Inactivation mechanism of Bacillus subtilis spores by ethanol extract of Torilis japonica fruit

To confirm the antimicobial mechanism of Torilis japonica, antimicrobial profile was observed on various spore conditions by combining 0.1% (3 mM) torilin with antimicrobial activity and 0.27% water fraction with germinants. A 75% ethanol extract of T. japonica fruit reduced Bacillus subtilis ATCC 6633 spore counts by 3 log cycles and reduced the vegetative cells to undetectable level (by about 6 log cycles) (both in terms of CFU/mL). Further fractionating the ethanol extract into n-hexane and water fractions revealed that the former reduced the spore count by 1 log cycle whereas the latter had no effect. The antimicrobial active compound was isolated and purified from the hexane layer, and identified as torilin (C₂₂H₃₂O₅). The water fraction of the ethanol layer did not show antimicrobial activity, whereas the antimicrobial effect of 0.1% (3 mM) torilin was significantly enhanced in the presence of the water fraction (0.27%). This result can be explained by synergistic effects of the water fraction containing considerable amounts of germinants such as L-alanine and K⁺ ions that triggered germination.