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Thompson 1993
Thompson 1993
ABSTRACT
Bacteria belonging to the genus Bacillus are capable of causing the food
spoilage condition in bread known as rope. This involves mainly Bacillus
subtilis, but also B. licheniformis, B. megaterium and B. cereus. Rope is
characterized by a sweet fruity odour, often described as resembling over-
ripe pineapples, together with patchy discolouration and softening of the
loaf crumb. Differentiation of microorganisms can be carried out in a
variety of ways. These include classical methods, for example the Gram
stain, commercially available kits, such as the Analytical Profile Index
(API) strip, and modern molecular methods, such as ribotyping. The
various methods used to differentiate the strains are an important aid in
highlighting the areas within the bakery processing line that are involved in
contributing to the food spoilage condition known as ropey bread.
INTRODUCTION
Bacteria belonging to the genus Bacillus are capable of causing the food
spoilage condition in bread k n o w n as rope (Voysey & Kaur, 1987;
Kirschner & Von Holy, 1989; Von Holy & Allan, 1990). Bacillus
mesentericus was initially described as the causative agent. This was later
referred to as B. subtilis (Barton-Wright, 1943). Other rope-inducing
species include B. licheniformis, B. megaterium and B. cereus (Collins
et al., 1991).
55
International Biodeterioration & Biodegradation 0964-8305/94J$07-00 © 1994 Elsevier
Science Limited, England. Printed in Great Britain.
56 Jackie M. Thompson, Christine E.R. Dodd, Will M. Waites
The genus Bacillus was first described by Ferdinand Cohn in 1872 (Collins
et al., 1991). Smith et al. (1946) carried out the first comprehensive study
on the taxonomy of Bacillus. Researchers have tried unsuccessfully to
divide the genus (Sneath, 1986). Recent studies using D N A base compo-
sition and D N A base reassociation have highlighted the broad nature of
the genus (Priest, 1981; Sneath, 1986). Small subunit (16S) ribosomal
RNA studies by Ash et al. (1991) have divided the genus into five distinct
clusters which Ash et al. suggested actually represent separate genera.
The use of classical tests to identify isolates is hampered by the need for
stringently controlled conditions. Colony characteristics of B. subtilis and
related species are known to vary considerably as a result of environ-
mental factors, such as agar depth and medium composition. For example,
colony variation can be influenced by the type of peptone used in the
medium. The degree of sporulation within the colony can also affect the
colony colour (Sneath, 1986). The use of the Analytical Profile Index
(API) 50CHB identification strips (BioMrrieux) have been shown to give
more reproducible results than classical methods (Logan & Berkeley,
1981, 1984; Collins et al., 1991). These strips examine the utilization of 49
carbohydrates, which is demonstrated by a colour change.
D I F F E R E N T I A T I O N OF B A C I L L U S ISOLATES
Ca) B.licheniformisl
B.eoagulans/ B .li¢heniformis/
B B..subfili~ B.pumilus 17.1% / B.eoagulans/
.purer|us u.o'~~ / B.subtilis 0.6%
~--~.-a------~ / ~,B .stearothermophilu s/
Non-identifiable _ / ~ ~ ~ / B.circulans/
3,7% ~ A , , \ \ \ \ \ \ \ \ " ~ ~ ~ 8.ma0~...24%
~,,,~~"~ ~ _ ~--'---'- B.stearothermophilu s/
~N,~'~,~'N~ ~ B.megaterium/
.
B.stearothermoph|lusl j~'///j,~ ~ I ~ B.sphaerieus/
-13 ......
B.cireulan,/~- ~ ~ ~ l~.orevts/3.,'r.
B.maeerans 3.5% / ~ -- ~ n ';,h,,i
B.licheniformis/
B.subtilis 9.4% B.eereus 6.5% B.eoagulansl 12.9%
(b)
B.lieheniformis 28.2%
~fll I I I I ] I I t/r'~,\,,,,,,'B-megaterium3.6%
~Ji"r' ~ 11'
] I I l lll
II I ) / ~ ' Y " " B ' p o i y m y x a 3.5%
. L1~B.macerans 3.0%
Fig. 1. Percentage distribution of 170 isolates of Bacillus using (a) classical methods and (b)
API 50CHB.
TABLE 1
Distribution of 170 Bakery Bacillus Isolates Identified Using API 50CHB
PROBLEMS WITH B A C I L L U S A N D M O L E C U L A R M E T H O D S
RIBOTYPING
12.2 K B
7.1 K B
--6.1 KB
~ 5.0 K B
--4.0 KB
°~ --3.0 KB
r~
....... - - 2 . 0 KB
~ 1.6 K B
g
Fig. 2. Ribotyping of B~lcillus species. (DNA digested with Cla 1). Lane 1:1 kb ladder; Lane 2: NCTC 3610 (B. subtilis); Lane 3: NCTC 6276
(ropey bread isolate) (B. subtilis); Lane 4: NCTC 7861 (B. suhtilis var. niger): Lane 5: NCTC 10073 (B. subtili~s var. globigii); Lane 6: NCIMB
10785 (B. amyloliqu~fociens); Lane 7: NCTC 10341 (B. liehen(formis); Lane 8: NCTC 962 (ropey bread isolate) (B. lieheni[brmis (B. mesen-
tericus)): Lane 9: NCTC 1024 (ropey bread isolate) (B. licheni/brmis (B. mesenterieus)); Lane 10: NCTC 1025 (ropey bread isolate) (B. liehe-
n([brmis (B. mesentericus)): Lane I1: NCTC 2599 (B. cereus): Lane 12: NCTC 926 (B. cereus var. mycoides); Lane 13: NCTC 2610
(B. circulans): Lane 14: NCTC 10337 (B. pumilus); Lane 15: NCTC 10335 (B.firmus); Lane 16: NCTC 10343 (B. polymyxa); Lane 17: NCTC
6355 (B. macerans); Lane 18: ATCC 9885 (B. megaterium); Lanc 19: I kb ladder.
64 Jackie M. Thompson, Christine E.R. Dodd, Will M. Waites
type patterns for B. subtilis N C T C 3610 and NCTC 6276. In some cases,
strains with the same API identifications were seen to have different
ribotypes. Two strains with similar patterns of carbohydrate utilization,
both identified as B. pumilus, gave different patterns when ribotyped, and
neither was identical to the banding obtained for B. pumilus NCTC 10337.
However, ribotypes identical or very similar to those shown by some of
the reference Bacillus strains were seen. For example, one isolate gave an
identical ribotype to that shown by NCTC 10341 (B. licheniformis).
CONCLUSIONS
Bacillus isolates that are capable of causing the spoilage of bread and
possibly food poisoning are important to both the manufacturer and the
consumer. Their differentiation by classical and molecular methods can
highlight the areas within the bakery processing line which need to be
controlled to prevent spoilage occurring.
ACKNOWLEDGEMENTS
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