Identification and characterization of antimicrobial peptide produced by indigenously isolated Bacillus paralicheniformis UBBLi30 strain

Survival and Germination of Bacillus clausii UBBC07 Spores in in vitro Human Gastrointestinal Tract Simulation Model and Evaluation of Clausin Production

“…Besides this, overnight grown single colonies of UBBC07 were each inoculated in 10 ml of MH (pH 7.3 ± 0.1) and clarified-MH broth (pH 7.8 ± 0.1) (Ahire et al, 2020a) and incubated shaking (160 rpm) at 37 • C for 24 h. After incubation, the culture was aseptically transferred to 100 ml of respective media and kept under the same conditions as described above. The 10-ml aliquots were removed every after 24 h for up to 196 h. The respective aliquots were centrifuged (Sorvall Legend 65 XTR, Thermo Fisher Scientific, United States) at 11,000 × g for 20 min at 4 • C and pH of the supernatant was recorded by using DPH 504 pH meter (Global, India).…”

“…In another experiment, 24-h-old UBBC 07 cultivated in 10 ml of clarified-MH broth as described above was mixed with 10 g of activated sterile XAD16N (Amberlite, Sigma-Aldrich, United States) beads (van Staden, 2015;Ahire et al, 2020a) and spread evenly on top of sterile clarified-MH agar plate (200 mm × 20 mm, Borosil, India). The plate was incubated at 37 • C for 6 days.…”

“…The XAD16N beads IPA extract was concentrated using Rotavapor (R-300, Buchi, Switzerland) and purified further by reversed-phase C 18 cartridge (Sep-Pak, Vac 35 cc, 10 g, Waters, United States) as described by Ahire et al (2020a). The eluted IPA gradients (10-90% containing 0.1% TFA, v/v/v) were analyzed on thin-layer chromatography (TLC) and antimicrobial activity against M. luteus.…”

“…One milligram of lyophilized sample was each mixed separately with 1 ml of proteinase K (1 mg/ml; ≥30 U/mg; Sigma-Aldrich, United States) and trypsin (1 mg/ml; 2500 USP/mg; HiMedia, India) prepared in 10 mM Tris-HCl (pH 8.0; Roche, Germany) and incubated at 37 • C for 4 h. Similarly, equal quantities of sample and pepsin (1 mg/ml; >3000 NFU/mg, HiMedia, India) were mixed in 0.1% TFA (v/v) water and incubated as described above. After incubation, the mixture was heated at 95 • C for 5 min and tested for antimicrobial activity against indicator M. luteus (Ahire et al, 2020a).…”

“…Aliquots of 0.1% TFA (v/v) water containing 0.1 mg/ml sample were each incubated at 40, 60, 80, and 100 • C water bath. At the interval of 10, 30 and 60 min, approximately 1 ml sample was transferred to cool and evaluated antimicrobial activity against M. luteus (Ahire et al, 2020a). In another experiment, a 10-ml aliquot was autoclaved at 121 • C for 15 min and checked for antimicrobial activity.…”