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Journal of Applied Microbiology 2001, 90, 761±770

Effect of sporulation and recovery medium on the heat

resistance and amount of injury of spores from spoilage bacilli

A.E. Cazemier, S.F.M. Wagenaars and P.F. ter Steeg

Microbiology & Preservation, Unilever Research Vlaardingen, Vlaardingen, The Netherlands

528/8/00: received 25 August 2000, revised 17 January 2001 and accepted 23 January 2001

A . E . C A Z E M I E R , S . F . M . W A G E N A A R S A N D P . F . T E R S T E E G . 2001.
Aims: To assess the in¯uence of sporulation media on heat resistance, and the use of stress
recovery media to measure preservation injury of spores of ®ve representative spoilage bacilli.
Methods and Results: Bacillus spores prepared on nutrient agar supplemented with Ca2+,
Mg2+, Mn2+, Fe2+ and K+ were more heat-resistant than spores obtained from nutrient agar
with Mn2+. This increased heat resistance correlated with a decrease in the protoplast water
content as determined by buoyant density sedimentation. The degree of preservation injury
severity could be assessed on media containing NaCl at moderate pH and organic acids at acid
pH. Ca-DPA, K+ or proline were added to the recovery media to demonstrate that heat
probably caused injury to both spore germination and the outgrowth system.
2 Signi®cance and Impact of the Study: The metal content of sporulation media can
strongly effect the validity of preservation resistance studies. The distinctive recovery
media developed here can be relevant for assessing and comparing new preservation
technologies.

botulinum; Stumbo 1973) than traditional sterilization (over-)

INTRODUCTION
Bacillus spores are common contaminants of vegetables and
fruits and may cause food spoilage (B. subtilis, B. coagulans,
B. licheniformis) or act as food-borne pathogens (B. cereus).
Bacterial spores are extremely resistant to heat and other
preservation treatments, such as irradiation, chemicals or
pressure, compared with vegetative cells. Sterilization of
foods is needed for complete inactivation of bacterial spores,
but the high temperatures and long heating times normally
employed in conventional static sterilization processes
adversely affect the nutritional and organoleptic qualities
of most foods. Therefore, pasteurization instead of steril-
ization, or alternative less detrimental preservation technol-
ogies such as ultra-high temperature (UHT) (Brown 1994),
3 rotary retort (Abbatemarco and Ramaswamy 1993) and high
hydrostatic pressure processing (HHPP) (Smelt 1998) are
desirable. Since alternative preservation technologies tend to
process closer to the actual food safety boundaries
(F0 ˆ 3 min at 121°C for a 12D reduction of Clostridium
Correspondence to: P.F. ter Steeg, Microbiology & Preservation, Unilever
Research Vlaardingen, PO Box 114, 3130 AC Vlaardingen, The Netherlands
(e-mail: Pieter-ter.Steeg@unilever.com).
processing, heat-resistant spores of spoilage bacilli may not
always be totally inactivated but may only be sublethally
injured by these technologies. However, a combination of
preservation technologies can then be applied to assure
microbial safety and stability of food products (Leistner
2000). To assess the ef®ciency of new preservation technol-
ogies, methods by which the amount and type of spore
injury can be determined are necessary. The extent of spore
injury could be established by studying spore recovery on
optimal media and media containing antimicrobials, such as
organic acids, sodium chloride or nitrite. Sublethally-injured
spores appear to have an increased sensitivity to these
antimicrobials (Roberts and Ingram 1966; Roberts et al.
1966; Foegeding and Busta 1981; Blocher and Busta 1983;
Cook and Pierson 1983; Faille et al. 1997).
The primary effect of sublethal injury to spores can be
(i) on the germination system or (ii) on the outgrowth and
growth of vegetative cells. The major targets for heat
inactivation or injury of spores are considered to be proteins
and enzymes (Marquis et al. 1994). Nevertheless, in many
spores it is not clear which enzymes or other proteins are the
critical targets for killing, and it is likely that functional
proteins in both the germination and outgrowth system
are injured at the same time. In addition, considerable

ã 2001 The Society for Applied Microbiology


762 A . E . C A Z E M I E R E T A L .
variability in the heat resistance of bacterial spores exists.
Therefore, the effectiveness of a heat treatment resulting in
spore injury will also depend on the type of spores present.
The thermal resistance of spores is to some extent species-
dependent, but it is also affected by sporulation conditions
such as the cationic environment, temperature and
pH (Marquis and Bender 1985; Raso et al. 1998; GonzaÂlez
et al. 1999; Palop et al. 1999ab). These factors in¯uence
the protoplast water content, which appears to be inversely
correlated to the spore heat resistance (Beaman et al. 1984;
Beaman and Gerhardt 1986). As a result, in the assessment
of new preservation technologies, the conditions used to
produce spore suspensions in the laboratory are critical.
In this study, spores of ®ve different Bacillus strains
were prepared on two sporulation media, supplemented
with different metal ions, to investigate the effect of the
composition of these sporulation media on spore heat
resistance and the water content of the spore protoplast.
In addition, the sensitivity of sublethally heat-injured spores
to various antimicrobials in the recovery media was exam-
ined, with the aim of predicting the amount of spore injury
in¯icted by a speci®c heat treatment and obtaining some
insight into the potential type of spore injury caused.
M A T E R I A LS A N D M E T H O D S
Strains and sporulation conditions
Bacillus subtilis A163, a mesophilic strain with an unusual
thermotolerance (D121 °C ˆ 1¢, personal communication),
B. subtilis AdHC1, an acid-tolerant organism, and B. lichen-
iformis VBBBa 03-25 were obtained from the culture
collection at Unilever Bestfoods Research Vlaardingen.
The other Bacillus strains studied were B. licheniformis
AS 1á813 and B. coagulans ATCC 10545.
Sporulation was obtained on nutrient agar supplemented
with MnSO4.H2O (3 ppm) (LoÂpez et al. 1996), or on a
slightly modi®ed sporulation medium, described by Faille
et al. (1997), containing the following ingredients (l±1):
nutrient broth (Difco), 13 g; MgSO4.7H2O, 0á51 g;
KCl, 0á97 g; CaCl2.2H2O, 0á2 g; MnSO4.H2O, 3 ´ 10±3 g;
4 FeSO4.7H2O, 0á55 ´ 10±3 g; and 1á5% (w/v) agar at pH 7á0
(13 instead of 8 g nutrient broth was used to obtain a higher
yield of spores). A single colony of a Bacillus strain was
suspended in physiological salt solution. A volume of 0á5 ml
of this spore suspension was spread onto plates with one of
the sporulation media. Plates were incubated right-side up
at 30°C (B. subtilis A163) or 37°C (B. subtilis AdHC1,
B. licheniformis VBBBa 03-25 and AS 1á813, and B. coagulans
ATCC 10545). More than 95% of the cells had sporulated
within 5±10 days of incubation on both media. Spores
from three plates were harvested with 10 ml physiological
salt solution, washed three times in physiological salt
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solution and stored at ±80°C. Spore suspensions contained
108±109 spores ml±1.
Preparation of coat-defective spores
To determine the protoplast water content by buoyant
density sedimentation, it was necessary to prepare coat-
defective (lysozyme-sensitive) spores. The different Bacillus
spores were made lysozyme-sensitive by treatment with 0á5%
SDS±0á1 mol l±1 dithiotreitol (DTT) in 0á1 mol l±1 NaCl, at
pH 10, for 2 h at 37°C, with continuous shaking (Lindsay
et al. 1985b). Treatments with 25% (v/v) thioglycollic acid,
pH 2á5±3, or 1% (v/v) thioglycollic acid in 8 mol l±1 urea,
pH 2á5±3, were also tested for B. subtilis AdHC1 spores
5 (Gould & Hitchins 1963). The effectiveness of the SDS±
DTT or thioglycollic acid treatments was checked micro-
scopically by following the appearance of phase dark spores
during incubation with 100 lg ml±1 lysozyme in 10 mmol l±1
phosphate buffer, pH 7á0, at 37°C. The viability of the spores
after the SDS±DTT and thioglycollic acid treatment was
checked on nutrient agar plates.
Determination of spore and protoplast
water content
The spore and protoplast wet densities were determined by
buoyant density sedimentation in Nycodenz (5-(N-2,3-
dihydroxypropylacetamido)-2,4,6-triiodo-N,N¢-bis(2,3-dihy-
droxypropyl)isophtalamide; Sigma) gradients, principally as
described by Lindsay et al. (1985a). The refractive index of
the Nycodenz solutions was determined in a precision
refractometer (Zeiss, Germany) at 20±21°C, and converted
to solution densities by use of a calibration curve and the
formula derived by Rickwood et al. (1982). A 100 ll volume
of coat-defective spores and 100 ll of untreated spores, both
containing 108±109 spores ml±1, were deposited on top of a
5 ml gradient containing 10, 0á5 ml layers of medium in a 10
ml polycarbonate tube. The tube was centrifuged to equilib-
rium in a Sorvall HS-4 swinging bucket rotor at 7000 g at
10°C for 45 min. The apparent wet density of the spore type
was determined by the position of the band at the interface
above and below a medium layer. Maximum precision
(<0á005 g l±1) was obtained by using successively narrower
gradients. Determinations were performed in duplicate. The
protoplast apparent wet densities were converted to protoplast
water contents by use of the linear correlation equation y ˆ
±0á00254x + 1á460, where y is the protoplast wet density and
x is the protoplast water content (Lindsay et al. 1985a).
Heat treatments
Heat treatments of spores were performed according to
the screw-cap tube method described by Kooiman (1973). A
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1 SPORULATION CONDITIONS, HEAT RESISTANCE AND INJURY OF SPOILAGE BACILLI 763

100 ll volume of a spore suspension was injected into 9á9 ml The effect of lysozyme on the recovery of heat-injured
pre-heated trypticase soy broth (TSB), at the desired pH B. subtilis A163 and B. subtilis AdHC1 spores was studied at
value, with a CR-700-200 Hamilton syringe. Heat treat- pH 5á9 and pH 4á5, respectively. Lysozyme (®nal concen-
ments were carried out in a water-bath (96±98°C) or a tration 10 and 100 lg l±1) was added to melted and cooled
glycerol bath (114°C) in 10 ml glass or metal screw-cap (to 50°C) TSA just before use.
tubes, respectively. After heating, the tubes were immedi-
ately cooled on ice/water and kept on ice/water until
RESULTS
plating. Heat treatments were performed in TSB at pH 5á9
for B. subtilis A163 (pH of the food product from which the In¯uence of the composition of the sporulation
strain was isolated), at pH 4á5 for B. subtilis AdHC1 and medium on the heat resistance of Bacillus spores
B. coagulans ATCC 10545 (approximate pH of tomato-based
The spores of different Bacillus strains prepared on nutrient
products), and at pH 7 for B. licheniformis AS 1á813 and
agar with the mix of metal ions (Ca2+, Mg2+, Fe2+, K+ and
VBBBa 03-25; the TSB was adjusted to the right pH with
Mn2+) all appeared to be more heat-resistant than the spores
5 mol l±1 NaOH and 4 mol l±1 HCl solutions.
prepared on nutrient agar with only Mn2+. The increase in
heat resistance was most pronounced for spores from
Recovery of heat-injured spores B. subtilis A163. A heat treatment at 114°C for 4 min did
not reduce spore counts when spores were prepared on
The quantity of heat-injured spores of B. subtilis A163 and
nutrient agar with a mix of metal ions, whereas a 5 log
B. licheniformis, strains AS 1á813 and VBBBa 03-25, was
reduction was found after this heat treatment when spores
estimated by comparing the spore recovery on trypticase soy
were prepared on nutrient agar with only Mn2+. For B. subtilis
agar (TSA) and on TSA containing 1, 6 and 9% (w/v) NaCl
AdHC1, differences in survival of between 0á6 and 2á2 logs
at pH 5á9 and pH 7, respectively. Since K+-ions and proline
were observed (Fig. 1a). Remarkably, spores from the
are known to be accumulated in B. subtilis cells under
B. coagulans ATCC 10545 prepared on nutrient agar with
osmotic stress (Whatmore and Reed 1990; Whatmore et al.
Mn2+ were not even able to outgrow at pH 4á5, although at
1990), the recovery of heat-injured spores from B. subtilis
pH 7á0 these spores did outgrow (results not shown). Results
A163 was also studied on TSA + 6% (w/v) NaCl + 10 m-
for B. licheniformis VBBBa 03-25 are presented in Fig. 1(b);
mol l±1 KCl, and TSA + 6% (w/v) NaCl + 10 mmol l±1
those for B. licheniformis AS 1á1813 were similar (not
KCl + 10 mmol l±1 proline, all at pH 5á9.
shown).
The quantity of heat-injured spores of B. subtilis AdHC1
and B. coagulans ATCC 10545 was estimated by comparing
spore recovery on TSA with that on TSA + 0á5% (w/v) citric Protoplast water content
acid, pH 4á5. In addition, the recovery of heat-injured Bacillus
An SDS±DTT treatment appeared to be effective for
sp. AdHC1 spores was studied on TSA with lactic acid (0á5%,
obtaining coat-defective spores. More than 95% of the
v/v), acetic acid (0á2%, v/v), and a combination of citric acid
treated spores turned phase dark within 30±60 min of
(0á5%, w/v) and acetic acid (0á2%, v/v). Furthermore, the
incubation with lysozyme in all spores tested except those of
effect of additional Ca2+ and Mg2+ ions (0á02 g l±1 CaCl2.
B. subtilis AdHC1. Only about 40% of the SDS±DTT-
2H2O and 0á05 g l±1 MgSO4.7H2O) in the recovery medium
treated B. subtilis AdHC1 spores turned phase dark after
with 0á5% (w/v) citric acid on the outgrowth of heat-injured
prolonged incubation with lysozyme. Treatment with 25%
B. subtilis AdHC1 spores was investigated.
(v/v) thioglycollic acid or 1% (v/v) thioglycollic acid in
8 mol l±1 urea (Gould and Hitchins 1963) did not increase
Effect of Ca-dipicolinic acid and lysozyme the sensitivity of the spores to lysozyme and reduced spore
on spore recovery viability considerably. Therefore, it was decided to use the
SDS±DTT treatment for the preparation of coat-defective
The recovery of heat-injured B. subtilis A163 and B. subtilis
spores of B. subtilis AdHC1 as well.
AdHC1 spores was examined on plates containing TSA +
The spore and protoplast wet densities, i.e., of the
Ca-dipicolinic acid (Ca-DPA, 50±40 mmol l±1; pH 5á9 for
untreated and coat-defective spores, respectively, deter-
both strains). The appropriate volume of a Na2-DPA
mined by Nycodenz buoyant density sedimentation, are
solution (0á5 mol l±1) was added to melted TSA, which
shown in Table 1. The wet densities of the B. licheniformis
was kept at 50°C until use; CaCl2.2H2O (2 mol l±1) was
and B. coagulans spores prepared on the two sporulation
added just before pouring the plates. In this way, no, or
media were in the same range. In contrast, the wet
only a limited amount of Ca-DPA precipitates were formed
densities of B. subtilis A163 and B. subtilis AdHC1 spores
during solidi®cation of the agar.
prepared on nutrient agar with the mix of metal ions were

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764 A . E . C A Z E M I E R E T A L .

Table 1 Wet densities, as determined by Nycodenz buoyant density

sedimentation, and protoplast water contents of several Bacillus spores
prepared on two different sporulation media

Wet density (g ml)1)* Water content

(g water 100 g)1
Sporoplast Protoplast protoplast) 

B. subtilis A163
NA Mnà 1á302 1á335 49á21
NA Mix 1á314 1á342 46á46
B. subtilis AdHC1
NA Mn 1á309 1á321 54á72
NA Mix 1á316 1á329 51á58
B. licheniformis AS 1á813
NA Mn 1á306 1á322 54á61
NA Mix 1á307 1á330 51á16
B. licheniformis VBBBa 03±25
NA Mn 1á306 1á325 53á47
NA Mix 1á306 1á338 48á03
B. coagulans ATCC 10545
NA Mn 1á305 1á320 54á96
NA Mix 1á303 1á325 53á17

* Data are the mean of duplicate measurements, variances were

between 0á002 and 0á006 g ml)1.
  The water content was calculated from the wet densities using the
linear correlation equation y = ±0á00254x + 1á460 (Lindsay et al.
1985a).
à NA Mn: nutrient agar with 3 ppm MnSO4.H2O; NA Mix: nutrient
agar with a mix of metal ions.

Effect of NaCl on the recovery of heat-injured

Fig. 1 Examples of the effect of the composition of the sporulation
medium on the heat resistance of Bacillus spores. Spores were prepared
on nutrient agar with 3 ppm MnSO4.H2O (n), or on nutrient agar with
a mix of metal ions (see Materials and Methods: h). Heat treatments
were performed in TSB at 114 °C and pH 5á9 for B. subtilis AdHC1
(a) and at 98 °C at pH 7á0 for B. licheniformis VBBBa 03-25 (b). The
recovery of spores after a heat treatment was studied on plates
containing TSA at a pH value similar to that of the heating
menstruum. Data are the mean of plate counts of duplicate heat
treatments; the variance is shown
signi®cantly higher (1á314 and 1á316 g ml±1, respectively)
than those of spores prepared on nutrient agar with Mn2+
(1á302 and 1á309 g ml±1, respectively). The protoplast
wet densities of all the tested spore types prepared on
nutrient agar with the mix of metal ions were higher, and
subsequently, the calculated protoplast water contents were
lower, than those of the spores prepared on nutrient agar
with only Mn2+.
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 761±770
Bacillus spores at moderate pH
To investigate the quantity of injured spores after a speci®c
heat treatment, the recovery of heated B. subtilis A163 spores
was determined on a rich medium (TSA) and a medium
containing different concentrations of NaCl at pH 5á9
(Fig. 2). The recovery of unheated spores was hardly affected
by the different NaCl concentrations used. In contrast, the
recovery of heated spores was reduced in the media with 6 and
9% (w/v) NaCl, but not with 1% (w/v) NaCl, compared with
the recovery on TSA. The more severe heat treatments
resulted in a relatively lower recovery of B. subtilis A163
spores (Fig. 2). The sensitivity to NaCl of heat-injured spores
was not in¯uenced by the heating temperature (98°C or
114°C, Fig. 2b). In addition, the composition of the sporu-
lation medium did not signi®cantly affect spore sensitivity to
NaCl, although longer heating times were necessary to cause a
similar amount of injury to spores prepared on nutrient agar
with a mix of metal ions, compared to those prepared on
nutrient agar with Mn2+ (Fig 2b,c). When heat-injured
B. licheniformis AS 1á813 and B. licheniformis VBBBa 03-25
 13652672, 2001, 5, Downloaded from https://ami-journals.onlinelibrary.wiley.com/doi/10.1046/j.1365-2672.2001.01302.x by Nat Prov Indonesia, Wiley Online Library on [22/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1 SPORULATION CONDITIONS, HEAT RESISTANCE AND INJURY OF SPOILAGE BACILLI 765

Fig. 2 Recovery of Bacillus subtilis A163 spores after a heat treatment in TSB at 98 °C (a, b) and 114 °C (b, c) at pH 5á9 on TSA (h, a, b, c) and TSA + 1
( , a), 6 (j, a, b, c) or 9% ( , a) (w/v) of NaCl (pH 5á9) after 3±7 days of incubation at 30 °C. Data are the mean of plate counts of duplicate heat
treatments; the variance is shown. Spores were prepared on nutrient agar with 3 ppm MnSO4.H2O (a,b) and on nutrient agar with a mix of metal ions (c)

spores were spread on plates with 6 and 9% (w/v) NaCl, a In contrast, an increased sensitivity of heat-injured
similar reduction in spore recovery was observed as for B. subtilis AdHC1 spores to organic acids in the recovery
B. subtilis A163 (results not shown). medium was observed. Compared with spore recovery on
To gain some further insight into the type of heat injury rich medium (TSA), a 0á5±1á5 log reduction in spore
that made B. subtilis A163 spores susceptible to NaCl in recovery was found on media containing 0á5% citric acid,
the recovery medium, KCl and proline, which are known to
be accumulated in B. subtilis cells under osmotic stress
Table 2 Effect of the addition of KCl and proline to recovery media
(Whatmore and Reed 1990; Whatmore et al. 1990), were
supplemented with 6% NaCl, on the recovery of heat injured Bacillus
added to the recovery medium with 6% (w/v) NaCl. A
subtilis A163 spores prepared on nutrient agar with a mix metal ions
slight increase in recovery of heat-injured spores was
observed (30±70%) when KCl and proline were added to % Survivors*
the recovery medium with 6% (w/v) NaCl (Table 2).
6 min 12 min
Recovery medium  Unheated 114 °C 114 °C
Effect of organic acids on the recovery TSA + 6% NaCl 100 100 100
of heat-injured spores at pH 4á5 TSA + 6% NaCl 119 ‹ 22 139 ‹ 7 125 ‹ 4
The use of NaCl did not appear to be suitable for + 10 mmol l)1 KCl
distinguishing heat-injured from unheated spores at TSA + 6% NaCl 80 ‹ 9 173 ‹ 35 149 ‹ 15
+ 10 mmol l)1 KCl + proline
pH 4á5. Both heat-injured and unheated spores of B. subtilis
AdHC1 were unable to outgrow at NaCl concentrations * Data are the mean of plate counts of three separate heat treatments in
>4% (w/v), and heat-injured spores showed no increased TSB, pH 5á9 ‹ S.D., after 10 days of incubation at 30 °C.
sensitivity to NaCl concentrations <4% (w/v, Fig. 3a).   The pH of the recovery medium was 5á9.

ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 761±770

766 A . E . C A Z E M I E R E T A L .
Fig. 3 (a) Recovery of heated Bacillus subtilis AdHC1 spores after
0 min (h), 10 min ( ) and 30 min (j) in TSB at pH 4á5 at 96 °C, on
TSA containing different concentrations of NaCl and citric acid,
pH 4á5, after 3±7 days of incubation at 37°. (b) Recovery of B. subtilis
AdHC1 spores after different heat treatments in TSB, pH 4á5, at 96 °C
on TSA, pH 4á5 (h), and TSA supplemented with 0á5 ( ) and 0á9%
(j) (w/v) citric acid. Data are the mean of plate counts of duplicate
heat treatments; the variance is shown. Spores were prepared on
nutrient agar with 3 ppm MnSO4.H2O
depending on the heat treatment applied (Fig. 3, Table 1).
A higher citric acid concentration (0á9%, w/v) did not
decrease B. subtilis AdHC1 spore recovery further (Fig. 3b).
When heat-injured spores of B. coagulans ATCC 10545 were
spread onto plates with 0á5% citric acid, a similar reduction
in spore recovery was observed as in the case of B. subtilis
AdHC1 (not shown).
Since citric acid acts partly as a sequestrant and may
reduce the availability of divalent metal ions (Gould 1989),
Ca2+ and Mg2+ ions were added to the recovery medium
with 0á5% (w/v) citric acid. The addition of these divalent
metal ions increased the recovery of injured B. subtilis
AdHC1 spores by about 0á5 log (Table 3).
When lactic acid and acetic acid were added to the
recovery medium, lower numbers of heat-injured B. subtilis
AdHC1 spores were able to outgrow than on the medium
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 761±770
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with citric acid (Table 3). Furthermore, the time until
outgrowth of spores when lactic acid and acetic acid were
added to the recovery medium was longer than on the
recovery medium containing citric acid. Citric acid was less
inhibitory than lactic acid, whereas lactic acid was less
inhibitory than acetic acid with respect to the outgrowth of
heat-injured B. subtilis AdHC1 spores at pH 4á5.
Repair of injury
The type of spore injury caused by heat was investigated
further by evaluating the effect of Ca-DPA (Ciarciaglini
et al. 2000) and lysozyme on spore recovery. The addition
of Ca-DPA to the recovery medium slightly increased the
recovery of heat-injured spores (0á5±1 log) of both B. subtilis
AdHC1 (Fig. 4) and B. subtilis A163 (results not shown). In
contrast, the addition of lysozyme to the recovery medium
decreased the recovery of heat-injured B. subtilis A163 and
B. subtilis AdHC1 spores at pH 5á9 and pH 4á5, respectively
(Fig. 5).
DISCUSSION
The heat resistance of bacterial spores can be attributed to
three main factors: protoplast dehydration, mineralization
and thermal adaptation. Since both protoplast mineralization
and a higher sporulation temperature result in more dehy-
drated spore protoplasts, it is assumed that protoplast water
content is the primary component determining spore heat
resistance (Beaman et al. 1984; Bender and Marquis 1985;
Marquis and Bender 1985; Nakashio and Gerhardt
1985; Beaman and Gerhardt 1986;Nakashio and Gerhardt
1985). The results shown here support this view. The
Bacillus spores prepared on nutrient agar with a mix of metal
ions (Ca2+, Mn2+, Mg2+, Fe2+ and K+) were more heat-
resistant and contained a lower protoplast water content
than the Bacillus spores prepared on nutrient agar with only
Mn2+ (Fig. 1, Table 1). Although the mineral content in the
spore protoplasts was not determined here, it is assumed
that the spores prepared on nutrient agar with a mix of metal
ions are more mineralized than the spores prepared on
nutrient agar with only Mn2+ (Beaman and Gerhardt 1986).
The protoplast water content of the Bacillus spores used in
this study were within the range of those described in the
literature, which varied between 26% and 57% (wet weight
basis; Nakashio and Gerhardt 1985; Beaman and Gerhardt
1986). Since spore integument contains more water (59%
and 86% wet weight basis; Nakashio and Gerhardt 1985),
the wet density of the entire spore is higher than that of the
spore protoplast (Table 1). It is not clear how the presence
of divalent metal ions in the sporulation medium affects the
ability observed in B. coagulans ATCC 10545 to outgrow at
low pH values.
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1 SPORULATION CONDITIONS, HEAT RESISTANCE AND INJURY OF SPOILAGE BACILLI 767

Table 3 Survival of Bacillus subtilis AdHC1 spores prepared on nutient agar with a mix of metal ions, on recovery media containing citric acid (CA),
lactic acid (LA) and/or acetic acid (Ac)

Log survivors ml)1*

Unheated 20 min, 98 °Cà 40 min, 98 °C

Recovery medium  3 days 10 days 17 days 3 days 10 days 17 days 3 days 10 days 17 days

TSA 7á3à 7á3 7á3 6á4 6á5 6á5 4á25 4á4 4á4
0á5% CA 7á2 7á3 7á3 5á9 5á9 5á9 3á05 3á2 3á2
0á5% CA + Mg/Ca§ 7á1 7á3 7á3 6á2 6á2 6á2 3á5 3á5 3á5
0á5% LA 0á1 6á5 6á5 0á1 4á1 4á1 0á1 2á4 2á5
0á5% CA + 0á1% Ac 0á1 0á1 5á8 0á1 0á1 2á0 0á1 0á1 0á5
0á1% Ac 0á1 0á1 7á3 0á1 3á7 3á9 0á1 0á1 1á7

* Data are the mean of the log survivors from duplicate heat treatments in TSB at pH 4á5, after 3, 10, and 17 days of incubation at 37 °C, counted on
agar plates.
  The recovery media consisted of TSA or TSA supplemented with the organic acids indicated, at pH 4á5.
à Heat treatments were performed in TSB pH 4á5.
§ Concentrations of MgSO4.7H2O and CaCl2.2H2O were 0á05 g l)1 and 0á02 g l)1, respectively.

The composition of the sporulation medium did not affect
the sensitivity of heat-injured spores of the different Bacillus
strains to antimicrobials. Although longer heating times or
higher heating temperatures were necessary to cause injury
to the Bacillus spores prepared on nutrient agar with a mix
of metal ions, the sensitivities to NaCl or citric acid in the
recovery medium were similar to those of spores prepared
on nutrient agar with Mn2+ (Figs 2, 3; Table 3). The higher
the NaCl concentration in the recovery medium, the lower
the quantity of heat-injured B. subtilis A163 spores able to
outgrow (Fig. 2a). Consequently, it is possible to distinguish
Fig. 4 Effect of Ca-DPA on the recovery of injured Bacillus subtilis
AdHC1. Spore suspensions were heated in TSB, pH 5á9, at 114 °C and
plated on TSA pH 5á9 (h) and TSA + Ca-DPA ( ). Data are the
mean of plate counts of duplicate heat treatments; the variance is
shown. The spores from both species were prepared on nutrient agar
with a mix of metal ions (see Material and Methods)
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 761±770
uninjured, injured and more heavily injured Bacillus spores
when they are plated on recovery media with different NaCl
concentrations.
The inhibitory effect of NaCl (1±10%) in the recovery
medium on heat-injured spores was earlier shown for several
Clostridium and Bacillus strains, although sensitivity to NaCl
may differ between strains (Roberts and Ingram 1966;
Roberts et al. 1966; Hutton et al. 1991). Nevertheless, the
level (germination or outgrowth) at which spores are injured
by heat resulting in an increased sensitivity to NaCl is not
clear. Gould (1964) showed that NaCl concentrations of
4±7% were inhibitory to the outgrowth level, and concen-
trations of 10±15% to the germination level, of a number of
uninjured Bacillus spores. In this study, it was shown that on
a medium with 6% NaCl, the addition of KCl and proline
slightly increased (30±70%) the recovery of heat-injured
spores, whereas KCl and proline hardly affected spore
recovery from unheated cells (Table 2). It is known that
B. subtilis cells have a turgor-sensitive K+-uptake system
which triggers the synthesis of proline used as a compatible
solute (Whatmore et al. 1990; Whatmore and Reed 1990). It
may be that in heat-injured spores, osmosensing proteins, or
proteins involved in proline synthesis in the forespore
(membrane), have been injured. The addition of K+ or
proline to the recovery medium with NaCl would enable the
vegetative cell to restore the internal osmotic pressure. This
may imply that the heat treatment applied has partly injured
spores at the outgrowth level.
At pH 4á5, a distinction between heat-injured and unin-
jured spores of B. subtilis AdHC1 was made by the addition
of citric acid to the recovery medium (Fig. 3; Table 3); this
was also established for B. coagulans ATCC 10545 (not
shown). A higher concentration of citric acid in the recovery
medium did not allow discrimination between different

768 A . E . C A Z E M I E R E T A L .
degrees of heat injury (Fig. 3b). In contrast, in the case of
B. subtilis AdHC1 spores, the addition of lactic acid and
acetic acid to the recovery medium decreased the recovery of
injured spores compared with that on medium with citric
acid (Table 3).
The inhibitory effect of acetic acid and lactic acid is partly
related to the interaction with the cell membrane to
neutralize the electrochemical potential. Citric acid may
also disturb the cell membrane, but it also acts as a
sequestrant, scavenging divalent metal ions (Table 3; Gould
1989). As heat-injured B. subtilis AdHC1 spores showed an
increased sensitivity to lactic acid and acetic acid compared
with citric acid (Table 3), a discrimination between injured
and more heavily injured spores is possible in principle.
However, it is not certain whether a similar type of injury is
6 in¯icted by the different organic acids.
From the sensitivities of injured Bacillus spores to NaCl
and organic acids, little can be concluded about the level
at which spore injury had occurred. Ca2+-DPA has been
shown to act as a chemical germinant capable of triggering
germination by a mechanism different to nutrient-induced
(L-alanine) germination (Paidhungat and Setlow 2000).
Since the addition of Ca2+-DPA to the recovery medium
caused a slight increase in the recovery of injured B. subtilis
A163 and B. subtilis AdHC1 spores (Fig. 4a,b), it is possible
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 761±770
13652672, 2001, 5, Downloaded from https://ami-journals.onlinelibrary.wiley.com/doi/10.1046/j.1365-2672.2001.01302.x by Nat Prov Indonesia, Wiley Online Library on [22/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Fig. 5 Sensitivity to lysozyme of heat-
injured Bacillus subtilis A163 (a) and B. subtilis
AdHC1 (b) spores. Bacillus subtilis A163
spores were heated at 98 °C and 114 °C in
TSB, pH 5á9, and plated on TSA pH 5á9
(h), TSA + 10 lg ml±1 ( ) and
TSA + 100 lg ml±1 (j). Bacillus subtilis
AdHC1 spores were heated at 96 °C in TSB,
pH 4á5, and plated on TSA pH 4á5 (h),
TSA + 10 lg ml±1 ( ) and TSA + 100 lg
ml±1 (j). Data are the mean of plate counts
of duplicate heat treatments; the variance
is shown. Spores from both strains were
prepared on nutrient agar + 3 ppm
MnSO4.H2O
that (receptor) proteins of the nutrient-induced germination
pathway had been inactivated by heat. Likewise, germina-
tion-speci®c lytic enzymes (GSLEs) may have been inacti-
vated by a heat treatment, the activities of which could be
replaced by lysozyme (Rasmussen and Labbe 1996; Faille
et al. 1997). However, lysozyme reduced the recovery of
heat-injured B. subtilis A163 and B. subtilis AdHC1 spores,
indicating that the spore coat had become permeable to the
enzyme by heat, but that it did not allow germination and
outgrowth of spores (Fig. 5).
In conclusion, it has been shown that the presence of
multiple metal ions in the medium during sporulation
considerably increased heat resistance of the Bacillus spores
tested. Since it is assumed that in their `natural' environ-
ment Bacillus spores are formed in the presence of multiple
metal ions, they should be prepared in the laboratory under
similar conditions to assess new preservation technologies
properly.
For the determination of the amount of uninjured, injured
and heavily injured spores of spoilage bacilli (B. subtilis A163
and two B. licheniformis strains) after a heat treatment at
moderate pH, the use of recovery media with different
concentrations of NaCl appeared to be effective. At pH 4á5,
recovery media containing different organic acids (citric,
lactic and acetic acid) may allow distinction between
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1 SPORULATION CONDITIONS, HEAT RESISTANCE AND INJURY OF SPOILAGE BACILLI 769

uninjured, injured and heavily injured spores of acid- Gould, G.W. ed. (1989) Mechanisms of Action of Food Preservation
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ATCC 10545). However it is not known whether a similar Gould, G.W. and Hitchins, A.D. (1963) Sensitisation of bacterial
7 type of injury is in¯icted by the different organic acids. With spores to lysozyme and to hydrogen peroxide with agents which
rupture disulphide bonds. Journal of General Microbiology 24,
the recovery media developed here, the extent of injury to
413±423.
spores of spoilage bacilli caused by new preservation
Hutton, M.T., Koskinen, M.A. and Hanlin, J.H. (1991) Interacting
technologies may be established. effects of pH and NaCl on heat resistance of bacterial spores. Journal
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accurate technique for the determination of the wet heat resistance of
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thank S.J.C.M. Oomes and A.M. van Duijvendijk for Leistner, L. (2000) Basic aspects of food preservation by hurdle
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