Experimental elucidation of an antimycobacterial bacteriocin produced by ethnomedicinal plant-derived Bacillus subtilis (MK733983)

Abstract

A bacteriocin from Bacillus subtilis (MK733983) originated from ethnomedicinal plant was purified using Preparative RP-HPLC. The HPLC fraction eluted with 65% acetonitrile showed the highest antimicrobial activity with Mycobacterium smegmatis as an indicator. Its specific activity and purification fold increased by 70.5% and 44%, respectively, compared to the crude bacteriocin. The bacteriocin showed stability over a wide range of pH (3.0–8.0) and preservation (− 20 °C and 4 °C), also thermal stability up to 80 °C for 20 min. Its proteinaceous nature was confirmed with complete loss of activity on its treatment with Trypsin, Proteinase K, and α-Chymotrypsin. Nevertheless, the bacteriocin retained up to 45% activity with Papainase treatment and was unaffected by salivary Amylase. It maintained ~ 95% activity on UV exposure up to 3 h and its activity was augmented by ethyl alcohol and metal ions like Fe 2+ and Mn 2+ . Most of the common organic solvents, general surfactants, preservatives, and detergents like Sulfobetaine-14, Deoxy-cholic-acid did not affect the bacteriocin’s action. Its molecular weight was estimated to be 3.4KDa by LC-ESI-MS/MS analysis. The bacteriocin is non-hemolytic and exhibited a broad inhibition spectrum with standard strains of Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Chromobacterium violaceum with MICs ranging 0.225 ± 0.02–0.55 ± 0.05 mg/mL. Scanning Electron Microscopy showed cell annihilation with pores in cell membranes of S. aureus and P. aeruginosa treated with the bacteriocin, implicating bactericidal mode of action. These promising results suggest that the bacteriocin is significant and has wide-ranging application prospects. Supplementary Information The online version contains supplementary material available at 10.1007/s00203-020-02173-7.

Chemical composition and antibacterial activity of ethyl acetate extract of Astragalus membranaceus aerial parts

“…(i) For thermal stability, the EAF solution with a concentration of 100 mg/mL was treated at 80°C, 100°C, and 121°C for 30 min, respectively, and then the filter paper method was conducted as mentioned above to obtain the DIZ values, where the untreated extract was used as the control (Kuang et al, 2018). The residual antimicrobial activity was also analyzed as an indicator of stability study, which was based on the ratio of DIZ values of treated to untreated (Santhi Sudha & Aranganathan, 2021). (ii) The effect of pH on antibacterial activity of EAF was tested by adjusting the pH of EAF solution (50 mg/mL) to 2.0, 4.0, 6.0, 8.0, and 10.0 with 5% HCl or 5% NaOH solution, respectively, and then performed the filter paper method to determine DIZ values and analyze the residual antimicrobial activity as previously mentioned, taking the untreated extract as the control (Qiao, Du, Wang, Han, & Zhou, 2020).…”

Purification, Characterization, Mode of Action, and Application of Jileicin, a Novel Antimicrobial from Paenibacillus jilinensis YPG26

“…The effect of sodium chloride on antimicrobial activity was determined by supplementing different concentrations of sodium chloride (3, 4, and 5%) to jileicin and incubating at 37 °C for 2 h according to the previously described with appropriately modifications, and then the residual antimicrobial activity was determined. The effect of metal ions on antimicrobial activity was determined by assaying the relative activity with the addition of 5 mM metal ions (Mg 2+ , Fe 2+ , Ca 2+ , and Ba 2+ ) according to the previously described methods with appropriate modifications . After incubation at 37 °C for 3 h, the residual antimicrobial activity was determined.…”

“…The effect of ultraviolet rays on antimicrobial activity was determined by measuring residual antimicrobial activity after different times (20, 40, 60 min) of ultraviolet radiation according to the previously described methods with appropriate modifications. 15 The effect of sodium chloride on antimicrobial activity was determined by supplementing different concentrations of sodium chloride (3,4, and 5%) to jileicin and incubating at 37 °C for 2 h according to the previously described with appropriately modifications, 16 and then the residual antimicrobial activity was determined. The effect of metal ions on antimicrobial activity was determined by assaying the relative activity with the addition of 5 mM metal ions (Mg 2+ , Fe 2+ , Ca 2+ , and Ba 2+ ) according to the previously described methods with appropriate modifications.…”

“…The thermal stability of jileicin was determined by exposure to temperatures of 60, 80, and 100 °C for 30 min in a thermostatic water bath, respectively, as well as to 121 °C for 20 min in an autoclave, and then, the residual antimicrobial activity was determined. The effect of ultraviolet rays on antimicrobial activity was determined by measuring residual antimicrobial activity after different times (20, 40, 60 min) of ultraviolet radiation according to the previously described methods with appropriate modifications . The effect of sodium chloride on antimicrobial activity was determined by supplementing different concentrations of sodium chloride (3, 4, and 5%) to jileicin and incubating at 37 °C for 2 h according to the previously described with appropriately modifications, and then the residual antimicrobial activity was determined.…”

“…The effect of metal ions on antimicrobial activity was determined by assaying the relative activity with the addition of 5 mM metal ions (Mg 2+ , Fe 2+ , Ca 2+ , and Ba 2+ ) according to the previously described methods with appropriate modifications. 15 After incubation at 37 °C for 3 h, the residual antimicrobial activity was determined. The effect of organic solvents and detergents on antimicrobial activity was determined by adding different organic solvents and detergents at their working concentrations: methanol, ethanol, butanol, acetone (10%, v/v), Tween 20, Triton X-100 (5%, v/v), SDS (5%, m/v), and EDTA (5 mM) to jileicin and incubated at 37 °C for 3 h according to the previously described with appropriate modifications, 10 and then the residual antimicrobial activity was determined.…”