Inactivation of Bacillus subtilis spores by high pressure CO2 with high temperature

https://doi.org/10.1016/j.ijfoodmicro.2015.04.012Get rights and content

Highlights

  • The spore clumping induced a protective effect on Bacillus subtilis spores.

  • The inactivation kinetics of Bacillus subtilis spores showed nonlinear behavior.

  • High pressure CO2 inactivated bacterial spores by damaging the inner membrane.

Abstract

The objective of this study was to investigate the inactivation of the Bacillus subtilis spores by high pressure CO2 combined with high temperature (HPCD + HT) and to analyze the clumping effect of the spores on their HPCD + HT resistance. The spores of B. subtilis were subjected to heat at 0.1 MPa and HPCD at 6.5–25 MPa, and 82 °C, 86 °C, and 91 °C for 0–120 min. The spores were effectively inactivated by HPCD + HT, but a protective effect on the spores was also found, which was closely correlated to the pressure, temperature and time. The spores treated by HPCD + HT at 6.5 and 10 MPa exhibited a two-stage inactivation curve of shoulder and log-linear regions whereas the spores at 15–25 MPa exhibited a three-stage inactivation curve of shoulder, log-linear and tailing regions, and these curves were well fitted to the Geeraerd model. Approximately 90% of pyridine-2,6-dicarboxylic acid (DPA) was released after HPCD + HT and the 90% DPA release time depend on the pressure and temperature. Moreover, the spore clumping in suspensions was examined by dynamic light scattering. The particle size of the spore suspensions increased with the increase of pressure, temperature and time, indicating the spore clumping. 0.1% Tween 80 as a surfactant inhibited the spore clumping and increased the inactivation ratio of the spores by HPCD + HT. These results indicated that the spore clumping enhanced the spores' resistance to HPCD + HT and induced a protective effect.

Introduction

Spores of Bacillus and Clostridium species formed in sporulation are metabolically dormant and extremely resistant to a variety of stress factors, including heat, desiccation, chemicals and radiation because of their unique structures (Setlow, 1995, Setlow, 2006). These spores in food are common agents that cause spoilage, foodborne illnesses, and detrimental changes to the organoleptic quality (Brown, 2000). The extreme resistance of bacterial spores to physical and chemical treatments makes them a significant problem in the food industry.

High pressure carbon dioxide (HPCD) was firstly shown to inactivate Escherichia coli cells in the 1950s by Fraser (1951). During the recent two decades, HPCD has been proposed as an alternative non-thermal pasteurization technique for foods (Spilimbergo et al., 2002). Besides the environmentally benign nature of the HPCD process (CO2 is nontoxic), the CO2 pressures applied for preservation purposes are much lower (generally lower than 30 MPa) compared to the high pressures (100–600 MPa) employed in high pressure processing (Garcia-Gonzalez et al., 2007), which makes it easier to manage pressure in HPCD technique. The bactericidal effects of HPCD have been reviewed by Damar and Balaban (2006), Garcia-Gonzalez et al. (2007), Perrut (2012), Spilimbergo and Bertucco (2003), and Zhang et al. (2006b). Previous studies indicated that the vegetative forms of pathogenic and spoilage bacteria, yeasts, and molds were completely inactivated by HPCD at pressures less than 30 MPa and at 20 to 40 °C. However, the use of HPCD at moderate temperatures (20–40 °C) is often insufficient to obtain a substantial reduction in bacterial spore counts due to their more complex structure as compared to vegetative cells. Limited studies indicated that bacterial spores were inactivated by HPCD with high temperature  60 °C (HPCD + HT), and the inactivation ratio was increased with the increase of temperature, pressure and time (Bae et al., 2009, Ballestra and Cuq, 1998, Enomoto et al., 1997, Hata et al., 1996, Spilimbergo et al., 2002, Watanabe et al., 2003). The inactivation kinetics of spores by HPCD + HT was described by first-order models (Enomoto et al., 1997, Hata et al., 1996, Qiu et al., 2009, Watanabe et al., 2003, White et al., 2006) and two-fraction models (Ballestra and Cuq, 1998). Different models were observed probably because of the incomprehensive HPCD conditions and insufficient experimental data numbers in these studies (Garcia-Gonzalez et al., 2007). Inactivation of spores by extensive HPCD + HT conditions and the inactivation kinetics were necessary to be studied. Meanwhile, as DPA (pyridine-2,6-dicarboxylic acid) was a major chemical component in the inner core of bacterial spores (~ 10% of spore dry weight) (Setlow, 2003) and its release was closely correlated to the damage of spores' inner membrane (Reineke et al., 2013, Zhang et al., 2006a, Zhang et al., 2007), DPA release was necessary to be studied to further understand the inactivation mechanism of bacterial spores.

Moreover, Enomoto et al. (1997) reported that a pressure-dependent protective effect on Bacillus megaterium spores was observed at 5.8–9.7 MPa by HPCD + HT at 1.9–9.7 MPa and 60 °C for 24 h, and these authors hypothesized that the protective effect was attributed to spore clumping. This hypothesis was later confirmed by Furukawa et al. (2006), who observed spores of Bacillus coagulans and Bacillus licheniformis clumping by HPCD at 6.5 and 30 MPa and 35 °C for 0–120 min by phase contrast microscopy, and indicated that the ratio of spore clumping increased with the increase of pressure and time. However, the spores were not effectively inactivated in the study since the temperature was far less than 60 °C, therefore the contribution of the study to understand the inactivation of the bacterial spores was very limited. Moreover, phase contrast microscopy cannot reflect the spore clumping of the spore suspensions in a full-scale. Due to drawback of the studies and the method on spore clumping investigation, the spore clumping by HPCD + HT should be in-depth studied.

In this study, the inactivation of Bacillus subtilis spores by extensive HPCD + HT conditions was performed and the inactivation kinetics was analyzed. DPA was analyzed to further understand the inactivation mechanism of B. subtilis spores by HPCD + HT. Moreover, the spore clumping by HPCD + HT at these extensive conditions and its impact on spores' resistance to HPCD + HT were investigated.

Section snippets

Strain and spore preparation

B. subtilis 168 was obtained from China General Microbiological Culture Collection Center (Beijing, China). Overnight cultures of Bacillus strains grown in nutrient broth (Beijing Aoboxing Biological Technology Co. Ltd., Beijing, China) were transferred to sporulation agar plates, nutrient agar (Beijing Aoboxing Biological Technology Co. Ltd., Beijing, China) containing 50 μg of Mn2 +/mL. After 1 week incubation at 37 °C, the spores were harvested in a sterile flask by flushing the surface of the

Spore inactivation at extensive pressures and temperatures

The inactivation of the B. subtilis spores subjected to HPCD + HT at 44–91 °C and 0.1–20 MPa for 60 min was shown in Fig. 1. When the temperatures were ≤ 77 °C (44 °C, 58 °C, 66 °C, 77 °C), a less than 1-log reduction in the number of spores was achieved. However, when the temperatures were ≥ 82 °C (82 °C, 86 °C, 91 °C), the inactivation ratio of the spores by HPCD + HT dramatically increased with the increase of the temperature or the pressure. The spores were totally inactivated (approximately 7-log cycle

Discussions

In this study, the inactivation ratios of B. subtilis spores treated by HPCD + HT at ≤ 77 °C did not exceed 1-log whereas it dramatically increased at ≥ 82 °C (Fig. 1), indicating that temperature played a significant role in the inactivation of the spores. Coleman et al. (2007) suggested that moist heat at 87–90 °C inactivated B. subtilis spores by damaging the proteins in the spores' inner membrane and rupturing the membrane. Therefore, it was guessed that the spores' inner membrane could not be

Acknowledgment

This work was supported by “Novel Technologies and Equipments of Food Non-thermal Processing” (Project No. 2011AA100801) of the 863 High-Tech Plan of China.

References (47)

  • M. Perrut

    Sterilization and virus inactivation by supercritical fluids (a review)

    J. Supercrit. Fluids

    (2012)
  • K. Reineke et al.

    The release of dipicolinic acid—the rate-limiting step of Bacillus endospore inactivation during the high pressure thermal sterilization process

    Int. J. Food Microbiol.

    (2013)
  • P. Setlow

    Spore germination

    Curr. Opin. Microbiol.

    (2003)
  • S. Spilimbergo et al.

    Microbial inactivation by high pressure

    J. Supercrit. Fluids

    (2002)
  • S. Spilimbergo et al.

    Inactivation of Bacillus subtilis spores by supercritical CO2 treatment

    Innovative Food Sci. Emerg. Technol.

    (2003)
  • A. White et al.

    Effective terminal sterilization using supercritical carbon dioxide

    J. Biotechnol.

    (2006)
  • E.Y. Wuytack et al.

    A study on the effects of high pressure and heat on Bacillus subtilis spores at low pH

    Int. J. Food Microbiol.

    (2001)
  • J. Zhang et al.

    On the mechanism of deactivation of Bacillus atrophaeus spores using supercritical carbon dioxide

    J. Supercrit. Fluids

    (2006)
  • J. Zhang et al.

    Sterilization using high-pressure carbon dioxide

    J. Supercrit. Fluids

    (2006)
  • J. Zhang et al.

    Supercritical carbon dioxide and hydrogen peroxide cause mild changes in spore structures associated with high killing rate of Bacillus anthracis

    J. Microbiol. Methods

    (2007)
  • G.R. Bender et al.

    Sensitivity of various salt forms of Bacillus megaterium spores to the germination action of hydrostatic pressure

    Can. J. Microbiol.

    (1982)
  • K.L. Brown

    Control of bacterial spores

    Br. Med. Bull.

    (2000)
  • W.H. Coleman et al.

    How moist heat kills spores of Bacillus subtilis

    J. Bacteriol.

    (2007)
  • Cited by (28)

    View all citing articles on Scopus
    View full text