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Food Microbiology, 2002, 19, 589^595

Available online at http://www.idealibrary.com on

doi:10.1006/yfmic.514

ORIGINAL ARTICLE

Enumeration, isolation and


characterization of Bacillus cereus
strains from Spanish raw rice
J. A. Sarr| as, M.Valero and M.C. Salmeron

Bacillus cereus was present in 61 samples of raw rice analysed representing unhusked, husked and
commercial origins. B. cereus in husked and white rice samples did not reach 102 cfu g 1, while in the
unhusked rice B. cereus densities exceeded 103 cfu g 1. Processing steps such as drying, husking and
polishing reduced the number of B. cereus in the nal product. Eight strains with typical morphology
of B. cereus on Polymyxin^Mannitol^EggYolk^Phenol Red Agar (PMYPA) were isolated. According to
ISO conrmatory tests, the API System tests and supplementary tests of motility, oxidase activity and
enterotoxin production, these isolates were characterized and identied as B. cereus. All strains were
motile, oxidase-negative and produced diarrheal enterotoxin inTSB. D and z-values were used to characterize heat resistance of spores obtained from the eight strains of B. cereus characterized. A large
diversity in heat resistance was observed among the isolates. At 901C, D-values ranged from 2?23 to
23?26 min, with ve groups of D-value means signicantly dierent at the 95% condence level. D95and D100 values calculated for the eight strains ranged from 0?69 to 5?17 min and from 0?43 to
1?09 min, respectively. Statistical analysis revealed that there was signicant dierence between the
D-value means obtained for the strains at each temperature.The z-values for the eight strains of B. cereus tested in this study ranged from 7?421C to 8?201C with an average of 7?71C.
# 2002 Elsevier Science Ltd. All rights reserved.

Introduction
Microbiological analysis of cereal grains is not
routinely performed, nevertheless, the determination and count of total mesophilic aerobes,
coliforms (Escherichia coli), moulds and yeast,
Bacillus cereus and Salmonella have been used
in order to ascertain hygienic-sanitary quality
(Pascual Anderson, 1992). The external surface
of the cereal grains are contaminated heavily
by saprophytes acquired during development

Corresponding author. Fax: 966749619;
E-mail: m.valero@umh.es

0740 -0020/060589+07 $35.00/0

of the plant along with contaminants from the


soil, air and animals (including humans). In
general, members of the Pseudomonadaceae,
coliforms, endospore-forming bacteria, yeast
and moulds are the most common members of
the rice microora.
Drying of grains destroys the majority of micro-organisms (approx. 99% of culturable aerobes) (Goel et al. 1970). However, spores of the
genus Bacillus are heat resistant and can withstand such heat treatment. Likewise, among
surviving microbes are moulds of the genera
Aspergillus and Penicillium which are able to
grow at low water activity (aw ). Under optimal
r 2002 Elsevier Science Ltd. All rights reserved.

Received:
7 January 2002
Departamento de
ProduccionVegetal y
Microbiolog| a,
Escuela Politecnica
Superior de Orihuela,
Universidad Miguel
Hernandez - Campus
de Orihuela, Carretera
de Beniel, km 3.2,
03312-Orihuela,
Alicante, Spain.

590 J. A. Sarr| as et al.

conditions of storage (temperature 14^151C


and relative humidity o14?5%), the water activity of grains such as rice is decreased to levels that inhibit microbial growth. Husking
eliminates most external microora while boiling further decreases the microbial counts in
white or commercial rice. Between 1992 and
1996 in England and Wales, 29 outbreaks of
food poisoning attributed to cooked rice contaminated with B. cereus or other Bacillus species were reported, with 164 people aected
(Nichols et al. 1999). Many outbreaks of a similar nature have occurred in Canada, Finland,
the Netherlands, Japan, and the USA (Beckers
1976, Raevuori et al. 1976, Schmitt et al. 1976,
Terranova and Blake 1978, Schinagawa et al.
1979, Khodr et al. 1994).
The purpose of this study was to establish B.
cereus contamination levels in raw rice at different stages of processing. Eight presumptive
B. cereus strains were isolated, conrmed, identied and characterized according to ISO 7932
procedure and API tests. Isolates were further
assayed for heat resistance in the temperature
range of 90^1001C.

with an irregular edge surrounded by a white


area were considered as positive and enumerated. At least ve colonies from eight samples
representing the four types of rice analysed
were subcultured in CeNAN selective agar
and were conrmed subsequently as B. cereus
by Gram-staining, shape and position of
endospores, and biochemical testing as specied in the ISO 7932:1993(F). Cultures were
then streaked in tryptone soya agar
(TSA, Scharlau Chemie, S.A.) slants and stored
refrigerated (41C) as pure cultures.
For detecting and enumerating B. cereus in
husked and white (commercial) rice, the statistical estimate of the most probable number
(MPN) was used. Three serial decimal dilutions from each sample were performed and
1 ml from each dilution was used to inoculate
three series of three tubes containing the selective enrichment broth base without mannitol
or indicator, and supplemented with 100 mg l 1
of polymyxin B sulphate. The tubes were incubated at 301C for 24 h.The dierential isolation
of the strains detected was performed following the procedure outlined above.

Sporulation of isolated strains

Materials and Methods


Enumeration and isolation
To enumerate B. cereus contamination in unhusked rice the method described by AFNOR
(1996) was used. Ten grams of rice was homogenized in a masticator with 90 ml of sterile 0?1%
peptone (Scharlau Chemie, S.A., Barcelona,
Spain). Detection and enumeration were performed by supercial streaking of 0?1 ml of the
suspension in Petri dishes containing selective
solid medium [Bacillus cereus CeNAN agar
base supplemented with 100 mg l 1 of polymyxin B sulphate and 100 ml l 1 of egg yolk sterile
solution (Scharlau Chemie, S.A.)]. This medium
(polymyxin^mannitol^egg yolk^phenol red
agar, PMYPA) was designed by Mossel et al.
(1967) and has been adopted by the Centro Nacional de Alimentacion y Nutricion (CeNAN
1982), since it permits a good dierentiation of
B. cereus colonies from a variety of food samples. Cultures were incubated at 301C for 24 h.
Colonies presenting a pink or purple colour

B. cereus strains were grown overnight in nutrient broth at 301C to achieve the stationary
phase. Culture volumes of 0?2 ml (corresponding to 3^9?5  107 cfu ml 1 ) were dispensed on
plates containing as sporulation medium, fortied nutrient agar (FNA) (Johnson et al. 1982,
Mazas et al. 1995), and incubated at 301C for 4
days. Sporulation was veried daily by microscopic examination. When at least 90% sporulation was reached, spores were harvested by
ooding the agar surface with sterile distilled
water, centrifuged, resuspended and stored at
41C until use (Valero et al. 2000).

Computer-assisted identication and


conrmation of B. cereus
Bacterial identication with APILAB Plus
software V3.2.2 Version B 01.93 (bioMerieux,
Marcy lEtoile, France) was used to conrm isolates as B. cereus. Identication was based on
the analysis of 61 biochemical tests of the API
50CH and API 20E strips (bioMerieux). The

Characterization of B. cereus strains from Spanish raw rice 591

oxidase activity on lter paper using a wooden


applicator to make the extension, motility in a
semi-solid medium [tryptone, 10 g; yeast extract, 2?5 g; dextrose, 5 g; Na2 HPO4, 2?5 g; agar,
3 g; distilled water, 1000 ml; pH 7?470?2 (ADSA
Micro, Barcelona, Spain)], and anaerobic
growth in an agaried medium [tryptone, 20 g;
NaCl, 5 g; sodium thioglycollate, 2 g; sodium
formaldehyde sulphoxylate, 1 g; agar, 15 g; distilled water, 1000 ml; pH 7?270?2 (ADSA Micro)] for anaerobes with neither glucose nor
indicator were also tested (Pascual Anderson
1992, Atlas and Parks 1993). Enterotoxin
production was tested in culture ltrates of
isolates grown at 301C in tryptone soya
broth (TSB, Scharlau Chemie, S.A.) using the
B. cereus enterotoxin reverse passive latex
agglutination (BCET-RPLA) test from Oxoid
(Basingstoke, Hampshire, England) following
the instructions of the manufacturer.
Inocula used for the biochemical tests were
prepared using 18 -h-old cultures grown in
TSB medium and incubated at 301C (Valero
et al. 2002). After 18 h of incubation a decimal
dilution was made of the cultures, and 0?1 ml
aliquots were distributed with a Drigalski spatula on the surface of Petri plates containing JAgar (Gordon et al. 1973, Claus and Berkeley
1986). Once the inocula had been reabsorbed,
plates were incubated at 301C for 16^18 h. Two
milliliters of sterilized saline solution (NaCl
0?85%) was then added to each of the plates
and the cultures were collected from the agar
surface with sterile pipettes. Suspensions were
prepared with the cultures with a turbidity
equivalent to a 2 MacFarland standard (bioMerieux). The API 50CH and API 20E galleries
were lled, inoculated, incubated and interpreted following the instructions specied for
each.

Heat treatment of B. cereus spores


Heat treatment of bacterial spore suspensions
was performed in 10 ml microhematocrite capillary tubes (Vitrex, MODULOHM A/S, Herlev,
Denmark) which ensure uniform heat transmission (Fernandez et al. 1999). Capillary tubes
were lled with bacterial spore suspensions in
sterilized water containing approximately 105 ^
106 spores ml 1. Spore suspensions were cen-

trally injected into glass capillary tubes with


a syringe, and the capillaries were then amesealed (Fujikawa et al. 2000).Three series of six
capillary tubes were lled from each condition
and strain assayed. They were submerged in a
stirred oil bath with immersion circulator
HAAKE DC5 (Gebrder HAAKE GmbH,
Karlsruhe, Germany) at 901C, 951C or 1001C
constant temperature. Subsequently, tubes
were removed at regular intervals and cooled
in ice-water, washed in soap solution, rinsed
in distilled water, and immersed in
ethanol. Spore heat treatments were done in
triplicate.
For each strain the initial spore concentration was determined after heating at 801C for
10 min. Plate count agar (PCA, Oxoid) was used
to recover heated spores. The log10 numbers of
surviving spores were plotted against time
and D-values were calculated from the slope
of the linear phase of the spore destruction as
the time in minutes needed to decreased the
population by one log10 cycle (ICMSF 1980).
The analysis of variance for D-values by strains
heated at each temperature and a multiple
comparison procedure (Fishers least signicant dierence, LSD) were performed using
Statgraphics Plus. z-values were determined
as the increase in temperature required to reduce the decimal reduction time (D) by one
log10 cycle when log10 D was plotted against
temperature.

Results
Enumeration of B. cereus in raw rice
All the unhusked rice samples examined were
positive for B. cereus with counts ranging from
1?2 to 3?5  103 cfu g 1. The levels of B. cereus in
all husked and elaborated rice samples were
low and were dicult to detect on PMYPA selective medium. The most probable numbers of
micro-organisms per gram estimated from
these types of rice are shown in Table 1.

Identication of isolates
Eight strains with typical morphology of B.
cereus on PMYPA were isolated. All were

592 J. A. Sarr| as et al.

Table 1. Numbers of Bacillus cereus in dierent phases of processing and commercialization of rice
Type of rice

No. of samples

Average counts 7 s.d. (cfu g 1 )

10
17
17
17

F
F
F
2061?57676?4

White or commercial (WR)


Husked (HR )
Husked (HR)
Unhusked (UR)


MPN g

7 s.d.

77?5724?4
24?576?6
26?974?5
F

Husked rice treated with phosphine (Tinarelli, 1989).

conrmed as Gram-positive and grew under


anaerobic conditions. Endospores were oval,
central or subterminal, and did not swell the
sporangium. These isolates were conrmed as
B. cereus by ISO 7932 and were further characterized and identied by API 50CH/20E phenotypic system using APILAB Plus software.
Most isolates belonged to the prole intermediate between B. cereus 1 and B. mycoides
(Table 2). Two strains with the API prole
B. cereus 1 were isolated from white (commercial) and husked rice, while a strain with a
prole intermediate between B. cereus 1
and B. anthracis was isolated from husked
rice treated with phosphine (Tinarelli 1989).
These three species together with B. thuringiensis are closely related and form one
subgroup of Bacillus, the B. cereus group.
Motility (Granum 1997) was a ne test to
distinguish B. cereus from B. mycoides and
B. anthracis. Also all strains were oxidase
negative conrming the identication of
isolates as B. cereus and ruled out the possibility of B. thuringiensis. The eight strains characterized by API strips were tested for
enterotoxin production, and all were positive
with the BCET-RPLA test from Oxoid.

Heat resistance
Distilled-water suspensions of spores prepared
from each one of the B. cereus strains were analysed for heat resistance over the temperature
range 90^1001C. D-value means obtained at different temperatures for the eight strains are
listed in Table 3. The D90 -values ranged from
3?23 to 23?26 min, with ve groups of D-value
means signicantly dierent at the 95% condence level. Strain EPSO- 41WR was the most
heat-resistant (D90 -values from 20?38 to
23?26 min), followed by the strains EPSO42WR, EPSO- 43WR and EPSO- 47HR with
D90 -values ranging from 17?24 to 13?41 min.
Strain EPSO- 45HR was the least heat-resistant, having D90 -values from 3?23 to 4?29 min.
D95 - and D100 -values calculated for the eight
strains ranged from 0?69 to 5?17 and from 0?43
to 1?09 min, respectively. Statistical analysis revealed that there were signicant dierences
among D-value means obtained for the strains
at each temperature (Table 3).
Figure 1 illustrates the linear regression
of thermal death time curves (log10 D-values
vs temperature) for spores of ve B. cereus
strains. The least-squares regression analysis

Table 2. Characterization of Bacillus cereus strains isolated from raw rice on the basis of 66 both physiological and biochemical properties
Strains
EPSO- 41WR
42WR
43WR
44HR*
45HR
46HR*
47HR
50UR

% Id.

T index

API name

Motility

Name

BCET-RPLA test

83?9
56?4/43?5
53?0/45?9
55?6/44?3
64?2/35?7
72?2/7?8
94?0
61?5/37?2

0?84
0?99/0?95
0?94/0?97
0?90/0?92
0?91/0?92
0?93/0?90
0?87
0?96/0?91

B. cereus
B. mycoides/B. cereus
B. cereus/B. mycoides
B. cereus/B. mycoides
B. cereus/B. mycoides
B. anthracis/B. cereus
B. cereus
B. mycoides/B. cereus

+
+
+
+
+
+
+
+

B. cereus
B. cereus
B. cereus
B. cereus
B. cereus
B. cereus
B. cereus
B. cereus

+
+
+
+
+
+
+
+

Characterization of B. cereus strains from Spanish raw rice 593

Table 3. Mean D-values (min) of three experiments 7s.d. obtained for spores of eight Bacillus cereus
strains heated at dierent temperatures
Strains
EPSO- 41WR
42WR
43WR
44HR*
45HR
46HR*
47HR
50UR

D85

D90

D95

D100

NT
NT
NT
NT
1375971?27a
NT
NT
NT

22?0571?49a
15?2170?31b
15?7172?03b
12?9670?48c
3?8470?55e
11?7670?20c
14?7471?21b
7?45 70?07d

5?0270?19a
3?54 70?05c
3?3170?27c
2?84 70?03d
0?7070?01f
2?75 70?16d
4?34 70?05b
2?28 70?11e

0?9970?09a
0?7670?01b
0?7670?02b
0?6270?01c
NT
0?5970?01c
0?7470?06b
0?4570?02d

NT, not tested.


Values within columns followed by the same letter are not signicantly dierent (P 0?05).
a
Mean of three experiments 7s.d.

Table 4. z-values for spores of eight Bacillus


cereus strains from four types of raw rice heated in
distilled water

1.6
1.4
1.2

Strains

Log D-values

1
0.8

EPSO- 41WR
42WR
43WR
44HR*
45HR
46HR*
47HR
50UR

0.6
0.4
0.2
0
-0.2
-0.4
-0.6
80

85

90

95

100

z-value (1C)

Coecient R2

7?42
7?68
7?60
7?57
7?76
7?69
7?70
8?20

0?999
0?999
0?999
1?000
0?993
0?999
0?989
0?992

105

Temperature (oC)

Figure 1. Least-squares regression analysis of


thermal death time curves for spores of ve Bacillus
cereus strains [EPSO- 41WR (*), EPSO- 42WR (*),
EPSO- 44HR (~), EPSO- 45HR (&) and EPSO50UR (&)] recovered in plate count agar (PCA).

of thermal death time curves for strains


EPSO- 42WR, EPSO- 43WR and EPSO- 47HR
were very similar; likewise the analysis for the
strains EPSO- 44HR and EPSO- 46HR were
similar. The z-values obtained for all strains
in the temperature range tested are listed in
Table 4. High determination coecients (R2 )
ranged from 1?000 to 0?989 were estimated for
all strains.

Discussion
Bacillus cereus was present in the 61 samples of
raw rice analysed. Numbers of B. cereus in the

husked and white rice samples did not reach


102 cfu g 1, while in the unhusked rice the numbers of B. cereus usually found were higher than
103 cfu g 1 (Table 1). Processing phases reduced
the number of B. cereus in the nal product.
All colonies isolated from raw rice on
selective PMYPA medium for B. cereus were
conrmed by Gram-staining, shape and position of endospores, and ISO 7932 procedure.
Species of isolates were determined by the
API 50CH and API 20E systems (Logan et al.
1985, Weber et al. 1988, Guinebretie're et al.
2001) and from supplementary tests. Three
API proles (Table 2) were found among
the presumptive B. cereus isolates but
additional tests of motility, oxidase activity
and enterotoxin production allowed for identication as B. cereus. All strains were motile,
oxidase-negative and produced diarrheal enterotoxin in TSB.

594 J. A. Sarr| as et al.

D- and z-values were used to characterize


heat resistance of spores obtained from the
eight strains of B. cereus characterized. A large
diversity in heat resistance was observed
among the isolates. At 901C, the range of heat
resistance found here (Table 3) was higher than
that reported by Shehata and Collins (1972),
Bassen et al. (1989), Dufrenne et al. (1995), Arinder et al. (1999) and Choma et al. (2000), but
very similar to that found by Dufrenne et al.
(1994). This latter group reported D90 -values
ranging from 4?6 to 14?0 min for B. cereus
strains isolated from dierent sources with
the capacity to grow at temperatures 91C.
D90 -values 4100 min for some B. cereus
isolates were also reported (Dufrenne et al.
1994, 1995).
The calculated D95 -values determined during this work ranged from 0?70 to 5?02 min
(Table 3). For comparative purposes, Parry
and Gilbert (1980) reported a range of
D95 -values from 2?5 to 36?2 min for strains
isolated from emetic foodborne illness
outbreaks, and Johnson et al. (1982) published
D95 -values from 1?2 to 20?2 min for spores
of B. cereus strains representing diarrheal,
emetic and atoxigenic origins. Bradshaw
et al. (1975) calculated D95 -values of 256?7
and 5122?3 min for two highly resistant strains
of B. cereus.
The D-values determined at 1001C ranged
from 0?45 to 0?99 min (Table 3). These D100 values are in agreement with those obtained
by Gonzalez et al. (1995) for the B. cereus strains
ATCC 4342 and ATCC 7004; the strain ATCC
9818 was more heat-resistant (D100 44 min).
The D85 (13?59 min), D95 (3?84 min) and D100
(0?7 min) values calculated for the strain
EPSO- 45HR, the least heat-resistant strain
assayed, are in agreement with those obtained
by Fernandez et al. (1999): 17?0, 4?1 and 0?98 min,
respectively.
The z-values for the eight strains of B. cereus
tested in this study ranged from 7?421C to
8?201C, with an average of 7?71C. The z-values
obtained by Gonzalez et al. (1995) for the three
strains studied were very similar ranging from
7?24 to 8?271C, with an average of 7?641C. Gilbert
et al. (1974) found z-values ranging from 6?71C
to 8?31C, and Johnson et al. (1982) reported
z-values of 6?8^13?91C.

Acknowledgements
This work was carried out with nancial support from FEDER and is an integral part of
the I+D Project IFD97-1005 -C04 coordinated
by CEBAS (Centro de Edafolog| a y Biolog| a
Aplicada del Segura). The authors are grateful
to Manuel Giner Pastor for his valuable technical assistance.

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