Biodegradation Of Aflatoxin B1 Research Articles

Biodegradation of aflatoxin B1 by Aspergillus terreus HNGD-TM15 and its degradation mechanism

Abstract The Aspergillus terreus strain HNGD-TM15 that was isolated from soil grown with rosemary was shown to efficiently degrade aflatoxin B1 (AFB1). Specifically, AFB1 was degraded by the strain’s fermentation broth at a maximum degradation rate of 98.3%. HPLC and LC-MS analyses detected a degradation product with an m/z ion value of 312.0636. Its molecular formula was C18H39NO3, which was tentatively identified as 2-amino-1, 3,4-octadecatriol. Based on LC-MS results and further analysis, it was revealed that a series of reactions, such as decomposition, reduction (lactone ring hydrogenation) and substitution (hydrolysis), occurred during the degradation of AFB1. Therefore, A. terreus HNGD-TM15 has a great potential for application in the detoxification of AFB1 contaminating food and feed products.

Biodegradation of aflatoxin B1 by Bacillus subtilis YGT1 isolated from yoghurt

Aflatoxin contamination of food products is recognised as a major food safety concern throughout the world because of its carcinogenic, mutagenic, and immunosuppressive effects on human health. Of the various types of aflatoxins, aflatoxin B1 (AFB1) is the ubiquitous and most threatening foodborne mycotoxin to humans. A wide range of detoxification methods is used to reduce the toxic effects of AFB1. In the present work, the ability of probiotics isolated from yoghurt (produced by bacterial fermentation of milk), “laban” (fermented milk beverage), and “idli” batter (fermented rice and black gram) in the detoxification of AFB1 was investigated under laboratory conditions. Among the four isolates from fermented foods evaluated, the isolate YGT1 from yoghurt showed the maximum (83.8%) degradation of AFB1 in Luria-Bertani (LB) liquid medium after 48 h of incubation at 30°C. The degradation of AFB1 by the probiotic isolate was further confirmed by liquid chromatography/mass spectrometry analysis. On the basis of 16S rRNA gene sequence analysis, the bacterial isolate YGT1 was identified as Bacillus subtilis. The culture supernatant and heat-treated culture supernatant (boiled for 30 min) of B. subtilis YGT1 also exhibited degradation of AFB1, thus suggesting the involvement of thermostable bioactive compound(s) in the degradation of AFB1. These results suggested that B. subtilis YGT1 isolated from yoghurt may be a promising candidate for exploitation in food and feed industries for the removal of AFB1.

Biodegradation of Aflatoxin B1 in Maize Grains and Suppression of Its Biosynthesis-Related Genes Using Endophytic Trichoderma harzianum AYM3

Aflatoxin B1 is one of the most deleterious types of mycotoxins. The application of an endophytic fungus for biodegradation or biosuppression of AFB1 production by Aspergillus flavus was investigated. About 10 endophytic fungal species, isolated from healthy maize plants, were screened for their in vitro AFs-degrading activity using coumarin medium. The highest degradation potential was recorded for Trichoderma sp. (76.8%). This endophyte was identified using the rDNA-ITS sequence as Trichoderma harzianum AYM3 and assigned an accession no. of ON203053. It caused a 65% inhibition in the growth of A. flavus AYM2 in vitro. HPLC analysis revealed that T. harzianum AYM3 had a biodegradation potential against AFB1. Co-culturing of T. harazianum AYM3 and A. flavus AYM2 on maize grains led to a significant suppression (67%) in AFB1 production. GC-MS analysis identified two AFB1-suppressing compounds, acetic acid and n-propyl acetate. Investigating effect on the transcriptional expression of five AFB1 biosynthesis-related genes in A. flavus AYM2 revealed the downregulating effects of T. harzianum AYM3 metabolites on expression of aflP and aflS genes. Using HepaRG cell line, the cytotoxicity assay indicated that T. harazianum AYM3 metabolites were safe. Based on these results, it can be concluded that T. harzianum AYM3 may be used to suppress AFB1 production in maize grains.

Biodegradation of Aflatoxin B1 in the Baijiu Brewing Process by Bacillus cereus

Aflatoxin is a potent mycotoxin and a common source of grain contamination that leads to great economic losses and health problems. Although distilled baijiu cannot be contaminated by aflatoxin, its presence in the brewing process affects the physiological activities of micro-organisms and reduces product quality. Bacillus cereus XSWW9 capable of degrading aflatoxin B1 (AFB1) was isolated from daqu using coumarin as the sole carbon source. XSWW9 degraded 86.7% of 1 mg/L AFB1 after incubation at 37 °C for 72 h and tolerated up to 1 mg/L AFB1 with no inhibitory effects. Enzymes in the cell-free supernatant of XSSW9 played a significant role in AFB1 degradation. The AFB1-degradation activity was sensitive to protease K and SDS treatment, which indicated that extracellular proteins were responsible for the degradation of AFB1. In order to investigate the AFB1-degradation ability of XSSW9 during the baijiu brewing process, AFB1 and XSWW9 were added to grain fermentation (FG-T) and normal grain fermentation without AFB1, while normal grain fermentation without AFB1 and XSWW9 was used as a control (FG-C). At the end of the fermentation, 99% AFB1 was degraded in the residue of fermented grains. The differences of microbial communities in the fermented grains showed that there were no significant differences between FG-T and FG-C in the relative abundance of dominant genera. The analysis of volatile compounds of their distillation showed that the contents of skeleton flavor components was similar between FG-T and FG-C. These results offer a basis for the development of effective strategies to reduce the effect of AFB1 on the brewing process and ensure that the production of baijiu is stable.

Growth Inhibition of Aflatoxigenic Molds and Biodegradation of Aflatoxin B1 by Certain Bacterial Isolates

AFLATOXIN B1 (AFB1) is mutagenic, carcinogenic, and harmful to humans and animals. Different physical and chemical methods have been proposed to reduce mycotoxins but few have been approved for practical application. This study investigated the possible use of certain bacterial species were isolated from different agricultural soils as biological control agents against the growth of toxigenic Aspergillus flavus and Aspergillus parasiticus isolated from sorghum and peanuts as well as biodegradation of aflatoxin B1 (AFB1). Out of these 30 bacterial isolates, 6 isolates showed that have antagonistic effects against the aflatoxigenic fungi but with varying efficiencies. The six different isolates were screened for their ability to degrade AFB1. Out of 6 isolates, one isolate showed high reduction activity of AFB1. Maximum antifungal activities were observed in one isolate was identified as Staphylococcus lentus. S. lentus completely prevented the A. flavus and A. parasiticus growth and completely degraded AFB1 in Liquid Culture. LC-MS/MS analysis revealed that S. lentus resulted in a 96.54% degradation of AFB1, but no products has been detected. In practical application, it was found that S. lentus at concentration of 10 and 25mL kg-1 were completely prevented the growth of A. flavus and A. parasiticus in sorghum and peanuts, respectively, consequently no aflatoxin were produced. According to our information, this is the first study to prove that Staphylococcus lentus has the capability to inhibit the growth of Aspergillus flavus and Aspergillus parasiticus by 100% and degrading AFB1 by 96%.

Biodegradation of aflatoxin B1 with cell-free extracts of Trametes versicolor and Bacillus subtilis

Aflatoxin B1 (AFB1) is one of the most common contaminants of poultry feed and has been linked to adverse effects on animal health and productivity. In this study, the degradation of AFB1 was studied with cell-free extracts (CFE) of Trametes versicolor and Bacillus subtilis using High-Performance Liquid chromatography (HPLC). CFE from B. subtilis and T. versicolor gave 60% and 34% of AFB1 degradation respectively, while heat-inactivated extracts showed no degradation. By-products obtained at the end of AFB1 degradation were analyzed by Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). After 96 h of incubation, by-products with lower m/z values were obtained with CFE from B. subtilis as compared to that from T. versicolor, indicating a higher degradation efficiency of the former. Additionally, the detection of a by-product which could correspond to AFB1-8,9 dihydrodiol - a less toxic derivative of AFB1 - after 72 and 96 h of incubation with CFE from B. subtilis, could indicate the simultaneous detoxification along with degradation of AFB1 by B. subtilis CFE.

Biological Degradation of Aflatoxin B₁ by Cell-Free Extracts of Bacillus velezensis DY3108 with Broad PH Stability and Excellent Thermostability.

(1) Background: Aflatoxin contamination in food and grain poses serious problems both for economic development and public health protection, thus leading to a focus on an effective approach to control it; (2) Methods: Aflatoxin B1 (AFB1) degrading bacteria were isolated using a medium containing coumarin as the sole carbon source, and the biodegradation of AFB1 by the isolate was examined by high performance liquid chromatography, and liquid chromatography mass spectrometry; (3) Results: a bacterial strain exhibiting strong AFB1 degradation activity (91.5%) was isolated and identified as Bacillus velezensis DY3108. The AFB1 degrading activity was predominantly attributed to the cell-free supernatant of strain DY3108. Besides, it was heat-stable and resistant to proteinase K treatment but sensitive to sodium dodecyl sulfate treatment. The optimal temperature for the maximal degradation of AFB1 was 80 °C. Even more notable, the supernatant showed a high level of activity over a broad pH (4.0 to 11.0) and exhibited the highest degradation (94.70%) at pH 8.0. Cytotoxicity assays indicated that the degradation products displayed significantly (p < 0.05) lower cytotoxic effects than the parent AFB1; (4) Conclusions: B. velezensis DY3108 might be a promising candidate for exploitation in AFB1 detoxification and bioremediation in food and feed matrices.

Complete genome sequence of Bacillus licheniformis BL-010

The biodegradation of Aflatoxin B1 (AFB1) is an industry of increasing importance. Bacillus licheniformis BL-010 was isolated from the aflatoxin contaminated corn feed storage, and was shown to degrade AFB1 efficiently. Here we present the complete genome sequence of BL-010, the genome comprises 4,287,714 bp in a circular chromosome with a GC content of 46.12% and contains genes encoding AFB1 degrading enzymes. The genome sequence displayed that this strain contains genes involved in production of laccase, aromatic ring-opening dioxygenase which could detoxify AFB1. Complete genome sequence of the strain BL-010 can further provide the genomic basis for the biotechnological application of strain BL-010 as an effective way to degrade AFB1.

Biodetoxification of aflatoxin B1 in cottonseed meal by fermentation of Cellulosimicrobium funkei in duckling diet

Two experiments were conducted to optimize the fermentation of cottonseed meal by Cellulosimicrobium funkei (C. funkei) for the ability of the bacteria to degrade aflatoxin B1 (AFB1) and then to evaluate the bacterial detoxification in ducklings. In experiment 1, the fermentation of cottonseed meal by C. funkei was improved by changing the inoculation amounts by 10% (108 cfu/mL), using a 1:0.5 material to water ratio at 35°C temperature for a 144 h reaction duration, which resulted in an 83.4% biodegradation of AFB1. In experiment 2, 112 one-day-old male Cherry Valley ducklings were randomly allocated to 4 experimental groups with 4 replicates of 7 birds each. For a period of 2 wk the controls received a base duckling diet (BD), a second group received a base diet contaminated with 10% AFB1 cottonseed meal (96.8 μg AFB1/kg), a third group was fed a base diet added with 5% unfermented and 5% fermented AFB1-contaminated cottonseed meal (57.0 μg AFB1/kg), and the fourth group was fed a base diet added with 10% AFB1-contaminated fermented cottonseed meal (16.0 μg AFB1/kg). The growth performance, relative organ weights, and serum biochemistry were analyzed. The results showed that the feed conversion ratio in the second group was lower than that of the controls at wk one and 2 (P < 0.05). Also, after 2 wk, group 2 ducklings had increased relative weights of the liver, kidneys, and spleen, increased activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), increased concentration of blood urea nitrogen (BUN) and creatinine (Crt), and decreased relative weight of Fabricius bursa (P < 0.05). In addition, the concentrations of total protein (TP) and albumin (ALB) in serum were also significantly higher at weeks one and 2 (P < 0.05). These alterations were attenuated or prevented when 5 or 10% fermented cottonseed meal substituted equal amounts of unfermented cottonseed meal in the diet. In conclusion, fermentation of AFB1-contaminated feed materials by C. funkei offers a new strategy to reduce the negative effects of aflatoxicosis in ducklings.

Biodegradation of aflatoxin-B1 and zearalenone by Streptomyces sp. collection

Aflatoxin B1 (AFB1) and zearalenone (ZON) are hazardous mycotoxins. AFB1 has cytotoxic, mutagenic and carcinogenic effects, whereas ZON can disrupt the endocrine system. Biodegradation by microbes is an effective method to eliminate these hazardous toxins. The aim of this work was to screen AFB1 and ZON biodegrading potential of one hundred and twenty-four Streptomyces strains deposited in the Actinomycetes strain collection of the Department of Environmental Safety and Ecotoxicology. Two different biotests were used for screening purposes: SOS-Chromotest was used to monitor genotoxicity and select microorganisms with the best AFB1 degrading potential. Estrogenic effect of ZON was measured with a yeast based bioluminescent test including human estrogen receptors Bioluminescent Yeast Estrogen System (BLYES). Biodegradation experiments were conducted with 1 mg l−1 AFB1 and 1 mg l−1 ZON concentration. On the base of the results, ten strains were selected for biodegradation experiments and Enzyme-linked Immunosorbent Assay tests (ELISA). The results of these tests Streptomyces cacaoi subsp. asoensis (K234) strain degraded AFB1 over 88 per cent and totally eliminated genotoxicity. Two strains of Streptomyces rimosus (K145, K189) degraded almost total amount of ZON and estrogenicity was not detected besides that.

Metabolomics of the Bio-Degradation Process of Aflatoxin B1 by Actinomycetes at an Initial pH of 6.0

Contamination of food and feed by Aflatoxin B1 (AFB1) is a cause of serious economic and health problems. Different processes have been used to degrade AFB1. In this study, biological degradation of AFB1 was carried out using three Actinomycete species, Rhodococcus erythropolis ATCC 4277, Streptomyces lividans TK 24, and S. aureofaciens ATCC 10762, in liquid cultures. Biodegradation of AFB1 was optimised under a range of temperatures from 25 to 40 °C and pH values of 4.0 to 8.0. An initial concentration of 20 µg/mL of AFB1 was used in this study. The amount of AFB1 remaining was measured against time by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC), coupled with UV and mass spectrometry (LC-MS). All species were able to degrade the AFB1, and no significant difference was found between them. AFB1 remained in the liquid culture for R. erythropolis, S. lividans and S. aureofaciens were 0.81 µg/mL, 2.41 µg/mL and 2.78 µg/mL respectively, at the end of the first 24 h. Degradation occurred at all incubation temperatures and the pH with the optimal conditions for R. erythropolis was achieved at 30 °C and pH 6, whereas for S. lividans and S. aureofaciens the optimum conditions for degradation were 30 °C and pH 5. Analysis of the degradative route indicated that each microorganism has a different way of degrading AFB1. The metabolites produced by R. erythropolis were significantly different from the other two microorganisms. Products of degradation were identified through metabolomic studies by utilizing high-resolution mass spectral data. Mass spectrometric analysis indicated that the degradation of AFB1 was associated with the appearance of a range of lower molecular weight compounds. The pathway of degradation or chemical alteration of AFB1 was followed by means of high resolution Fourier transform mass spectrometry (HR-FTMS) analysis as well as through the MS2 fragmentation to unravel the degradative pathway for AFB1. AFB1 bio-degradation was coupled with the accumulation of intermediates of fatty acid metabolism and glycolysis. A plausible mechanism of degradation of AFB1 by Rhodococcus was hypothesized.

Biodegradation of aflatoxin B1 in contaminated rice straw by Pleurotus ostreatus MTCC 142 and Pleurotus ostreatus GHBBF10 in the presence of metal salts and surfactants.

Aflatoxin B1 (AFB1) is a highly toxic fungal metabolite having carcinogenic, mutagenic and teratogenic effects on human and animal health. Accidental feeding of aflatoxin-contaminated rice straw may be detrimental for ruminant livestock and can lead to transmission of this toxin or its metabolites into the milk of dairy cattle. White-rot basidiomycetous fungus Pleurotus ostreatus produces ligninolytic enzymes like laccase and manganese peroxidase (MnP). These extracellular enzymes have been reported to degrade many environmentally hazardous compounds. The present study examines the ability of P. ostreatus strains to degrade AFB1 in rice straw in the presence of metal salts and surfactants. Laccase and MnP activities were determined spectrophotometrically. The efficiency of AFB1 degradation was evaluated by high performance liquid chromatography. Highest degradation was recorded for both P. ostreatus MTCC 142 (89.14 %) and P. ostreatus GHBBF10 (91.76 %) at 0.5 µg mL(-1) initial concentration of AFB1. Enhanced degradation was noted for P. ostreatus MTCC 142 in the presence of Cu(2+) and Triton X-100, at toxin concentration of 5 µg mL(-1). P. ostreatus GHBBF10 showed highest degradation in the presence of Zn(2+) and Tween 80. Liquid chromatography-mass spectrometric analysis revealed the formation of hydrated, decarbonylated and O-dealkylated products. The present findings suggested that supplementation of AFB1-contaminated rice straw by certain metal salts and surfactants can improve the enzymatic degradation of this mycotoxin by P. ostreatus strains.

The Role of Extracellular Enzymes Produced by Aspergillus oryzae KKB4 in Biodegradation of Aflatoxin B1

Previous research showed that Aspergillus oryzae KKB4 able to degrade aflatoxin B1 (AFB1) during its growth. According to detoxification test, it was known that extracellular enzymes produced by this mold was able to detoxify AFB1. In this study, the role of these enzymes in biodegradation of AFB1 were further studied. Biodegradation products of AFBI were analyzed using HPLC and the destruction of functional groups were analyzed using IR spectrophotometer. Molecular weight and enzymes activity were also determined. The result showed that extra cellular enzymes of Aspergillus oryzae KKB4 consist of five fractions with molecular weight of 14.2, 20, 23, 29 and 36 kDalton. All enzyme fractions were able to degrade AFB1 with the highest specific activity 3.79 ig AFBl/mg enzyme protein/20 hrs. The HPLC analysis indicated that biodegradation product of AFB1 was different from untreated AFB1. The infrared spectrum suggest that enzymes degraded lactone ring, cyclo-pentanone reduction and openined difuran ring.

Factors influencing the inhibition of aflatoxin production in corn by Aspergillus niger.

Aspergillus niger, a mold commonly associated with Aspergillus flavus in damaged corn, interferes with the production of aflatoxin when grown with A. flavus on autoclaved corn. The pH of corn-meal disks was adjusted using NaOH-HCl, citric acid-sodium citrate, or a water extract of A. niger fermented corn. Aflatoxin formation was completely inhibited below pH 2.8-3.0, irrespective of the system used for pH adjustment. When grown in association with A. flavus NRRL 6432 on autoclaved corn kernels, A. niger NRRL 6411 lowered substrate pH sufficiently to suppress aflatoxin production. The biodegradation of aflatoxin B1 or its conversion to aflatoxin B2a were eliminated as potential mechanisms by which A. niger reduces aflatoxin contamination. A water extract of corn kernels fermented with A. niger caused an additional inhibition of aflatoxin formation apart from the effects of pH.