Trypticase soy agar

De Man, Rogosa, and Sharpe (MRS) broth, potato soluble starch, glucose yeast extract agar, trypticase soy agar, eosin methylene blue agar (EMB Agar), glucose yeast extract agar (GYEA) were purchased from HiMedia, Mumbai, India. Lysozyme, pepsin, fructooligosaccharide (FOS), pancreatin from porcine, bile salt, copper sulfate, and SCFAs standards (acetic, propionic and butyric acids) were procured from Sigma‐Aldrich (St. Louis, MO, USA). Oxyrase was procured from Oxyrase, Inc, Mansfield, OH, USA. Probiotic bacteria B. coagulans MTCC 5856 is a patented strain of Sami Labs Limited and deposited to Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India. E. coli ATCC 25922 was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). trypticase soy agar (TSA; Becton Dickinson) was used for culturing of bacteria, and culture stocks were maintained in aqueous glycerol (15%, v/v) at −80°C.

To investigate the activities of the EntP peptide in the presence of high salt concentrations, the MIC was determined as described above, except that fixed concentration of NaCl was added to each well of the microtiter plate. Overnight cultures of S. aureus (ATCC 25923) and E. coli (ATCC 25922) as Gram-positive and Gram-negative model strains were incubated in MHB with 0, 50, 100, or 150 mM NaCl for 4 h. Bacteria were serially diluted and plated in triplicate on Trypticase soy agar (HiMedia, India) plates. CFUs were counted after 24 h of incubation at 37 °C. The stability of EntP peptide in 50% human plasma was evaluated as previously described (Hou et al. 2011 (link)). The human plasma was determined to contain no antimicrobial activity before the test. Then 800 μg/ml of recombinant EntP peptide was diluted 1:1 with fresh human plasma and incubated at 37 °C for 0, 3, or 6 h. After incubation, the antimicrobial activity of each sample was determined by MIC assays with S. aureus (ATCC 25923) and E. coli (ATCC 25922).