino de frango foram incubados com 100 ppb AFB1 em meio aquoso, e o efeito de diferentes parâmetros (frações celulares, tempo, temperatura, pH) na desintoxicação foi determinado por CLAE. O efeito melhorador de Lactobacillus salivarius (LS) contra AFB1 foi estudado em frangos de corte. Os resultados revelaram que LS (CR. 4) apresentou os melhores resultados (in vitro) em comparação com outros isolados [L. salivarius (CR. 3, CR. 4), L. agilis (CE. 2.1, CE. 3.1) e L. crispatus (CE. 28)]. Detritos celulares de CR. 4 mostraram desintoxicação significativamente maior (P < 0.05). A quantidade máxima de AFB1 foi desintoxicada a 30 °C (97%), pH 4.0 (99%) e 6 h (99,97%). O estudo in vivo mostrou que AFB1 diminuiu o ganho de peso (1,269 ± 0.04 g / ave), alimento consumido (2,161 ± 0.08 g / ave), proteína total sérica (2.42 ± 0.34 g / dl), albumina sérica (0.5 ± 0.22 gm / dl) e título de anticorpo (4.2 ± 0.83). As enzimas da função hepática foram encontradas (alanina transaminase (ALT): 32 ± 10.7 U / L) e aspartato transaminase (AST): 314.8 ± 27 U / L) elevadas em AFB1 alimentados com frangos. O tratamento com 1% LS não só diminuiu os efeitos tóxicos de AFB1 (grupo D), mas também melhorou a saúde geral dos frangos devido aos seus efeitos probióticos (p < 0.05) em comparação com o controle negativo (grupo A). A capacidade de desintoxicação do LS foi melhor do que o aglutinante comercial (CB) (0.2% Protmyc). Concluiu-se que a desintoxicação de AFB1 por Lactobacillus foi dependente da cepa, temperatura, pH e tempo. LS tem capacidade de desintoxicação contra AFB1 in vivo.

Contamination status and exposure risk of mycotoxins in Coicis Semen / 中国中药杂志

By investigating the contamination status and predicting the exposure risk of mycotoxin in Coicis Semen, we aim to provide guidance for the safety supervision of Chinese medicinal materials and the formulation(revision) of mycotoxin limit standards. The content of 14 mycotoxins in the 100 Coicis Semen samples collected from five major markets of Chinese medicinal materials in China was determined by UPLC-MS/MS. The probability evaluation model based on Monte Carlo simulation method was established after Chi-square test and One-way ANOVA of the sample contamination data. Health risk assessment was performed on the basis of margin of exposure(MOE) and margin of safety(MOS). The results showed that zearalenone(ZEN), aflatoxin B_1(AFB_1), deoxynivalenol(DON), sterigmatocystin(ST), and aflatoxin B_2(AFB_2) in the Coicis Semen samples had the detection rates of 84%, 75%, 36%, 19%, and 18%, and the mean contamination levels of 117.42, 4.78, 61.16, 6.61, and 2.13 μg·kg~(-1), respectively. According to the limit standards in the Chinese Pharmacopoeia(2020 edition), AFB_1, AFs and ZEN exceeded the standards to certain extents, with the over-standard rates of 12.0%, 9.0%, and 6.0%, respectively. The exposure risks of Coicis Semen to AFB_1, AFB2, ST, DON, and ZEN were low, while 86% of the samples were contaminated with two or more toxins, which needs more attention. It is suggested that the research on the combined toxicity of different mycotoxins should be strengthened to accelerate the cumulative exposure assessment of mixed contaminations and the formulation(revision) of toxin limit standards.

The synergism of hepatitis B and aflatoxicosis in hepatocellular carcinoma development: A mini-review

Hepatocellular carcinoma (HCC) is the twelfth most common cancer and the fifth leading cause of worldwide cancer related death. Chronic hepatitis B infection, caused by the hepatitis B virus (HBV) and exposure to aflatoxins is fundamental in the formation of HCC in developing countries. This review of scientific publications aims to establish the detrimental effects of aflatoxin-contaminated foods and highlights the correlation between aflatoxin and hepatitis B viral-associated hepatocellular carcinoma. Research has shown a significant increase in the occurrence of HCC in HBV-infected individuals exposed to fungal toxins. HBV demonstrates the ability to integrate and bind to p53 protein in the host DNA and propagate hepatocyte vulnerability through carcinogenic aflatoxin B1 (AFB1) damage. Although there has been clear evidence about the synergistic interaction of exposure to AFB1 and HBV infection in the induction of HCC, other literature has shown otherwise, mainly because incomplete and vague findings and hypotheses were made in regions where AFB1 and HBV pose a public health risk. Vaccination against hepatitis B and measures such as robust food safety systems to avoid hepatotoxicity and hepatocellular carcinogenesis induced by AFB1 is the most effective methods in the prevention of HCC induced by HBV and AFB1

Rapid detection of aflatoxin B_1 in Chinese herbal medicines by indirect and direct competitive enzyme-linked immunosorbent assays: a comparative analysis / 中国中药杂志

The indirect competitive enzyme-linked immunosorbent assay(ic-ELISA) and direct competitive enzyme-linked immunosorbent assay(dc-ELISA) were performed for the rapid detection of aflatoxin B_1(AFB_1) in Astragali Radix, Zingiberis Rhizoma Recens, Jujubae Fructus, and Nelumbinis Semen with self-made antigens and antibodies. Different extraction methods were investigated to reduce the matrix effects of different medicinal parts in Chinese herbal medicines. The sensitivity of dc-ELISA method was improved by optimizing the molar ratio of AFB_1 to horseradish peroxidase(HRP). In this study, the sensitivity(IC_(50)) of ic-ELISA and dc-ELISA was 0.046 and 0.023 ng·mL~(-1), with the limit of detection(LOD) of 0.007 and 0.004 ng·mL~(-1), respectively. The detection time was 3 h and 50 min for ic-ELISA and dc-ELISA, respectively. The recovery rates were within the range of 62.96%-104.4%, with RSDs of less than 10%. Confirmed by LC-MS/MS, three positive samples of Nelumbinis Semen were detected from 53 samples. Two ELISA methods established in this study were accurate, rapid and sensitive, and can be used for rapid screening of AFB_1 in Chinese herbal medicines such as Astragali Radix, Zingiberis Rhizoma Recens, Jujubae Fructus, and Nelumbinis Semen. In addition, the advantages and limitations of the two methods were compared and discussed, which can provide a reference for the testing institutions to choose the proper method.

Saccharomyces cerevisiae as a probiotic agent and a possible aflatoxin B1 adsorbent in simulated fish intestinal tract conditions / 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.(AU)

Objetivou-se, com esta pesquisa, avaliar in vitro o potencial probiótico e adsorvente de Saccharomyces cerevisiae para aflatoxina B1 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 AFB1. 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 AFB1 avaliadas neste estudo (10 e 25ng. mL-1). Dessa forma, conclui-se que as cepas de Saccharomyces cerevisiae possuem potencial probiótico e adsorvente de AFB1.(AU)

Biopanning the mimotopes of aflatoxin B1 and their immunogenicity / Panning de mimotopo aflatoxina B1 e sua imunogenicidade

Utilizando um anticorpo monoclonal contra a aflatoxina B1 (AFB1) como ligante, foi identificado um mimotopo específico de aflatoxina B1 após se realizarem quatro ciclos de seleção biológica de 7-peptídeos aleatórios em biblioteca de fago exibida. O mimotopo é denominado P10, e sua sequência de aminoácidos é YRRHEKD. O soro imunológico de ratos Balb/c imunizados com P10 foi especificamente ligado à aflatoxina B1-albumina, indicando que o anticorpo era específico ao AFB1. Esses resultados sugerem que é possível desenvolver a vacina baseada em mimotopo associado à toxina.(AU)

Protective Effect of Procyanidin B2 on Acute Liver Injury Induced by Aflatoxin B in Rats / 生物医学与环境科学（英文）

Objective@#This study aimed to explore the protective effect of procyanidin B2 (PCB2) on acute liver injury induced by aflatoxin B (AFB ) in rats.@*Methods@#Forty Sprague Dawley rats were randomly divided into control, AFB , AFB + PCB2, and PCB2 groups. The latter two groups were administrated PCB2 intragastrically (30 mg/kg body weight) for 7 d, whereas the control and AFB groups were given the same dose of double distilled water intragastrically. On the sixth day of treatment, the AFB and AFB + PCB2 groups were intraperitoneally injected with AFB (2 mg/kg). The control and PCB2 groups were intraperitoneally administered the same dose of dimethyl sulfoxide (DMSO). On the eighth day, all rats were euthanized: serum and liver tissue were isolated for further examination. Hepatic histological features were assessed by hematoxylin and eosin-stained sections. Weight, organ coefficient (liver, spleen, and kidney), liver function (serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, and direct bilirubin), oxidative index (catalase, glutathione, superoxide dismutase, malondialdehyde, and 8-hydroxy-2'-deoxyguanosine), inflammation factor [hepatic interleukin-6 (IL-6) mRNA expression and serum IL-6], and bcl-2/bax ratio were measured.@*Results@#AFB significantly caused hepatic histopathological damage, abnormal liver function, oxidative stress, inflammation, and bcl-2/bax ratio reduction compared with DMSO-treated controls. Our results indicate that PCB2 treatment can partially reverse the adverse liver conditions induced by AFB .@*Conclusion@#Our findings indicate that PCB2 exhibits a protective effect on acute liver injury induced by AFB .

Preparation of highly sensitive monoclonal antibody against aflatoxin B_1 and its application in rapid detection of contamination in Ziziphi Spinosae Semen / 中国中药杂志

A highly sensitive monoclonal antibody against aflatoxin B_1(AFB_1) was prepared and an indirect competition enzyme-linked immunosorbent assay(ic-ELISA) was established based on the antibody which was used for high-throughput and rapid screening of AFB_1 contamination in Chinese herbal medicines to ensure the safety of medication. In this study, the structure of AFB_1 was modified by improved oxime method, and the carrier protein was coupled by EDC-NHS method to obtain the complete antigen of AFB_1, which was more convenient and environmental friendly. The Balb/c female mice were immunized using increasing the immunization dose and various ways of injection, and finally the AFB_1 monoclonal antibody was prepared. The AFB_1 monoclonal antibody belongs to IgG_(2 b) immunoglobulin by identifying its immunological characteristics, and its sensitivity(IC_(50)) can reach 0.15 μg·L~(-1), and the affi-nity is 2.81×10~8 L·mol~(-1). The cross-reaction rates of AFB_2, AFG_1, and AFG_2 were 35.07%, 8.75%, and 1.15%, respectively, and there was almost no cross-reactivity with other mycotoxins. Based on the high sensitivity and specificity of the antibody, an ic-ELISA method was established and applied to the determination of AFB_1 contamination in Ziziphi Spinosae Semen. According to the matrix matching standard curve, the linear concentration range for AFB_1 was 0.05-0.58 μg·L~(-1)(R~2=0.992), the recoveries were 88.00%-119.0%, and the detection limit was 1.69 μg·kg~(-1). The AFB_1 in 33 batches of Ziziphi Spinosae Semen samples was determined by ic-ELISA, and the contamination level was 3.62-206.58 μg·kg~(-1). The linear correlation coefficient between the detection results of ic-ELISA and UHPLC-MS/MS was 0.996, and there were no false positive and false negative cases. It indicates that the established ic-ELISA is accurate and reliable, and could provide a simple and effective technique for fast screening of AFB_1 contamination in Ziziphi Spinosae Semen, and also could be considered as the reference for the detection and monitoring of AFB_1 contamination in other Chinese herbal medicines.

Anti-injury effect of hydrogen-enriched water in a rat model of liver injury induced by aflatoxin B / 生理学报

The purpose of this study was to investigate the anti-injury effect and protective mechanism of hydrogen-enriched water in a rat model of acute liver injury induced by aflatoxin B (AFB). Healthy male Sprague-Dawley (SD) rats were randomly divided into control group, model group (AFB group) and hydrogen-enriched water treatment group (AFB+H group). The rat model of acute liver injury induced by AFB was established by single intragastric administration of AFB (2.0 mg/kg), and then the rats were treated with hydrogen-enriched water intragastrically. HE staining was used to observe the pathological changes of liver tissue. Blood samples were taken from vena cava to measure serum liver function indexes. Live tissue was sampled to detect malondialdehyde (MDA) and reduced glutathione (GSH) contents. Western blot was used to detect phosphorylation levels of MAPK signaling pathway proteins (ERK, JNK and p38 MAPK). The results showed that, compared with the AFB group, the AFB+H group exhibited increased body weights, alleviated acute liver injury, decreased activities of serum glutamic-pyruvic transaminase and glutamic oxaloacetic transaminase, as well as total bilirubin level in the serum. Meanwhile, hydrogen-enriched water decreased MDA content and increased GSH content in liver tissue. AFB-increased phosphorylation levels of ERK, JNK and p38 MAPK in liver tissue were down-regulated significantly by hydrogen-enriched water treatment. These results suggest that hydrogen-enriched water can alleviate liver injury induced by AFB, and its mechanism may be related to the reduction of oxidative stress and the inhibition of MAPK signal transduction pathway activation.

Validation of a liquid chromatography/tandem mass spectrometry method for the detection of aflatoxin B1 residues in broiler liver / Validación de una técnica para detectar aflatoxina B1 en hígado de pollo por cromatografía líquida de alta performance acoplada a espectrómetro de masas en tándem

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= 0.050 ng/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.

La aflatoxina B1 (AFB1) es una micotoxina carcinogénica y mutagénica producida principalmente por Aspergillus flavus y Aspergillus parasiticus. Es la principal toxina que contamina las materias primas utilizadas para la elaboración de alimentos balanceados destinados a la alimentación de pollos parrilleros. El objetivo de este trabajo fue desarrollar un método nuevo y optimizado para detectar y cuantificar bajos niveles de AFB1 en hígado de pollo, usando limpieza por extracción en fase sólida (SPE) y cromatografía líquida acoplada a detección por espectrometría de masa en tándem con ionización por electrospray (LC-ESI-MS/MS). Se validaron la linealidad, la exactitud, la precisión, el límite de detección (LOD) y el límite de cuantificación (LOQ). El método resultó tener linealidad (r²>0,99), exactitud y precisión muy satisfactorias (4,57% RSD intradía; 14,65% RSD interdía), un alto porcentaje de recupero (99 ± 13%) y la sensibilidad más alta lograda para la detección de AFB1 en muestras de origen animal (LOQ=0.050 ng/g y LOD = 0.017). El método fue muy efectivo para detectar y cuantificar bajos niveles de AFB1 en hígados de pollos parrilleros. Este método podría potencialmente utilizarse para la detección de esta toxina en otros tejidos y subproductos de origen animal luego de su exposición a AFB1 con una mayor sensibilidad y precisión.

In vivo amelioration of aflatoxin B1 in broiler chicks by magnetic carbon nanocomposite

In this study an Iron oxide/carbon nanocomposite from maize straw was prepared and was characterized by XRD, SEM, EDX, FTIR, TG/DTA and Surface area analyzer. The adsorbent was fed to different groups of poultry birds along with aflatoxin B1. Different physiological and blood parameters were monitored in order to study the efficacy of the prepared adsorbent for binding of aflatoxin B1 in the gastrointestinal tract of chickens. It was found that adsorbent at dose of 0.3%/ kg feed was highly effective in detoxifying aflatoxin B1 in gastrointestinal tract of poultry birdswith no harmful effects. The high doses given to groups E and F; 0.4% and 0.5% respectively showed slight variation in tested parameters from group A. No negative symptoms associated with the use of activated carbon as previously reported were observed for the adsorbent under study.(AU)

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)

Effect of Saccharomyces cerevisiae yeast on ruminal detoxification of aflatoxin B[1]

Background: Yeasts are microorganisms that have the ability to absorb aflatoxins

Objectives: The effect of the yeast Saccharomyces cerevisiae [PTCC 5177] on aflatoxin B] detoxification and absorption of toxin in in vitro [the cow rumen] was investigated

Methods: For this purpose, the yeast used in various treatments [live-treated, autoclave, heat-treated, treated with acid 100 °C] was prepared and added to the rumen of cattle. Af-latoxin E in different doses [0, 5, 10,20] ppb in the rumen were added and were incubated at 37°C for one and two hours. The amount of toxin residues was measured by ELISA using Europroxima kits. RESULTS: The results showed that microorganisms that have been treated in an autoclave have the highest amount of toxin removal [90.5%] [p<0.05]. Also, with increases in the incubation time, the amount of toxin absorbed significantly increased [78%] [p<0.05] and with increasing concentrations of toxin in vitro the yeast's ability to absorb toxin increases. These results demonstrate that the major toxin is absorbed by the yeast cell wall and therefore non-living microorganisms shown an ability to absorb higher. This is because the composition of the yeast cell wall mannoprotein that are effective at absorb in toxin

Conclusions: As a strategy for the animal feed industry the use of glycomannan yeast cell wall can be useful for reducing aflatoxin B

Efectos de la temperatura, la actividad de agua y el tiempo de incubación en el crecimiento fúngico y la producción de aflatoxina B1 por aislados toxicogénicos de Aspergillus flavus en sorgo / Effects of temperature, water activity and incubation time on fungal growth and aflatoxin B1 production by toxinogenic Aspergillus flavus isolates on sorghum seeds

Sorghum, which is consumed in Tunisia as human food, suffers from severe colonization by several toxigenic fungi and contamination by mycotoxins. The Tunisian climate is characterized by high temperature and humidity that stimulates mold proliferation and mycotoxin accumulation in foodstuffs. This study investigated the effects of temperature (15, 25 and 37 °C), water activity (a w, between 0.85 and 0.99) and incubation time (7, 14, 21 and 28 d) on fungal growth and aflatoxin B1 (AFB1) production by three Aspergillus flavus isolates (8, 10 and 14) inoculated on sorghum grains. The Baranyi model was applied to identify the limits of growth and mycotoxin production. Maximum diameter growth rates were observed at 0.99 a w at 37 °C for two of the isolates. The minimum a w needed for mycelial growth was 0.91 at 25 and 37 °C. At 15 °C, only isolate 8 grew at 0.99 a w. Aflatoxin B1 accumulation could be avoided by storing sorghum at low water activity levels (≤0.91 a w). Aflatoxin production was not observed at 15 °C. This is the first work on the effects of water activity and temperature on A. flavus growth and AFB1 production by A. flavus isolates on sorghum grains.

El sorgo, que se consume en Túnez como alimento humano, puede sufrir la colonización severa de varios hongos toxicogénicos, con la consiguiente bioacumulación de micotoxinas. Además, el clima de Túnez, caracterizado por las altas temperaturas y humedad, estimula el crecimiento fúngico y la acumulación de micotoxinas en los productos alimenticios. Este estudio investigó los efectos de la temperatura (15, 25 y 37 °C), la actividad de agua (a w) (entre 0,85 y 0,99) y el tiempo de incubación (7, 14, 21 y 28 días) sobre el crecimiento y la producción de aflatoxina B1 (AFB1) de 3 aislados de Aspergillus flavus (designados como 8, 10 y 14) que se inocularon sobre granos de sorgo. El modelo Baranyi se aplicó para identificar los límites del crecimiento y la producción de micotoxinas. Las tasas máximas de crecimiento para 2 de los aislados se observaron en la combinación 0,99 a w y 37 °C. La a w mínima necesaria para el crecimiento del micelio fue de 0,91 a 25 °C y 37 °C. A 15 °C, solo el aislado 8 creció a 0,99 a w, pero fue incapaz de producir la aflatoxina B1. Es posible evitar la acumulación de aflatoxina B1 en el sorgo almacenándolo a baja actividad de agua (≤ 0,91 a w). Este es el primer trabajo que ha estudiado el efecto de la actividad del agua y la temperatura sobre el crecimiento de aislados de A. flavus y su producción de aflatoxina B1 en granos de sorgo.

Docosahexaenoic acid inhibits aflatoxin B1-induced migration and invasion in hepatocellular carcinoma cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of docosahexaenoic acid (DHA) on invasiveness of aflatoxin B1 (AFB1)-induced hepatocellular carcinoma cells in vitro.</p><p><b>METHODS</b>HepG2.2.15 cells were exposed to different concentrations of AFB1 and DHA plus AFB1. The cell migration and invasion were assessed using wound-healing and Transwell assay, and flow cytometry was used to analyze the cell cycle changes. The ultrastructural changes of the cells were observed by transmission electron microscopy.</p><p><b>RESULTS</b>Compared with the control group, the cells exposed to2 µmol/L AFB1 showed obviously enhanced migration and invasion with decreased cell ratio in G1/G1 phase and increased cell ratio in G2/M phase but no changes in S phase cells; transmission electron microscopy revealed the presence of multiple nucleoli and significantly increased mitochondria and Golgi apparatus in the exposed cells. Compared with AFB1-exposed cells, the cells treated with DHA and AFB1 showed decreased migration and invasion abilities, and the G1/G1 phase cells increased and G2/M phase cells decreased significantly; ultrastructurally, the cells contained single nucleoli with decreased mitochondria and vacuolization occurred in the cytoplasm.</p><p><b>CONCLUSION</b>DHA can significantly inhibit AFB1-induced enhancement of cell migration and invasion in hepatocellular carcinoma cells in vitro.</p>

In vitro ability of beer fermentation residue and yeast-based products to bind aflatoxin B1

This study aimed to verify the in vitro ability of beer fermentation residue (BFR) containing Saccharomyces cerevisiae cells and five commercial products that differed in the viability and integrity of S. cerevisiae cells to remove aflatoxin B1 (AFB1) from a citrate-phosphate buffer solution (CPBS). BFR was collected at a microbrewery and prepared by drying and milling. The commercial yeast-based products were as follows: inactive intact yeast cells from beer alcoholic fermentation, inactive intact yeast cells from sugarcane alcoholic fermentation, hydrolyzed yeast cells, yeast cell walls and active yeast cells. Adsorption assays were performed in CPBS spiked with 1.0 μg AFB1/mL at pH 3.0 and 6.0 for a contact time of 60 min at room temperature. Analysis of AFB1 in the samples was performed by high performance liquid chromatography. AFB1 adsorption by the products ranged from 45.5% to 69.4% at pH 3.0 and from 24.0% to 63.8% at pH 6.0. The higher percentages (p < 0.05) of AFB1 binding at both pH values were achieved with products containing hydrolyzed yeast cells or yeast cell walls rather than intact cells. The AFB1 binding percentages of BFR were 55.0 ± 5.0% at pH 3.0 and 49.2 ± 4.5% at pH 6.0, which was not significantly different (p > 0.05) from commercial products containing inactive intact yeast cells. The results of this trial indicate that the yeast-based products tested, especially the BFR, have potential applications in animal feeds as a suitable biological method for reducing the adverse effects of aflatoxins.

Estudo sobre a ocorrência de fungos e aflatoxina B1 na dieta de bovinos leiteiros em São Paulo / Study on the occurrence of fungi and aflatoxina B1 in the diet of dairy cattle in São Paulo, Brazil

A qualidade da dieta ofertada às vacas em lactação é uma preocupação dos agentes de saúde devido à possibilidade da detecção de micotoxinas prejudiciais a saúde humana e animal. Os objetivos do trabalho foram avaliar o perfil da micobiota, determinar a atividade de água (Aa) e a ocorrência natural de aflatoxina B1 (AFB1) em dietas ofertadas a vacas em lactação de fazendas leiteiras no estado de São Paulo, Brasil. As amostragens das dietas foram realizadas diretamente dos cochos de lote de 15 vacas, em dois dias consecutivos com intervalos de 24h e a cada 15 dias, perfazendo um período de 45 dias de amostragens por fazenda. A purificação e determinação de AFB1 foram realizadas em colunas de imunoafinidade e Cromatografia Líquida de Alta Eficiência (CLAE). O estudo da micobiota presente nas amostras das dietas (288) revelou que as leveduras foram predominantes em todas as dietas (83,97 a 99,98%). Foram isolados 15 gêneros de fungos filamentosos, com os gêneros Aspergillus spp (20,09%), Fusarium spp (14,16%) e Penicillium spp (11,48%) os mais prevalentes. As contagens de Unidades Formadoras de Colônias por grama de alimento (UFC. g-1) variaram de 102 a 1011. A atividade de água das amostras variou entre 0,91 a 0,98. Foi detectada a presença de AFB1 em 31,44% das amostras com teores entre 1,68 a 194,51μg.kg-1. Medidas de boas práticas de produção, estocagem e utilização devem ser tomadas para diminuir a ocorrência de AFB1 nas dietas ofertadas às vacas em lactação...

The quality of the diet offered to lactating cows is a concern to health officials the possibility of detecting mycotoxins harmful to human and animal health. The objectives were to evaluate the profile of mycoflora, determine the water activity (Aw) and the natural occurrence of aflatoxin B1 (AFB1) in diets offered to lactating cows from dairy farms in the state of São Paulo, Brazil. Samples of the diets were taken directly from the troughs batch of 15 cows, on two consecutive days at intervals of 24 hours and every 15 days with a period of 45 sampling days per farm. Purification and determination of AFB1 were performed on immunoaffinity columns and High Performance Liquid Chromatography (HPLC). The study of mycobiota present in samples of diets (288) revealed that yeast cells were predominant in all diets (83.97 to 99.98%). 15 genera were isolated from filamentous fungi, with Aspergillus spp (20.09%), Fusarium spp. (14.16%) and Penicillium spp. (11.48%) the most prevalent. The counts of colony forming units per gram of food (UFC.g-1) ranged from 102 a1011. The water activity of the samples ranged from 0.91 to 0.98. We have detected the presence of AFB1 in 31.44% of samples with levels between 1.68 a 194.51μg.kg-1. Measures of good production, storage and use should be taken to reduce the occurrence of aflatoxin B1 in the diet offered to lactating cow...

Contamination level of aflatoxin B1 in lotus seeds rapid screening by indirect competitive ELISA method / 中国中药杂志

A simple and cost-effective indirect competitive enzyme-linked immune sorbent assay (ic-ELISA) was developed to rapidly screen the content of aflatoxin B1 (AFB1) in lotus seeds, and the results were confirmed by ultra-fast liquid chromatography-tandem mass spectrometry( UFLC-MS/MS). Matrix-matched calibration expressed a good linearity ranging from 0. 171 to 7. 25 µg · L(-1) for AFB, with R2 > 0.978. The medium inhibitory concentration( IC50 ) for AFB1 was 1.29 µg · L(-1), the recovery for AFB1 was 74.73% to 126.9% with RSD < 5%, and the limit of detection (IC10) was 0.128 µg · L(-1). The developed ic-ELSIA method was applied to rapid analysis of AFB, in 20 lotus seeds samples and the results indicated that the contents of AFB, in samples 1-15 were in the range of 1. 19- 115. 3 µg · kg(-1) and in 40% of the samples exceeded the legal limit(5 µg · kg(-1)), while the contamination rate of AFB, in samples 16-20 was 40%. Pearson correlation coefficient(r) reached 0.997 for AFB1 content in the samples detected by ic-ELSIA and UFLC-MS/MS methods. The results proved that the developed ic-ELISA method is simple, sensitive and reliable, and can be used for rapid and high-throughput screening of AFB1 in lotus seeds

Development of an aptamer/fluorescence dye PicoGreen-based method for detection of fumonisin B1 / 生物工程学报

Fumonisin B1 (FB1) is a carcinogenic mycotoxin found in commodities such as corn and corn-originated products. An aptamer-based method for detection of FB1 was developed using the fluorescent dye PicoGreen, which can recognize and bind double-stranded DNA. A peak fluorescence of PicoGreen was obtained in 15 min in the presence of FB1 aptamer, which formed a double-stranded hybridizer DNA with its complementary strand. The excitation and emission wavelengths for PicoGreen detection were 480 nm and 520 nm, respectively. The sensitivity of this aptamer/PicoGreen-based method was 0.1 μg/L. This method showed a good linearity for FB1 concentration ranging from 0.1 to 1 μg/L. The entire detection procedure for FB1 could be completed within 40 min. No cross reactions were observed with any other mycotoxins against aflatoxin B1, ochratoxin A, citrinin and zearalenone, demonstrating high specificity towards FB1 aptamer. Agreement between commercial, antibody-based enzyme-linked immunosorbent assay (ELISA) kit and aptamer method was excellent with a kappa value of 0.857. Taken together, this aptamer/PicoGreen-based method is more cost-effective, time-saving and useful than ELISA for detection of FB1.