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Previous Article | Next Article 
Applied and Environmental Microbiology, January 2001, p. 317-322, Vol. 67, No. 1
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.1.317-322.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Effect of Water Activities of Heating and Recovery
Media on Apparent Heat Resistance of Bacillus cereus
Spores
Louis
Coroller,
Ivan
Leguérinel, and
Pierre
Mafart*
Laboratoire Universitaire de Microbiologie
Appliquée de Quimper Pôle Universitaire, Creach Gwen,
29000 Quimper, France
Received 24 April 2000/Accepted 5 October 2000
 |
ABSTRACT |
Spores of Bacillus cereus were heated and recovered in
order to investigate the effect of water activity of media on the
estimated heat resistance (i.e., the D value) of spores. The water
activity (ranging from 0.9 to 1) of the heating medium was first
successively controlled with three solutes (glycerol, glucose, and
sucrose), while the water activity of the recovery medium was kept near 1. Reciprocally, the water activity of the heating medium was then kept
at 1, while the water activity of the recovery medium was controlled
from 0.9 to 1 with the same depressors. Lastly, in a third set of
experiments, the heating medium and the recovery medium were adjusted
to the same activity. As expected, added depressors caused an increase
of the heat resistance of spores with a greater efficiency of sucrose
with respect to glycerol and glucose. In contrast, when solutes were
added to the recovery medium, under an optimal water activity close to
0.98, a decrease of water activity caused a decrease in the estimated D
values. This effect was more pronounced when sucrose was used as a
depressor instead of glycerol or glucose. When the heating and the
recovery media were adjusted to the same water activity, a balancing
effect was observed between the protective influence of the solutes
during heat treatment and their negative effect during the recovery of injured cells, so that the overall effect of water activity was reduced, with an optimal value near 0.96. The difference between the
efficiency of depressors was also less pronounced. It may then be
concluded that the overall protective effect of a decrease in water
activity is generally overestimated.
| |
INTRODUCTION |
It has been recognized that the heat
resistance of bacterial spores depends on the medium in which the
spores are heated. The maximum thermostability of most microorganisms
was found in the range of between 0.2 and 0.4 water activity (1,
3, 27, 28, 29). In typical ranges of water activities which are
found in foodstuffs (aw > 0.8), the heat resistance
of microorganisms generally increases at decreasing water activities.
However, the apparent effect of the water activity of the medium on
spores or vegetative cells is complicated by the specific effect of
solutes which are used as depressors. It is generally agreed that the occurrence of such solutes in the medium reduces the heat resistance of
microorganisms. This antagonism between the protective effect of an
increase in water activity and the opposite specific effect of
depressors can explain conflicting data from various authors.
The influence of salt on the thermostability of microorganisms is
disputed and depends on the heated type of microorganism. Some authors
found no effect of the sodium chloride concentration on the heat
resistance of bacteria (9, 29, 32, 42). Others observed a
reduced heat resistance of microorganisms at increasing salt
concentrations (7, 12, 22, 23). On the contrary, a
protective effect of salt was found in several studies (6, 14,
26, 35, 38, 39, 40). Corry (14) deduced from his
data that sodium chloride had a thermal protective effect on most
heat-sensitive bacteria and the opposite effect on most heat-resistant
species. Other solutes show the same opposite influence between their
common depressor character which protects spores against heat and their
specific effect which, on the contrary, reduces their heat resistance.
It has been observed (21) that an increase of the thermal
resistance of spores was more pronounced when the decrease of the
medium water activity was generated by drying instead of an addition of
glycerol, sodium chloride, lithium chloride, or glucose. Baird-Parker
et al. (5) could not find any correlation between the heat
resistance D (values) of salmonellae and the water activity of heating
media when sodium chloride or glycerol were used as depressors.
However, these researchers observed a clear protective effect of
sucrose that was more pronounced for most heat-sensitive strains. It is
generally recognized that sucrose is the most protective depressor,
while glucose, sodium chloride, and lithium chloride show a clearly
lower influence or even an opposite effect. Glycerol shows an
intermediate behavior (13, 19, 20, 26, 37). Interactions
between the influences of water activity and heating temperature were
often observed. An increase of D values generated by a reduced water
activity of the heating medium is generally related to an increase of z values. Moreover, several workers have demonstrated that the effect of
the water activity of the heating medium depended on the treatment temperature; for example, in Staphylococcus epidermidis
(39) or Listeria monocytogenes
(37), the protective effect of decreasing water activity
is more pronounced at a higher treatment temperature, while the
opposite trend was observed for Staphylococcus aureus (38). A few predictive models describing the effect of the
water activity of the heating medium on the heat resistance of spores were developed (8, 18, 31).
The nature of the recovery medium in which surviving heated cells are
incubated has a great influence on their apparent heat resistance,
i.e., their estimated D value (24). It is generally agreed
that there is an optimum temperature of incubation for the cell ratio
of recovery (16, 36) and the apparent D value (10). Acidification of the recovery medium causes also a
reduction in spore recovery and in apparent heat resistance (11,
17, 33, 34, 41). The addition of sodium chloride in the recovery medium causes effects similar to those observed with acidification: a
reduction of the viability of cells and a lower apparent D value (7, 12, 22, 30, 32). However, as far as we know, the effect of reducing the water activity of the recovery medium by depressors other than sodium chloride has never been investigated.
The purpose of this work was to investigate and to describe based on a
predictive model the influence of the water activity of the recovery
medium with glycerol, glucose, and sucrose used as depressors upon the
apparent D value of Bacillus cereus spores.
| |
MATERIALS AND METHODS |
Microorganism and spore production.
The strain B. cereus CNRZ 110 was obtained from the Institut National de
Recherche Agronomique (Paris, France). Spores were kept in distilled
water at 4°C. Cells were precultivated at 37°C for 24 h in
brain heart infusion (Difco). The preculture was used to inoculate
nutritive agar plates (Biokar Diagnostics BK021) with MnSO4
(40 mg liter
1) and CaCl2 (100 mg
liter1) on the surface area. Plates were incubated at
37°C for 5 days. Spores were then collected by scraping the surface
of the agar, suspended in sterile distilled water, and washed three
times by centrifugation (10,000 × g for 15 min)
(Bioblock Scientific model Sigma 3K30). The pellet was then resuspended
in 5 ml of distilled water and 5 ml of ethanol. The obtained suspension
was kept at 4°C for 12 h to eliminate vegetative nonsporulated
bacteria and then washed again three times by centrifugation.
Lastly, the final suspension (ca. 1010 spores
ml1) was distributed into sterile Eppendorf microtubes
and kept at 4°C.
Thermal treatment of spore suspension.
D values in
citrate-phosphate buffers were determined at 95°C with one replicate
at each aw value ranging from 1 to 0.89.
Three solutes (glycerol, glucose, and sucrose) were used to adjust the
water activity value. The previous molarities of the different solutes
were determined using curves from the model UNIFAC-LARSEN
(2). The heating medium was sterilized by filtration, and
the aw values were controlled with an aw meter
(FA-st1 GBX; France Scientific Instrument).
First, 30 µl of spore suspension was diluted in 3 ml of heating
medium. Capillary tubes of 25 µl (Vitrex) were filled with 10 µl of
sample and subjected to a thermal treatment in a thermostated oil bath.
After being heated, the tubes were cooled in an ice-water bath, washed
in a solution of soap, and rinsed with sterile distilled water.
Finally, the ends were flamed with ethanol. The capillary tubes were
broken at both ends, and their contents poured into a tube containing 9 ml of sterile tryptone salt broth (Biokar Diagnostics) by rinsing with
1 ml of tryptone salt broth contained in a needle-equipped syringe.
Recovery conditions.
Viable spores were counted by duplicate
plating at different aw values in nutritive agar (10 g of
tryptone, 5 g of meat extract, 5 g of sodium chloride, and
15 g of agar per 1,000 ml) (Biokar Diagnostic) and incubated at
25°C for 6 to 21 days. The aw ranging from 1 to 0.92 was
adjusted with glycerol, glucose, or sucrose. To adjust aw
values, the previous molarities of the different solutes were
determined using curves from the model UNIFAC-LARSEN (2).
Nutritive agar was sterilized by autoclaving, and glycerol, glucose, or
sucrose solutions were sterilized by filtration to avoid the Maillard
reaction. After sterilization, the two solutions were mixed, the pH was
adjusted to 7, and the aw value was controlled. The
correspondence between the water activity and the concentrations of
each depressor is shown in Table 1.
Data analysis.
D values were determined on the straight
portion of curves obtained when the log number of survivors was plotted
against time. The parameters of the models were estimated by simple
linear regression carried out with MINITAB software. The goodness of
fit of the model was evaluated by using the percent variance
R2 value.
| |
RESULTS |
Effect of the water activity of the heating medium.
For
identical heat treatment, the recovery conditions influence the
apparent heat resistance of bacterial spores. A clear protective effect
on spores of B. cereus heated at 95°C and at pH 7 was
observed when solutes were added to the heating medium for the three
types of depressors (Fig. 1 and
2).
However, it can be seen that the effect of sucrose is more pronounced
than that of glycerol and glucose. While a D95°C of about
4 min at a water activity close to 1 was found, at a water activity of
0.9 the observed D95°C became close to 11.2, 10.5, and 27 min for glycerol, glucose, and sucrose, respectively.
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FIG. 1.
Log UFC versus the heating time for B. cereus
CNRZ 110 heated at 95°C at pH 7 with an aw of 1 ( ) or
0.9 ( ) adjusted with sucrose. The aw value of the
recovery condition is equal to 1.
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FIG. 2.
(A) B. cereus D95°C value
versus the aw of the heated medium adjusted with glycerol.
Symbols: , experimental data;  , calculated values of
aw according to equation 1. (B) B. cereus
D95°C value versus the aw of the heated
medium adjusted with glucose. Symbols: , experimental data; ,
calculated values according to equation 1. (C) B. cereus
D95°C value versus the aw of the heated
medium adjusted with sucrose. Symbols:  , experimental data; ,
calculated values according to equation 1.
|
|
The three sets of data corresponding to each depressor were fitted
according to the Gaillard et al. model (18) which, under isothermal conditions and at a fixed pH of the heating medium, can be
reduced to:
|
(1)
|
where D(aw,1) is the estimated D value at
a water activity of the heating medium aw (which is the
controlled variable) and a water activity of the recovery medium of 1. zaw* corresponds to the decrease in water
activity of the treatment medium which would cause a 10-fold reduction
of the decimal reduction time, with a water activity of the recovery
medium of 1. The estimated parameter values are presented in Table
2.
Effect of the water activity of the recovery medium.
Whatever
the solute used as depressor in the recovery medium, heated spores show
the same maximum apparent heat resistance (a D95°C value
of ca. 5 min) at an optimum water activity close to 0.98 (see Fig. 3
and 4).
Under this optimal value, an increasing concentration of the three
depressors causes a decrease in the apparent heat resistance of the
spores. Sucrose presents the most pronounced effect, followed in turn
by glucose and glycerol. At water activity of 0.92, the estimated
D95°C values were 0.9, 1.9, and 2.5 min with
sucrose, glucose, and glycerol, respectively.
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FIG. 3.
Log UFC versus the heating time for B. cereus
CNRZ 110 heated at 95°C at pH 7 with an aw of 1 incubated
at 25°C in recovery medium at an aw of 1 ( ) or an
aw of 0.92 ( ) adjusted with sucrose.
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FIG. 4.
(A) B. cereus D95°C value
versus the aw of the recovery medium adjusted with
glycerol. Symbols: , experimental data; , calculated values of
aw according to equation 2. (B) B. cereus
D95°C value versus the aw of the recovery
medium adjusted with glucose. Symbols: , experimental data; ,
calculated values according to equation 2. (C) B. cereus
D95°C value versus the aw of the recovery
medium adjusted with sucrose. Symbols:  , experimental data; ,
calculated values according to equation 2.
|
|
We tried to adapt the model describing the influence of the pH of the
recovery medium to the apparent thermal resistance of spores, which was
tested in our laboratory (15) by substituting pH for water
activity, leading to the following equation:
|
(2)
|
where D'(1,aw) is the estimated D value at
a water activity of the recovery medium a'w (which is the
controlled variable) and a water activity of the heating medium of 1. z'aw* corresponds to the decrease of water
activity of the recovery medium which would cause a 10-fold reduction
of the decimal reduction time, with a water activity of the heating
medium of 1. The estimated parameter values are presented in Table
3.
Overall effect of water activity of foods on the apparent heat
resistance of spores.
Since foods make up both the heating medium
and the recovery medium, a third set of experiments was carried out in
which spores were recovered at the same water activity as those of the
heating menstruum (Fig. 5). With respect
to the second set of data in which the water activity of the heating
medium was kept to 1, some noteworthy differences appear. First, the
overall influence of water activity becomes relatively slight, while
the differences in the curve patterns according to the depressors used
are less pronounced than those of Fig. 4. Second, a shift of the
optimum water activity from 0.98 toward 0.96 can be observed, with a
maximum D95°C value of close to 8 min instead of 5 min.
Equations 1 and 2 cannot directly be combined in order to build a model which would take into account the overall effect of the food water activity because they were developed by keeping the water activity of
the recovery medium at 1 for equation 1, and keeping the water activity
of the heating medium at 1 for equation 2. The accuracy of this third
set of data was too poor to allow suitable modeling.
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FIG. 5.
(A) B. cereus D95°C value
versus the aw of both heated and recovery medium adjusted
with sucrose. Symbols: , experimental data; , calculated values of
aw according to equation 2. (B) B. cereus
D95°C value versus the aw of both heated and
recovery medium adjusted with glucose. Symbols: , experimental data;
, calculated values according to equation 2. (C) B. cereus
D95°C value versus the aw of both heated and
recovery medium adjusted with sucrose. Symbols: , experimental data;
, calculated values according to equation 2.
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|
| |
DISCUSSION |
It has been confirmed that an increase in the water activity
produces opposite effects on the apparent heat resistance of spores
according to whether it concerns the heat treatment or the recovery
medium. Inside the investigated water activity range (0.9 to 1), which
corresponds to that of most typical foods, a clear protective effect of
an increasing water activity of the heating medium can be observed. For
a fixed water activity, the degree of protection depends on the type of
depressor used; according to our investigations, sucrose showed a more
effective protective effect than glycerol and glucose, a finding which
is in agreement with observations by other authors (5, 19, 26,
37). As in the case of NaCl, the protective influence of an
increase of the water activity could partly be balanced by a specific
antagonistic and toxic effect of glycerol and glucose. Moreover, the
plasmolyse which is partly responsible for the heat protection of
spores is limited by the penetration of glycerol and glucose inside the cells. This limitation does not exist when the depressor is not uptaken
inside cells, which is the case with sucrose. Another explanation for
the protective effect of a depressor in the heating medium could be an
inhibition of spore germination. Anagnastopoulos and Sidhu
(4) observed for Bacillus stearothermophilus
that the percentage of germination decreases when the water activity decreased and that, at the same water activity, the percentage of
germination was lower in a nutrient broth supplemented with sucrose
than in a broth supplemented with glycerol. Hydration of spore
protoplast is an important condition of spore activation and initiation
of germination. Germination is inhibited in the absence of moisture or
in a concentrated solution of nonpenetrating solute. The spores which
do not germinate are protected during heat treatment and can germinate
and grow during recovery. This explanation is in agreement with our
results: when the water activity decreases, the heat resistance of
spores increases and, at the same water activity, sucrose, a
less-penetrating solute in protoplast than glycerol and glucose, shows
a greater protective effect than these two solutes. Moreover, according
to Anagnastopoulos and Sidhu, the zaw values
for glycerol and sucrose (0.28 and 0.13, respectively) correspond to
the aw value difference which leads to a 10-fold reduction
of the percentage of germination of B. stearothermophilus at
75°C with glycerol and sucrose (0.31 and 0.11, respectively).
It is recognized that the addition of sodium chloride to the recovery
medium causes both a reduction of viability of cells and a lower
apparent D value of the spores (7, 12, 16, 22, 32).
However, as far as we know, the influence of the water activity of the
recovery medium and the types of depressors used upon the estimated D
values of spores had never been investigated. In our experimental
conditions, a maximal apparent heat resistance of spores appeared at an
optimal water activity close to 0.98. Below this value, a decrease in
the water activity of the recovery medium causes a reduction of the
estimated D value of the spores. It is interesting to note regarding
this trend that the depressors appear at the same increasing order of
effectiveness as was found regarding their protective effect in the
heating medium: glycerol, glucose, and sucrose, respectively. This
observation is consistent with the behavior of germinated (i.e.,
nonrefractile) spores, which are rapidly permeable to glycerol,
permeable to glucose by active transport, and permeable to sucrose to a
lower extent. This order also corresponds to the increasing order of
molecular weights and to the decreasing degree of penetration inside
the cells. Particularly, the absence of uptake of sucrose by cells keeps a sharp gradient of osmotic pressure between the cell inside and
the outside medium, which reduces the viability of surviving cells.
The overall influence of water activity of a single medium which makes
up both the heating menstruum and the recovery medium upon the apparent
D values of spores had not yet been investigated. Our results show that
the protective effect of a decrease in the water activity of the medium
during heat treatment is more or less offset by a reduction of the
viability of surviving cells during the recovery. Moreover, because
depressors, which are most effective in the heat protection of spores,
are also responsible for the maximum loss of viability of injured
cells, their overall difference in efficiency is greatly reduced.
Whereas most authors have determined the optimal water activity of the
maximum heat resistance of spores to be between 0.2 and 0.4 because the
surviving cells were recovered under optimal conditions when the media
of the heat treatment and of the incubation are the same, as for heat-processed foods, the optimal water activity is actually near 0.96.
Investigating separately the influence of water activity of the heating
medium on the heat resistance of spores on the one hand and the effect
of water activity of the recovery medium on the viability of surviving
cells on the other hand obviously provides very interesting and useful
data: these two effects must be regarded as two different factors that
interact with each other. However, when the heating medium is also the
recovery medium, it is worth investigating the overall influence of
water activity on the apparent heat resistance of the spores, which
reflects both their immediate thermal resistance during heating and
their ability to grow in a recovery medium.
In the framework of predictive microbiology, we could develop two
separate models for describing the effect of the water activity of the
heating menstruum on the one hand and that of water activity of the
recovery medium on the other hand upon the apparent heat resistance of
the spores. However, further works would be needed in order to develop
an overall predictive model adapted for heat-processed food
calculations. Such a model would take into account both opposite effects of water activity and would allow us to improve of heat treatment optimization.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire
Universitaire de Microbiologie Appliquée de Quimper Pôle
Universitaire, Creach Gwen, 29000 Quimper, France. Phone:
33(0)2-98-10-00-61. Fax: 33(0)2-98-10-00-01. E-mail:
pierre.mafart{at}univ-brest.fr.
| |
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Applied and Environmental Microbiology, January 2001, p. 317-322, Vol. 67, No. 1
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.1.317-322.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
