| Summary: | Exopolysaccharides (EPSs) produced by <i>Bacillus</i> species have recently emerged as promising commercial antioxidants in various industries, such as pharmaceutics and biomedicine. However, little is known about EPS production and function from <i>Bacillus velezensis</i> so far. In the present study, the effect of sugar sources on EPS production by <i>B. velezensis</i> VTX20 and the genetic biosynthesis, characteristics, and antioxidant activity of the resulting EPS were evaluated. The strain VTX20 produced the maximum EPS yield of 75.5 ± 4.8 g/L from an initial 200 g/L of sucrose after a 48-h cultivation. Through genomic analysis, <i>ls</i>-<i>levB</i> operon was found, for the first time, to be responsible for the levan-type EPS production in <i>B. velezensis</i>. Biochemical and structural characterization further confirmed the majority of levan, followed by an extremely low level of dextran biopolymer. The water solubility index and water holding capacity of the EPSs were 81.9 ± 3.4% and 100.2 ± 3.4%, respectively. In vitro antioxidant activity analyses showed strong scavenging activity for 1,1-diphenyl-2-picrylhydrazyl and hydroxyl radical values of 40.1–64.0% and 16.0–40%, respectively. These findings shed light on the EPS biosynthesis of <i>B. velezensis</i> at both structural and genetic levels and the potential application of EPS as a natural antioxidant for pharmaceutical and biomedical industries.
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