Food Microbiology 45 (2015) 45e53

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Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Bacterial spoilers of food: Behavior, tness and functional properties

Benot Remenant a, b, Emmanuel Jaffrs a, b, Xavier Dousset a, b, Marie-France Pilet a, b,
Monique Zagorec a, b, *
a
b

INRA, UMR 1014 Secalim, F-44307 Nantes, France

LUNAM Universit, Oniris, F-44307 Nantes, France

a r t i c l e i n f o

a b s t r a c t

Article history:
Available online 24 March 2014

Most food products are highly perishable as they constitute a rich nutrient source for microbial development. Among the microorganisms contaminating food, some present metabolic activities leading to
spoilage. In addition to hygienic rules to reduce contamination, various treatments are applied during
production and storage to avoid the growth of unwanted microbes. The nature and appearance of
spoilage therefore depend on the physiological state of spoilers and on their ability to resist the processing/storage conditions and ourish on the food matrix. Spoilage also relies on the interactions between the microorganisms composing the ecosystems encountered in food. The recent rapid increase in
publicly available bacterial genome sequences, as well as the access to high-throughput methods, should
lead to a better understanding of spoiler behavior and to the possibility of decreasing food spoilage. This
review lists the main bacterial species identied as food spoilers, their ability to develop during storage
and/or processing, and the functions potentially involved in spoilage. We have also compiled an inventory of the available genome sequences of species encompassing spoilage strains. Combining in silico
analysis of genome sequences with experimental data is proposed in order to understand and thus
control the bacterial spoilage of food better.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Bacterial genomes
Meat products
Seafood products
Dairy products

1. Introduction
Food spoilage caused by microorganisms results from microbial
metabolism leading to the production of molecules that alter the
sensory quality of the products, in particular the aspect (including
texture and color) and odor (Gram et al., 2002). There is thus a large
diversity of spoilage: from volatile compound production, which
provokes off-odors and/or blown-pack spoilage of vacuum-packed
foods, to slime or liquid production, or various color deteriorations
(Borch et al., 1996; Brightwell et al., 2007; Dainty et al., 1989;
Nychas et al., 2008). In most studies, spoilage characteristics are
assessed by sensory analyses and/or by measurement of various
physicochemical parameters such as the amount or nature of
different metabolites or gases, color, pH, etc., which are then
correlated to the presence of microorganisms (Diez et al., 2009;
Jaffrs et al., 2011; Mac et al., 2013; Paarup et al., 2002). It is

* Corresponding author. INRA, Oniris, UMR 1014 Secalim, rue de la Graudire,

CS82225, F-44322 Nantes cedex 3, France. Tel.: 33 2 51 78 55 42; fax: 33 2 51 78
55 20.
E-mail address: monique.zagorec@oniris-nantes.fr (M. Zagorec).
http://dx.doi.org/10.1016/j.fm.2014.03.009
0740-0020/ 2014 Elsevier Ltd. All rights reserved.

often accepted that the bacteria responsible for spoilage are those
belonging to the major population in the spoiled product. A large
majority of studies reported in the literature rst used classical
plating and phenotyping methods to characterize the bacterial
population of spoiled products (Dainty and Mackey, 1992;
Rattanasomboon et al., 1999). More recently, an increasing number of articles have reported species identication through DNA
extraction, followed by various types of molecular identication
such as Denaturing Gradient Gel Electrophoresis (DGGE) or Temporal Temperature Gel Electrophoresis (TTGE) performed on 16S
rRNA gene portions amplied by PCR (Doulgeraki and Nychas,
2013; Ercolini et al., 2006; Jaffrs et al., 2009). The use of highthroughput 16S rDNA pyrosequencing or metagenomics relying
on the whole bacterial food ecosystem, including spoilers, is still
scarce. By such approaches, many different bacterial species have
been reported as specic spoiler organisms (Nieminen et al., 2012).
The involvement of these putative spoilers has sometimes been
proven by re-inoculation of candidate strains into food model
systems to reproduce spoilage. Yet, only a few studies have successfully proved the exact nature of spoilage and the bacterial
functions involved in producing the spoiling molecules
(Jskelinen et al., 2013).

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B. Remenant et al. / Food Microbiology 45 (2015) 45e53

2. The main bacterial species known as food spoilers, their

spoiling effects and tness
Many studies have reported the main bacterial species associated with various types of spoilage from a large range of foodstuffs.
The ability of some species to grow under or resist the harsh conditions encountered during processing and storage has been
described. For various foods of animal origin, Gram-positive bacteria, and especially Firmicutes, have often been reported as
spoilers (Bron and Kleerebezem, 2011). In particular, lactic acid
bacteria (LAB), encompassing Lactococci, Lactobacilli, Leuconostoc,
Weissella, and Carnobacteria species, as well as Enterococci are
frequently associated with spoilage. In fact, although LAB are
generally benecial for food and are used for the fermentation of a
variety of food and raw materials, where they contribute to avor,
texture and shelf-life (Bron and Kleerebezem, 2011), some species
can play a signicant role in food spoilage and decay. This is the
case, for instance, of Lactobacillus alimentarius, known as a specic
spoilage organism in marinated herring (Lyhs et al., 2001), and of
Lactobacillus sakei and Lactobacillus curvatus, also found in the
spoilage microbiota of herring (Lyhs and Bjorkroth, 2008). L. sakei
was reported as predominant in the spoilage microbiota of sliced,
vacuum-packed, smoked, oven-cooked turkey breast llets, which
developed sour spoilage avors (Samelis et al., 2000). L. curvatus
was also found to be one of the specic spoilers in cold-smoked
salmon (Jrgensen et al., 2000), as was L. sakei (Stohr et al., 2001;
Joffraud et al., 2006). The latter was also isolated in the spoilage
microbiota of brined and drained shrimp stored under modied
atmosphere packaging (Mejlholm et al., 2012, 2008). Carnobacterium is another LAB genus commonly involved in the spoilage
process of food, with notably Carnobacterium divergens and Carnobacterium maltaromaticum known to dominate the spoilage
microbiota of different meat and seafood products, particularly
those packed under vacuum or modied atmosphere (Laursen
et al., 2005; Leisner et al., 2007; Vasilopoulos et al., 2008). Carnobacterium sp. was shown to be an important contributor to the
spoilage-related microbiota of freshly prepared chicken products
stored aerobically under refrigeration (Liang et al., 2012).
C. maltaromaticum is considered a strong spoiler of raw salmon
(Salmo salar) llets stored under modied atmosphere packaging
(Mac et al., 2013), and also of cooked and peeled shrimps, where it
can be responsible for cheese/feet, sour/fermented and milkyboiled off-odors (Jaffrs et al., 2011). Other LAB genera and species have also been incriminated in the spoilage process of various
food products. Enterococcus faecalis and Enterococcus faecium can
be involved in the greening of meat products (Foulqui-Moreno
et al., 2006) while E. faecalis is also a spoilage species of artisantype cooked ham under modied atmosphere packaging
(Vasilopoulos et al., 2008). Leuconostoc species like Leuconostoc
gelidum and Leuconostoc gasicomitatum are known to produce
slime on marinated herring (Lyhs et al., 2004). The role of
L. gasicomitatum in gas and slime formation, the development of
pungent acidic and buttery off-odors, and the greening of beef have
been proven by challenge tests (Johansson et al., 2011). The species
Leuconostoc mesenteroides was recovered as the predominant
spoiler from the microbial community of blown packs of vacuumpackaged beef (Yang et al., 2009). Vihavainen et al. (2008) reported the spoilage potential of several Leuconostoc species on
vegetable sausage products. They demonstrated that L. gelidum,
L. gasicomitatum, and L. mesenteroides were the predominant LAB in
commercial vegetable sausages. The inoculation of these species
into vegetable sausages resulted in the formation of gas, slime, and
a sour off-odor. The Weissella genus, notably Weissella confusa, was
also found to be one of the major spoilers of oyster gill stored at
10  C (Chen et al., 2013). Diez et al. (2009) highlighted that the

sensory and volatile changes during the anaerobic cold storage of

morcilla de Burgos, a typical blood sausage from Spain, could be
induced to a great extent by Weissella viridescens and
L. mesenteroides. Both species have also been associated together in
the spoilage of other food products such as sliced vacuum-packed
cooked ham after high-pressure treatment (Han et al., 2011).
In addition to LAB species, other Gram-positive bacteria can play
a signicant role in food spoilage. One of the most prominent is the
psychrotrophic species Brochothrix thermosphacta, known as an
important spoiler bacterium of various food matrixes
(Rattanasomboon et al., 1999; Russo et al., 2006). B. thermosphacta
is an ubiquitous microorganism throughout the meat production
chain, from animal to food. It has been isolated from beef carcasses
during boning, dressing and chilling. Moreover, lairage slurry, cattle
hair, rumen content, walls of slaughterhouses, hands of workers, air
in the chill room, neck and skin of the animals as well as the cut
muscle surfaces have all been shown to be contaminated by this
organism (Nychas et al., 2008). In meat products packed under
vacuum or modied atmosphere, B. thermosphacta can dominate
the spoilage microbiota at the expense of other genera, such as
Carnobacterium, Lactobacillus or Leuconostoc (Borch et al., 1996;
Ercolini et al., 2006). B. thermosphacta was also found in the
spoilage microbiota of raw turkey breasts (Samelis et al., 2000),
pork meat (Bohaychuk and Greer, 2003) and cooked ham (Samelis
et al., 1998). It was identied as the dominant spoiler in freshly
prepared chicken products (Liang et al., 2012) and was responsible
for cheesy/dairy off-odor and discoloration of chilled vacuumpackaged lamb meat (Gribble and Brightwell, 2013). Seafood
products also provide a favorable niche for B. thermosphacta
development, i.e. neutral pH, high water activity (Aw) and high
content of low molecular weight compounds such as free amino
acids and nucleotides (Jeyasekaran et al., 2006). Nickelson et al.
(1980) isolated this species from minced sh esh, at various production stages. It was also isolated from sh (Archosargus probatocephalus) packed under CO2 (Lannelongue et al., 1982) and frozen
sh such as cod (Gardner, 1981). On refrigerated tuna (Thunnus
alalunga) steaks, packed under modied atmosphere, the growth of
B. thermosphacta was not affected by CO2, as long as O2 was also
present at a concentration above 0.2% (Lopez-Galves et al., 1995).
More recently, B. thermosphacta was found to be predominant in
meat and coral of king scallops (Pecten maximus), during storage
under air at low temperature (Coton et al., 2013). The growth of
B. thermosphacta in vacuum-packed meat products depends largely
on the amount of available oxygen remaining in the package.
B. thermosphacta may become the dominant spoilage species when
oxygen is present, but is displaced by Lactobacillus species under
anaerobic conditions. The spoilage potential of B. thermosphacta in
vacuum-packed meat products is thus inuenced by factors that
control the level of oxygen in the pack like, for example, the lm
permeability, the residual gas composition and the pack integrity
(Gribble and Brightwell, 2013). In vacuum packs, there is often
enough residual oxygen for B. thermosphacta to metabolize offensive off-odors. The anaerobic growth of B. thermosphacta at chill
temperatures is also reduced by meat pH values of less than 5.8;
consequently B. thermosphacta is most often associated with the
early spoilage of high pH meat cuts of lamb and beef (Bell, 2001;
McClure et al., 1993).
Several species have been reported as responsible for the socalled blown-pack defect of vacuum-packed food due to gas production. This defect is mostly associated with chilled fresh meat
particularly beef, lamb and venison, but was also reported for
cooked meat products (Broda et al., 1996). The causative agents
include psychrotrophic Enterobacteriaceae or Clostridium species
such as Clostridium estherteticum (Brightwell et al., 2007;
Hernandez-Macedo et al., 2012; Yang and Badoni, 2013). Other

B. Remenant et al. / Food Microbiology 45 (2015) 45e53

Clostridium species (Clostridium algidixylanolyticum, Clostridium

frigidicarnis, Clostridium frigoris, Closdridium gasigenes, and Clostridium algidicarnis) may also cause blown pack spoilage of
vacuum-packed beef (Silva et al., 2011; Yang and Badoni, 2013). In
addition, the acid tolerant species Clostridium perfringens, Clostridium barati, and Clostridium butyricum have been reported as the
causative agents of the spoilage of canned pasteurized mung bean
sprouts, stored under acidic conditions (de Jong, 1989).
Gram-negative bacteria with species belonging to the genera
Serratia, Hafnia, and Pseudomonas have also often been incriminated. Some species like Shewanella baltica and Photobacterium
phosphoreum, typical of sea water or sediment, have been specifically reported in seafood spoilage. Shewanella sp., notably Shewanella putrefaciens or S. baltica, are typical spoilers of fresh marine
sh (Gram and Huss, 1996; Broekaert et al., 2011), such as halibut
(Hovda et al., 2007b), sea bass (Papadopoulos et al., 2003), mullet
(Pournis et al., 2005), swordsh (Pantazi et al., 2008), salmon lets
(Mac et al., 2013), and sea bream (Trynopoulou et al., 2007).
Shewanella have also been found on smoked sh such as blue cod or
salmon (Penney et al., 1994; Joffraud et al., 2001).
P. phosphoreum is a bioluminescent, psychrotrophic, halophilic
and CO2-resistant bacterium (Baumann and Baumann, 1984; Gram
and Huss, 1996) widely present in sea water or various marine
ecological niches. P. phosphoreum has been identied as the specic
spoilage organism of several seafood products mostly coming from
cold water, in contrast to sh from warmer temperature waters, e.g.
Mediterranean species such as sea bream and sea bass. It has also
been reported in chilled marine sh including cod, garsh, halibut,
saithe, salmon, and also in shrimp (Dalgaard et al., 1997, 2006;
Hansen et al., 2009; Hovda et al., 2007a; Hovda et al., 2007b;
Lpez-Caballero et al., 2002; Mac et al., 2013; Olafsdottir et al.,
2005; Paarup et al., 2002; Reynisson et al., 2009). Another Photobacterium species, Photobacterium iliopiscarium closely related but
phylogenetically distinct from P. phosphoreum (Urbanczyk et al.,
2011), has been associated with the spoilage microbiota of seafood products, notably vacuum-packed cold-smoked salmon
(Olofsson et al., 2007). However, its spoilage potential has not yet
been completely elucidated.
Enterobacteriaceae (enterobacteria) can also play a key role in
food spoilage due to their ability to metabolize amino acids to
malodorous volatile compounds, such as foul-smelling diamines
and sulfuric compounds (Baylis, 2006; Borch et al., 1996; GarcaLpez et al., 1998; Samelis, 2006). A range of enterobacteria may
grow on chilled meats: Hafnia alvei, Pantoea agglomerans, Rahnella
spp., Serratia spp., and Yersinia enterocolitica are frequently reported at the end of chilled storage (Sade et al., 2013). Among these
meat-associated enterobacteria, H. alvei, Serratia liquefaciens and
P. agglomerans have been reported to cause various defects such as
objectionable odors (Dainty et al., 1989; Garca-Lpez et al., 1998;
Stanbridge and Davies, 1998), gaseous distension of vacuum packages (Brightwell et al., 2007; Chaves et al., 2012), and green
discoloration (Dainty et al., 1989; Stanbridge and Davies, 1998). In a
recent publication, Sade et al. (2013) reported that in modiedatmosphere packaged meat and poultry products, enterobacteria
were found in the bacterial community of 60% of the analyzed
samples. They identied the predominant genera and species as
40% Hafnia spp., with H. alvei and Hafnia paralvei, and 42% Serratia
spp., with Serratia fonticola, Serratia grimesii, S. liquefaciens, Serratia
proteamaculans, and Serratia quinivorans. In addition, 6% of the
isolates were identied as Rahnella spp., 3% as Yersinia spp., and 1%
as Buttiauxella spp. Other food matrixes can be spoiled by enterobacteria. For instance, S. liquefaciens has been isolated from seafood spoilage microbiota in smoked sh (Gonzalez-Rodriguez et al.,
2002; Joffraud et al., 2006), and cooked and peeled shrimps (Jaffrs
et al., 2011, 2009). This species has also been isolated from milk and

47

dairy products (Kagkli et al., 2007; Martin-Platero et al., 2009;

Morales et al., 2004), egg products (Hidalgo et al., 2008), and pork
sausages, where it contributes to spoilage (Garcia-Fontan et al.,
2007; Lorenzo et al., 2010). Serratia spp., Yersinia intermedia,
H. alvei, and Buttiauxella gaviniae were isolated from the spoilage
microbiota of raw salmon steaks stored under vacuum or modied
atmosphere packaging (Mac et al., 2012).
Pseudomonas spp., particularly Pseudomonas uorescens, Pseudomonas putida, and Pseudomonas fragi, also contribute to a large
extent to the spoilage process of food. These are the predominant
spoilers of proteinaceous raw foods stored under aerobic refrigerated conditions (Nychas et al., 2008), especially aerobically chillstored beef (Doulgeraki and Nychas, 2013; Liu et al., 2006), seafood (Gram and Huss, 1996; Mac et al., 2013; Reynisson et al.,
2008), poultry (Dominguez and Schaffner, 2007; Mellor et al.,
2011), and milk (Quigley et al., 2013; Stevenson et al., 2003). In
fact, as already well described by Arslan et al. (2011), Pseudomonas
spp. may cause spoilage of dairy products through the production
of extracellular enzymes that are heat-stable and can therefore
remain active during pasteurization or ultra-high-temperature
treatments. P. uorescens also plays an important role in the
spoilage of aerobically stored chicken meat by producing biosurfactants which provide a competitive advantage to producing
strains in a number of ways e.g. enhancement of water-immiscible
substrate bioavailability and of adhesion to surfaces, antibiotic activity or motility facilitation (Mellor et al., 2011). P. fragi is involved
in the spoilage of fresh food products, such as milk, stored under
refrigerated aerobic conditions (Ercolini et al., 2007) where it can
produce thermo-resistant extracellular enzymes, causing spoilage
and structural defects in pasteurized and ultra-high-temperaturetreated milk (De Jonghe et al., 2011; Dogan and Boor, 2003;
Marchand et al., 2009). Other Pseudomonas species were reported
to express a signicant spoilage potential of milk, such as Pseudomonas gessardii, Pseudomonas lundensis, and P. uorescens-like (De
Jonghe et al., 2011; Marchand et al., 2009). In vegetables, several
species of Pseudomonas have been identied as spoilage bacteria,
including Pseudomonas chlororaphis, particularly effective on many
vegetables such as lettuce, red mustard seeds, broccoli, onion, potato, or carrot, by producing plant cell wall-degrading enzymes
(PCWDEs) like pectate lyase (Lee et al., 2013). Some Pseudomonas
species can also produce biolm on food and food-processing
equipment (Kerekes et al., 2013). The biolm-forming bacterial
cells can communicate by the density-dependent cell-to-cell
communication mechanism, quorum sensing, notably by releasing
signaling molecules, N-acyl homoserine lactones (AHLs), into their
environment. Gram et al. (2002) reported the widespread occurrence of AHLs in sh products, poultry and vacuum-packed meat.
AHL production is a widespread phenomenon in food-spoiling
bacteria.
3. Functional properties of spoilers: spoilage as the result of
metabolic activities
It is often difcult to correlate food spoilage characteristics to
the functional properties of spoilers. As described in the previous
section, spoilage microorganisms are responsible for various sensory defects (off-odors, discoloration) but these sensory descriptors
are not easily associated with enzymatic functions or metabolic
pathways. Several studies have reported the detection or measurement of molecules (volatile compounds, organic acids, biogenic
amines) in food spoiled by known microorganisms. However,
correlating the production of these molecules responsible for or
associated with spoilage appearance to the functions of spoilage
microorganisms is not always possible. There are several reasons
for this limitation; spoilage may result from a large variety of

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B. Remenant et al. / Food Microbiology 45 (2015) 45e53

processes. It can be the consequence of a complex succession of

enzymatic reactions, potentially associated with non-enzymatic
reactions, such as meat discoloration, or with enzymatic reactions
originating from both the spoilage organisms and the food matrix
(like lytic activities of enzymes from muscle cells). Another
example is metabiosis, a phenomenon involved in food spoilage
which results from enzymatic reactions successively carried out by
different microorganisms, as reported by Gram et al. (2002).
Spoilage can also result from reactions catalyzed by enzymes that
are not well identied. Finally, some molecules responsible for
spoilage can be produced by many different enzymes and identifying those that produce them in food may be a challenge. Many
studies reported by the scientic community were devoted to understand the virulence of foodborne pathogens or the metabolic
functions of microorganisms used for bioremediation or food
fermentation. Comparatively, the studies investigating the metabolism and physiology of bacteria responsible for food spoilage are
much less abundant. Clearly, there is a lack of knowledge about the
metabolic properties of spoilage microorganisms. Nevertheless, a
few examples can be cited.
The mechanism of biogenic amine production through decarboxylation of various amino acids has been reported for several
species. The enzymes responsible for biogenic amine synthesis are
known, particularly in LAB but also in other species, with the
description of histidine decarboxylases (histamine production),
tyrosine decarboxylases (tyramine production), ornithine decarboxylases, and agmatine deiminases (putrescine production). Some
bacterial amino acid and biogenic amine transporters have also
been described. L. curvatus was shown to be responsible for the
production of large amounts of tyramine, putrescine, cadaverine
and phenylethylamine on sausages (Latorre-Moratalla et al., 2012).
In addition, C. divergens can produce tyramine on cold-smoked
salmon (Connil et al., 2002a; Connil et al., 2002b) or on fresh
pork sausages (Curiel et al., 2011) while Enterococcus malodoratus is
considered a spoiler in Italian goat cheese through tyramine and
histamine production (Bonetta et al., 2008). S. putrefaciens can also
produce putrescine and cadaverine on penaeid shrimps or Mediterranean hake (Baixas-Nogueras et al., 2005; Benner et al., 2004).
This species along with S. liquefaciens and Serratia marcescens are
the main producers of cadaverine in vegetables such as spinach
(Lavizzari et al., 2010). As a last example, P. phosphoreum can cause
histamine sh poisoning incidents (Dalgaard et al., 2006; Emborg
et al., 2005; Kanki et al., 2004, 2007). In these cases, the spoilage
microorganism functions can be associated with a food product
defect (presence of biogenic amines) because the producing pathways or enzymes are known.
Some examples associating food color defects with known reactions can also be cited: Carnobacterium viridans is responsible for
the green discoloration of vacuum-packed cooked cured Bologna
sausage due to the production of hydrogen peroxide, which then
reacts with myoglobin to give green cholemyoglobin (Holley et al.,
2002; Peirson et al., 2003). However, enzymes producing hydrogen
peroxide can be very diverse and identifying the C. viridans protein(s) involved in Bologna sausage spoilage is difcult. Spoilage by
Pseudomonas spp. leading to color defect can also result from the
ability of some species to produce pigments on food like pyocyanin,
a blue-green phenazine derivative, and yellow-green uorescent
pigments (Martin et al., 2011; Moore et al., 2006). This pigment
production explains the involvement of P. uorescens in the Italian
Blue Mozzarella event, when Italian consumers widely reported a
blue pigmentation of this dairy product (Nogarol et al., 2013).
In other cases, sensory alteration related to off-odors can be
associated with the production of known metabolites, like volatile
compounds coming from various metabolic pathways, including
glycolysis, carbon catabolism or more complex metabolic routes. In

the spoilage process of morcilla de Burgos, when W. viridescens and

L. mesenteroides were found together they appeared to perform
different roles and to increase some signs of spoilage (vacuum loss
and slime production) (Diez et al., 2009). Indeed, L. mesenteroides
can grow faster than W. viridescens, due to its more energy-efcient
metabolism, which signicantly inuences pH drop, milky exudate
production and sour smell. This can be associated with the
appearance of aldehydes and acids, especially hexanal and acetic
acid, whereas W. viridescens inuences vacuum loss and the
appearance of alcohols and ketones, especially ethanol, diacetyl
and acetoin. In another example, cheesy off-odors caused by
B. thermosphacta isolated on chill-stored meat, were related to the
production of acetoin, diacetyl and 3-methylbutanol (Dainty and
Mackey, 1992; La Storia et al., 2012). On cold-smoked salmon,
B. thermosphacta was also able to produce butter/plastic/rancid,
blue-cheese, sour/pungent off-odors, due to the high release of
chemical compounds such as 2-heptanone and 2-hexanone
(Joffraud et al., 2001; Stohr et al., 2001). On cooked and brined
shrimps too, B. thermosphacta was found to be responsible for a fast
and strong spoilage, notably with the production of cheese/feet and
sour/fermented off-odors related to the chemical compounds 3methyl-1-butanal, 2,3-butanedione of 2,3-heptanedione (Jaffrs
et al., 2009, 2011). Ercolini et al. (2010) reported that ethylhexanoate, ethyloctanoate and ethyldecanoate were the most
commonly detected molecules in air-stored beef meat inoculated
with strains of P. fragi and that the ester release was associated with
the sensory perception of fruity off-odors of meat. In these examples, although molecules associated with spoilage could be identied, the bacterial functions involved in their production were too
complex to be predicted.
The specic spoilage of seafood products has been associated
with amine compound or sulfur metabolism: S. putrefaciens can use
trimethylamine N-oxide (TMAO) as a terminal electron acceptor
during anaerobic respiration, which induces TMAO reduction to
trimethylamine (TMA), known to be responsible for the typical
strong shy, urine and ammonia-like off-odors (Gram and Huss,
1996; Lopez-Caballero et al., 2001). TMA production by
P. phosphoreum has also been reported and is approximately 30fold higher than that of S. putrefaciens (Dalgaard, 1995). The production of hydrogen sulde gas (H2S) has been described as
responsible for putrid off-odors in various sh products. It was
shown to be related to the presence of S. putrefaciens or S. baltica
(Fonnesbech Vogel et al., 2005). Other species of the Shewanella
genus, isolated from marine sh of the Baltic Sea (cod, plaice or
ounder), can produce H2S: Shewanella hafniensis, Shewanella
morhuae, Shewanella glacialipiscicola, and Shewanella algidipiscicola
(Satomi et al., 2006, 2007). Several enzymes involved in the
metabolism of sulfur amino acids (cysteine and methionine) may
produce H2S and therefore be involved in this spoilage.
Spoilage may also result from enzymatic activities with many
different consequences: in milk products, P. fragi produces proteases that degrade casein, resulting in bitterness in milk, gelation
of ultra-high-temperature sterilized milk and decreased yields of
soft cheese. Lipases, also produced by P. fragi, catalyze the hydrolysis of triglycerides to free fatty acids and glycerol, which lead to
soapy and rancid avors (Cleto et al., 2012).
4. Toward the description of spoiling mechanisms through
genome analysis
The recent development of high-throughput sequencing has led
to a deeper analysis of food microbial ecosystems. These approaches, mostly focused on 16S rDNA, have conrmed previous
studies performed with classical cultural methods about the nature
of bacteria found in foods. In addition, the data obtained by direct

B. Remenant et al. / Food Microbiology 45 (2015) 45e53

analysis of DNA extracted from food matrixes have enabled a better

description of the ecosystems at genus or even species level, and
have also revealed that the involvement of some species in food
spoilage might have been underestimated (Parlapani et al., 2013;
Rasolofo et al., 2010; Schirmer et al., 2009). Moreover, this
method has conrmed that spoilage may result from the presence
of several bacterial species and is not always only due to the
dominant one. Using the genomics of spoilage microorganisms and
the metagenomics of food ecosystems combined with other
omics, particularly metabolomics, to determine the volatile
compounds or molecules putatively responsible for spoilage should
pave the way to identifying the bacterial metabolic pathways
involved in the appearance of spoilage. To date, however, only one
example of the spoilage ability of a microorganism, L. gasicomitatum, and a deep analysis of the genomic functions responsible for it
as well as a metagenomics approach, has been reported
(Jskelinen et al., 2013; Johansson et al., 2011; Nieminen et al.,
2012; Vihavainen and Bjorkroth, 2007, 2009).
Thus, a better understanding of the functions involved in
spoilage appearance is expected in the near future from global
analyses of spoiler genomes, as was the case for bacteria of relevance to food fermentations (Zagorec et al., 2008). Supplementary
Table 1 summarizes the genome sequences of bacterial species that
may encompass spoiling strains, which are or should soon become
publicly available. Interestingly, many large metagenomics projects
aimed at describing various communities (from sea, soil, human or
animal microbiota) have led to the release of complete or draft
genome sequences of strains (often type strains from public collections) known to be present in these communities. For many
species mentioned as food spoilers in this paper, at least one
genome sequence is available, or announced as sequencing in
progress, or targeted (see Supplementary Table 1). For some
species, the number of genomes should be high enough to enable a
signicant comparison of the functional properties between
different strains in order to highlight some putatively associated
with spoilage. For instance, almost 20 complete or draft
P. uorescens genomes have been published and this species has
been incriminated in the blue coloration defect in Mozzarella
cheese. Although these genomes come from environmental strains
or strains isolated from vegetables, searching for genes putatively
involved in pigment production may elucidate whether the potential for a color defect in food is widespread in this species.
Several S. baltica and S. putrefaciens genomes have been sequenced,
such as environmental strains. Although the strains whose genomes were sequenced were not isolated as seafood spoilers, it is
nevertheless important to analyze their genome content to search
for putative spoiling activities. Indeed, the microbial population
that can ourish in food products and spoil them mostly originates
from the environment and from animal gut microbiota. Spoiling
ability might be strain-dependent (i.e. environmental or gut strains
are not all able to grow and act as a spoilers in food). Nevertheless,
genome sequences of environmental or gut microbiota species
susceptible to colonize food, whatever the spoiler status of the
strain might be informative about the functions potentially
responsible for spoilage.
As mentioned above, S. putrefaciens has been reported to be
associated with putrescine and cadaverine production on seafood
products (Baixas-Nogueras et al., 2005; Benner et al., 2004). The
genome sequence of S. putrefaciens soil strains has been determined and is publicly available from several genome databases (see
Supplementary Table 1). To illustrate the present review we
therefore searched in the genome annotation results, accessible
from databases, for putative pathways that could lead to the synthesis of putrescine or cadaverine. Data mining revealed that the
genomes of S. putrefaciens CN32 and 200, two soil isolates, may

49

encode an L-ornithine decarboxylase (EC 4.1.1.17) producing putrescine from L-ornithine. However, the arginine deiminase
pathway, known to produce L-ornithine by the degradation of
arginine, an amino acid present in sh esh, is absent from both
S. putrefaciens genomes. Interestingly, we noticed that an alternative pathway may lead to N-acetylornithine from glutamate (see
Fig. 1) and that only strain 200 may encode an aminocyclase (EC
3.5.1.14, encoded by gene sput200DRAFT_3630) which can convert
N-acetylornithine to L-ornithine, leading to a complete pathway. In
addition, strain CN32 may encode a putative spermidine synthase
(EC 2.5.1.16, gene sputcn32_3089) that converts putrescine into
spermidine. This in silico analysis led to the hypothesis that some
S. putrefaciens strains may indeed produce putrescine (like strain
200) and that some may lack this functional property due to their
inability to produce L-ornithine, a putrescine precursor, and/or to
their ability to degrade it into spermidine. Although in silico analyses need experimental validation, such approaches may help to
prove the spoiling ability of isolates. For example, the presence or
expression of genes encoding putative spoiling enzymes may be
searched for or tools to detect specic functions may be developed.
The synthesis of some compounds involved in spoilage may also
result from different enzymatic reactions and correlating a particular metabolic pathway to spoilage can be problematic. As an
example, although shrimp spoilage by S. baltica has been associated
with metabolites from carbon metabolism, including acetate (Mac
et al., 2014), too many enzymes putatively producing acetate are
encoded by S. baltica genomes to identify those responsible for
spoilage. Conversely, genome analysis may fail to detect simple
pathways although metabolites have been experimentally detected. Acetoin (3-hydroxybutanone) can be produced from diacetyl
by diacetyl reductase (EC 1.1.1.4/EC 1.1.1.303) or from acetolactate by
a-acetolactate decarboxylase (EC 4.1.1.5). Although S. baltica
spoilage has been associated with acetoin production, no gene
encoding any of the above-mentioned enzymes is present in the
S. baltica genomes available in public databases. This may be
because acetoin is produced by as yet unknown enzymes or
because the environmental strains whose genomes are available do
not have spoiling properties.
Many species including isolates that have been described as
spoilers also encompass strains used for food fermentation or
technology. This is particularly true for lactic acid bacteria. This
group has been investigated for its fermentative and protective or
probiotic properties and many genome sequences are publicly
available. It should therefore be of interest to search, in these genomes, for the ability to express metabolic pathways involved in
the production of molecules described in spoilage. Ideally, genome
comparison between strains of technological interest and strains
proven as spoilers should lead to the identication of spoilagespecic functions.
Finally, many studies have reported that the presence of several
species may have a synergistic effect, increasing the extent of
spoilage when compared to the presence of only one spoilage
microorganism. It would thus be interesting to investigate the
pathway complementarity between putative spoiler species.
Several databases can present schematic views of metabolic pathways, based on the presence of enzyme-encoding genes from
sequenced genomes. Some also allow genome comparison through
construction of metabolic pathways presenting enzymatic steps
that are common, unique, or missing in several genomes. In this
way, it should be possible to investigate metabiosis phenomena,
looking for the possibility of several strains cooperating to produce
molecules responsible for spoilage.
The list of genome sequences of species encompassing putative
spoiler strains is already huge and will certainly increase rapidly,
since many genomes are announced as in progress or targeted.

50

B. Remenant et al. / Food Microbiology 45 (2015) 45e53

Fig. 1. Schematic representation of a putative pathway producing putrescine from glutamate in S. putrefaciens strains 200 and CN-32. Substrates and products are indicated.
Putative enzyme EC numbers deduced from genome annotation available in public databases are provided. Gray or dashed arrows indicate enzymes for which a putative encoding
gene is present in only one of the two strains whose genome has been sequenced.

Unspoiled

Comparison

Spoiled

Genomics Metagenomics
Presence/absence of putative spoilers
Putative spoilage functions
Metatranscriptomics
Expression of putative spoilage functions
Metabolomics
Presence/absence of putative spoilage molecules

Description of:
Spoiler organisms
Spoiler molecule
synthesis
Spoiler metabolic
functions
Control of:
Spoiler organisms
Spoilage

Fig. 2. Schematic representation of the expected output of studies combining omics and high throughput methods for diminishing food spoilage.

Yet, the genome sequences of some major spoiler bacteria

(B. thermosphacta, P. phosphoreum, and some Weissella species for
example) have not been determined. Nevertheless, a mass of information concerning the putative functional properties of spoilers
is already available in genome databases and has not yet been fully
exploited. Metagenomic and metatranscriptomic studies in food
are also emerging. The few articles in this eld were published
during the past two years and mainly focus on fermented products,
with the aim of elucidating fermentation and deciphering the dynamics and roles of various species involved in this process. There is
no doubt that a similar approach will be used in the future to understand the spoilage processes of various food products and the
spoiler functions responsible for food losses. Fig. 2 summarizes how
combining how coupling genomics, metagenomics, transcriptomics
and metabolomics to spoilage analysis may help understanding
and then controlling food spoilage.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.fm.2014.03.009.

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