The double-agar layer method was used; B. coagulans BDU3 was
streaked onto the surface of MRS agar and after incubation at 30
◦
C
overnight, 10 mL of soft Trypticase soy agar (TSA) seeded with an
overnight culture of indicator strain (S. typhi MTCC 733), was overlayed. After agar solidification, the plates were incubated for 18 h at
37
◦
C and examined for the presence of inhibition zones. Inhibition
halos indicate a putative bioactive compound production.
Further, the antimicrobial potency of the culture free supernatant was verified. Briefly, 10 mL of overnight culture of BDU3 was
inoculated into 500 mL MRS broth. The culture was centrifuged at
15,000 rpm at 4
◦
C, and the supernatant was filter sterilized. The
cell free supernatant (CFS) thus obtained was screened for bioactive
potential by agar well diffusion method [20]. A panel of microbes
was used to test the antimicrobial potency of CFS. An overnight culture of the test strains was inoculated on MRS soft agar. 100 L CFS
were poured into the wells and the plates were incubated at 37
◦
C.
After 24 h of incubation, the diameter of the zone of inhibition was
measured and scored.