Temperature-dependency on the inactivation of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles

  • Fumiyuki KobayashiEmail author
  • Sachiko Odake
Original Article


Temperature-dependency on cell membrane injury and inactivation of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles (MBCO2) was investigated. The number of surviving S. pastorianus cells after MBCO2 treatment detected with yeast and mould agar (YMA, an optimum agar) was higher than that with YMA adding 2.5 g/L sodium chloride and yeast nitrogen base agar (a minimum agar). However, the decrease of the surviving number by thermal treatment was not changed among above agars used. The fluorescence polarization (FP), which indicated the phase transition of the membrane of S. pastorianus cells treated with MBCO2 increased with increasing temperature. The activity of the alkaline phosphatase (AP), a periplasmic enzyme, in S. pastorianus cells after MBCO2 and thermal treatments increased with the FP but was reduced by further increasing temperature. The FP and AP activities after MBCO2 treatment increased at a temperature lower than the temperature of the thermal treatment. In addition, intracellular pH of S. pastorianus decreased by the MBCO2 treatment at lower temperature with increasing pressure. Therefore, it was revealed that phase transition of the cell membrane and inactivation of S. pastorianus was caused by MBCO2 treatment at lower temperature than thermal treatment and that the effect was induced by the dissolved CO2 and increased with increasing pressure.


Cell membrane injury Inactivation Intracellular pH Low-pressure carbon dioxide microbubbles Saccharomyces pastorianus 



We thank Riho Takanashi and Yuhei Kameda of the Faculty of Applied Life Science, Nippon Veterinary and Life Science University (Musashino, Japan) for experimental assistance. A part of this study was financially supported by Mishima Kaiun Memorial Foundation (Tokyo, Japan) in 2016.


  1. Amaral GV, Silva EK, Cavalcanti RN, Cappato LP, Guimaraes JT, Alvarenga VO, Esmerino EA, Portela JB, Sant’Ana AS, Freitas MQ, Silva MC, Raices RSL, Meireles MAA, Cruz AG (2017) Dairy processing using supercritical carbon dioxide technology: theoretical fundamentals, quality and safety aspects. Trends Food Sci Technol 64:94–101CrossRefGoogle Scholar
  2. Barba FJ, Koubaa M, Prado-Silva L, Orlien V, Sant’Ana AS (2017) Mild processing applied to the inactivation of the main foodborne bacterial pathogens: a review. Trends Food Sci Technol 66:20–35CrossRefGoogle Scholar
  3. Bertoloni G, Bertucco A, de Cian V, Parton T (2006) A study on the inactivation of micro-organisms and enzymes by high pressure CO2. Biotechnol Bioeng 95:155–160CrossRefGoogle Scholar
  4. Bothun GD, Knutson BL, Strobel HJ, Nokes SE (2005) Liposome fluidization and melting point depression by pressurized CO2 determined by fluorescence anisotropy. Langmuir 21:530–536CrossRefGoogle Scholar
  5. Garcia-Gonzalez L, Geeraerd AH, Spilimbergo S, Elst K, Van Ginneken L, Debevere L, Van Impe JF, Devlieghere F (2007) High pressure carbon dioxide inactivation of microorganisms in foods: the past, the present and the future. Int J Food Microbiol 117:1–28CrossRefGoogle Scholar
  6. Giulitti S, Cinquemani C, Quaranta A, Spilimbergo S (2011) Real time intracellular pH dynamics in Listeria innocua under CO2 and N2O pressure. J Supercrit Fluids 58:385–390CrossRefGoogle Scholar
  7. Guimarāes JT, Silva EK, de Freitas MQ, Meireles MAA, de Cruz AG (2018) Non-thermal emerging technologies and their effects on the functional properties of dairy products. Curr Opin Food Sci 22:62–66CrossRefGoogle Scholar
  8. Hong SI, Pyun YR (2001) Membrane damage and enzyme inactivation of Lactobacillus plantarum by high pressure CO2 treatment. Int J Food Microbiol 63:19–28CrossRefGoogle Scholar
  9. Howladar MS, French WT, Shields-Menard SA, Amirsadeghi M, Green M, Rai N (2017) Microbial cell disruption for improving lipid recovery using pressurized CO2: role of CO2 solubility in cell suspension, sugar broth, and spent media. Biotechnol Prog 33:737–748CrossRefGoogle Scholar
  10. Hurst A, Hughes A, Beare-Rogers JL, Collins-Thompson DL (1973) Physiological studies on the recovery of salt tolerance by Staphylococcus aureus after sublethal heating. J Bacteriol 116:901–907Google Scholar
  11. Katsui N, Tsuchido T, Hiramatsu R, Fujikawa S, Takano M, Shibasaki I (1982) Heat-induced blebbing and vesiculation of the outer membrane of Escherichia coli. J Bacteriol 151:1523–1531Google Scholar
  12. Kim SR, Rhee MS, Kim BC, Kim KH (2007) Modeling the inactivation of Escherichia coli O157:H7 and generic Escherichia coli by supercritical carbon dioxide. Int J Food Microbiol 118:52–61CrossRefGoogle Scholar
  13. Kobayashi F, Odake S (2015) Quality evaluation of unfiltered beer as affected by inactivated yeast using two-stage system of low pressure carbon dioxide microbubbles. Food Bioprocess Technol 8:1690–1698CrossRefGoogle Scholar
  14. Kobayashi F, Odake S (2017) Intracellular acidification and change of cellular membrane fluidity of Saccharomyces pastorianus by low pressure CO2 microbubbles. Food Cont 71:360–370CrossRefGoogle Scholar
  15. Kobayashi F, Odake S (2018) The relationship between intracellular acidification and inactivation of Saccharomyces pastorianus by a two-stage system with pressurized carbon dioxide microbubbles. Biochem Eng J 134:88–93CrossRefGoogle Scholar
  16. Kobayashi F, Ikeura H, Odake S, Sakurai H (2014) Quality evaluation of sake treated with a two-stage system of low pressure carbon dioxide microbubbles. J Agric Food Chem 62:11722–11729CrossRefGoogle Scholar
  17. Li P, Takahashi M, Chiba K (2009a) Enhanced free-radical generation by shrinking microbubbles using a copper catalyst. Chemosphere 77:1157–1160CrossRefGoogle Scholar
  18. Li P, Takahashi M, Chiba K (2009b) Degradation of phenol by the collapse of microbubbles. Chemosphere 77:1371–1375CrossRefGoogle Scholar
  19. Li H, Deng L, Chen Y, Liao X (2012) Inactivation, morphology, interior structure and enzymatic activity of high pressure CO2-treated Saccharomyces cerevisiae. Innov Food Sci Emerg Technol 14:99–106CrossRefGoogle Scholar
  20. Li J, Wang A, Zhu F, Xu R, Hu S (2013) Membrane damage induced by supercritical carbon dioxide in Rhodotorula mucilaginosa. Ind J Microbiol 53:352–358CrossRefGoogle Scholar
  21. Liao H, Zhang F, Liao X, Hu X, Chen Y, Deng L (2010) Analysis of Escherichia coli cell damage induced by HPCD using microscopies and fluorescent staining. Int J Food Microbiol 144:169–176CrossRefGoogle Scholar
  22. Nikerson WJ, Krugelis EJ, Andresen N (1948) Localization of alkaline phosphatase in yeast. Nature 162:192–193CrossRefGoogle Scholar
  23. Silva EK, Alvarenga VO, Bargas MA, Sant’Ana AS, Meireles MAA (2018) Non-thermal microbial inactivation by using supercritical carbon dioxide: synergic effect of process parameters. J Supercrit Fluids 139:97–104CrossRefGoogle Scholar
  24. Spilimbergo S, Foladori P, Mantoan D, Ziglio G, Della Mea G (2010a) High-pressure CO2 inactivation and induced damage on Saccharomyces cerevisiae evaluated by flow cytometry. Process Biochem 45:647–654CrossRefGoogle Scholar
  25. Spilimbergo S, Quaranta A, Garcia-Gonzalez L, Contrini C, Cinquemani C, Van Ginneken L (2010b) Intracellular pH measurement during high-pressure CO2 pasteurization evaluated by cell fluorescent staining. J Supercrit Fluids 53:185–191CrossRefGoogle Scholar
  26. Straka RP, Stokes JL (1959) Metabolic injury to bacteria at low temperature. J Bacteriol 78:181–185Google Scholar
  27. Takahashi M, Chiba K, Li P (2007a) Free-radical generation from collapsing microbubbles in the absence of a dynamic stimulus. J Phys Chem B 111:1343–1347CrossRefGoogle Scholar
  28. Takahashi M, Chiba K, Li P (2007b) Formation of hydroxyl radicals by collapsing ozone microbubbles under strongly acidic conditions. J. Phys Chem B 111:11443–11446CrossRefGoogle Scholar
  29. Tamburini S, Anesi A, Ferrentino G, Spilimbergo S, Guella G, Jousson O (2014) Supercritical CO2 induced marked changes in membrane phospholipids composition in Escherichia coli K12. J Membr Biol 247:469–477CrossRefGoogle Scholar
  30. Tsuchido T, Katsui N, Takeuchi A, Takano M, Shibasaki I (1985) Destruction of the outer membrane permeability barrier of Escherichia coli by heat treatment. Appl Environ Microbiol 50:298–303Google Scholar
  31. Watanabe T, Furukawa S, Kitamoto K, Takatsuki A, Hirata R, Ogihara H, Yamasaki M (2005) Vacuolar H+-ATPase and plasma membrane H+-ATPase contribute to the tolerance against high-pressure carbon dioxide treatment in Saccharomyces cerevisiae. Int J Food Microbiol 105:131–137CrossRefGoogle Scholar
  32. Wu Y, Yao SJ, Guan YX (2007) Inactivation of microoganisms in carbon dioxide at elevated pressure and ambient temperature. Ind Eng Chem Res 46:6345–6352CrossRefGoogle Scholar

Copyright information

© Association of Food Scientists & Technologists (India) 2019

Authors and Affiliations

  1. 1.Faculty of Applied Life ScienceNippon Veterinary and Life Science UniversityMusashinoJapan

Personalised recommendations