Applied Microbiology and Biotechnology

, Volume 103, Issue 5, pp 2329–2338 | Cite as

Effect of ultrasonication and thermal and pressure treatments, individually and combined, on inactivation of Bacillus cereus spores

  • Ruiling Lv
  • Mingming Zou
  • Thunthacha Chantapakul
  • Weijun Chen
  • Aliyu Idris Muhammad
  • Jianwei Zhou
  • Tian Ding
  • Xingqian Ye
  • Donghong LiuEmail author
Applied microbial and cell physiology


Bacillus cereus spores are a concern to the food industry due to their high resistance to processing and their ability to germinate to vegetative cells under suitable conditions. This research aimed to elucidate the mechanisms of Bacillus cereus spore inactivation under ultrasonication (US) combined with thermal (thermosonication, TS) treatments, with pressure (manosonication, MS) treatments, and with thermal and pressure (manothermosonication, MTS) treatments. Electronic microscopy, dipicolinic acid (DPA) release, and flow cytometric assessments were used to investigate the inactivation effect and understand the inactivation mechanisms. The sporicidal effects of the US and thermal treatment were slight, and the MS and TS also showed little inactivation effect. However, ultrasonication promoted the detachment of the exosporium, thereby reducing the spore’s ability to adhere to a surface, while the thermal treatment induced a decrease in the electron density in the nucleoid of bacterium, which retained a relatively intact exosporium and coat. MS caused 92.54% DPA release, which might be due to triggering of the germinant receptors or releasing of ions and Ca2+-DPA. In addition, the morphological changes such as core hydration and cortex degradation were significant after treatment with MS. The release of DPA and the morphological changes were responsible for the reduction in thermal resistance. The MTS showed a remarkable inactivation effect of 3.12 log CFU/mL reductions after 30 min of treatment. It was the most effective treatment and exhibited a large fraction of damage. In addition, the MTS had a significant impact on the intracellular structure of the spores, with the coat destroyed and the cortex damaged. These results indicated that ultrasonication combined with thermal and pressure treatments had a significant sporicidal effect on Bacillus cereus spores and could be a promising green sterilization technology.


Bacillus cereus spores Ultrasonication Thermosonication Manothermosonication Synergistic inactivation 



This study was funded by the National Key Research and Development Program of China (grant number 2016YFD0400301).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants performed by any of the authors.


  1. Alvarez I, Manas P, Sala FJ, Condon S (2003) Inactivation of Salmonella enterica serovar enteritidis by ultrasonic waves under pressure at different water activities. Appl Environ Microbiol 69(1):668–672. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Alvarez I, Manas P, Virto R, Condon S (2006) Inactivation of Salmonella Senftenberg 775W by ultrasonic waves under pressure at different water activities. Int J Food Microbiol 108(2):218–225. CrossRefPubMedGoogle Scholar
  3. Ansari JA, Ismail M, Farid M (2017) Investigation of the use of ultrasonication followed by heat for spore inactivation. Food Bioprod Process 104:32–39. CrossRefGoogle Scholar
  4. Arnesen LPS, Fagerlund A, Granum PE (2008) From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev 32(4):579–606. CrossRefGoogle Scholar
  5. Black EP, Koziol-Dube K, Guan DS, Wei H, Setlow B, Cortezzo DE, Hoover DG, Setlow P (2005) Factors influencing germination of Bacillus subtilis spores via activation of nutrient receptors by high pressure. Appl Environ Microbiol 71(10):5879–5887. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bottone EJ (2010) Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev 23(2):382-+ doi:
  7. Butz P, Tauscher B (2002) Emerging technologies: chemical aspects. Food Res Int 35(2–3):279–284. CrossRefGoogle Scholar
  8. Cai R, Yuan YH, Wang ZL, Guo CF, Liu B, Yue TL (2015) Reduction of Alicyclobacillus acidoterrestris spores on apples by chlorine dioxide in combination with ultrasound or shaker. Food Bioprocess Technol 8(12):2409–2417. CrossRefGoogle Scholar
  9. Carstensen EL (1986) Biological effects of acoustic cavitation. Ultrasound Med Biol 12(9):703–704. CrossRefGoogle Scholar
  10. Chemat F, Zill e H, Khan MK (2011) Applications of ultrasound in food technology: processing, preservation and extraction. Ultrason Sonochem 18(4):813–835. CrossRefPubMedGoogle Scholar
  11. Chemat F, Rombaut N, Meullemiestre A, Turk M, Perino S, Fabiano-Tixier AS, Abert-Vian M (2017a) Review of green food processing techniques. Preservation, transformation, and extraction. Innovative Food Sci Emerg Technol 41:357–377. CrossRefGoogle Scholar
  12. Chemat F, Rombaut N, Sicaire AG, Meullemiestre A, Fabiano-Tixier AS, Abert-Vian M (2017b) Ultrasound assisted extraction of food and natural products. Mechanisms, techniques, combinations, protocols and applications. A review. Ultrason Sonochem 34:540–560. CrossRefPubMedGoogle Scholar
  13. Coleman WH, De C, Li YQ, Cowan AE, Setlow P (2007) How moist heat kills spores of Bacillus subtilis. J Bacteriol 189(23):8458–8466. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Condon-Abanto S, Arroyo C, Alvarez I, Condon S, Lyng JG (2016) Application of ultrasound in combination with heat and pressure for the inactivation of spore forming bacteria isolated from edible crab (Cancer pagurus). Int J Food Microbiol 223:9–16. CrossRefPubMedGoogle Scholar
  15. Diaz M, Herrero M, Garcia LA, Quiros C (2010) Application of flow cytometry to industrial microbial bioprocesses. Biochem Eng J 48(3):385–407. CrossRefGoogle Scholar
  16. Evelyn E, Silva FVM (2015) Thermosonication versus thermal processing of skim milk and beef slurry: modeling the inactivation kinetics of psychrotrophic Bacillus cereus spores. Food Res Int 67:67–74. CrossRefGoogle Scholar
  17. Evelyn, Silva FVM (2015a) Inactivation of Byssochlamys nivea ascospores in strawberry puree by high pressure, power ultrasound and thermal processing. Int J Food Microbiol 214:129–136. CrossRefPubMedGoogle Scholar
  18. Evelyn, Silva FVM (2015b) Use of power ultrasound to enhance the thermal inactivation of Clostridium perfringens spores in beef slurry. Int J Food Microbiol 206:17–23. CrossRefPubMedGoogle Scholar
  19. Evelyn, Silva FVM (2016a) High pressure processing pretreatment enhanced the thermosonication inactivation of Alicyclobacillus acidoterrestris spores in orange juice. Food Control 62:365–372. CrossRefGoogle Scholar
  20. Evelyn, Silva FVM (2016b) High pressure thermal processing for the inactivation of Clostridium perfringens spores in beef slurry. Innovative Food Sci Emerg Technol 33:26–31. CrossRefGoogle Scholar
  21. Evelyn, Silva FVM (2018) Differences in the resistance of microbial spores to thermosonication, high pressure thermal processing and thermal treatment alone. J Food Eng 222:292–297. CrossRefGoogle Scholar
  22. Ferrario M, Alzamora SM, Guerrero S (2015) Study of the inactivation of spoilage microorganisms in apple juice by pulsed light and ultrasound. Food Microbiol 46:635–642. CrossRefPubMedGoogle Scholar
  23. Henriques AO, Moran CP (2007) Structure, assembly, and function of the spore surface layers annual review of microbiology. Ann Rev Microbiol 61:555–588CrossRefGoogle Scholar
  24. Janssen FW, Lund AJ, Anderson LE (1958) Colorimetric assay for dipicolinic acid in bacterial spores. Science 127(3288):26–27. CrossRefPubMedGoogle Scholar
  25. Jimenez-Sanchez C, Lozano-Sanchez J, Segura-Carretero A, Fernandez-Gutierrez A (2016) Alternatives to conventional thermal treatments in fruit-juice processing. Part 1: techniques and applications. Crit Rev Food Sci Nutr 57(3):501–523. CrossRefGoogle Scholar
  26. Khanal SN, Anand S, Muthukumarappan K (2014) Evaluation of high-intensity ultrasonication for the inactivation of endospores of 3 bacillus species in nonfat milk. J Dairy Sci 97(10):5952–5963. CrossRefPubMedGoogle Scholar
  27. Lee HI, Zhou B, Liang W, Feng H, Martin SE (2009) Inactivation of Escherichia coli cells with sonication, manosonication, thermosonication, and manothermosonication: microbial responses and kinetics modeling. J Food Eng 93(3):354–364. CrossRefGoogle Scholar
  28. Lee H, Kim H, Cadwallader KR, Feng H, Martin SE (2013) Sonication in combination with heat and low pressure as an alternative pasteurization treatment - effect on Escherichia coli K12 inactivation and quality of apple cider. Ultrason Sonochem 20(4):1131–1138. CrossRefPubMedGoogle Scholar
  29. Li J, Ahn J, Liu DH, Chen SG, Ye XQ, Ding T (2016) Evaluation of ultrasound-induced damage to Escherichia coli and Staphylococcus aureus by flow cytometry and transmission Electron microscopy. Appl Environ Microbiol 82(6):1828–1837. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Li J, Ding T, Liao XY, Chen SG, Ye XQ, Liu DH (2017) Synergetic effects of ultrasound and slightly acidic electrolyzed water against Staphylococcus aureus evaluated by flow cytometry and electron microscopy. Ultrason Sonochem 38:711–719. CrossRefPubMedGoogle Scholar
  31. Lv RL, Chantapakul T, Zou MM, Li M, Zhou JW, Ding T, Ye XQ, Liu DH (2018) Thermal inactivation kinetics of Bacillus cereus in Chinese rice wine and in simulated media based on wine components. Food Control 89:308–313. CrossRefGoogle Scholar
  32. Magnusson M, Christiansson A, Svensson B (2007) Bacillus cereus spores during housing of dairy cows: factors affecting contamination of raw milk. J Dairy Sci 90(6):2745–2754. CrossRefPubMedGoogle Scholar
  33. Mathys A, Chapman B, Bull M, Heinz V, Knorr D (2007) Flow cytometric assessment of Bacillus spore response to high pressure and heat. Innovative Food Sci Emerg Technol 8(4):519–527. CrossRefGoogle Scholar
  34. Milani EA, Silva FVM (2017) Ultrasound assisted thermal pasteurization of beers with different alcohol levels: inactivation of Saccharomyces cerevisiae ascospores. J Food Eng 198:45–53. CrossRefGoogle Scholar
  35. Neppiras EA (1980) Acoustic cavitation. Phys Rep Rev Sect Phys Lett 61(3):159–251. Google Scholar
  36. Oomes S, van Zuijlen ACM, Hehenkamp JO, Witsenboer H, van der Vossen J, Brul S (2007) The characterisation of Bacillus spores occurring in the manufacturing of (low acid) canned products. Int J Food Microbiol 120(1–2):85–94. CrossRefPubMedGoogle Scholar
  37. Plesset MS (1972) Temperature effects in cavitation damage. J Basic Eng 94(3):559. CrossRefGoogle Scholar
  38. Raso J, Barbosa-Canovas GV (2003) Nonthermal preservation of foods using combined processing techniques. Crit Rev Food Sci Nutr 43(3):265–285. CrossRefPubMedGoogle Scholar
  39. Raso J, Pagan R, Condon S, Sala FJ (1998a) Influence of temperature and pressure on the lethality of ultrasound. Appl Environ Microbiol 64(2):465–471PubMedPubMedCentralGoogle Scholar
  40. Raso J, Palop A, Pagan R, Condon S (1998b) Inactivation of Bacillus subtilis spores by combining ultrasonic waves under pressure and mild heat treatment. J Appl Microbiol 85(5):849–854. CrossRefPubMedGoogle Scholar
  41. Rayman A, Baysal T (2011) Yield and quality effects of electroplasmolysis and microwave applications on carrot juice production and storage. J Food Sci 76(4):C598–C605. CrossRefPubMedGoogle Scholar
  42. Reineke K, Mathys A, Heinz V, Knorr D (2013a) Mechanisms of endospore inactivation under high pressure. Trends Microbiol 21(6):296–304. CrossRefPubMedGoogle Scholar
  43. Reineke K, Schlumbach K, Baier D, Mathys A, Knorr D (2013b) The release of dipicolinic acid - the rate-limiting step of Bacillus endospore inactivation during the high pressure thermal sterilization process. Int J Food Microbiol 162(1):55–63. CrossRefPubMedGoogle Scholar
  44. Scherba G, Weigel RM, Obrien WD (1991) Quantitative assessment of the germicidal efficacy of ultrasonic energy. Appl Environ Microbiol 57(7):2079–2084PubMedPubMedCentralGoogle Scholar
  45. Sevenich R, Reineke K, Hecht P, Frohling A, Rauh C, Schluter O, Knorr D (2015) Impact of different water activities (a(w)) adjusted by solutes on high pressure high temperature inactivation of Bacillus amyloliquefaciens spores. Front Microbiol 6:689.
  46. Soni A, Oey I, Silcock P, Bremer P (2016) Bacillus spores in the food industry: a review on resistance and response to novel inactivation technologies. Compr Rev Food Sci Food Saf 15(6):1139–1148. CrossRefGoogle Scholar
  47. Stewart GC (2015) The Exosporium layer of bacterial spores: a connection to the environment and the infected host. Microbiol Mol Biol Rev 79(4):437–457. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Suslick KS (1990) Sonochemistry. Science 247(4949):1439–1445. CrossRefPubMedGoogle Scholar
  49. Tseng S, Abramzon N, Jackson JO, Lin WJ (2012) Gas discharge plasmas are effective in inactivating Bacillus and Clostridium spores. Appl Microbiol Biotechnol 93(6):2563–2570. CrossRefPubMedGoogle Scholar
  50. Ugarte-Romero E, Feng H, Martin SE (2007) Inactivation of Shigella boydii 18 IDPH and Listeria monocytogenes Scott A with power ultrasound at different acoustic energy densities and temperatures. J Food Sci 72(4):M103–M107. CrossRefPubMedGoogle Scholar
  51. Wang LP, Xia Q, Li YF (2017) The effects of high pressure processing and slightly acidic electrolysed water on the structure of Bacillus cereus spores. Food Control 79:94–100. CrossRefGoogle Scholar
  52. Wells-Bennik MHJ, Eijlander RT, den Besten HMW, Berendsen EM, Warda AK, Krawczyk AO, Groot MNN, Xiao YH, Zwietering MH, Kuipers OP, Abee T (2016) Bacterial spores in food: survival, emergence, and outgrowth. In: Doyle MP, Klaenhammer TR (eds) Annual review of food science and technology, vol 7. Annual review of food science and technology, vol 7, pp 457–482Google Scholar
  53. Williams G, Linley E, Nicholas R, Baillie L (2013) The role of the exosporium in the environmental distribution of anthrax. J Appl Microbiol 114(2):396–403. CrossRefPubMedGoogle Scholar
  54. Wood RJ, Lee J, Bussemaker MJ (2017) A parametric review of sonochemistry: control and augmentation of sonochemical activity in aqueous solutions. Ultrason Sonochem 38:351–370. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Ruiling Lv
    • 1
  • Mingming Zou
    • 1
  • Thunthacha Chantapakul
    • 1
  • Weijun Chen
    • 1
  • Aliyu Idris Muhammad
    • 1
  • Jianwei Zhou
    • 1
    • 2
  • Tian Ding
    • 1
  • Xingqian Ye
    • 1
  • Donghong Liu
    • 1
    • 3
    Email author
  1. 1.College of Biosystems Engineering and Food Science, National-Local Joint Engineering Laboratory of Intelligent Food Technology and Equipment, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang R and D Center for Food Technology and EquipmentZhejiang UniversityHangzhouChina
  2. 2.Ningbo Institute of TechnologyZhejiang UniversityNingboChina
  3. 3.Fuli Institute of Food ScienceZhejiang UniversityHangzhouChina

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