4.3. Extraction of Antimicrobial Compounds (AMCs)

L. coryniformis BCH-4 was cultured in 6L of MRS broth (pH 6.4 ± 0.2) at 37 °C for 72 h with constant stirring at 120 rpm in the fermenter (BioFer-010, ICCC, Islamabad, Pakistan). After 72 h, cell-free supernatant (CFS) of L. coryniforims BCH-4 was prepared by centrifugation (Z326K, Hermle, Wehingen, Germany) at 6000 rpm (4430× g) for 10 min at 4 °C and subsequently filtered through sterilized 0.22 μm pore-size filters (Advantec Toyo Kaisha, Ltd. Tokyo, Japan). AMCs were extracted from filter-sterilized CFS by the solvent phase extraction according to Wang et al. [56], with a few modifications. Ethyl acetate was used as an extracting solvent and AMCs were extracted by mixing same ratio of CFS and solvent [150 mL:150 mL] in an Erlenmeyer flask followed by shaking at 120 rpm on table-top shaker (Phoenix RS-OS 10, Irmeco, Lütjensee, Germany) for 2 h at room temperature. Mixture was poured in a separating funnel and allowed to stand until organic and aqueous phases were separated. The organic phase was collected, and the aqueous phase was further used to extract the maximum amount of AMCs in CFS. This process was repeated three times and organic phase was combined, concentrated by rotary evaporator (Rotavapor^® R-2100, Buchi, Flawil, Switzerland) under vacuum at lower than 40 °C. Ethyl acetate was evaporated and extracted dark brown concentrated (viscous) compounds were further evaluated for their bioactive potential.