Objective: To evaluate the ability of Bacillus spp. as direct-fed microbials(DFM) to biodegrade al atoxin B1(AFB1) by using an in vitro digestive model simulating in vivo conditions.Methods: Sixty-nine Bacillus isolat...Objective: To evaluate the ability of Bacillus spp. as direct-fed microbials(DFM) to biodegrade al atoxin B1(AFB1) by using an in vitro digestive model simulating in vivo conditions.Methods: Sixty-nine Bacillus isolates were obtained from intestines, and soil samples were screened by using a selective media method against 0.25 and 1.00 μg/m L of AFB1 in modii ed Czapek-Dox medium. Plates were incubated at 37 °C and observed every two days for two weeks. Physiological properties of the three Bacillus spp. candidates were characterized biochemically and by 16 S r RNA sequence analyzes for identii cation. Tolerance to acidic p H, osmotic concentrations of Na Cl, bile salts were tested, and antimicrobial sensitivity proi les were also determined. Bacillus candidates were individually sporulated by using a solid fermentation method and combined. Spores were incorporated into 1 of 3 experimental feed groups: 1) Negative control group, with unmedicated starter broiler feed without AFB1; 2) Positive control group, with negative control feed contaminated with 0.01% AFB1; 3) DFM treated group, with positive control feed supplemented with 109 spores/g. After digestion time(3:15 h), supernatants and digesta were collected for high-performance liquid chromatography l uorescence detection analysis by triplicate.Results: Three out of those sixty-nine DFM candidates showed ability to biodegrade AFB1 in vitro based on growth as well as reduction of l uorescence and area of clearance around each colony in modii ed Czapek-Dox medium which was clearly visible under day light after 48 h of evaluation. Analysis of 16S-DNA identii ed the strains as Bacillus amyloliquefaciens, Bacillus megaterium and Bacillus subtilis. The three Bacillus strains were tolerant to acidic conditions(p H 2.0), tolerant to a high osmotic pressure(Na Cl at 6.5%), and were able to tolerate 0.037% bile salts after 24 h of incubation. No signii cant dif erences(P > 0.05) were observed in the concentrations of AFB1 in neither the supernatants nor digesta samples evaluated by highperformance liquid chromatography with l uorescence detection between positive control or DFM treated groups. Conclusions: In vitro digestion time was not enough to confirm biodegradation of AFB1. Further studies to evaluate the possible biodegradation ef ects of the BacillusDFM when continuously administered in experimentally contaminated feed with AFB1, are in progress.展开更多
Aim:Protective effects of aqueous extract of Amaranthus hybridus against afl atoxin B1(AFB_(1))and/or fumonisin B1(FB_(1))on the H4IIE-luc cell line were determined by use of the methyl thiazol tetrazolium viability a...Aim:Protective effects of aqueous extract of Amaranthus hybridus against afl atoxin B1(AFB_(1))and/or fumonisin B1(FB_(1))on the H4IIE-luc cell line were determined by use of the methyl thiazol tetrazolium viability assay and disruption of DNA integrity.Methods:H4IIE-luc cells were incubated with different concentrations of AFB_(1) and/or FB_(1) for 24 and 48 h with or without aqueous extract of A.hybridus.Results:AFB_(1) decreased the viability of cells after 24 and 48 h of exposure.EC_(50)values for AFB_(1) were 10.5 and 1.8μmol/L for the two periods,respectively.When the 48 h exposure to mycotoxin repeated with a pre-treatment of 20 and 40μg/mL extract of A.hybridus,the EC_(50)changed to 3.88 and 7.67μmol/L,respectively.H4IIE-luc cells exposed to FB_(1) for 24 h responded more than those incubated for 48 h.Cells treated with a combination of AFB_(1) and FB_(1) were less viable with a signifi cant decrease in the greater concentration.The mixture of AFB_(1) and FB_(1) resulted in a signifi cant threat to H4IIE-luc as indicated by the absence or appearance of new bands in random amplifi ed polymorphic DNA analysis,which demonstrated damage to DNA.The protective effects were probably due to greater content of total phenolics,carotenoids,β-carotene,folic-,linolenic-,linoleic and palmitic acids,as well as calcium,magnesium,iron,zinc,and selenium observed in the extract.Conclusion:Exposure to 40μg/mL of extract of A.hybridus protected cells from damage to DNA by stabilizing DNA.展开更多
基金Supported by the Autogenous Vaccine Research Project of the Poultry Health Laboratory,Poultry Science Department,University of Arkansas
文摘Objective: To evaluate the ability of Bacillus spp. as direct-fed microbials(DFM) to biodegrade al atoxin B1(AFB1) by using an in vitro digestive model simulating in vivo conditions.Methods: Sixty-nine Bacillus isolates were obtained from intestines, and soil samples were screened by using a selective media method against 0.25 and 1.00 μg/m L of AFB1 in modii ed Czapek-Dox medium. Plates were incubated at 37 °C and observed every two days for two weeks. Physiological properties of the three Bacillus spp. candidates were characterized biochemically and by 16 S r RNA sequence analyzes for identii cation. Tolerance to acidic p H, osmotic concentrations of Na Cl, bile salts were tested, and antimicrobial sensitivity proi les were also determined. Bacillus candidates were individually sporulated by using a solid fermentation method and combined. Spores were incorporated into 1 of 3 experimental feed groups: 1) Negative control group, with unmedicated starter broiler feed without AFB1; 2) Positive control group, with negative control feed contaminated with 0.01% AFB1; 3) DFM treated group, with positive control feed supplemented with 109 spores/g. After digestion time(3:15 h), supernatants and digesta were collected for high-performance liquid chromatography l uorescence detection analysis by triplicate.Results: Three out of those sixty-nine DFM candidates showed ability to biodegrade AFB1 in vitro based on growth as well as reduction of l uorescence and area of clearance around each colony in modii ed Czapek-Dox medium which was clearly visible under day light after 48 h of evaluation. Analysis of 16S-DNA identii ed the strains as Bacillus amyloliquefaciens, Bacillus megaterium and Bacillus subtilis. The three Bacillus strains were tolerant to acidic conditions(p H 2.0), tolerant to a high osmotic pressure(Na Cl at 6.5%), and were able to tolerate 0.037% bile salts after 24 h of incubation. No signii cant dif erences(P > 0.05) were observed in the concentrations of AFB1 in neither the supernatants nor digesta samples evaluated by highperformance liquid chromatography with l uorescence detection between positive control or DFM treated groups. Conclusions: In vitro digestion time was not enough to confirm biodegradation of AFB1. Further studies to evaluate the possible biodegradation ef ects of the BacillusDFM when continuously administered in experimentally contaminated feed with AFB1, are in progress.
基金The Morogo Research Program gratefully acknowledges the National Research Foundation of South Africa(Focus Area Grant FA2004050600064)National Research Center,Cairo,Egypt Project No.10070112 for financial support of this study.Prof.Giesy was supported by the Canada Research Chair program,a Visiting Distinguished Professorship in the Department of Biology and Chemistry and State Key Laboratory in Marine Pollution,City University of Hong Kong,the 2012“High Level Foreign Experts”(No.GDT20143200016)program,funded by the State Administration of Foreign Experts Affairs,the P.R.China to Nanjing University and the Einstein Professor Program of the Chinese Academy of Sciences.
文摘Aim:Protective effects of aqueous extract of Amaranthus hybridus against afl atoxin B1(AFB_(1))and/or fumonisin B1(FB_(1))on the H4IIE-luc cell line were determined by use of the methyl thiazol tetrazolium viability assay and disruption of DNA integrity.Methods:H4IIE-luc cells were incubated with different concentrations of AFB_(1) and/or FB_(1) for 24 and 48 h with or without aqueous extract of A.hybridus.Results:AFB_(1) decreased the viability of cells after 24 and 48 h of exposure.EC_(50)values for AFB_(1) were 10.5 and 1.8μmol/L for the two periods,respectively.When the 48 h exposure to mycotoxin repeated with a pre-treatment of 20 and 40μg/mL extract of A.hybridus,the EC_(50)changed to 3.88 and 7.67μmol/L,respectively.H4IIE-luc cells exposed to FB_(1) for 24 h responded more than those incubated for 48 h.Cells treated with a combination of AFB_(1) and FB_(1) were less viable with a signifi cant decrease in the greater concentration.The mixture of AFB_(1) and FB_(1) resulted in a signifi cant threat to H4IIE-luc as indicated by the absence or appearance of new bands in random amplifi ed polymorphic DNA analysis,which demonstrated damage to DNA.The protective effects were probably due to greater content of total phenolics,carotenoids,β-carotene,folic-,linolenic-,linoleic and palmitic acids,as well as calcium,magnesium,iron,zinc,and selenium observed in the extract.Conclusion:Exposure to 40μg/mL of extract of A.hybridus protected cells from damage to DNA by stabilizing DNA.
