Addition of ethanol to supercritical carbon dioxide enhances the inactivation of bacterial spores in the biofilm of Bacillus cereus

Hyong Seok Park, Hee Jung Choi, Myoung Dong Kim, Kyoung Heon Kim

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27 Scopus citations


Supercritical carbon dioxide (SC-CO2) was used to inactivate Bacillus cereus spores inside biofilms, which were grown on stainless steel. SC-CO2 treatment was tested using various conditions, such as pressure treatment (10-30MPa), temperature (35-60°C), and time (10-120min). B. cereus vegetative cells in the biofilm were completely inactivated by treatment with SC-CO2 at 10MPa and at 35°C for 5min. However, SC-CO2 alone did not inactivate spores in biofilm even after the treatment time was extended to 120min. When ethanol was used as a cosolvent with SC-CO2 in the SC-CO2 treatment using only 2-10ml of ethanol in 100ml of SC-CO2 vessel for 60-90min of treatment time at 10MPa and 60°C, B. cereus spores in the biofilm were found to be completely inactivated in the colony-forming test. We also assessed the viability of SC-CO2-treated bacterial spores and vegetative cells in the biofilm by staining with SYTO 9 and propidium iodide. The membrane integrity of the vegetative cells was completely lost, while the integrity of the membrane was still maintained in most spores. However, when SC-CO2 along with ethanol was used, both vegetative cells and spores lost their membrane integrity, indicating that the use of ethanol as a cosolvent with SC-CO2 is efficient in inactivating the bacterial spores in the biofilm.

Original languageEnglish
Pages (from-to)207-212
Number of pages6
JournalInternational Journal of Food Microbiology
Issue number2
StatePublished - 2 Sep 2013

Bibliographical note

Funding Information:
This work was supported by grants from the Advanced Biomass R&D Center of Korea ( 2011-0031353 ) funded by the Korean Government (MEST) and from the financial support of the Ministry for Oceans and Fisheries ( 12128295500 ). A Korea University Grant for H. S. Park is also acknowledged. Experiments were carried out using the facilities of the Institute of Biomedical Science and Food Safety at the Food Safety Hall, Korea University.


  • Biofilm
  • Cosolvent
  • Spore
  • Sterilization
  • Supercritical carbon dioxide


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