Research in Microbiology 168 (2017) 379e387

www.elsevier.com/locate/resmic

Spore-forming bacteria responsible for food spoilage

Stephane Andre a,*, Tatiana Vallaeys b, Stella Planchon a
a
Centre technique pour la Conservation des Produits agricoles (CTCPA), Unite Expertise dans la Maitrise du Risque
industriel en Thermoresistants sporules (EMaiRIT'S), 449 Avenue Clement Ader, 84911, Avignon, France
b
Laboratoire ECOSYM-UMR 51119, Universite de Montpellier, Montpellier, France
Received 7 February 2016; accepted 17 October 2016
Available online 27 October 2016

Abstract

This review explores the main spore-forming bacteria involved in the spoilage of various processed foods. Bakery products are specifically
spoiled by Bacillus species, the dominant one being Bacillus amyloliquefaciens, while different Clostridium species classically contaminate
refrigerated vacuum-packed meats. These two genera have also been isolated from milk products, even when milk is pasteurized, sterilized,
dehydrated or fermented, according to heat treatment and storage temperature. Finally, the most heat-resistant microorganisms are isolated in
low-acid canned foods, the three predominant species being Geobacillus stearothermophilus, Moorella thermoacetica and Thermoanaer-
obacterium spp.
© 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Spoilage; Bacillus; Clostridium; Geobacillus; Morella; Thermoanaerobacterium

1. Introduction three distinct orders, adding the more recently described

Spore-forming bacteria are considered a major threat in
heat-treated food plants. Spores show typical resistance to
both chemicals such as disinfectants and physical treatments
(thermal or non-thermal) used in the food processing industry
[1]. Endospore formers may thrive in different parts of the
food processing plant. Surviving spores can thus germinate
and grow out in the product under suitable conditions (time
and temperature during process, nutrients, and physical and
chemical properties) [2].
Endospore-forming bacteria were long classified into two
orders: Bacillales for aerobic rods and Clostridiales for strictly
anaerobic bacteria, with Bacillus and Clostridium as repre-
sentative genera, respectively [3]. The evolution of molecular
tools, and particularly 16S-based molecular classification of
bacterial species, has led to reconsideration of food spoilage
flora diversity. Food spoilage bacteria are now classified into
* Corresponding author.
E-mail address: sandre@ctcpa.org (S. Andre).
Thermoanaerobacterales [4]. Food spoilage Bacillales mem-
bers are typically assigned to the Bacillus, Geobacillus,
Anoxybacillus, Alicyclobacillus, and Paenibacillus genera,
while for Clostridiales, cases of contamination by species of
the Clostridium and Desulfutomaculum genera have been re-
ported [5]. The vast majority of “flat sour” spoilage species
belong to the Bacillus genus and its close relatives, with a few
exceptions. These two genera also include several species with
recognized pathogenic properties such as Bacillus cereus and
Clostridium botulinum. As our purpose was specifically to
address conventional industrial food spoilage flora, and given
that a large number of reviews have been specifically dedicated
to pathogenic species, we did not include these in this review.
The Thermoanaerobacterales order mainly includes ther-
mophilic anaerobes, previously classified under Clostridiales,
but also some now obsolete genera such as Acetogenium and
Thermobacteroides. This order has wide diversity, with case
reports of contaminations by Moorella (formerly known as
Clostridium species), Thermoanaerobacter, Thermoanaer-
obacterium, Caldanaerobacter, Caldanaerobius and Gelria [6].

http://dx.doi.org/10.1016/j.resmic.2016.10.003
0923-2508/© 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
380 S. Andre et al. / Research in Microbiology 168 (2017) 379e387
Classification of food spoilage bacteria is, however, an
emerging area, and more molecular work is required to
investigate the potential uncultivable microbial diversity of
this habitat.
Food spoilage is due to spore germination and outgrowth,
key factors in the stability and non-stability of heat-treated
foods. Food spoilage is characterized by changes in texture
and odor, pH variation and gas production, and depends
mainly on species and food matrix (plant or animal). This
review illustrates the diversity of food spoiling microflora
(bread-making, meat and milk products and different types of
canned foods). Links between different domains can be
established, and it is noteworthy that some species seem to be
product-specific, while others are ubiquitous.
2. Bakery products and aerobic flora
In the early 1980s, various species of Bacillus, such as
Bacillus mesentericus (now Bacillus pumilus) and Bacillus
subtilis, were identified as responsible for spoilage of bakery
products. This spoilage is generally characterized by a stringy
crumb, discolored crust and a melon-like odor. It could be
explained by decreased use of chemical preservatives in bak-
ery product manufacturing [7]. Spoilage was often associated
with the presence of Bacillus spp. in raw materials. Their
spores withstand cooking temperatures close to 100
C for
several minutes. The decimal reduction time D, which ex-
presses the resistance to moist heat, was studied for the three
main species involved. Its values lay between 10 and 56 min at
100
C [8]. In the 1990s, several studies showed the domi-
nance of B. subtilis and Bacillus licheniformis as spoilage
species. Given the low accuracy of identification in the 1990s,
Valerio et al. (2012) conducted a new study on raw materials
composing bakery products, using Fourier-transform near-
infrared spectrometry (FT-NIRS) [9]. Among the thirteen
species identified, three Paenibacillus and ten Bacillus species
were present in more than 50% of the samples, with concen-
trations between 1 and 100 spores g1. The authors identified
Bacillus amyloliquefaciens as the major species instead of B.
Subtilis, as claimed in earlier studies. Special attention has
been directed to antimicrobial substances in order to control
these spoilage micro-organisms [10,11].
3. Refrigerated vacuum-packed meats: anaerobic niche
bacteria
McBride in 1911, and later Sturges and Drake in 1926,
identified Clostridium as the main genus responsible for
spoilage of refrigerated meat called ‘blown pack’, and in
particular, the species Clostridium putrefaciens [12]. New
methods of molecular biology available in the 1990s identified
further species: Clostridium estertheticum, Clostridium algi-
dicarnis, Clostridium frigidicarnis, Clostridium gasigenes,
Clostridium algidixylanolyticum, Clostridium frigoris and
Clostridium bowmanii. These species are particularly associ-
ated with refrigerated and vacuum-packed meat products.
Meanwhile, many studies regularly identified C. botulinum or
other Clostridium species that have since been renamed (as
described in the Introduction). In all cases, spoilage is char-
acterized by an abundant production of gas, which causes the
plastic packaging to swell, a strong smell, and loss of meat
color and texture. This spoilage can result from very low
initial contamination. Clemens et al. (2010) showed that as
little as one spore of C. estertheticum per product could spoil
beef and lamb during storage [13]. These species are classified
according to their growth temperature into psychrotrophes
(optimum at 12
C and growth at 37
C) and psychrophiles
(optimum at 8e12
C and no growth above 30
C). Spoilage
by C. gasigenes has sometimes been observed even at 1
C
or 2
C [14]. Although many species are regularly isolated,
the main ones (cited above) are usually found in the same
sample, suggesting that there may be a synergistic effect be-
tween species [15]. The combination of psychrotolerant and
anaerobic characteristics makes these species notoriously hard
to isolate and handle. Many studies have been published that
propose new isolation media [16]. In parallel, some work on
defining methods of rapid identification have been published
by Pichner et al. (2012) with a PCR for C. estertheticum sensu
stricto or C. estertheticum-like [16]. In the CTCPA laboratory,
C. algidicarnis has been predominantly found in recent years
in spoiled pasteurized “foie gras” (fat duck liver). The years
2011e2012 were one period when C. bowmanii critically
affected several industrial operators (personal data). In this
particular matrix, the same defects were observed: strong
smell and gas production.
Sources of contamination were sought by several authors.
Broda et al. (2002) found that soil particles on carcass skins,
together with feces, needed to be monitored, with particular
care in health control procedures to reduce slaughterhouse
contamination by C. gasigenes or C. estertheticum [17].
Moschonas et al. (2009) worked on 1680 samples collected in
beef abattoirs in the course of a year, and tracked C. ester-
theticum and C. gasigenes in particular [18]. They found that
up to 38% of the samples taken at the slaughterhouse in May
were positive. Ten to fourteen percent of the soil samples
analyzed contained both species. For example, Silva et al.
(2011) identified only C. gasigenes and C. algidicarnis in both
spoiled and compliant beef samples, and at the slaughterhouse
on skin samples [19]. The specific localization of Clostridium
spp. on meat surfaces could result from cross-contamination.
This was confirmed in washing tests with both hot and cold
water by Adam et al. (2013) [20]. Simple washing increased
shelf life by 12 and 13 days relative to untreated samples.
Feces were also characterized as a vector of contamination by
psychrophilic Clostridium: high counts of C. estertheticum and
C. gasigenes were found [18].
4. Dairy products: diversified spore-forming flora
4.1. Fresh milk: the basic source
Studies on the spoilage of fresh milk include spoilage of
end-products such as dairy products and/or contamination of
dairy processing lines.
 S. Andre et al. / Research in Microbiology 168 (2017) 379e387
Dairy products, whether refrigerated or stored at room
temperature, can also be spoiled by spore-forming bacteria,
and when milk is fermented, the ‘spore hazard’ remains. In
many studies, high spore concentrations, up to more than 5000
spores ml1, were found in fresh milk [21]. In a literature
review, Quigley et al. (2013) confirmed that B. cereus is the
microorganism most often responsible for organoleptic
spoilage of refrigerated dairy products, besides being a path-
ogen [22]. In 1994, Sutherland and Murdoch identified it
throughout the production line from dairy farm to refrigerated
end-product [23].
Lücking et al. (2013) studied both milk spoilage flora and
the dairy production environment [24]. In their study, 43
different species were detected, 75% of which came from
spoiled products and 25% from processing lines. The B. cereus
group and B. licheniformis species occurred most often. The B.
subtilis and B. amyloliquefaciens or Bacillus smithii and Geo-
bacillus pallidus species predominated, respectively, among
mesophilic and thermophilic flora after selection of highly heat-
resistant spores (HHRS) by heat treatment at 80
C for 10 min.
The Geobacillus stearothermophilus species prevailed among
heat-resistant flora (treatment at 100
C for 10 min). This broad
diversity is due to variability in the products analyzed, which
came from widely differing processes. Other authors have re-
ported seasonal variations in mesophilic or psychrophilic
spores contaminating pasteurized milk [23]. Winter is the most
favorable season for mesophilic micro-organisms, with summer
and autumn favoring the proliferation of psychrophilic Bacil-
lus. This pattern may be related to the flora associated with
cattle food, based either on fresh grass or hay and silage. In
addition, Muir et al. (1986) linked processing-plant contami-
nation directly to contamination at the previous step, i.e. the
dairy farm [25]. All the studies cited concern only aerobic
species, but anaerobic flora is also probably involved, as strictly
anaerobic genera (Clostridium spp.) have been identified in
cheese spoilage, as discussed below. This confirms that
occurrence studies are valid only for the micro-organisms
investigated. Moreover, the methods applied are not universal,
and so whole sections of microbial ecology can be passed over
merely by the method of isolation used. Molecular biology can
be used directly on the environment or food sample to detect/
quantify microbial flora, reducing the analytical bias of culture-
dependent methods. In the case of cheese spoilage by swelling,
specific studies on anaerobic organisms have shown a direct
link between the quality of the milk, related in particular to
silage, and spoilage of the finished product [26,27]. For Das-
gupta and Hull (1989), butyric Clostridium species were
observed in milk all year round. However, autumn and winter
were the periods when more than 50% of samples contained at
least 1 spore per 5 ml of milk. The authors correlate these
values with the higher frequency of seasonal spoilage of Gouda
or Swiss cheese by Clostridium tyrobutyricum [28]. In another
study, the summer period was found to be most favorable for
contamination of milk with anaerobic spore-forming bacteria,
and consequently for spoilage of Manchego cheese [29]. These
authors reported that 97% of samples were contaminated, with
on average 14.5 spores ml1 in this season.
381
Species diversity is due to broad variety in dairy products
and their production processes. Dairy products are accordingly
separated into four categories in this review: (i) processed and
refrigerated products such as pasteurized milk, (ii) sterilized
milk and (iii) milk powder, both stable at ambient temperature,
and (iv) fermented products, i.e. cheeses.
4.2. Pasteurized milk: refrigerated milk
Pasteurization, or high temperature short time (HTST)
treatment, is performed at 72
C for 15 s (destruction of
Coxiella burnetti). It does not destroy spores of mesophilic
species that can grow during storage. With this treatment, or
any equivalent treatment, the product shelf life is between 2
and 3 weeks.
Since 2004, several authors such as Ivy et al. (2012) have
shown the preponderance, among psychrophilic aerobic spore-
forming bacteria, of the genus Paenibacillus (>50%) relative
to the genus Bacillus (>30%) in fresh and pasteurized milk
[30]. Occurrence of these new species is due to improved
identification methods that allow differentiation of the two
genera using phenotyping methods. In the genus Bacillus, the
species B. licheniformis and B. subtilis observed in fresh milk
are still largely predominant among mesophilic species, while,
for psychrophilic species, the B. cereus group is the largest
(with B. cereus or Bacillus weihenstephanensis/mycoides). For
the genus Paenibacillus, Paenibacillus odorifer was the main
species identified in this study, followed by Paenibacillus
amylolyticus (respectively, 62% and 25% of isolates). Ranieri
and Boor (2009) observed changes in the micro-organisms
present during the microbiological lifetime of pasteurized
milk [31]. From the first day to 17 days of storage, Bacillus
predominated, whereas Paenibacillus dominated at the end of
shelf life.
Taking into account the entire process, the relation between
the flora of fresh milk and that of pasteurized milk was
demonstrated by similar alleles of isolates based on the rpoB
DNA sequence-based subtyping method from the two ends of
the process [32]. For Scheldeman (2005), who studied the
diversity of aerobic HHRS directly in 17 Belgian dairy farms,
7 genera were identified in 700 aerobic isolates. B. lichen-
iformis and G. pallidus were the predominant species, due to
their high degree of resistance to heat treatment [33].
4.3. Sterilized milk: milk stable at ambient temperature
Sterilization treatment is performed at 130
C for 4 s,
termed the ultra-high temperature (UHT) schedule. Species
responsible for spoilage of sterilized milk differ from those in
pasteurized milk because of the higher temperature of heat
treatment. In 1979, Id and Schaal often isolated aerobic spores,
whereas anaerobic spores were scant [34]. Although Bacillus
coagulans and G. stearothermophilus have been isolated from
this type of product, several research teams have worked
specifically on Bacillus sporothermodurans in the last few
years [2,35]. The presence of G. stearothermophilus would be
expected after UHT treatment, as it is a highly resistant spore,
382 S. Andre et al. / Research in Microbiology 168 (2017) 379e387
but finding B. sporothermodurans is surprising. Huemer et al.
(1998) and later Scheldeman (2006) confirmed that this spe-
cies belongs to the HHRS group, with a D value at 140
C of
up to 5 s [36,37]. Accurate identification of the origin of this
species is still difficult because it has been detected only in the
finished product, i.e. in UHT milk [37]. These species were
thus selected by heat treatment, and they can grow and damage
the product unhindered by competition with other flora.
4.4. Dehydrated milk: milk powder
Aerobic flora has also been exclusively identified in milk
powder. Milk powder is mainly considered as a vector of
spores: spoilage occurs when milk powder is used because of
spore germination in a final product with a higher water
activity.
In a study conducted in 18 countries, Rueckert et al. (2004)
found G. stearothermophilus and Paenibacillus flavithermus to
be the most frequently occurring micro-organisms in milk
powder [38,39]. Those authors studied the distribution of these
thermophilic spores in milk powder for infants in China. They
identified both species cited above, along with B. lichen-
iformis. These three species represented more than 80% of the
isolates. B. licheniformis was the species most frequently
found in the samples in which spore contamination was mostly
less than 1000 CFU g1 but exceptionally reached
10,000 CFU g1. For Murphy et al. (1999), G. stear-
othermophilus and B. licheniformis predominated among milk
thermophilic flora, with concentrations from 30 to
300 CFU ml1 [40]. This thermophilic flora is considered a
good indicator of the purity of finished products. When spore
concentrations exceed 104 spores g1 of milk powder, the
cleanness of the process can be challenged, in particular
because thermophilic species increase during the process
[41,42].
Scott et al. (2007) showed two particular areas of prolif-
eration along the production line: the plate exchanger for
preheating, and the evaporator [39]. As early as the preheating
stage, the quantity of thermophilic spores can increase by 1e4
logarithmic units and peak in the evaporator. This concentra-
tion can remain at this level or decrease according to the final
process. Murphy et al. (1999) identified the evaporator as an
area of proliferation of thermophilic spore-forming bacteria,
also favored by the preheating step [40].
4.5. Fermented milk: cheeses
In cheeses, only strictly anaerobic spore-forming bacteria
are responsible for spoilage, characterized by abundant gas
production. All the Clostridium species in phylogenetic group
I can be responsible for ‘late swelling’ spoilage. They can
cause mostly hard cheeses to split open (Comte, Emmental,
Beaufort, Gouda, etc.) [43,44]. The defect arises from the
breakdown of lactate by bacteria whereby butyric fermentation
produces two gases (CO2 and H2) along with butyric acid,
which confers an unwanted rancid taste [28,45]. Hence,
analysis of volatile components such as butyric acid can be
used to detect abnormal fermentation during cheese-making.
Many studies have been conducted to characterize spoilage
flora. However, only four species are frequently responsible
for this defect: Clostridium beijerinckii, Clostridium butyr-
icum, Clostridium sporogenes and C. tyrobutyricum [27e29].
Clostridium cochlearium has occasionally been isolated [46].
These species are able both to withstand pasteurization of milk
through forming spores, and then to grow in the product,
causing damage to cheeses. Cases of spoilage can arise from
small numbers of spores (200 spores l1 of milk), are frequent
above 400 spores l1 and more generally occur above 1000
spores l1 [26].
Methods based on molecular biology have been developed
for faster detection on industrial lines or in products for the
species most often involved in spoilage (C. beijerinckii, C.
butyricum, C. sporogenes and C. tyrobutyricum) [27].
5. Canned foods: the most heat-resistant micro-organisms
Food canning is a simple process producing several types
of end-products according to pH. The first threshold, starting
from neutral pH, is 4.6 (international) or 4.5 (in some coun-
tries in Europe). Above this pH, canned foods are considered
non-acid, and the risk of botulism is not considered as
controlled unless: (i) there is a sterilization treatment at a
temperature above 100
C; and (ii) the treatment time reaches
a sterilizing value (F0) of at least 3 min, calculated at a
reference temperature of 121.1
C and a z value (temperature
leading to a 10-fold change in D value) of 10
C in most
canned foods. This time span (F0) is needed to reduce 12 logs
(12D) of spore population of C. botulinum. Below pH 4.6,
canned foods are classified as acid, and pasteurization may be
sufficient. A pasteurization value is calculated at a reference
temperature of 93.3
C and a z value of 8.89
C. C. botulinum
spores are not destroyed by pasteurization, but cannot
germinate at this pH. A secondary classification can be made,
with moderately acidic canned foods up to pH 3.8, in which
some rare acid-tolerant spore-forming species can still grow.
Below this pH, canned foods are considered very acidic and
only one acidophilic spore-producing genus can cause
spoilage.
5.1. Acid canned foods
Fruit juices and concentrates belong to the strongly acid
canned foods. The pH of these products is generally around
3.5, but some fruits such as blackcurrant give pH values of 2.5.
At these pH values, Alicyclobacillus is the only acidophilic
spore-producing genus described up to now as a cause of
spoilage. This genus was characterized by Wisotzkey et al. in
1992 [47]. Many authors later identified Alicyclobacillus spp.
as responsible for the spoilage of acidic canned juice and
concentrates [48e50]. Some strains in the genus Alicycloba-
cillus have greater spoilage ability because they can produce
large amounts of gaiacol. This substance, even at very low
concentrations of 2 ppb, adversely affects the odor of the
product [51].
 S. Andre et al. / Research in Microbiology 168 (2017) 379e387
In apple juice, the Alicyclobacillus genus was identified in
35% of spoiled juice, and authors detected a seasonal variation
in the spoilage of juice, with a peak in spring and summer
[49]. In orange juice, 11 out of 75 samples were contaminated
by the same genus [48]. In other work, 6.1% of tropical fruit
juice concentrates contained Alicyclobacillus, with 81% of
Alicyclobacillus acidoterrestris and 19% of Alicyclobacillus
acidocaldarius [51]. A. acidoterrestris was isolated in much
greater amounts than A. acidocaldarius in fruit concentrates,
wash water and soil [52].
5.2. Moderately acid canned foods
Few bacterial species can spoil products when the pH of the
food matrices is in the range 3.8e4.5. Such foods include
canned fruit, such as peaches or pears in syrup, and tomatoes.
In this last type of spoiled product, Townsend isolated the
species Clostridium pasteurianum in 1939 [53]. Deligaris et al.
(1996) isolated different Clostridium species from process
water on a peach canning line [54]. Although C. sporogenes
and C. beijerinckii have been isolated, the latter represented
84% of isolates. Spoilage by C. pasteurianum species displays
the greatest hazard because of its growth limit pH of 3.8e3.9.
The specific capacity of C. pasteurianum to grow at low pH
was confirmed more recently by Bocchi and Previdi (2004).
Those authors isolated three species of Clostridium from
different canned foods: C. pasteurianum (from peaches and
pears), C. tyrobutyricum from tomatoes and C. beijerinckii
from acidified mushrooms [55]. However, only C. pasteur-
ianum showed the ability to germinate at pH values below 4.2.
The lower pH limit observed by this team was 3.5 in a peach
puree. Several authors also isolated this species from an acidic
matrix such as orange and apple juice [56]. Although few
studies have addressed its ecology in the production environ-
ment, some articles on its heat resistance confirmed the
importance of considering C. pasteurianum in canned foods
with a pH between 3.7 and 4.2 [50,56].
Some authors have identified strains that are particularly
acid-resistant. Everis and Betts (2001) found a growth-
limiting pH value of 4.3 for isolates of Paenibacillus poly-
myxa and C. tyrobutyricum in low-acid canned foods [57].
Bacillus coagulans, which can grow at pH 4.2e4.3, is the
species most often identified in spoilage of moderately acidic
canned foods such as pasteurized tomatoes or “ratatouille”, a
south of France vegetable recipe [[58], personal data]. It was
clearly identified only by Gordon et al. (1973) after earlier
isolation of the species Bacillus thermoacidurans by Berry
(1933) in spoiled canned tomatoes, and by Hammer (1915) in
coagulated milk powder [59e61]. Many synonyms have
been used, e.g. Bacillus dextrolacticus, Bacillus thermoaci-
dificans and Lactobacillus cereale. Today, B. coagulans is
referred to as Lactobacillus sporogenes, a well-known pro-
biotic in the field of food additives. Ecological data on this
species are scarce, probably because pH and heat treatment
can be enough to control contamination by it. No specific
ecological study has been undertaken for this species in a
canned food.
383
5.3. Low-acid canned foods
Pirone and La Pietra analyzed 1800 samples of non-stable
(spoiled) canned foods between 1991 and 2001 [62]. When
spoilage was of microbiological origin, only 20% was due to
insufficient heat treatment. For mesophilic micro-organisms,
identification was limited to the genera Bacillus and Clos-
tridium. For incubations at high temperature, the micro-
organisms responsible for spoilage were Thermoanaer-
obacterium thermosaccharolyticum, G. stearothermophilus
and, occasionally, Desulfutomaculum nigrificans. In another
10-year study in France, Andre et al. (2013) observed that 70%
of the bacteria responsible for spoilage of canned non-stable
foods (cooked meals, vegetables) after incubation at 55
C
belonged to only two species: Moorella thermoacetica (36%)
and G. stearothermophilus (34%) [6]. Moorella is a spore-
producing anaerobic genus described as highly heat-resistant
[6,63]. Its growth in canned foods causes acidification and
sometimes swelling [64]. This genus has also been identified
in canned ‘shiruko’ and coffee [65]. G. stearothermophilus has
also been claimed to cause spoilage of canned food in many
other studies since 1920 [66,67]. The Thermoanaerobacterium
and Bacillus genera have been identified in fewer than 10% of
spoiled samples at 55
C with different species (T. thermo-
saccharolyticum, B. coagulans, B. smithii and B. lichen-
iformis). The other genera accounted for fewer than 5% of
spoiled samples. Some of them were never identified in can-
ned foods or in food such as Caldanaerobius, Gelria, Anox-
ybacillus, Paenibacillus, Thermoanaerobacter and
Clostridium. Other studies have identified a different ecology
in spoiled cans. Presland et al. (2004) implicated three genera
in the spoilage of canned food (Geobacillus, Bacillus and
Moorella) [68]. Dotzauer et al. (2002) identified Thermoa-
naerobacterium and Thermoanaerobacter, but not Moorella
[69]. Desulfutomaculum, not detected by Andre et al. (2013),
was described as causing spoilage of canned food such as
'shiruko' and vegetables [6,65].
Given the heat treatments applied to stabilize canned foods,
the most heat-resistant micro-organisms would be expected to
be among those responsible for spoilage. In the food industry,
M. thermoacetica is the most highly heat-resistant species,
with D values at 121
C up to 30 min [6,65]. The D value at
121
C obtained for G. stearothermophilus ranged between 1
and 6 min [70].
In the last five years, some researchers have started to
develop simple detection tools using PCR, or more complex
ones using micro-arrays [71,72]. Surprisingly, only one com-
plete sequenced genome has so far been reported for G.
stearothermophilus, despite its strong impact and the many
studies devoted to it (3671 references in Pubmed). Two studies
recently addressed ecology in canning lines for green beans
and peas using the G. stearothermophilus species as a marker
[73]. They found that some individual unit steps, such as
blanching, increased the spore population.
Few data are available on spoilage of canned foods at
ambient temperature or after incubation in mesophilic condi-
tions, as the causes may be varied (e.g. insufficient heat
384 S. Andre et al. / Research in Microbiology 168 (2017) 379e387
Table 1
Main species responsible for spoilage of different food types and their physiological characteristics.
Food Species Growth temperature and pH range Heat resistance (D value)
Bakery products Bacillus amyloliquefaciens 15
C to 50
C, opt. 30e40
Ca 2,5 min at 110
C, z ¼ 12,8
Cb
and aerobic flora 2,1 min at 115
C, z ¼ 7,4
Cc
Refrigerated Clostridium algidicarnis >4 to 37
C, opt 25e30
Cd 230 min at 90
C, z ¼ 10,5
Ce
vacuum-packed meats 5 min at 95
Cc
Clostridium putrefaciens <5 a 30
C opt 15e25
Cf 14 min at 80
Ch
<0
Ce<37
Cg
Clostridium estertheticum 1
C to 15
Ci 48 s a 100
Cj
Clostridium gasigenes 1,5 a 26
C, opt 20e22
Ck No data available
Pasteurized milk Bacillus No type species
Paenibacillus
Sterilized milk Bacillus sporothermodurans 0 to 50
Cl 6 s at 140
Cm
Dehydrated milk Geobacillus stearothermophilus 40 to 70
C, pH mini 5,0n 3 min at 121
C, z ¼ 9.1
Co
T5D < 0,5 min to 12,1 min at 120
Cn
Anoxybacillus flavithermus 43 to 62
C, opt 57
Cp 2 min at 110
C, z ¼ 13
Cp
Fermented milk Clostridium tyrobutyricum <25 to 45
C, opt 30e37
Cf 0,053 min at 120
C, z ¼ 14,5
Cr
<12e15
C to 40.2e43.3
Cq
Clostridium butyricum 8e11
C-NDs 0,045 min at 120
C, z ¼ 11,7
Cr
10
C-ND opt 30e37
Cf 4,7 min 100
Ct
Clostridium sporogenes <25 to >45
C, opt 30e40
Cf 1,28 min at 121
C, z ¼ 11,1
Cu
<11
C-NDv 0,8 to 2,2 min at 105
C, z ¼ 6,6e7,8
Cw
Clostridium beijerinckii 25 a 45
C opt 37
Cx No data available
Acidic canned foods Alicyclobacillus acidoterrestris <35
Ce>55
Cy 1.7 min at 95
C, z ¼ 7.6
Caa
25
Ce60
C pH 2.2e5.8z 2.3 min at 102
Cab
Moderately acidic Clostridium pasteurianum pH limit: 3,5ac or 4,3ad/c 1,9 to 4,31 min at 100
C, z ¼ 5,05 to 10,8 in foodae
canned foods 4,4 min at 90
C, z ¼ 11
Cad
13,6 min a 90
C, 3,8 min at 95
Cc
Bacillus coagulans <30 to <61
C, opt 40e57
C, 1.1 to 92,3 min at 110
C, z ¼ 6,8 to 9,6
Cag
pH limit 4.0f 0,05 to 38,6 min a 110
C, z ¼ 8,3 to 5,7
Cc
30
C to 55
Caf
Low acid canned foods Geobacillus 40 to 70
C, pH mini 5,0n D ¼ 3 min at 121
C, z ¼ 9.1
Co
stearothermophilus T5D < 0,5 min to 12,1 min a 120
Cn
Moorella thermoacetica 1 to 65
C, pH mini 5,7ah D ¼ 1e10 min at 130
Cah
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 S. Andre et al. / Research in Microbiology 168 (2017) 379e387
treatment or recontamination after heat treatment due to leaky
packaging). In some isolated studies taking into account all
spoilage, Richardson (1972) found that 64% of microbial
spoilage came from recontamination through faulty packaging
[74]. The other cases were due to defective sterilization, but
the species were unfortunately not identified. However, since
1922, C. botulinum, representing the main threat in canned
foods, with the greatest impact on consumers, has remained
the reference micro-organism for many canned foods, with the
destruction of 12 log CFU ml1 (12D) as a minimal process
requirement [75]. The “12D rule” for controlling the botulism
hazard has come under criticism in studies on the recovery of
surviving spores [76,77]. In addition, there exist many more
heat-resistant spoilage micro-organisms, which are used as
references to determine scales to assess the stabilization of
canned foods.
6. Conclusion
Spoilage due to spore-forming bacteria causes high eco-
nomic losses in the food and feed industry. Sterilization was
long considered a widely generalized means of food conser-
vation, and thermophilic or thermotolerant bacteria were the
common agents in food spoilage. Today, consumer demand for
preservation of organoleptic, nutritional and health properties of
food is rising. Accordingly, the food industry has made tech-
nological modifications to materials (replacement of metal cans
by heat-resistant plastics, can lining, etc.) and processes
(pasteurization, dehydration, etc.). These modifications entail
inherently different selective pressures that may cause existing
contaminants to adapt, or novel contaminants to emerge. The
scientific community therefore needs increased knowledge of
bacteria ecology and bacteria physiology to address new threats.
There are several ways in which the impact of spore for-
mers in food spoilage can be lessened, such as focusing on the
source of contamination and using combined treatments of
food matrices to induce germination, but care should be taken
to avoid microbial growth. The factors controlling spore
forming, spore germination and food-damaging mechanisms
need to be well known if they are to be inhibited, and novel
whole-genome expression studies may help. At the same time,
the list of contaminants may be incomplete: progress in met-
agenomics approaches, combined with diversification of food
conservation tools and means, will doubtlessly go on to reveal
unexpected uncultivable resistant forms.
In conclusion, this review provides food microbiologists
with an overview of spore-forming bacterial species respon-
sible for spoilage. The main species isolated from each
product group (baked goods, meat, milk and canned foods)
detailed here and their main physiological characteristics
(growth temperature and pH, heat resistance) are found in
Table 1. This review presents technology and advances in
knowledge concerning spoilage in different food matrices.
Conflict of interest
None.
385
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