Inactivation Mechanism of Bacillus subtilis Spores by Ethanol Extract of Torilis japonica Fruit

  • Published : 2009.04.30

Abstract

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 ($C_{22}H_{32}O_5$). 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.

Keywords

References

  1. Banks JG, Morgan S, Stringer MF. Inhibition of heated Bacillus spores by combinations of potassium sorbate, sodium benzoate, pH, and organic acids. Lebensm. -Wiss. Technol. 21: 250-255 (1988)
  2. Chaibi A, Ababouch L, Busta FF. Inhibition of bacterial spores and vegetative cells by glycerides. J. Food Protect. 59: 716-722 (1996) https://doi.org/10.4315/0362-028X-59.7.716
  3. Heo CY, Cho SH. Antimicrobial activity of polylysine produced by Streptomyces sp. J. Agric. Life Sci. 36: 47-52 (2002)
  4. Kim HY, Lee YJ, Kim SH, Hong KH, Kwon YK, Lee JY, Ha SC, Cho HY, Chang IS, Lee CW, Kim KS. Studies of the development of natural preservation from natural products. Korean J. Food Sci. Technol. 31: 1667-1678 (1999)
  5. Kida N, Mochizuki Y, Taguchi F. An effective sporicidal reagent against Bacillus subtilis spores. Microbiol. Immunol. 47: 279-283 (2003) https://doi.org/10.1111/j.1348-0421.2003.tb03396.x
  6. Ko EM, Kim BY. Antimicrobial activity of $\varepsilon$-polylysine mixtures against food-borne pathogens. J. Korean Soc. Food Sci. Nutr. 33:705-710 (2004) https://doi.org/10.3746/jkfn.2004.33.4.705
  7. Oscroft CA, Banks JG, McPhee S. Inhibition of thermally-stressed Bacillus spores by combinations of nisin, pH, and organic acids. Lebensm. -Wiss. Technol. 23: 538-544 (1990)
  8. Ruth GH. Combining bicontrol agents to reduce the variability of biological control. Phytopathology 91: 621-627 (2001) https://doi.org/10.1094/PHYTO.2001.91.7.621
  9. Setlow B, Loshon CA, Genest PC, Cowan AE, Setlow C, Setlow P. Mechanisms of killing spores of Bacillus subtilis by acid, alkali, and ethanol. J. Appl. Microbiol. 92: 362-375 (2002) https://doi.org/10.1046/j.1365-2672.2002.01540.x
  10. Kim HS, Shin JO. Isolation and antimicrobial activity of Xanthium strumarium L. extract. Korean J. Appl. Microbiol. Biotechnol. 25:183-188 (1997)
  11. Nishina A, Tezuka H, Matsunaga M. Inhibition of the development and reduction of heat resistance of bacterial spores by coffee residue extract. J. Antibacter. Antifung. Ag. 23: 681-684 (1995)
  12. Chaibi A, Ababouch L, Belasri K, Boucetta S, Busta FF. Inhibition of germination and vegetative growth of Bacillus cereus T and Clostridium botulinum 62A spores by essential oils. Food Microbiol. 14: 161-174 (1997) https://doi.org/10.1006/fmic.1996.0075
  13. Lee MC, Ryu KS. Studies of the constituents in the fruits of Torilis japonica DC. Bull. KH Pharma. Sci. 6: 61-67 (1978)
  14. Hamouda T, Shin AY, Baker Jr JR. A rapid staining technique for the detection of the initiation of germination of bacterial spores. Lett. Appl. Microbiol. 34: 86-90 (2002) https://doi.org/10.1046/j.1472-765x.2002.01047.x
  15. Lee JY, Yoon JW, Kim CT, Lim ST. Antioxidant activity of phenylpropanoid esters isolated and identified from Platycodon grandiflorum A. DC. Phytochemistry 65: 3033-3039 (2004) https://doi.org/10.1016/j.phytochem.2004.08.030
  16. Lim SN, Cheung PC, Ooi VE, Ang PO. Evaluation of antioxidative activity of extracts from a brown seaweed, Sargassum siliquastrum. J. Agr. Food Chem. 50: 3862-3866 (2002) https://doi.org/10.1021/jf020096b
  17. Kang SS, Lee EB, Kim TH, Kim KR, Jung JH. The NMR assignments of torilin from Torilis japonica. Arch. Pharm. Res. 17:284-286 (1994) https://doi.org/10.1007/BF02980463
  18. Kim MS, Lee YM, Moon E-J, Kim SE, Lee JJ, Kim KW. Antiangiogenic activity of torilin, a sesquiterpene compound isolated from Torilis japonica. Int. J. Cancer. 87: 269-275 (2000) https://doi.org/10.1002/1097-0215(20000715)87:2<269::AID-IJC19>3.0.CO;2-W
  19. Ryu JH, Jeong YS. A new guaiane type sesquiterpene from Torilis japonica. Arch. Pharm. Res. 24: 532-535 (2001) https://doi.org/10.1007/BF02975160
  20. Moriyama R. Molecular mechanism of cortex hydrolysis during bacterial spore germination. Nippon Nogeik. Kaishi 72: 1455-1461 (1998) https://doi.org/10.1271/nogeikagaku1924.72.1455
  21. Russell AD. Bacterial spores and chemical sporicidal agents. Clin. Microbiol. Rev. 3: 99-119 (1990) https://doi.org/10.1128/CMR.3.2.99