Optimization and purification of a novel bacteriocin-like substance from Tarhana-originated <i>Enterococcus faecium</i>

This research was carried out to examine the <i>in vitro</i> probiotic characteristics of <i>Enterococcus faecium</i> T49 isolated, to optimize bacteriocin-like substances (BLIS) production using the Box-Behnken design, and also to purify and characterize BLIS. <i>E. faecium</i> T49 exhibited high resistance to low pH, bile salts (118.23-120.90%), pepsin (102.17%), pancreatin (89.80%), and phenol (137.56%). The strain demonstrated different levels of susceptibility to antibiotics. The amounts of exopolysaccharide, hydrogen peroxide and diacetyl produced by <i>E. faecium</i> T49 were 119.90 µg/mL, 0.25 µg/mL and 87.88 ng/mL, respectively. The maximum BLIS production was detected under conditions where incubation temperature was 36 °C, pH value was 8, and incubation time was 18 h. Optimization of the growth parameters resulted in a 4.03 fold increase in BLIS activity by <i>E. faecium</i> T49. BLIS, which was purified using ammonium sulfate precipitation, dialysis and solid phase extraction, demonstrated high stability to pH, heat and enzyme, while the activity was entirely inactivated by proteolytic enzymes. The molecular weight of BLIS was specified between 3.496 and 6.500 kDa. BLIS could be provisionally classified as a Class II bacteriocin, based on its antimicrobial activity against <i>Listeria monocytogenes</i>, heat resistance, and molecular weight under 10 kDa; however, this classification remains tentative in the absence of molecular structural or genetic sequence data. These properties suggest its potential application as a natural bio-preservative in food products and in strategies to control foodborne pathogens. The current study is very important as it introduces novel BLIS to scientific literature.