International Journal of Food Microbiology 181 (2014) 10–18

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International Journal of Food Microbiology

The combined effect of pasteurization intensity, water activity, pH and

a r t i c l e i n f o a b s t r a c t

Article history: The objective of the study was to evaluate the combined effects of pasteurization intensity (no heat treatment
Received 4 November 2013 and 10 min at 70, 80 and 90 °C), water activity (aw) (0.960–0.990), pH (5.5–7.0) and storage temperature (7
Received in revised form 24 March 2014 and 10 °C) on the survival and outgrowth of psychrotolerant spores of Bacillus cereus FF119b and Bacillus pumilus
Accepted 11 April 2014
FF128a. The experiments were performed in both artificial media and a validation was performed on real food
Available online 21 April 2014
products (cream, béchamel sauce and mixed vegetable soup). It was determined that in general, heat treatments
Keywords:
of 10 min at 70 °C or 80 °C activated the spores of both B. cereus FF119b and B. pumilus FF128a, resulting in faster
Spore-formers outgrowth compared to native (non-heat treated) spores. A pasteurization treatment of 10 min at 90 °C generally
Activation resulted in the longest lag periods before outgrowth of both isolates. Some of the spores were inactivated by this
Heat treatment heat treatment, with more inactivation being observed the lower the pH value of the heating medium. Despite
Béchamel sauce this, it was also observed that under some conditions the remaining (surviving) spores were actually activated
Vegetable soup as their outgrowth took place after a shorter period of time compared to native non-heated spores. While the
Cream response of B. cereus FF119b to the pasteurization intensity in cream and béchamel sauce was similar to the
trends observed in the artificial media at 10 °C, in difference, outgrowth was only observed at 7 °C in both
products when the spores had been heated for 10 min at 80 °C. Moreover, no inactivation was observed in
cream or béchamel sauce when the spores were heated for 10 min at 90 °C in these two products. This was
attributed to the protective effect of fat in the cream and the ingredients in the béchamel sauce. The study
provides some insight into the potential microbial (stability and safety) consequences of the current trend
towards milder heat treatments which is being pursued in the food industry.
© 2014 Published by Elsevier B.V.

1. Introduction The commonly used performance standard for heating chill stored
foods is 10 min at 90 °C (Gould, 1999). This treatment results in a 6D
Over the last two decades the demand for products of high sensorial, reduction of non-proteolytic Clostridium botulinum. Although the processes
nutritional and textural quality, including refrigerated ready-to-eat or mentioned above to heat produce high quality products such as REPFEDS
cook food products has rapidly increased (Membré et al., 2006). Refrig- are sufficient to inactivate the vegetative microbial flora of the products,
erated ready-to-eat or ready-to-cook food products are often referred to they may actually activate surviving spores by increasing the percentage
as refrigerated products of extended durability (REPFEDS). The food of spores that germinate (Collado et al., 2003, 2006; Turnbull et al., 2007).
industry has relied on the use of milder (heat) treatments, primarily Heat activation is defined as a sub-lethal short heat treatment that poten-
high temperature short time (HTST) pasteurization, but also ultra- tiates and synchronizes the germination of N 90% of a spore population
high temperature (UHT), rotary retort and high hydrostatic pressure (Zhang et al., 2009). The activation of spores can also be induced by
processing (Cazemíer et al., 2001) to produce products with these aging (Powell, 1950), aqueous alcohols (Holmes and Levinson, 1967),
desired properties. and reducing agents such as mercaptoethanol and thioglycolate
(Keynan et al., 1964). The heat activation of metabolically dormant spores
has been reported to take place when sub-lethal temperatures are
applied to spores i.e. 30 min at 65 °C and 70 °C for Bacillus cereus and
⁎ Corresponding author. Tel.: +32 9 264 9902; fax: +32 9 225 5510. Bacillus subtilis, respectively (Cook and Brown, 1965; Martin and
E-mail address: Simbarashe.Samapundo@UGent.be (S. Samapundo). Blackwood, 1971). The temperature and duration of heating required

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.04.018
0168-1605/© 2014 Published by Elsevier B.V.
 S. Samapundo et al. / International Journal of Food Microbiology 181 (2014) 10–18 11

for optimal heat activation vary widely between different species and 2. Material and methods
even between spore preparations of the same strain (Curran and Evans,
1942; Keynan et al., 1964). 2.1. Experimental set-up
Although several studies have been performed to elucidate the mech-
anism of heat activation, the mechanism is not yet fully known (Keynan The study was divided into two parts. In the first part the effect of
et al., 1964; Zhang et al., 2009). However, a number of mechanisms pasteurization intensity, water activity (aw), pH and incubation temper-
have been suggested including reversal of the breakage of bonds i.e. ature was evaluated in artificial media. In the second part the effect of
hydrogen bonds in some proteins (Zhang et al., 2009). In particular, pasteurization intensity was evaluated in four commercial products.
partial denaturation of proteins may be associated with the breakage of
disulfide bonds during activation (Gould and Hitchins, 1962). Heat activa- 2.2. Isolates and preparation of spore inoculum
tion has been observed to coincide with the release of calcium dipicolinic
acid (Ca-DPA) and protein from spores (Alimova et al., 2006). Additionally Two psychrotolerant spore formers, B. pumilus FF128a and B. cereus
a transition of the spore core from a glass-like state in the dormant spore FF119b, isolated from meatloaf and ready-to-eat chicken curry, respec-
to a rubbery-like state has been observed to occur upon heat activation tively, were used in this study. These isolates were previously identified
(Ablett et al., 1999). Heat activation is reversible (Keynan et al., 1964; by (GTG)5-PCR finger printing according to the method of De Jonghe
Issahary et al., 1970) with the spores usually reverting to their original et al. (2008) and are maintained in the culture collection of the Laboratory
dormant state when they are stored at low temperatures (Keynan et al., of Food Microbiology and Food Preservation (Ghent University, Belgium).
1964; Foster and Johnstone, 1989). As heat activation increases the rate Spores of these two isolates were produced largely according to the
and extent of germination and reduces the lag period before outgrowth method of Coroller et al. (2001). In brief, inoculum was collected sepa-
is apparent (Hyatt and Levinson., 1968; Vary and Halvorson, 1965), it rately from tryptone soy agar (TSA, Oxoid, Basingstoke, UK) slants using
could potentially lead to reduced microbial stability and/or safety of prod- sterile inoculation loops and used to inoculate test-tubes with 9 ml of
ucts. However, this will only occur when the food intrinsic properties, sterile tryptone soy broth (TSB, Oxoid, Basingstoke, UK). After incubation
packaging and storage conditions are adequate for outgrowth of the at 30 °C for 24 h, ca. 100 μl of the first cultures was separately spread on
activated spores. the surface of strengthened Nutrient Agar (sNA) plates [28 g Nutrient
Consumer pressure for clean label products or products with agar (Oxoid, Basingstoke, UK) + 0.04 g MgCl2 (Sigma-Aldrich, Seelze,
reduced levels of traditional chemical preservatives such as benzoate, Germany) + 0.10 g CaCl2 (VWR International, Leuven, Belgium) per
sorbate and nitrite has been mounting over the last two decades. Indeed liter of water]. Four plates were prepared per isolate and incubated for
some of these preservatives have been determined to be detrimental to at least 6 days at 30 °C. After incubation 4 ml of sterile salt solution
human health i.e. benzoates have been associated with ADHD in (8.5% NaCl) was transferred to the surface of each sNA plate. The spores
children (McCann et al., 2007) and the use of nitrite has been associated were collected by gently rubbing the surface of the flooded plates using
with the formation of potent carcinogenic N-nitroso compounds (nitrosa- an inoculation needle or a sterile spatula. A further 4 ml was used to
mines) in processed meat and fish products (Eichholzer and Gutzwiller, rinse the surface of the plates to remove remaining spores. The suspen-
1998; Knekt et al., 1999). The germination and outgrowth (and where sions collected from the four plates were combined in a 50 ml Falcon
applicable the production of toxin) of heat activated spores of important tube. The spores were collected by centrifuging at 10,000 g for 15 min
psychrotolerant spoilage and pathogenic sporeformers such as B. cereus at 4 °C (Sigma 4K15 Centrifuge, Sigma, Germany). The supernatant was
and C. botulinum during refrigerated storage would be expected to take discarded leaving the spore pellet, which was then suspended in 10 ml
place more readily and extensively in these types of food products. of the sterile salt solution and centrifuged again. This process was repeat-
Therefore, the new processing methods based on mild heat treatments ed once more, after which the resultant pellet was suspended in 10 ml of
could compromise the safety of these products (Collado et al., 2006). ethanol–water (1/1) and incubated at 2–4 °C for 12 h to eliminate any
The present study had the major objective of determining the remaining vegetative cells. The centrifugation process was then repeated
combined effects of aw, pH, heat treatment and storage temperature on three more times to wash-off the ethanol–water, with resuspension of
the survival and subsequent outgrowth of spores of psychrotolerant the pellet (in between each centrifugation step) in 10 ml of sterile
B. cereus and Bacillus pumilus strains previously isolated from ready-to- distilled water. The final pellet was then suspended in 10 ml sterile
eat chicken curry and meat loaf, respectively, in artificial media. A valida- distilled water and kept at 2 °C. The number of spores collected was
tion was performed on a few real food products. The results observed in enumerated by serially diluting the spore suspension in peptone physio-
this study are of importance to the food industry as to date most studies logical saline (PPS) [1 g bacteriological peptone (Oxoid) and 8.5 g NaCl
found in the literature have focused on the effect of mild heat treatments per liter], plating out on TSA and an incubation period of 24 h at 30 °C.
on vegetative pathogens and spoilage organisms rather than spores. The Typical yields were 108–9 spores ml−1 suspension. This was confirmed
studies which have been performed on spores include Gaillard et al. by microscopic counts done on a Bürker counting chamber (Superior,
(2005) and Daelman et al. (2013a, 2013b). Gaillard et al. (2005) evaluat- Mareinfeld, Lauda-Könisghofen, Germany) using a Carl Zeiss Axio Imager
ed the lag-time for regrowth of heated B. cereus spores as a function of A1 microscope (Carl Zeiss, Göttingen, Germany).
temperature and duration of heating and the pH of the recovery medium.
They reported that a linear relationship existed between the lag time of 2.3. Combined effect of pasteurization intensity, pH, aw and incubation
growth and the duration of the heat treatment when the heat treatment temperature on the outgrowth of B. cereus FF119b and B. pumilus FF128a
corresponded to a two log10 reduction of the surviving population of spores in artificial media
spores. Daelman et al. (2013a) developed a growth/no growth model
for heat treated spores of B. cereus under cold storage at 10 °C while A full factorial experimental design was used to determine the
Daelman et al. (2013b) developed a time-to-detect growth model for combined effect of pasteurization intensity (no heat treatment, and
heat treated spores of B. cereus when storage was done at 8–30 °C. In 10 min at 70 ± 1 °C, 80 ± 1 °C or 90 ± 1 °C), pH (5.50 ± 0.01 and
both of these studies the effects of aw, pH, and heat treatment were de- 6.20 ± 0.01), aw (0.960 ± 0.001 and 0.990 ± 0.001) and incubation
scribed in the models developed, however, no validation was performed temperature (7.0 ± 0.01 and 10.0 ± 0.01 °C) on the outgrowth of
in real food products. Therefore, this study is one of the few to report the B. cereus FF119b and B. pumilus FF128a.
combined effects of aw and pH (during the heat treatment and storage), Spores of B. cereus 119b and B. pumilus 128a were serially diluted to
heat treatment intensity and storage temperature on the outgrowth of ca. 106–7 spores/ml in TSB adjusted to the desired combination of aw
spores in both artificial media and the effect of heat treatment intensity and pH. A 1 or 2 ml sample was taken to determine the spore counts
in real food products. before the heat treatment was done. 1 ml each of the diluted spore
12 S. Samapundo et al. / International Journal of Food Microbiology 181 (2014) 10–18

suspensions was then used to inoculate 99 ml of pre-heated (70 ± 1 °C, The spores of B. cereus 119b used to inoculate the food products
80 ± 1 °C or 90 ± 1 °C) TSB in Schott bottles which had the same aw and were first serially diluted in PPS to a level of ca. 107 spores/ml. 1 ml of
pH. For each combination of conditions two bottles were prepared. The the diluted spores was then used to inoculate 99–100 g of pre-heated
spores were homogenously distributed in the pre-heated TSB by manu- (heated to 70 ± 1 °C, 80 ± 1 °C or 90 ± 1 °C) product in duplicate.
ally shaking the bottles. The bottles were then kept in a hot-water bath The inoculum was then distributed homogeneously into the product
at the desired temperature for 10 min after which they were cooled by means of a sterile sampling spoon. This gave a level of ca.
under running cold tap water. 1 ml samples were then aseptically 105 spores/g of product without significantly changing the water activity.
drawn from the bottles to determine if any changes in the spore counts The inoculated product was then kept for 10 min at the desired heating
(inactivation) had taken place as a result of the heat treatment. The heat temperature after which it was cooled to ca. 20–22 °C by placing it
treated spore suspensions were then used to inoculate (by spread under running tap water for 2–3 min. Cooling was promoted by manually
plating 100 μl aliquots per plate) 20 plates of TSA adjusted to the same rotating the containers. 1 ± 0.1 g of the heat treated inoculated product
aw and pH per incubation temperature. 10 of the 20 plates were inocu- was then used itself to inoculate each of six sterile 150 ml Falcon flasks
lated with heated spores from one of the two bottles prepared per containing 99–100 g of product. This gave a final inoculation level of ca.
condition evaluated and the other 10 with heated spores from the 103 spores/g, if the spores were not inactivated. The inoculum was
other bottle. The plates had ca. 20 ± 0.1 g of agar and each plate was distributed homogeneously into the product by means of a sterile
considered as a replicate. These plates were then incubated aerobically sampling spoon before aerobic incubation was done at 7 °C and 10 °C
at 7 °C and 10 °C for a maximum of 55 days. During incubation two for a maximum of 60 days. The exact initial inoculation levels were
plates were randomly selected on a regular basis and ca. 10–12 g of determined by spread plating appropriate serial dilutions from duplicate
their contents (inoculated agar) was aseptically transferred to sterile 10–15 g samples of each inoculated product on TSA. Resultant colonies
stomacher bags. These were then serially diluted and depending on were counted after incubation for a minimum of two days at 22 °C.
the stage of the experiment, appropriate decimal dilutions in PPS were During storage two of the six Falcon flasks from each condition were
spread plated on TSA. Resultant colonies were counted after 1–2 days randomly selected on a regular basis and ca. 10–15 g was aseptically
of incubation of the spread plates at 22 °C. transferred to a sterile stomacher bag and decimally diluted in PPS
before homogenization in a stomacher. Further serial decimal dilutions
were then prepared from the primary decimal dilution in PPS and these
2.4. Effect of heat intensity on the outgrowth of B. cereus FF119b spores in were spread plated on TSA. Resultant colonies were counted after
selected food products 1–2 days of incubation at 22 °C. On alternate sampling days the serial
dilutions were also pour plated on MRS agar (lactic acid bacteria) and
Three products – full fat cream, mixed vegetable soup and béchamel RCA (total anaerobic counts), and spread plated on YGC (yeasts and
sauce (water, milk, wheat flour, margarine, modified starch and NaCl) – molds) to determine if other microorganisms other than the inoculated
were evaluated in this study. These were supplied by companies partic- B. cereus spores were also growing.
ipating in the study. The aw and pH of these products were determined
in triplicate by means of a AW SPRINT TH-500 SPRINT aw meter 3. Results and discussion
(Novasina, Switzerland) and a SevenEasy pH meter (Mettler-Toledo,
Germany), respectively. The physico-chemical properties of the prod- 3.1. Combined effect of pasteurization intensity, pH, aw and incubation
ucts are shown in Table 1. The effect of heat treatment intensity was temperature on the outgrowth of B. cereus FF119b and B. pumilus FF128a
evaluated only on B. cereus FF119b spores, which was selected over on artificial media
B. pumilus FF128a as a result of the known adverse food safety and
food spoilage capacities of B. cereus spp. Before the products were The results obtained in this part of the study are shown in Figs. 1 and
used in the study their initial microbiological quality was determined. 2 for B. cereus 119b and Figs. 3 and 4 for B. pumilus FF128a.
This was done as follows. 10–15 g of each product was collected in ster- It can be observed in Fig. 1 that at the highest aw (0.990) evaluated
ile stomacher bags in duplicate. These were then decimally diluted in the lag period before outgrowth (time until the start of exponential
sterile physiological peptone saline (PPS = 1 g peptone + 8.5 g NaCl growth) of spores of B. cereus FF119b was shortest for the spores that
per liter). The serial decimal dilutions were then spread plated on TSA received a heat treatment of 10 min at 70 °C or 80 °C. This was regardless
and yeast–glucose–chloramphenicol (YGC, Oxoid, Basingstoke, UK) of the pH of the medium and the incubation temperature. This faster
agar, respectively, to determine the initial total aerobic and yeast counts, outgrowth when these two pasteurization intensities are applied is a
respectively. The serial decimal dilutions were also pour plated (with an result of heat activation and indicates that under the conditions investi-
additional over-layer) on both de Man Rogosa Sharpe (MRS, Oxoid, gated the most heat activation for these two spore formers takes place
Basingstoke, UK) agar and reinforced clostridial agar (RCA, Oxoid, when a heat treatment of 10 min at 70 to 80 °C is applied. The optimal
Basingstoke, UK) to determine the initial counts of lactic acid bacteria temperature–time combination for heat activation has been reported to
and anaerobes, respectively. The RCA pour plates were incubated in vary widely among different species and sometimes even among differ-
anaerobic jars together with oxygen scavenging AnaeroGen 3.5 liter ent spore preparations of the same strain (Keynan et al., 1964). The
sachets (Oxoid, Basingstoke, UK). Incubation was done at both 22 °C only exception to the general trend at aw 0.990 was observed for
and 30 °C for up to five days. Except for one TSA plate with one colony B. cereus FF119b spores that were inoculated on agar of pH 7 and incubat-
on which a decimal serial dilution from béchamel sauce had been ed at 10 °C, where outgrowth of the native spores (non-heat treated) was
plated, no colonies developed on any of the plates indicating that the similar to that of the spores that received a heat treatment of 10 min at
food products used had b1 bacteria per 10 g of product. 70 °C or 80 °C. This exception was attributed to the more favorable
combination of aw (0.990), pH (7) and storage temperature (10 °C),
which enabled the spores of B. cereus FF119b to germinate and grow-
out spontaneously.
Table 1
Water activity and pH values of the food products evaluated. It was observed that some of the spores of B. cereus FF119b were
partly inactivated upon heating at 90 °C (see Fig. 1) in TSB adjusted to
Product aw pH
aw 0.990 and at all pH values evaluated. The initial spore load reduced
Full fat cream 0.953 ± 0.001 6.73 ± 0.02 by ca. 1.5, 2 and 2.5 log10 CFU/g when the spores were heated at 90 °C
Vegetable soup 0.996 ± 0.001 5.50 ± 0.01 in TSB with pH values of 7, 6.2 and 5.5, respectively. It is well known
Béchamel sauce 0.993 ± 0.001 6.53 ± 0.01
that spores of B. cereus become more heat labile when the pH is
 S. Samapundo et al. / International Journal of Food Microbiology 181 (2014) 10–18
Fig. 1. Effect of heat intensity (○ no heat treatment, 10 min at 70 °C, ▲ 10 min at 80 °C and △ 10 min at 90 °C), incubation temperature (a, b, c = 10 °C; d, e, f = 7 °C) and pH (a, d = 7; b, e = 6.2; c, f = 5.5) on the outgrowth of spores of B. cereus
FF119b at aw 0.990. Points on the graph where log10 CFU/g = 0 indicate where ≤1 colony was observed on the plates. The standard errors are not shown as they were very small.

13
14 S. Samapundo et al. / International Journal of Food Microbiology 181 (2014) 10–18

7 7

6 6
a b
5 5
log10 CFU/g

log10 CFU/g
4 4

3 3

2 2

1 1

0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
time (d) time (d)

6
c
5
log10 CFU/g

0
0 10 20 30 40 50 60
time (d)

Fig. 2. Effect of heat intensity (○ no heat treatment, 10 min at 70 °C, ▲ 10 min at 80 °C and △ 10 min at 90 °C) and pH (a = 7; b = 6.2; c = 5.5) on the outgrowth of spores of B. cereus
FF119b at aw 0.960 and 10 °C. Points on the graph where log10 CFU/g = 0 indicate where ≤1 colony was observed on the plates. The standard errors are not shown as they were very small.

decreased (Samapundo et al., 2011). This initial reduction resulted in before outgrowth of the native spores at aw 0.990 became longer than
the spores heated at 90 °C having what appeared at first to be the that of spores heated for 10 min at 70 °C and 80 °C. As an example of
longest times to outgrowth. While this was true on agar adjusted to the effect of pH, it can be observed from Fig. 1a and b that, although (as
pH 7 and incubated at both 10 and 7 °C, it can be seen in Fig. 1b and e mentioned above) no difference occurred in the lag phase before
that at the less optimal pH of 6.2, the native spores actually had longer outgrowth of native spores and the spores heated for 10 min at 70 °C or
lag phases before outgrowth than spores heat treated for 10 min at 80 °C and incubated at 10 °C, when the pH was lowered to 6.2, the time
90 °C. This implies that although a pasteurization of 10 min at 90 °C is to outgrowth was only 2 days for the spores heated for 10 min at 70 °C
sufficient to inactivate a part of the spore population, it actually or 80 °C while it was ca. 7 days for the native spores. Furthermore it can
activates the remaining (surviving) spores as they take a shorter time also be seen that outgrowth of the spores of B. cereus FF119 at aw 0.990
to grow out than native spores. This finding supports the original obser- and pH 5.5 only took place from spores that had been heat treated for
vations by Curran and Evans (1942) that the overwhelming majority of 10 min at 70 °C or 80 °C over a 28 day incubation period at 10 °C and
spores of Bacillus coagulans, B. subtilis and Bacillus calidolactis which sur- 55 days at 7 °C. The native spores and those pasteurized for 10 min at
vive a severe heating are potentiated (activated) to germinate as they 90 °C did not grow out at pH 5.5. This clearly shows that under sub-
are able to germinate at lower temperatures than native spores. Addi- optimal conditions for germination and outgrowth of spores, mild heat
tionally, it has been reported that spores even from the same species (pasteurization) treatments can actually activate the spores and enable
will vary in their heat resistance giving rise to bimodal populations them to germinate and grow out where native spores would normally
(Shull et al., 1963) or sub-populations with greater heat resistance not readily germinate. Therefore, these treatments have the potential to
than the general spore (Ababouch et al., 1987). This has been thought compromise the microbial stability and safety of those products if they
to partly contribute to the occurrence of tailing on some thermal are contaminated by spores from bacilli.
death curves (Ababouch et al., 1987). It is therefore the heat sensitive Reduction in aw from 0.990 to 0.960 had a very large impact on the
fraction that was most likely inactivated in our study, while the more ability of the spores of B. cereus FF119b to germinate and grow out. As
heat resistant fraction survived and was activated. can be seen in Fig. 2, where outgrowth took place at aw 0.960 during
Differences in outgrowth between the various pasteurizations incubation at 10 °C, it occurred after at least 35 days of incubation.
applied became larger as the pH of the agar was reduced from 7 to 5.5 Additionally, at 7 °C (results not shown) outgrowth did not take place
and/or when the incubation temperature was reduced for 10 °C to at aw 0.960 under all combinations of aw and pH evaluated in this
7 °C. As an example of the effect of the incubation temperature, it was study. It can be seen that no difference occurred in the outgrowth of
observed in Fig. 1a that the lag phase before outgrowth of native spores the native spores and spores that had received a pasteurization of
did not differ with that of spores heated for 10 min at 70 °C or 80 °C on 10 min at 70 °C or 80 °C and incubated at 10 °C on media with favorable
agar of aw 0.990 and pH 7, when incubation was done at 10 °C. However, aw 0.990 and pH values of 7 and 6.2. However, it has to be noted that
when the temperature was lowered to 7 °C (see Fig. 1d) the lag phase very little growth (b2 log CFU/g) had taken place at pH 6.2 over a 52
 S. Samapundo et al. / International Journal of Food Microbiology 181 (2014) 10–18
Fig. 3. Effect of heat intensity (○ no heat treatment, 10 min at 70 °C, ▲ 10 min at 80 °C and △ 10 min at 90 °C), incubation temperature (a, b, c = 10 °C; d, e, f = 7 °C) and pH (a, d = 7; b, e = 6.2; c, f = 5.5) on the outgrowth of spores of B. pumilus
FF128a at aw 0.990. Points on the graph where log10 CFU/g = 0 indicate where ≤1 colony was observed on the plates. The standard errors are not shown as they were very small.

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Fig. 4. Effect of heat intensity (○ no heat treatment, 10 min at 70 °C, ▲ 10 min at 80 °C and △ 10 min at 90 °C), incubation temperature (a, b, c = 10 °C; d, e, f = 7 °C) and pH (a, d = 7; b,
e = 6.2; c, f = 5.5) on the outgrowth of spores of B. pumilus FF128a at aw 0.960. Points on the graph where log10 CFU/g = 0 indicate where ≤ 1 colony was observed on the plates.
The standard errors are not shown as they were very small.

day incubation period. In addition, it was also observed that the initial 0.960, the outgrowth of B. pumilus FF128a was only inhibited over a
reduction (inactivation) observed at aw 0.990 (see Fig. 1) when the 39 day period when inoculation was done at pH 5.5 and incubation
spores were heated at 90 °C was much reduced at aw 0.960; where was done at 7 °C. Additionally, it can be seen in Fig. 4c that at aw
the largest reduction was only ca. 0.5 log10 CFU/g. This was attributed 0.960, pH 5.5 and 10 °C, native spores of B. pumilus FF128a grew out
to the thermo-protective effect low aw values are known to have on after a much shorter time than those heated for 10 min at either 70
spores (Samapundo et al., 2011). and 80 °C, indicating that at this pH and a w, these pasteurization
As can be seen in Figs. 3 and 4, in comparison to B. cereus FF119b, intensities were no longer activating the spores. The longer time to
B. pumilus FF128a responded mostly in a similar way to the combined outgrowth indicates that the pasteurizations most likely injured
effect of heat treatment, medium aw and pH, and incubation tempera- the spores sub-lethally, which would have required the spores to
ture. In agreement with the results observed for B. cereus FF119b, it repair the damage before outgrowth.
was observed that the lag phase before the outgrowth of B. pumilus The ability of the spores of B. pumilus FF128a to grow at aw 0.960 and
128a at aw 0.990 was for all conditions evaluated shortest for the spores 7 °C enabled us to gain more insight into the effect of the pasteurization
that received a heat treatment of 10 min at 70 °C or 80 °C, while that of intensity on the survival, heat activation and/or outgrowth of spores
native spores was longer and that of the spores that were heated at under adverse conditions. It can be seen in Fig. 4, that outgrowth of
90 °C was generally the longest. This confirms that heat activation B. pumilus FF128a at aw 0.960 takes place when the spores are heated
appears to occur when a mild pasteurization is performed at 70–80 °C. for 10 min at 90 °C and inoculated on agar of pH 6.2 and incubated at
As for B. cereus FF119b, the effect of the pasteurization intensity with 10 °C (Fig. 4b). When incubation is done at the less conducive temper-
regard to the heat activation of the spores became more evident at the ature of 7 °C (Fig. 4e), the spores die-off steadily during incubation
lower pH values investigated. This can clearly be seen in Fig. 3c and f, until they cannot be recovered after 23 days of incubation. It can also
where outgrowth at pH 5.5 is fastest for the spores of B. pumilus be seen in Fig. 4c and f that at aw 0.960 and pH 5.5, only the spores of
FF128a heated for 10 min at 70 or 80 °C and slowest for the native B. pumilus FF128a heated for 10 min at 90 °C die-off during incubation
non-heated spores. Additionally, it can be seen in the same figures at both 7 and 10 °C, while those heated for the same duration at 70 or
that although the spores heated for 10 min at 90 °C were initially 80 °C do not change in their numbers when incubation is done at 7 °C
reduced by up to 4 log10 CFU/g at aw 0.990 and pH 5.5, the surviving or increase slightly when incubation is done at 10 °C. This indicates
spores were actually heat activated as they grew out faster than the that B. pumilus FF128a is only able to recover from heat injury at 90 °C
native non-heated spores. This was observed at a higher pH value of when then storage or medium conditions (temperature and pH in this
6.2 for B. cereus FF119b. case) are favorable. These findings are in agreement with Issahary
Some differences occurred between the response of B. cereus FF119b et al. (1970) who reported that the heat activation of B. cereus spores
and B. pumilus FF128. B. pumilus FF128a was generally more tolerant to at a very low pH value of 1.0 was followed by gradual loss of viability,
adverse combinations of aw, pH and temperature than B. cereus FF119b. whereas the counts of spores activated in distilled water remained
It can be seen in Fig. 4 that at aw 0.960, B. pumilus FF128a was able to unchanged. Issahary et al. (1970) also stated that heat activation at
grow at 7 °C, all pH values and almost all pasteurization intensities low pH values differs from that at neutral pH as it involves two
evaluated. In difference, B. cereus FF119b did not grow out after superimposed processes: enhanced activation and death; both of
52 days at aw 0.960 when the temperature was reduced to 7 °C. At aw which were observed in the present study.
 S. Samapundo et al. / International Journal of Food Microbiology 181 (2014) 10–18 17

3.2. Effect of heat intensity on the outgrowth of B. cereus FF119b spores in more complex than simple broths or buffer systems and they potentially
food products contain components that can offer protection to the spores.
At 7 °C a very interesting effect of storage temperature is observed
The results obtained in this part of the study are shown in Fig. 5. on the outgrowth of B. cereus FF119b in both cream and béchamel
Germination and outgrowth of spores of B. cereus FF119b only took sauce. While outgrowth took place during storage in inoculated cream
place in cream and béchamel sauce. No growth was observed in the and béchamel sauce at 10 °C irrespective of the heat treatment applied,
mixed vegetable soup after 34 days of incubation at both 10 and 7 °C. outgrowth at 7 °C only took place from the spores that were heated for
The mixed vegetable soup had a low pH value of 5.50 ± 0.01 which 10 min at 80 °C. The inability of the spores of B. cereus FF119b heated for
contributed to inhibition of the outgrowth of the spores of B. cereus 10 min at 70 °C in both cream and béchamel sauce to grow out at 7 °C
FF119b. This observation was in agreement with the results observed in could possibly be due to the presence of protective ingredients in
artificial medium with similar aw (0.990) and pH (5.5) values. Cream these products which resulted in insufficient activation energy being
had more favorable aw and pH values of 0.953 ± 0.01 and 6.73 ± 0.02, transferred to the spores to enable germination to occur at 7 °C. By
respectively. Béchamel sauce also had favorable aw and pH values of inference, 10 min at 80 °C provided sufficient activation energy for
0.993 ± 0.001 and 6.53 ± 0.01, respectively. germination and outgrowth at 7 °C. The inability of the spores heated
It can be seen in Fig. 5 that the effect of the pasteurization intensity on at 90 °C to grow out in the cream and béchamel stored at 7 °C may be
outgrowth of the spores of B. cereus FF119b appeared to be largely depen- a result of failure to recover or repair non-lethal injuries in these
dent on the storage temperature. At 10 °C (Fig. 5a), it can be seen that the products at this temperature, as can be seen by the steady rate at
outgrowth of the spores in cream was instantaneous irrespective of the which they die during storage. So while some similarities do occur
pasteurization intensity and reached maximum population densities of between the responses of B. cereus FF119b in artificial media and real
7.3–7.4 for spores heated for 10 min at 80 °C, and 6.4–6.5 log10 CFU/g products of approximately the same aw and pH, some striking differ-
for the spores heated for 10 min at 70 °C and 90 °C after just 11 days of ences also occur. It therefore would be worthwhile to determine in
incubation. In béchamel sauce a similar trend was observed with regards future studies the role different food components may have on the
to outgrowth at 10 °C. The outgrowth of B. cereus FF119b spores that effect of heat intensity on the germination and outgrowth of spores.
were heated for 10 min at 90 °C took a slightly longer time (ca. 3 days) Busta and Ordal (1964) already demonstrated that the presence of
at 10 °C as was observed on artificial media. Additionally, it was noted simple monomers such as glucose, NaCl, xylose and ribose does not
that the outgrowth of B. cereus FF119b spores in artificial media at aw have an influence on the heat activation of B. subtilis at 75 °C. It is
and pH combinations closest to that of cream (aw 0.960 and pH 6.2 and therefore most likely that the macromolecular compounds in foods i.e.
7.0), took a much longer time of at least 35 days. This we believe is largely fat, proteins, and starch may influence the effect of a heat treatment
a result of the differences in heat conductivity between the TSB used for on the survival or outgrowth of spores in food products.
the studies in artificial media and the food products evaluated. The fat
in the cream may have had a thermo-protective on the spores owing to 4. Conclusions
its lower thermal conductance. It can also be seen that, in difference to
the results observed in artificial media, no inactivation of some of the The results of this study demonstrated that the potential conse-
spores took place when the spores where heated in cream and béchamel quences of the intensity of a (mild) heat treatment on the survival and
sauce 90 °C. This further illustrates the protective effect of the fat in milk outgrowth of psychrotolerant spores of B. cereus and B. pumilus are highly
and the various ingredients in béchamel sauce. Leguérinel et al. (2005) storage temperature, aw and pH dependent. This further implies that
and Jagannath and Tsuchido (2003) both stated that food products are certain combinations of pasteurization intensity, storage temperature,

Fig. 5. Effect of heat intensity ( 10 min at 70 °C, ▲ 10 min at 80 °C and △ 10 min at 90 °C) on the outgrowth of spores of B. cereus FF119b in cream at (a) 10 °C and (b) 7 °C and in béchamel
sauce at (c) 10 °C and (d) 7 °C. Points on the graph where log10 CFU/g = 0 indicate where ≤1 colony was observed on the plates. The standard errors are not shown as they were very
small.
18 S. Samapundo et al. / International Journal of Food Microbiology 181 (2014) 10–18

aw and pH could be used to limit the potential outgrowth of spores in food Daelman, J., Vermeulen, A., Willemyns, T., Ongenaert, R., Jacxsens, L., Uyttendaele, M.,
Devlieghere, F., 2013a. Growth/no growth models for heat-treated psychrotrophic
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conditions evaluated, greatest activation of the spores generally took Daelman, J., Sharma, A., Vermeulen, A., Uyttendaele, M., Devlieghere, F., Membré, J.-M.,
place when the spores of both B. cereus and B. pumilus were heated for 2013b. Development of a time-to-detect growth model for heat-treated Bacillus
cereus spores. Int. J. Food Microbiol. 165, 231–240.
10 min at 70 °C or 80 °C in artificial media. Species dependency was De Jonghe, V., Coorevits, A., Vandroemme, J., Heyrman, J., Herman, L., De Vos, P.,
also observed as differences were observed in some cases between the Heyndrickx, M., 2008. Intraspecific genotypic diversity of Bacillus species from raw
responses of B. cereus and B. pumilus to the effects of pasteurization inten- milk. Int. Dairy J. 18, 496–505.
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and real food products indicates that in addition to aw and pH, food Smith, I., Slepecky, R.A., Setlow, P. (Eds.), Regulation of Proharyotic Development.
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Gaillard, S., Leguérinel, I., Savy, N., Mafart, P., 2005. Quantifying the combined effects of the
heat treatment. A heat treatment of 10 min at 80 °C clearly provided heating time, the temperature and the recovery medium pH on the regrowth lag time
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