Isolation, screening and identification of Bacillus spp. as direct-fed microbial candidates for aflatoxin B1 biodegradation

• Published in: Asian Pacific Journal of Tropical Biomedicine
• Main Authors: Rosario Galarza-Seeber, Juan David Latorre, Xochitl Hernandez-Velasco, Amanda Drake Wolfenden, Lisa Renee Bielke, Anita Menconi, Billy Marshall Hargis, Guillermo Tellez
• Format: Article in Journal/Newspaper
• Language: English
• Published: Wolters Kluwer Medknow Publications 2015
• Subjects: Aflatoxin B1; Bacillus; Direct-fed microbials; Biodegradation; Broiler feed; Arctic medicine. Tropical medicine; RC955-962; Biology (General); QH301-705.5; Arctic
• Online Access: https://doi.org/10.1016/j.apjtb.2015.07.014; https://doaj.org/article/9f10b6f3908f42258a3a4fb9a286e254

Objective: To evaluate the ability of Bacillus spp. as direct-fed microbials (DFM) to biodegrade aflatoxin 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/mL of AFB1 in modified 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 16S rRNA sequence analyzes for identification. Tolerance to acidic pH, osmotic concentrations of NaCl, bile salts were tested, and antimicrobial sensitivity profiles 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 fluorescence 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 fluorescence and area of clearance around each colony in modified Czapek-Dox medium which was clearly visible under day light after 48 h of evaluation. Analysis of 16S-DNA identified the strains as Bacillus amyloliquefaciens, Bacillus megaterium and Bacillus subtilis. The three Bacillus strains were tolerant to acidic conditions (pH 2.0), tolerant to a high osmotic pressure (NaCl at 6.5%), and were able to tolerate 0.037% bile salts after 24 h of incubation. No significant differences (P > 0.05) were observed in the concentrations of AFB1 in neither the supernatants nor digesta ...