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doi:10.1006/yfmic.514
ORIGINAL ARTICLE
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
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.
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).
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
Table 1. Numbers of Bacillus cereus in dierent phases of processing and commercialization of rice
Type of rice
No. of samples
10
17
17
17
F
F
F
2061?57676?4
MPN g
7 s.d.
77?5724?4
24?576?6
26?974?5
F
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
+
+
+
+
+
+
+
+
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
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)
Discussion
Bacillus cereus was present in the 61 samples of
raw rice analysed. Numbers of B. cereus in the
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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