International Journal of Food Microbiology 72 (2002) 239 242

www.elsevier.com/locate/ijfoodmicro

Short communication

High-pressure inactivation of Bacillus cereus spores

in the presence of argon
Keiko Fujii a,*, Ayami Ohtani b, Junko Watanabe b, Hiro Ohgoshi b,
Tomoyuki Fujii c, Kazuo Honma d
a
Faculty of Education, Yamagata University, Yamagata, Yamagata 990-8560, Japan
Department of Food and Nutrition, Japan Womens University, Bunkyo-ku, Tokyo 112-0015, Japan
c
Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
d
Research Institute of QP Corporation, Fuchu, Tokyo 183-0034, Japan
b

Received 18 June 2001; received in revised form 3 September 2001; accepted 10 September 2001

Abstract
We investigated the high-pressure inactivation of Bacillus cereus spores in water containing argon. At the pressure of 600
MPa, addition of argon accelerated the inactivation of spores at 20 C, but had no effect on the inactivation at 40 C. The
influence of added argon on inactivation of the spores was marked under conditions with a strong water ordering effect. The
pressure resistance of B. cereus spores was thus shown to be affected by water ordering. D 2002 Elsevier Science B.V. All
rights reserved.
Keywords: High pressure; Bacillus cereus; Water ordering; Inactivation; Argon

1. Introduction
When a hydrocarbon or rare gas is dissolved in
water under appropriately selected temperature and
pressure conditions, highly ordered iceberg-like
form around solute molecules in aqueous solution
due to hydrophobic hydration (Davidson, 1973). In
some cases, a crystalline clathrate hydrate is formed in
this way. The phenomenon whereby the crystallinity
of the liquid structure of water increases is known as
water ordering. A clathrate hydrate containing argon
as the guest molecule can remain stable, for example

Corresponding author. Fax: +81-23-628-4353.

E-mail address: ep250@kdw.kj.yamagata-u.ac.jp (K. Fujii).

at 124 C or below at less than 0.1 MPa (Barrer and

Edge, 1967) or at 0.8 C or below at less than 8.7
MPa (Marshall et al., 1964).
Since high-pressure treatment has recently become
applicable to biochemical and food processing (Knorr,
1993; Cheftel, 1995), we have considered the combined use of water ordering with high pressure,
achieved by dissolving a rare gas in the water contained in foods or bioproducts. This combined effect
might make it possible to establish novel non-heating
processing/pasteurizing techniques without altering
the quality of bioproducts.
While Bacillus cereus is a cause of food poisoning,
Bacillus is used as a research model in biotechnology
to study bacterial nutrition, cell wall formation, sporogenesis, gene regulation and plasmid expression,

0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 6 0 5 ( 0 1 ) 0 0 7 0 0 - 0

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K. Fujii et al. / International Journal of Food Microbiology 72 (2002) 239242

production of insecticides (Agaisse and Lereclus,

1995), immunogenesis in vaccine production (Medina
and Guzman, 2001), and resistance to extreme conditions (Nicholson et al., 2000). Inactivation of spores
by high pressure is a two-step process: spore germination followed by inactivation of the germinated
forms (Clouston and Wills, 1969). The influence of
physicochemical agents on the initiation of germination and inactivation of B. cereus spores under high
pressure has been documented (Raso et al., 1998).
Although pressure-induced germination of B. subtilis
spores has been recently studied (Wuytack et al.,
1998), the biological responses of microorganisms
under high pressure are still unclear (Abee and
Wouters, 1999). We investigated the behaviour of B.
cereus spores inactivated by high pressure, and
addressed it from the viewpoint of water ordering
under high pressure achieved by dissolution of a rare
gas.

2.3. High-pressure treatment

Each sample containing dissolved argon was treated under high pressure of 500 and 600 MPa at 20, 30
and 40 C for 30 min with a Mitsubishi Heavy
Industries high-pressure generator model MFP-7000.
The time to reach final pressure desired was within 90
s. Decompression could be completed within 15 s.
2.4. Enumeration of spore survival
After the pressure treatment, each sample was
inoculated onto plates containing a nutrient agar
medium (0.3% Difco Bacto Beef Extract/0.5% Difco
Bacto peptone) at pH 6.8 and incubated at 30 C for
48 h. The survival was calculated by dividing the
viable count after pressurization by that before pressurization.

3. Results and discussion

2. Materials and methods
2.1. Preparation of spore suspension
A culture (24 h, 30 C) of B. cereus IAM 12605 in
nutrient broth (Difco, Detroit, MI, USA) was spread
on a plate of standard agar medium (Eiken Chemical,
Tokyo, Japan) and incubated at 30 C for 2 weeks.
The culture was collected with a glass spatula and was
suspended in 1/15 M phosphate buffer solution (pH
7.1). After harvesting, it was heated to 80 C for 30
min to exterminate all vegetative cells. The obtained
suspension containing spores (about 105 spores/ml)
was referred to as the standard spore suspension.

Fig. 1 shows the effects of treatment temperature

on the survival of B. cereus spores at 600 MPa. The
argon-free sample showed a 2-log10 reduction of spore
numbers after pressurization for 30 min at 40 C,
although inactivation was not observed at 20 C. The
argon-containing sample showed a 1-log10 reduction
of spore numbers after pressurization for 30 min at 20

2.2. Dissolution of argon

The standard spore suspension was pipetted in 20ml portions into aluminum pouches (3 cm in diameter,
22 cm in length; three-layered structure consisting of
polyester, aluminum foil and special high-density
polyethylene), and then argon was dissolved in the
suspension by bubbling. The bubbling was carried out
for 10 min, because a preliminary experiment had
shown that this produces no change in the viable
count.

Fig. 1. Effect of added argon on inactivation of B. cereus spores at a

treatment pressure of 600 MPa.

K. Fujii et al. / International Journal of Food Microbiology 72 (2002) 239242

Fig. 2. Effect of added argon on inactivation of B. cereus spores at a

treatment temperature of 30 C.

C. Both the argon-free and argon-containing samples

showed a 2-log10 reduction of spore numbers after
pressurization for 30 min at 40 C, thus showing no
effect of added argon. The argon-containing sample
showed lower survival than the argon-free sample at
20 C, different from the case at 40 C. Although
addition of argon had no effect on the inactivation of
spores at 40 C, it effectively accelerated the inactivation at 20 C. The results of the treatment at 500
MPa were almost the same as those obtained by
treatment at 600 MPa (data not shown).
Values were calculated by dividing the survival in
the presence of argon by that in the absence of argon
(i.e., relative survival), to summarize the effects of the
added argon. A relative survival of less than 1 means
that added argon accelerated the inactivation. At the
treatment pressure of 600 MPa, the decrease in
relative survival was 0.91, 0.31 and 0.068, at 40, 30
and 20 C, respectively. Relative survival was close to
1 at 40 C, showing that addition of argon scarcely
affected the inactivation effect. The addition of argon
enhanced the inactivation of the spores at 20 C.
Fig. 2 shows the effect of pressure on the survival
of B. cereus spores at 30 C. The survival in argonfree samples decreased with increasing pressure. The
influence of added argon on inactivation of the spores
at 400 MPa was superior to that achieved at 600 MPa.
When a hydrocarbon is dissolved in water under high
pressure, the solubility pressure curve under constant

241

pressure generally shows a peak (Sawamura et al.,

1989). The hydrophobic hydration is elevated with an
increase in pressure at the low-pressure side ( < 100
150 MPa). At the high-pressure side, in contrast, the
hydrophobic-hydration shell around the hydrocarbon
is destroyed due to the water-ordering deformation
resulting from compression. The results of this study
show that argon has an inactivation accelerating effect
within the low-temperature and low-pressure region
where hydrophobic hydration generally arises.
It is anticipated that under a high pressure of 500
MPa or above, where proteins would be denatured,
high-pressure denaturation is enhanced with increased
pressure. However, hydrophobic hydration is weakened with increased pressure. Therefore, it is considered that the effects of added argon on spore inactivation would differ due to the overlapping effects of
these two phenomena. Although its roles in the initiation of germination or inactivation are not obvious,
hydrophobic hydration would affect bacterial spore
pressure resistance. Finally, it is expected that if a novel technique involving a combination of water ordering and high pressure could be established, it would
be useful for the processing of bioproducts and foods.

Acknowledgements
This study was supported in part by The SKYLARK Food Science Institute of Japan.

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