Screening for Probiotic Properties In Vitro

This study was designed as in a model gastrointestinal tract experiment to investigate the survival of B. subtilis. Overnight cultures of B. subtilis were centrifuged at 3,000 × g for 10 min, washed twice with phosphate buffer saline, and resuspended in a new liquid medium (10⁸ CFU mL⁻¹). The new media were adjusted to pH 2.5 by adding a hydrochloric acid solution with 1% pepsin or to pH 7.2 with 1% trypsin and incubated at 37°C. At 0, 1, and 2 h of incubation, 100 μL of the cultures were removed and spread on modified nutrition agar for cell number estimation. Respectively, the bacterial suspensions were inoculated into modified nutrition broth with 0.3% bile salt and cultured at 37°C for 24 h. Optical density at 600 nm (OD600) was measured and compared to a control culture without bile salts.

These isolates were screened for neutral and alkaline protease production. Overnight cultures of B. subtilis strains were centrifuged at 6,000 × g for 15 min, and then the supernatant was assayed for protease activity according the SB/T 10317-1999 (22).

The well diffusion method was performed on agar using cultured broth. Briefly, the target strain (10⁶ CFU mL⁻¹) was incorporated into agar (1% w/v) plates, which were mixed for uniformity and poured onto plates to solidify. Overnight cultures of B. subtilis (10⁸ CFU mL⁻¹) were transferred to holes (5-mm diameter) punched into the agar palates. The plates were then incubated as anaerobic or aerobic at 37°C for 24 h, depending on the target strain, and the antimicrobial ability was recorded as being in the inhibition zone (23).

Bacillus subtilis suspensions were spread on Muller–Hinton agar plates, onto which antibiotic disks of penicillin, kanamycin, doxycycline, tetracycline, gentamicin, lincomycin, erythromycin, cefotaxime, and streptomycin were placed. After 24 h incubation at 37°C, the inhibition zone was measured (24).