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Contamination flows of Bacillus cereus and spore-forming aerobic

bacteria in a cooked, pasteurized and chilled zucchini


puree processing line
M.H. Guinebretiere
*
, H. Girardin, C. Dargaignaratz, F. Carlin, C. Nguyen-The
Institut National de la Recherche Agronomique, UMR A408 Securite et Qualite des Produits dOrigine Vegetale, INRA,
Domaine Saint-Paul, Site Agroparc, F-84914 Avignon Cedex 9, France
Received 25 August 2001; received in revised form 10 May 2002; accepted 26 June 2002
Abstract
A food processing plant producing pasteurized purees and its zucchini puree processing line were examined for
contamination with aerobic and facultative anaerobic bacterial spores during a days operation. Multiplication of spores was
also monitored in the product stored under different conditions. High concentrations of Bacillus cereus spores were found in the
soil in which the zucchinis were grown (4.6 F0.3 log CFU/g), with a background spore population of 6.1 F0.2 log CFU/g. In
the processing plant, no B. cereus or psychrotrophic bacterial spores were detected on equipment. B. cereus and psychrotrophic
bacterial spores were detected after enrichment in all samples of raw zucchinis, washed zucchinis, of two ingredients (starch and
milk proteins) and in processed puree at each processing step. Steam cooking of raw zucchinis and pasteurization of puree in the
final package significantly reduced spore numbers to 0.5 F0.3 log CFU/g in the processed food. During storage, numbers of
spore-forming bacteria increased up to 7.8 F0.1 log CFU/g in puree after 5 days at 2025 jC, 7.5 F0.3 log CFU/g after 21
days at 10 jC and 3.8 F1.1 log CFU/g after 21 days at 4 jC. B. cereus counts reached 6.4 F0.5 log CFU/g at 2025 jC,
4.6 F1.9 log CFU/g at 10 jC, and remained below the detection threshold (1.7 log CFU/g) at 4 jC. Our findings indicate that
raw vegetables and texturing agents such as milk proteins and starch, in spite of their low levels of contamination with bacterial
spores and the heat treatments they undergo, may significantly contribute to the final contamination of cooked chilled foods.
This contamination resulted in growth of B. cereus and psychrotrophic bacterial spores during storage of vegetable puree. Ways
to eliminate such contamination in the processing line are discussed.
D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Bacillus cereus; Bacterial spores; Cooked chilled foods; Vegetables; Processing line
1. Introduction
Cooked chilled foods supply a rapidly expanding
market in Europe. Consumption of chilled prepared
meals increased by 10% to 37% according to the
product in 1997, while at the same time frozen
prepared meals fell by 4% (Hauben, 1999). The main
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S0168- 1605( 02) 00307- 0
* Corresponding author. Tel.: +33-4-32-72-25-24; fax: +33-4-
32-72-24-92.
E-mail address: guinebre@avignon.inra.fr.
(M.H. Guinebretiere).
www.elsevier.com/locate/ijfoodmicro
International Journal of Food Microbiology 82 (2003) 223232
objective of processors of cooked chilled foods is to
offer a ready-to-use product with optimized sensory
qualities. A large proportion of cooked chilled foods
are pasteurized in their final packaging to eliminate
hazards from vegetative pathogenic bacteria. How-
ever, this pasteurization does not kill bacterial spores,
which can survive the process and develop during
storage before consumption. The development of
cooked chilled foods may thus result in an increased
exposure of consumers to pathogenic spore-forming
bacteria.
The spore-forming bacterium Bacillus cereus has
been incriminated in many cases of food poisoning
(Granum, 1997). Recently, vegetable purees were
the cause of a severe B. cereus outbreak in a French
nursing home for elderly persons (Lund et al., 2000;
De Buyser, personal communication). B. cereus is a
ubiquitous bacterium found in soil and in many raw
and processed foods such as rice, milk and dairy
products, spices, and vegetables (Roberts et al.,
1982), meat products and farinaceous foods (Kramer
and Gilbert, 1989). Its ubiquitous nature and the
heat resistance of its endospores, associated with
psychrotrophic properties, make it a potential con-
taminant of cooked chilled foods. B. cereus was
isolated from 80% to 100% of samples of cooked
pasteurized and chilled vegetable purees of leek,
zucchini, broccoli, split pea, carrot and potato
purees stored at 10 jC (Carlin et al., 2000). Among
these vegetable purees, zucchini puree supported the
most rapid bacterial growth and B. cereus was
significantly present in products stored at 10 jC
(3.6 to 5.2 log CFU per gram) at use-by date. The
zucchini puree thus called for a more thorough
investigation. It received a mild heat treatment
(pasteurization in final packaging) that cannot be
increased to levels sufficient to kill B. cereus spores
and psychrotrophic spores. Therefore, a knowledge
of the flows of contamination through the process-
ing line should help identify critical steps in the
process for B. cereus and psychrotrophic contami-
nation, and so help improve safety by reducing or
eliminating contamination at source.
The objective of the present study was to deter-
mine the possible sources of contamination for
zucchini purees and the impact of processing on
this contamination, with special reference to B.
cereus and psychrotrophic spores. For this purpose,
we monitored the flows of contamination during the
production of one batch of cooked and pasteurized
zucchini purees from their hypothetical sources to
the final stored product. Possible primary sources of
contamination were the zucchinis used for process-
ing, the soil where the zucchinis were grown and
the ingredients (flavouring and texturing agents)
blended with the puree. Possible secondary sources
of contamination were the surfaces of equipment
and air in contact with the product during process-
ing. B. cereus and psychrotrophic spore-forming
bacteria were tested for all these possible contami-
nation sources, in the product at each stage of
processing, and in the puree packs stored until
consumption. Only spores were enumerated or de-
tected along the processing line, because pasteuriza-
tion eliminates vegetative cells in the final pack-
aging of the product.
2. Materials and methods
2.1. Cooked pasteurized and chilled vegetable puree
Zucchini puree was manufactured in a processing
plant located in Normandy, France. Raw zucchinis
were harvested in a field in the same area. The raw
zucchinis (300 kg) were washed, cut, steam cooked
for a few minutes, ground, and mixed with 44 kg of
ingredients (UHT milk cream, milk proteins, starch
and salt) (Fig. 1). The resulting puree was dispensed
into preformed polypropylene trays in 400-g units
using an industrial liquid food dispenser, then vac-
uumed and covered with a 90-Am thick polypropy-
lene-polyamide film sealed on the tray. The packed
zucchini puree was then pasteurized in a water bath
to achieve a core temperature of 80 jC during 10
min. The equipment used to process the cooked
zucchinis comprised a grinder, a blender, the distri-
bution system previously described and three con-
tainers for the transport of food materials between
the different items of equipment. There was a phys-
ical separation between the reception areas (raw
vegetable reception area, ingredient storage area)
and the puree production area (vegetable washing
room, production room, pasteurization room, cold
storage room for final products). Six hundred and
sixty-eight puree packs of 400 g were produced
M.H. Guinebretiere et al. / International Journal of Food Microbiology 82 (2003) 223232 224
during our study, corresponding to one batch of
zucchini puree.
2.2. Sampling strategy
Sampling was performed on: cultivation soil of
zucchinis, raw zucchinis, washed zucchinis, processed
zucchinis (cooked and ground zucchinis, cooked/
ground zucchinis mixed with ingredients, zucchini
purees pasteurized in their final package), the ingre-
dients (UHT cream, starch, milk proteins, salt), the tap
water used to dilute ingredients, the surfaces of equip-
ment in contact with the product, and production room
air. Packs of puree were also analyzed after storage at
the temperature recommended in France for cooked
chilled foods (4 jC), and at 10 and 2025 jC,
representing defective storage temperatures that may
occur in consumer refrigerators and when the cold
chain is broken.
All these processing points were studied over 1 day
of production. The production involved large volumes
of foods ( f280 kg) and large equipment surfaces.
We expected low levels of B. cereus spores on
samples during processing from the results of Carlin
et al. (2000) and Choma et al. (2000). We therefore
chose to analyze a large volume of samples, covering
approximately 10% of the food used for processing
and 50% of the equipment surfaces. This approach
reduced the accuracy of counts but allowed a better
detection of B. cereus.
2.3. Sampling procedure for soil
Soil samples were taken from two different field
plots, on three rows for each plot. Dry soil was
randomly sampled with sterile spoons (stainless steel
spoons purchased from the local supermarket) on the
plastic lining covering the cultivated soil and around
the zucchini stems from a given row. Six samples of
500 g, one per row, were collected in sterile stomacher
bags (Laboratoire Humeau, La Chapelle-sur-Erdre,
France). The bags were closed hermetically with clips
(Laboratoire Humeau, La Chapelle-sur-Erdre).
2.4. Sampling procedure for equipment, wash water
and air
Samples from equipment surfaces were collected
immediately before the start of processing to esti-
mate the potential transfer of contamination to the
zucchini puree. Ten pieces of equipment were
sampled. For each piece of equipment, one half of
Fig. 1. Flow diagram of the processing line producing cooked, pasteurized and chilled zucchini puree.
M.H. Guinebretiere et al. / International Journal of Food Microbiology 82 (2003) 223232 225
the surface was swabbed with a sterile cotton swab;
these were ready-to-use chiffonnette ATL (40 40
cm), impregnated with a sterile buffer containing
neutralizers against disinfectant solutions (Labora-
toire Humeau, La Chapelle-sur-Erdre) and were used
as recommended by the manufacturer. The surfaces
studied were the grinder screw (2800 cm
2
), the
grinder inner surface (14,600 cm
2
), the inner surface
of the three stainless steel transport containers
(18,000 cm
2
each), the blender grid (4800 cm
2
),
the blender inner surface (22,500 cm
2
), the rotating
mixing blade (8700 cm
2
) and the outlet pipe of the
distribution system.
Two 70-ml samples of the water used to clean the
distribution pipe were collected in sterile vials (Labo-
ratoire Humeau), one before and one at the end of
puree distribution.
Viable airborne B. cereus spores were determined
by the sedimentation method. Nineteen open Petri
dishes (90 mm in diameter) containing B. cereus
selective medium MYP (van Netten and Kramer,
1992) were left open near equipment for 30 min
during grinding and blending operations. The dishes
were then sealed with ParafilmR and incubated at 28
jC for 48 h.
2.5. Sampling procedure for zucchinis, processed
zucchinis, and ingredients
Six raw zucchinis per box were randomly selected
to obtain a total of 120 zucchinis ( f30 kg of
zucchinis). One slice ( f25 g) was cut from each
zucchini with a sterile scalpel and collected in a
stomacher bag. One sample unit was composed of
24 slices ( f600 g), a total of five samples was
collected. The same sampling procedure was used
for washed zucchinis. This sampling strategy allowed
the survey of approximately 10% of the raw and
washed zucchinis used for processing.
For processed zucchinis, samples of cooked/
ground zucchinis ( f1 kg each) were collected
directly in stomacher bags at the outlet of the grinder.
Five samples were collected at regular intervals from
the start to the end of the grinding operation. Five
samples of processed zucchinis mixed with ingre-
dients ( f1 kg each) were collected in stomacher
bags with a sterile ladle after the transfer of the food
into the transport container. Forty packs of processed
purees (400-g unit) were randomly selected at the end
of the processing line.
For ingredients, a total of approximately 800 g of
milk proteins, 600 g of starch, and 150 g of salt were
collected with sterile spoons in stomacher bags (Table
1). Three liters of UHT cream were also collected
(Table 1).
Additionally, two 70-ml samples of tap water used
to dilute dehydrated ingredients were collected in
sterile vials.
2.6. Sample maintenance and transport
In the processing plant, hydrated samples (zuc-
chinis, processed zucchinis, liquid samples, surface
samples) were immediately cooled and kept in melt-
ing ice until the end of the production. All the
samples were maintained in a cold room at 4 jC
overnight, and at 2 jC during a 24-h transport step
and in the laboratory until they were analyzed.
During analysis, samples were maintained in melting
ice.
Processed zucchinis (cooked/ground zucchinis,
cooked zucchinis mixed with ingredients and packs
of nonstored pasteurized purees) were analyzed on
arrival at the laboratory. Some packs of pasteurized
puree were incubated at 4 jCF0.0, 10 jCF0.5 for
21 days and at ambient temperature (2025 jC) for 5
days. The incubation time at low temperature (4 and
10 jC) corresponded to the usual shelf life applicable
to cooked chilled foods in France; the incubation
time at 2025 jC was the time at which spoilage
was first visible. Ten packs were stored in each storage
condition.
Raw and washed zucchinis, liquid samples and
surface samples were analyzed within 24 h after their
arrival in the laboratory. Soil samples and dehydrated
ingredients were analyzed within 2 weeks after their
arrival in the laboratory.
2.7. Microbial analyses
Spores were enumerated on J-agar (Claus and
Berkeley, 1986), and B. cereus spores on MYP agar
(van Netten and Kramer, 1992). B. cereus spores were
detected after enrichment of samples at 28 jC for 24 h
in both modified MYP broth (MYP broth without egg
yolk and mannitol) and Claus medium (Claus and
M.H. Guinebretiere et al. / International Journal of Food Microbiology 82 (2003) 223232 226
Berkeley, 1986). Psychrotrophic bacterial spores were
detected after enrichment in J-broth (Claus and Ber-
keley, 1986) at 10 and 4 jC for 4 and 14 days,
respectively.
In a preliminary step, samples of zucchinis, soil
and cotton swabs were homogenized for 2 min as
described in Table 1, using, respectively, a rotating
blender (Kinematica, Luzern, Switzerland), a conical
flask containing a sterile magnetic rod and a Stom-
acher (Seward Medical, London, UK).
For soil, raw and processed zucchini(s), swabs, and
water, each sample was divided up into five portions
in stomacher bags; one was used for bacterial counts
and the remainder for bacterial enrichments (Table 1).
For ingredients, samples were distributed between
direct counts and enrichments as described in Table 1.
The stomacher bags were heat-sealed (Impulse
sealer TISH-300, Bioblock, Illkirch, France) and sub-
jected to a pasteurization. Heat treatment at 80 jC for
10 min is a common procedure used for the pasteu-
rization of samples (Claus and Berkeley, 1986). In our
study, most samples represented large volumes after
dilution (Table 1). Therefore, stomacher bags were
immersed in a water bath (Polystat 55, Bioblock) for
15 min at 80 jC, to allow heat diffusion. Small
samples (cotton swabs and water samples) were
heat-treated at 80 jC for 10 min. All samples were
cooled in melting ice. The bags for enrichments were
Table 1
Number, size and preparation of samples
Samples Number Sample Direct counts
a
Enrichments
a
of
samples
size
F2%
Preliminary
homogenate,
containing for
one sample
Number
of samples
Sample
size
Sterile
distilled
Number
of
Sample
size
Enrichment
broth
Sterile
phosphate
Sterile
distilled
H
2
O added
(ml)
samples added
(ml)
buffer
b
(ml)
H
2
O
(ml)
BCE
c
CE
c
Soil 6 500 g 1000 6 200 ml
d
12 12 200 ml
d
200
Raw courgettes 5 600 g 475 5 200 ml
d
10 10 175 ml
d
25 (4 )
e
Washed zucchini(s) 5 600 g 475 5 200 ml
d
10 10 175 ml
d
25 (4 )
Cotton swabs 10 1 unit 45 10 20 ml
d
20 20 7.5 ml
d
2.5 (4 )
CG zucchini(s)
f
5 1000 g 5 200 g 150 10 10 200 g 150
CGZ with
ingredients
f
5 1000 g 5 200 g 150 10 10 200 g 150
Water pipe
cleaning
2 70 ml 2 10 ml 4 4 15 ml 5 (4 )
Tap water 2 70 ml 2 10 ml 4 4 15 ml 5 (4 )
UHT cream 15 200 ml 3 200 ml 200 4 8 200 ml 200
Starch 29 20 g 3 20 g 380 14 12 20 g 380
Milk proteins 7 3 g 3 3 g 20 2 2 3 g 200
Milk proteins 6 130 g 2 4 130 g 240
Salt 29 3 ou 5 g 3 3 g 100 14 12 5 g 500
Pasteurized
purees
10 50 g 5 100 g 200
Stored purees
(for each storage
condition)
10 50 g 5 100 g 400
a
For soil, raw and processed zucchini(s), swabs, and water, each sample was divided up into five portions; one was used for bacterial counts
and the remainder for bacterial enrichments. For ingredients, samples were distributed between direct counts and enrichments.
b
Phosphate buffer, K
2
HPO
4
8.5 g/l, KH
2
PO
4
6.75 g/l, pH 6.8.
c
BCE, B. cereus enrichments in MYP and Claus medium; CE, cold enrichments in J-broth at 4 and at 10 jC.
d
Sub-sample of the preliminary homogenate.
e
4 , Enrichment medium 4 concentrated.
f
CG, cooked and ground; CGZ, cooked and groung zucchini(s).
M.H. Guinebretiere et al. / International Journal of Food Microbiology 82 (2003) 223232 227
then incubated at the desired temperature/time con-
ditions.
Before plating, the bags were homogenized for 1
min using a Stomacher (Seward Medical). Homoge-
nates were serially diluted and 100 Al of each dilution
were spread plated on J-agar and MYP agar. For plate
counts, 4 to 9 100 Al and 1 500 Al of the non-
diluted homogenate were also spread plated on each
medium to lower the detection thresholds. MYP plates
and J-agar plates were incubated at 28 jC for 24 and
48 h, respectively.
For the pasteurized puree, the content of each pack
was homogenized with sterile spoons. Approximately
50 g of puree per pack was collected with a sterile
spoon and samples from two packs were pooled and
weighed in sterile stomacher bags ( f100 g per bag).
The five resulting samples were used for counts as
previously described. For stored purees (puree packs
stored at 4, 10 and 2025 jC), only one 100 Al aliquot
of the nondiluted homogenate was spread plated.
2.8. Isolation and confirmation of B. cereus isolates
For each sample, three to five colonies were
randomly selected from MYP plates bearing 20 to
50 colonies. Only colonies with the typical mannitol
negative and lecithinase positive characteristics of B.
cereus were taken into account (van Netten and
Kramer, 1992).
The presumptive B. cereus cultures were grown on
J-agar plates to check purity, and kept frozen in 30%
(vol/vol) glycerol solution at 20 and 80 jC.
Identification of isolates to the B. cereus group was
confirmed by shape and position of endospore under
contrast phase microscopy ( 1000) and XP V 08-
058 procedure (Anonymous, 1996).
2.9. Statistical analysis and counts
Counts were expressed as log CFU per gram or per
milliliter of food samples, or CFU per piece of equip-
ment. Variation among replicate samples was
expressed by standard deviation. To evaluate the
effect of washing, cooking, addition of ingredients
and pasteurization on spore levels in processed zuc-
chinis, means before and after each operation were
compared using Students t-test (Systat version 9,
SPSS Chicago).
3. Results and discussion
3.1. Confirmation of B. cereus isolates
A total of 196 presumptive B. cereus isolates
were obtained from the different samples. Subse-
quent confirmation showed that 100% of the isolates
had the typical spore morphology of B. cereus.
Among the 196 isolates, 20 had atypical characters
(hemolysis negative or weakly positive), but on
MYP plates and J-agar plates, they exhibited char-
acteristic rhizoid outgrowths of B. mycoides that
belongs to the B. cereus group. Thus, 100% of the
isolates was regarded as belonging to the B. cereus
group.
3.2. Contamination levels in soil
Levels of B. cereus spores in the cultivation soil
of the zucchinis were quite high: 4.6 F0.3 log CFU/
g. Spore counts were 6.1 F0.2 log CFU/g. It is
generally accepted that the primary habitat of most
Bacillus species is the soil where they are considered
as forming the greater part of the metabolically
active bacterial flora (Felske et al., 1998). The levels
and ratio of spores to total cell numbers of Bacillus
spp. vary according to irrigation period, fertilizer
practice, season and climate (Von Stetten et al.,
1999; Watanabe and Hayano, 1995). In soil, spore
number is usually high; Ueda and Kuwabara (1986)
found levels of 4.3 F0.4 to 5.1 F0.5 log CFU/g in
rice field soils, and Watanabe and Hayano (1995)
found 10
5
to 10
7
log CFU/g in rice and wheat
rotation soils, consistent with our results. In temper-
ate soils, the abundance of B. cereus spores ranged
from 10
5
to 10
6
CFU/g according to Von Stetten et
al. (1999) and 10
2
to 10
5
CFU/g according to Te
Giffel et al. (1995).
Zucchinis are the aerial part of the plant. However,
many fruits are in contact with soil or aerosols of soil
particles. The high levels of spore-forming bacteria
measured in soil samples where the zucchinis were
grown suggest that the cultivation soil constituted a
significant source of contamination for zucchinis with
B. cereus and other Bacillus spp. Soil is also consid-
ered as a source of B. cereus contamination for other
foods such as milk (Andersson et al., 1995; Te Giffel
et al., 1995).
M.H. Guinebretiere et al. / International Journal of Food Microbiology 82 (2003) 223232 228
3.3. Contamination from equipment surfaces and air
No bacterial spores were detected in the piped
water used to clean the puree distribution system.
The total number of spores collected on half the total
surface of equipment before the start of production
ranged from 160 to 19,000 spores according to the
piece of equipment. The highest contamination
occurred on rotating equipment such as the screw
and mixing blade (Table 2). As surfaces of equip-
ment came into contact with 280 kg of zucchini
puree, we can consider that contamination potentially
transferred by equipment surfaces to the final prod-
uct was low.
No B. cereus spores or psychrotrophic bacteria
spores were detected in samples from equipment
surfaces.
Current procedures for cleaning the distribution
system included three successive steps of washing
with hot water. Equipment surfaces were cleaned with
hot water under high pressure before and after each
production step. At the end of each processing day,
surfaces were cleaned with disinfectant solutions
(Penngar neige, Penngar, Le Mans, France; Anios
W4, Laboratoire Anios, Lilles, France) containing a
fungicide, bactericides and notably one B. cereus
sporicide (Norm NF T 72-300). Wirtanen et al.
(1996) showed that the high mechanical forces of
the cleaning flow, heat treatment and chemical treat-
ment were efficient in removing Bacillus biofilms on
equipment surfaces. Given the low levels of contam-
ination measured on equipment in our study, we can
conclude that the cleaning procedures employed were
effective and satisfactorily controlled B. cereus on
equipment surfaces.
On MYP plates exposed to the air of the process-
ing room for 30 min, only one viable B. cereus
particle was detected among 19 plates. Counts of
these bacteria in air are poorly documented. Ueda et
al. (1988) found averages of airborne bacterial
counts ranging from 0.2 to 10.7 CFU per liter of
air in mill plants, composed of 26% to 33% of viable
B. cereus particles. In our study, the product was in
contact with air mainly during its transport in the
three containers. Contamination transferred by air to
the final product was thus presumably very low.
3.4. Contamination levels of ingredients
In the ingredients, spore counts were close to or
below the limit of detection, except for milk proteins
and starch, which carried 2.8 F0.6 and 1.6 F0.1 log
CFU per gram (Table 3), respectively. B. cereus spore
Table 2
Counts of bacterial spores on equipment surfaces before the
beginning of production
a
Total (CFU)
b
B. cereus
(CFU)
b
Grinder screw 5.3 10
3
V1.6 10
2
Grinder inside surface 2.3 10
3
V1.6 10
2
Blender grid 1.7 10
3
V1.6 10
2
Rotating mixing blade 1.9 10
4
V1.6 10
2
Blender inside surface 1.7 10
2
V1.6 10
2
Container inside surface no. 1 3.3 10
2
V1.6 10
2
Container inside surface no. 2 V1.6 10
2
V1.6 10
2
Container inside surface no. 3 1.7 10
2
V1.6 10
2
Distributor tip 1.3 10
3
V1.6 10
2
Distributor conveyor belt 1.7 10
2
V1.6 10
2
Pipe cleaning water A
c
V2 V2
Pipe cleaning water B
c
V2 V2
a
Enrichments for B. cereus spores and for psychrotrophic
bacterial spores were all negative.
b
Numbers are the number of spores present on half of the
surface of each piece of equipment and the number of spores per
milliliter for water samples.
c
A, before the beginning of production; B, after the end of the
production.
Table 3
Counts and detection of bacterial spores in ingredients added to the
puree
Spore counts
a
Enrichments
b
Total B. cereus B. cereus Psychrotrophs
(log CFU/g) (log CFU/g)
10 jC 4 jC
UHT cream < 0.6 < 0.6 0/4 0/4 0/4
Starch 1.6 F0.1 < 1.6 4/4 2/2 2/2
Milk proteins 2.8 F0.6 < 1.4 4/4 3/3 3/3
Salt < 1.2 < 1.2 0/4 0/2 0/2
Tap water < 0.3 < 0.3 0/4 0/2 0/2
a
Means Fstandard deviation (n =3).
b
Number of positive samples relative to the total number of
enriched samples; for starch and salt, the 26 enriched samples were
pooled during analyses so as to obtain four analyses for B. cereus
enrichment and four analyses for cold enrichments; samples sizes
were 200 ml for UHT cream, 15 ml for tap water, 60 to 80 g for
starch (pooled samples), 15 to 20 g for salt (pooled samples), and 3
to 130 g for milk proteins.
M.H. Guinebretiere et al. / International Journal of Food Microbiology 82 (2003) 223232 229
counts were below the level of detection in all the
ingredients. After enrichment, B. cereus and psychro-
trophic bacterial spores were detected in all the
samples of starch and milk proteins.
Of the ingredients, milk proteins and starch con-
stituted the principal sources of spore contamination.
B. cereus and psychrotrophic spore-forming bacteria
have often been considered as contaminants of farina-
ceous foods, dried foods, dehydrated milk and milk
products (Blakey and Priest, 1980; Kramer and Gil-
bert, 1989; Roberts et al., 1982; Wong et al., 1988).
Levels of B. cereus generally measured on milk
powder and dried products ranged from 10 to 10
5
CFU/g (Te Giffel, 1997).
3.5. Contamination of zucchinis, processed zucchinis
and stored product
On raw zucchinis, spore counts were 1.5 F0.2
log CFU/g and washing operations failed to reduce
the contamination on washed zucchinis (Table 4).
Contamination level significantly decreased after
cooking according to Students t-test ( P= 0.02) and
increased after the addition of ingredients ( P=
0.001). Finally, pasteurization resulted in a signifi-
cant decrease in spore counts ( P = 0.001) to
0.5 F0.3 log CFU/g. Purees were pasteurized in
their final package. The marked effect of the pasteu-
rization may be explained by the germination of a
proportion of spores during the distribution and
packaging of the puree. The resulting contamination
level in unstored pasteurized product represented 640
to 2,500 spores per puree pack of 400 g. Spore-
forming bacteria were able to multiply in the product
stored at cold temperatures, reaching, after 21 days,
3.8 F1.1 log CFU/g at 4 jC and 7.5 F0.3 log CFU/g
at 10 jC (Table 5).
B. cereus counts were less than or equal to 0.4
log CFU/g on raw and processed zucchinis (Table
4). However, B. cereus and psychrotrophic bacterial
spores were detected after enrichment in most
samples at all stages of processing. The number
of positive samples did not increase after the
addition of ingredients. Thus, levels of B. cereus
contamination transferred by starch and milk pro-
teins were probably of the same order of magnitude
as that transferred by zucchinis after cooking. On
packaged purees, B. cereus counts were less than or
equal to 0.2 log CFU/g (Table 4). This level was
nevertheless sufficient to initiate growth of B.
cereus in products stored at 10 and 2025 jC.
Counts of B. cereus reached 4.6 F1.9 log CFU/g in
purees stored at 10 jC for 21 days (Table 5),
higher than the upper limit of 4.0 log CFU/g
recommended in France for some processed vege-
tables (Jouve, 1996).
In purees maintained at 4 jC, no significant
growth of B. cereus was measured. As 4 jC is the
recommended storage temperature for these products
in France (Anonymous, 1991), B. cereus should not
be a hazard provided refrigeration is properly main-
tained throughout the cold chain (cold storage room
Table 4
Counts and detection of bacterial spores in raw zucchini(s) and processed zucchinis
Spore counts
a
Enrichments
b
Total B. cereus B. cereus Psychrotrophs
(log CFU/g) (log CFU/g)
10 jC 4 jC
Raw zucchini(s) 1.5 F0.2 < 0.4 10/10 5/5 5/5
Washed zucchini(s) 1.7 F0.6 < 0.4 10/10 5/5 5/5
Cooked/ground zucchini(s) 0.9 F0.4 < 0.3 2/10 4/5 2/5
Cooked/ground
zucchini(s) mixed
with ingredients
3.2 F0.8 < 0.4 2/10 4/5 2/5
Pasteurized purees 0.5 F0.3 < 0.2 nt nt nt
a
Means Fstandard deviation (n = 5).
b
Number of positive samples relative to the total number of enriched samples; sample weighs were 100 to 200 g.
M.H. Guinebretiere et al. / International Journal of Food Microbiology 82 (2003) 223232 230
of the processing plant, transport, distribution, and
consumer refrigerator). Fluctuations of storage before
consumption of the product, most often occurring
during distribution of the product or in the refriger-
ator of consumers, may induce growth of B. cereus,
as some strains from pasteurized vegetable purees
were able to grow at temperatures below 10 jC
(Choma et al., 2000).
4. Conclusion
B. cereus and psychrotrophic bacterial spores were
detected at many points along the processing line of
zucchini puree and in particular in all the expected
primary sources of contamination (soil, zucchini(s),
ingredients). The efficient cleaning procedure used for
equipment surfaces and especially the use of B. cereus
sporicide prevented the installation of B. cereus on
equipment surfaces. Also, salt and UHT cream added
to the puree did not carry B. cereus and psychrotro-
phic spores. Raw material (zucchinis) and texturing
agents (milk proteins and starch) used to process the
puree were, therefore, the main sources contributing
to the contamination with B. cereus and psychrotro-
phic bacterial spores. They both transferred a low
level of B. cereus spores, but that was sufficient to
induce bacterial growth in the stored product. Pre-
vious work (Carlin et al., 2000; Choma et al., 2000)
showed that the low levels of contamination recovered
in unstored vegetable purees did not vary significantly
according to season.
Our work shows that secondary sources of contam-
ination (equipment surfaces) can be effectively
avoided. Research and efforts from processors should
therefore be focused on means to significantly reduce
primary sources of contamination. The final pasteuri-
zation is applied to a complex mixture of vegetables,
dairy cream and texturing agents, and any increase in
temperature over 80 jC, or any increase in treatment
time, would result in unacceptable adverse changes in
the product. In addition, heat transfer through the
packs of puree is slow and this further reduces any
possibility for intensifying the pasteurization treat-
ment. In contrast, there are more possibilities for
strengthening heat treatment applied during the cook-
ing of zucchinis. They can be cut into small pieces to
permit rapid heat transfer. Improved steam cooking
technologies should enhance the heat treatment with-
out causing unwanted alterations (Varoquaux and
Nguyen-The, 1994). This would permit a further
reduction in the number of spores carried by zucchinis.
However, such reduction would be worthwhile only if
milk proteins and starch free of B. cereus spores could
be obtained, as no step except the final pasteurization
could reduce this source of contamination.
This work represents the first analysis of contam-
ination flows occurring during processing of cooked
chilled foods. It shows that although texturing agents
carry a very low number of B. cereus spores, they
represent a critical step for contamination and pre-
sumably limit reduction of the incidence of B. cereus
in cooked chilled foods. It emphasizes the importance
of hygienic processing of dehydrated ingredients for
the general safety of the cooked chilled foods sector.
Acknowledgements
This work was supported by research project FAIR
CT 97-3159 (Commission of the European Commun-
ities). The authors thank the food company involved
for active co-operation, and Dr Cindy Morris (Station
de Pathologie Vegetale, INRA, Avignon, France) for
the help in designing the sampling strategy.
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