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THERMAL RESISTANCE OF BACILLUS CEREUS SPORES AS

AFFECTED BY ADDITIVES IN THE RECOVERY MEDIUM

ISAAC GONZALEZI,MERCEDES LOPEZ~,MARGARITAW A S , JOSEFA


G O N Z k E Z I and ANA BERNARD01.3

'Dpto. de ffigiene y Tecnoiogia de Ios Alimentos


Universidad de Ledn, Spain

2Area Tecnologia de 10s Alimentos


Universidad de Vigo, Spain

Received for Publication November 25, 1996


Accepted for Publication January 22, 1997

ABSTRACT

The effects of the addition of starch, glucose, sodium chloride, sodium citrate,
monopotassium phosphate and disodium phosphate to the recovery medium on
apparent heat resistance of Bacillus cereus spores (ATCC 4342, 7004 and 9818)
were investigated. Sodium citrate, monopotassium and disodiurn phosphate at
concentrations of 0. I% were efective inhibitory agentsfor heat injured B. cereus
spores especially for strain 9818, although only monopotassium and disodium
phosphate caused a signifcant reduction ($0.05) in D-values obtainedfor strain
9818. Sodium chloride also had a marked efect on the recovery of heat injured
spores. Concentration as low as 0.5% caused a significant reduction in the
recovery ratesfor strains 9818 and 7004. In all cases, increasing the salt levels
from 0.5 to 4% resulted in a progressive decrease in spore recovery. D-values
gradually decreased as the salt content increased, although the concentrations
which produced statistically significant differences (p<0.05) varied among strains.
The addition of starch at 0.I % resulted in a significant increase in the countsfor
strains 9818 and 7004. In contrast, glucose (0. I %), did not significantly mod& the
counts obtained. Neither of these compound aflected decimal reduction times. No
statistical signlficance (PO. 05) diferences were detected among z-values for the
spores of the three strains recovered in the presence of dflerent additives assayed.
z- Values rangedfiom 6.67 to 8.32, with a mean value of 7.56 f 0.46C.

'Correspondence to: Dr. h a Bemardo, Dpto. de Higiene y TecnologIa de 10s Alimentos, Facultad de
Veterinaria, Universidad de Le6n, Campus de Vegazanash, 24071-Le611, Spain. Phone: 34 87 291 182;
Fax: 34 87 291284.

Journal of Food Safety 17 (1997) 1-12. AN Rights Reserved.


OCopyright 1997 by Food d; Nutrition Press, Inc., Trumbull, CT06611 1
2 I . GONZALEZ ETAL.

INTRODUCTION

Bacillus cereus causes food-borne illness and is frequently isolated from


pasteurized dairy products. It is also occasionally isolated from processed foods
(Franklin 1970; Bradshaw et al. 1975; Mostert et al. 1979). Despite the important
role of B. cereus in heat processed foods, data on its thermal resistance
characteristics are very scarce, Furthermore, the influence of environmental factors
on its heat resistance is practically unknown. Our group studied the effects of some
recovery conditions on parameters determining thermal resistance of this organism
such as recovery medium and temperature (Gonziilez et al. 1995) and pH of the
recovery medium ( G o d l e z et ul. 1996). No information is available on the effects
of components normally present in foods (starch, glucose, sodium chloride) and
certain stabilizing additives (phosphates and citrates) could have on the behavior
of heated B. cereus spores. These compounds are used extensively in the food
industry and may affect the recovery of surviving heat damaged spores.
For other sporeformersthere is sufficient information on the beneficial effect of
starch in recovery media on the recovery of heated spores. Some authors have
reported that heat resistance is greater in media containing starch (Cook and Gilbert
1968; Mallidis and Scholefield 1986). Thus, this compound may promote recovery
of thermally injured B. cereus spores.
It is also well established that the presence of sodium chloride in the recovery
medium influences heat resistance of spores. However, the effect exerted by salt
seems to vary among the species. Thus, while for B. stearothermophilus and
Clostridium sporogenes, most authors agree that as the concentration of salt
increases both the recovery rates and the D-values drop (Jarvis et al. 1976; Labbe
1979; Chumey and Adams 1980; Feeherry et al. 1987; Hutton et ul. 1991; L6pez
et al. 1994); for B. subtilis and B. pumilus spores, the recovery is better when the
medium contained this ingredient (Briggs and Yazdany 1970).
The aim of this work was to determine the influence of several compounds added
to the recovery medium (starch, glucose, sodium chloride, phosphates and citrates)
upon recoverability and D and z-values of three B. cereus strains.

MATERIALS AND METHODS

Microorganisms and Spore Preparation


Three strains of B. cereus (ATCC 4342,7004 and 98 18) were used in this study.
Spores were produced in nutrient agar (NA, Difco, Detroit, MI) supplemented with
1 ppm Mn" at 32C as described by Gondlez et ul. (1 995). Stock suspensions were
stored in McIlvaine buffer -citric aciddisodium hydrogen phosphate- (Mckenzie
and Dawson 1969) pH 7.0 at 0-5C for hrther use.
HEAT RESISTANCE OF B. CEREUS 3

Thermal Treatments
Thermal treatments were carried out in a thermoresistometer TR-SC (Condbn et
a/. 1989)over a wide range of temperatures (92-1 14C). Buffer McIlvaine (400 mL)
pH 7.0 was used as a heating menstruum and was inoculated with 0.2-1 mL of
spore suspensions of each strain. Intervals between viability determinations ranged
from 0.5 s to 2 min, depending on the heat resistance of the strain and treatment
temperature.

Recovery Conditions
Aliquots of 0.1-0.5 mL of the heated spore suspensions were sampled at different
treatment times and were directly placed on petri dishes poured with nutrient agar.
Parallel counts were also made on NA containing 0.1% (w/v) of glucose (Difco,
Detroit, MI), starch (Difco, Detroit, MI), monopotassium phosphate (Panreac,
Montplet & Esteban SA, Barcelona, Spain), disodium phosphate (Panreac,
Montplet & Esteban SA, Barcelona, Spain) or sodium citrate (Panreac, Montplet
& Esteban SA, Barcelona, Spain). The media in experiments performed with
monopotassium phosphate and disodium phosphate were adjusted to pH 6.8 with
1N NaOH (Panreac, Montplet & Esteban SA, Barcelona, Spain) and 1N HCI
(Panreac, Montplet & Esteban SA, Barcelona, Spain), respectively.
Finally, sodium chloride was tested by adding to the plating medium
concentrations of 0.5, 1,2,3 and 4% (wh) of NaCl (Panreac, Montplet & Esteban
SA, Barcelona, Spain).
Plates were incubated at 30C for 20-24 h, but plates containing sodium chloride
were incubated for up to 3 days. Longer incubation time did not result in a
significant increase in the number of CFU/plate.
Colonies of strains ATCC 4342 and 7004 were counted using the method
described by Cond6n et ul. (1987). Strain 9818, due to its overspread, required
suitable dilutions to determine the counts. Spore level recovery was determined as
described by Gonzhlez et ul. (1 995).

D and Z-values
D-values were calculated as the negative reciprocals of the slopes of the
regression lines plotted with the straight portion values of the survival curves (log
population versus time). Strain 7004 exhibited tails at all temperatures tested.
Decimal reduction times were obtained from the first portion of their survival
curves. D-values were determined in triplicate at 1OOC. Student’s “t” test (Steel and
Torrie 1960) was used to check the significance on the slope value. z-Values were
determined as the negative reciprocals of the lines of regression of thermal death
time curves (log D-values versus temperature). In order to compare z-values, the
homogeneity test was used, calculated as described by Steel and Torrie (1960).
4 I. GONZALEZ ETAL

RESULTS AND DISCUSSION

Effect of Starch and Glucose on Heat Resistance


The results presented in Table 1 show that the three strains tested showed
increase in recovery percentages when starch was added to the recovery medium,
although with strain 4342, it was not statistically significant (p0.05). The addition
of glucose did not significantly modify the recovery efficiency obtained with any
of the strains studied.
'The average D,, values and z-values obtained in different assayed conditions are
summarized in Table 2. Neither decimal reduction times nor z-values were affected
by the addition of these compounds to the recovery medium (p>0.05).
The positive effect of starch on the recovery has already been reported for other
sporeformers, such as Clostridium botulinum (Olsen and Scott 1950; Sugiyama
1951) and Bacillus steurothermophilus (Cook and Gilbert 1968; Labbe 1979;

TABLE 1.
EFFECT OF STARCH AND GLUCOSE ON THE RECOVERY* OF BACILLUS CEREUS
SPORES

Experimental Condition Recovery (%)* on


Strain H.T. Time (min) NA NA+Starch' NA+Glucose'

4342 IOOC 1.66 1oo.oi 9.2" 107.8* 13.6" l04.1* 9.65'

2.66 lOO.oi12.4" 129.8i18.6" 116.2*15.6"

3.66 100.oil2.9" 134.7*21.4" I09.7*13.6"

7004 96C 0.33 100.M13,6ab 132.7*17.9" 72.4* 13 .7b

0.66 1oo.oi 9.2" 15 1.5i16.9b 91.3* 7.3"

I .oo 100.oi16.8" 158.8*27.9b 84.1*12.9"

9818 104C 1.66 100.M 7.1" 139.2*17.6b 125.9*18.4"b

2.66 lOO.O* 7.9" 142.6*24.6b 116.6*17.0°b

3.66 IOO.Ok 6.8" 172.9*36.gb l10.7*12.4"

NA:nutrient agar.
I Starch and glucose were added at 0.1% (w/v).
H.T. Heating time
Mean of three experiments f C.V.
Values in the same row which have the same superscript were not significantly different at 5% level.

CFU per plate N A o r N A + S o r N A + G )


'Expressed as: plating efficiency = x 100
CFU per plate (NA)
HEAT RESISTANCE OF B. CEREUS 5

Mallidis and Scholefield 1986), although some authors (Cook and Gilbert 1968;
Mallidis and Scholefield 1986) found that this compound exerted considerably
greater stimulatory effect and that such protection caused an increase in D-values
obtained. There seems to be no information published on the effect of glucose into
the plating medium on heat resistance of B. cereus spores. For other Bacillus
species, authors disagree on the influence of this compound. Although Richardson
(1965) reported that the addition of glucose had a protective effect on B. subtilis,
others (Zechman and Pflug 1991 for B. stearothermophilus) described that the
recovery was less effective using a medium which contained glucose. These
authors proposed as a possible explanation the formation of inhibitory brown
compounds or intermediary reducing compounds during medium preparation and
sterilization due to the presence of glucose and phosphate. In our experimental
conditions and although the medium was darker after sterilization, it was free of
potassium phosphate, which exerts an inhibitory effect on recovery of B. cereus
(see below). Recently, L6pez (personal communication) observed a similar effect
on B. stearothermophilus.

TABLE 2.
EFFECT OF STARCH AND GLUCOSE IN THE RECOVERY MEDIUM ON THE APPARENT
HEAT RESISTANCE OF BACILLUS CEREUS SPORES
D,, (min) AND z-VALUES (C)
Thermal resistance parameters obtained on

NA NA+Starch' NA+Glucose'

Strain D-value* z-value D-value* z-value D-value* z-value


4342 1.26iO.15" 7.77 1.45k0.05" 8.14 1.43*0.15" 8.11

7004 0.29*0.03 Ib 7.75 0.31i0.024b 7.80 0.28*0.034b 7.88

9818 4.52*0.36' 7.39 4.99i0.50" 7.61 4.76k0.49" 7.43

NA: nutrient agar.


I Starch and glucose were added at 0.1% (w/v).

* Mean of three experiments f S.D.


""Values obtained for each strain with the same superscript were not significantly different at 5% level.

Effect of Sodium Chloride on Heat Resistance


Figure 1 shows one of the survival curves obtained for B. cereus strains using as
plating medium nutrient agar with different NaCl concentrations. These curves are
representative examples of those obtained in all the range of temperatures studied.
The presence of NaCl in the recovery medium had a marked effect on the thermal
6 I . GONZALEZ ETAL.

characteristicsof B. cereus spores. While for unheated spores, salt concentrations


of 3% for strain 4342, 4% for 7004 and 6% for 9818 were required to cause
significant differences in counts (data not shown), for heated spores, salt
concentrations as low as 0.5% caused a significant reduction in the recovery
efficiency (see Fig. 1). As the salt concentration in the medium rose, the
recoverability efficiency progressively dropped for all strains tested. The tail
population of 7004 strain was also affected by the level of salt in the recovery
medium, but this second most resistant fraction did not disappear even at a
concentration as high as 3% (w/v).
Table 3 shows the D,, values average obtained for the different strains at the
previously cited conditions. The salt content in the recovery medium also
influenced D-values obtained which progressively decreased as the NaCl
concentration increased. The extent of this reduction and the salt concentrations in
which statistically differences (p<0.05) were obtained varied among the strains.
Thus, D-values for 7004 strain were reduced at concentrations as low as 0.5%
while for the other two strains, at least 2% salt was necessary to have significant
effect on decimal reduction times. However, at higher levels (3%), the drop
observed was much more marked for strain 9818.
Figure 2 shows the thermal death time curves obtained when sodium chloride
was added to the recovery medium. The corresponding statistical analysis showed
that in no case were the z-values affected (p>0.05).
These fmdings show that in order to improve the effectiveness of media used
for detection and enumeration of B. cereus in heat processed food, it would be wise
to bear in mind the inhibitory effect caused by sodium chloride on the recovery of
surviving heat damaged spores. This is of particular importance given that the most
of media used for the isolation and identification of B. cereus contain salt in fairly
high concentrations. This could have a considerable inhibitory effect. On the other
hand, in the practical canning operation it is common to use salt and taking into
account its inhibitory effect, it could be expected that this compound plays a critical
role in the germination and/or outgrowth of B. cereus, regardless of the temperature
of treatment.

Effect of Different Stabilizing Additives on Heat Resistance


Table 4 shows the recovery percentages obtained for each strain at different
treatment times after the addition of sodium citrate, monopotassium phosphate and
disodium phosphate. None of these compounds had any significant effect (p>0.05)
in the counts of unheated spores. After heating, the addition of these compounds
resulted in a drop in the recovery efficiency of the three strains, being more
pronounced for the more heat-tolerant strain (98 18 ATCC) when disodium
phosphate and monopotassium phosphate were used giving way to significant
lower D-values (Table 5). z-Values obtained in medium containing these additives
were 7.82 f 0.26C for strain 4342, 8.10 f 0.18C for strain 7004 and 6.95 f 0.05C
for strain 98 18.
HEAT RESISTANCE OF B. CEREUS 7
J 51

ATCC 7oW
4

-_
s 7 j
z
P
y 3.
3
co
2.5

FIG. 1 . SURVIVAL CURVES FOR BACILLUS CEREUS SPORES RECOVERED IN NUTRlENT


AGAR WITH DIFFERENT SODIUM CHLORIDE LEVELS: 0%(x), 0.5% (A), 1% (0).2% (o),
3% (B) AND 4% (+)
8 I. WNZALEZ ETAL

TABLE 3.
EFFECT OF SODIUM CHLORIDE IN THE RECOVERY MEDIUM ON THE APPARENT
HEAT RESISTANCE OF BACILLffSC E E U S SPORES
NaCl D,,* (min)
~

concentration (%) strain 4342 strain 7004 strain 981 8


0 1.26i0.15' 0.29i0.03 1" 4.52i0.36"
0.5 1.12i0.10' 0.16*0.03k 4.09i0.40"
1.o 1.04i0.09" 0.13i0.008' 3.95*0.34"
2.0 0.68*0.06b N.T. 1.15io.l o b
3.0 0.24i0.02" 0.056*0.002d 0.48*0.05"
4.0 0. 15*0.005d N.T. 0.19=t0.01d
' Mean of three experiments * S.D.
N.T.No tested.
'dValues obtained for each strain with the same superscript were not significantly different at 5% level.

TABLE 4.
EFFECT OF DIFFERENT STABILIZING ADDITIVES ON THE RECOVERY' OF BACILLUS CEREUS SPORES
Strain H.T. Time (rnin) NA NA+C NA+PK NA+PNa
4342 lOOC 0 100.h13.6' 91.3* 7.6" 89.6i11.6n 94.1*15.7"
0.66 100.O.t 9.8" 82.7*12.6"b 73.3*1 1.2h 75.65*10.9b
1.33 100.O.t10.2" 84.9.t 8.Qb 73.4* 7Sb 72.95* 8.8b
2.0 1 O O . h 7.9' 68.5i~12.7~ 62.4* 5.6b 57.7* 6.Sb
7004 96C 0 1OO.o-k 9.4' 108.o-kl3.3" 101.3i 9.6' 105.5i13.6'
0.33 lOO.o-kl4.2" 8 4 3 7Snb 77.7i 8.4b 74.8i 8.6b
0.66 1OO.o-kl1.4" 80.7*13.6°b 75.7*10.2b 76.6* 9.4b
1 .o 1OO.o-k 9.1" 76.3* 6.9b 79.9i 8.9b 77.0i10.0b
9818 104C 0 1O0.o-k16.l0 81.o-k 7.9' 92.7i12.6" 97.5* 9.8"
1 .o 100.0.tlS.2° 8 0 . 8 i 7.6" 45.7* 5.6b 48.5* 4.1b
1.66 1OO.O.t 8.9" 62.1i 5.4b 21.4i 2.0" 28.1* 3.1d
2.33 1O0.O.t13.Oa 54.2* 4.8b 14.7* 1.7" 20.7*2.1d
3.0 1OO.o-k 4.9" 53.3i 7.1b 9 . 2 1.0" 16.7k 1.9d
NA:nutrient agar; NA+C: nutrient agar supplemented with 0.1% of sodium citrate; NA+PK: nutrient
agar supplemented with 0.1% of monopotassium phosphate; NA+PNa: nutrient agar supplemented with
0.1% of disodium phosphate.
H.T.: Heating time
Mean of three experiments i C.V.
"Values with the same superscript within rows and strain were not significantly different at 5% level.
* Expressed as:
CFU per PIate ( N A ~ ~ N A ~ c ~ ~ N A ~ P K ~ ~ N A + P N . )
plating efficiency = XI00
CFU per plate (NA)
HEAT RESISTANCE OF B. CEREUS 9
'1
ATCC 4342

7.74
-1.5 7.88

.5
.2s
0.....
5.25.
- -.s
0

-.75,
-1.

~I .25
-1.5
-1.75'
92 94 96 98 100 102 104 106 108
Temperature (T)

1.251

ATCC 9818

- ".,,
98 100 102 104 106 10R 110 llZ 114 116
Temperature (T)

FIG. 2. THERMAL DEATH TIME CURVES FOR BACILLUS CEREUS SPORES RECOVERED
IN NUTRIENT AGAR WITH DIFFERENT SODIUM CHLORIDE LEVELS: 0% (x), 0.5% (A),
1% ( O ) , 2% (m), 3% (m) AND 4% (+)
10 I . GONZALEZETAL

TABLE 5.
EFFECT OF DIFFERENT STABILIZING ADDITIVES IN THE RECOVERY MEDIUM ON THE
APPARENT HEAT RESISTANCE OF 5AClLLUS CEREUS SPORES

Dim* (min)
Strain NA NA+C NA+PK NA+PNa
4342 1.26*0.15" 1.19*0.11" I .07*0.06' I. 18*0.07'
7004 0.2%0.031" 0.32*0.043" 0.34*0.058' 0.3W0.040'

9818 4.52*0.36" 5.34*0.56" 3.79i0.29b 3.9W0.23b

- ..
NAmutrient agar: NA+C: nutrient agar suaalemented
I ,
with 0.1%of sodium citrate: NA+PK: nutrient
agar supplemented with 0. I% of monopotassium phosphate; NA+PNa: nutrient agar supplemented with
0.1%of disodium phosphate.
*
* Mean of three experiments S.D.
a-bValuesobtained for each strain with the same superscript were not significantly different at 5% level.

The heat resistance of different B. cereus spores showed a considerable range.


In this study we found that the D,,, values obtained in standard conditions ranged
from 0.29 min for ATCC 7004 to 4.52 min for ATCC 9818. From our data and
others (Burgos et al. 1972; Johnson et al. 1982; Wong et al. 1988; Dufrenne et al.
1994) it could be concluded that thermal sterilizationtreatments would be sufficient
to guarantee a good degree of safety with regard to the B. cereus in sterilized food.
Furthermore, results obtained here and the data previously published by our group
(Gonzblez et al. 1995; Gonzhlez et al. 1996) showed that heat resistance of B.
cereus is influenced by recovery conditions, especially medium pH and presence
of salt. We have found that D-values for the strain that exhibits the higher heat
resistance (ATCC 98 18) decreased 4-fold when the medium was acidified up to pH
5.4 (Gonzhlez et al. 1996) and about 25-fold in presence of 4% of sodium chloride.
This effect could give an additional security margin in certain products. However,
one of the strains tested (ATCC 7004) exhibits subpopulations with greater heat
resistance. The existence of tails has been reported for others strains of B. cereus
(Johnson et al. 1982; Rajkowski and Mikolajcik 1987). Although these tails
represented a minority lkaction of the population, they were present in all recovery
conditions studied.

ACKNOWLEDGMENTS

This work was supported by CICYT, project ALI 90-0555 and by a grant to I.G.
from the Gobiemo Vasco.
HEAT RESISTANCE OF B. CEREUS 11

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