Meat Science 89 (2011) 296–302

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Meat Science
j o ur nal ho mep ag e : www.el sevier. com /l ocate / m e ats c i

Review

Biodiversity and dynamics of meat fermentations: The contribution of molecular

methods for a better comprehension of a complex ecosystem
Luca Cocolin ⁎, Paola Dolci, Kalliopi Rantsiou
DIVAPRA, Agricultural Microbiology and Food Technology Sector, Faculty of Agriculture, University of Turin, Italy

a r t i c l e in fo abstract

Article history: The ecology of fermented sausages is complex and includes different species and strains of bacteria, yeasts and
Received 25 February 2011 molds. The developments in the field of molecular biology, allowed for new methods to become available, which
Received in revised form 8 April 2011 could be applied to better understand dynamics and diversity of the microorganisms involved in the production
Accepted 12 April 2011
of sausages. Methods, such as denaturing gradient gel electrophoresis (DGGE), employed as a culture -
independent approach, allow to define the microbial dynamics during the fermentation and ripening. Such
Keywords:
approach has highlighted that two main species of lactic acid bacteria, namely Lactobacillus sakei and Lb.
Sausages
Ecology curvatus, are involved in the transformation process and that they are accompanied by Staphylococcus xylosus,
Molecular methods as representative of the coagulase-negative cocci. These findings were repeatedly confirmed in different regions
Culture-dependent methods of the world, mainly in the Mediterranean countries where dry fermented sausages have a long tradition and
Culture-independent methods history. The application of molecular methods for the identification and characterization of isolated strains
from fermentations highlighted a high degree of diversity within the species mentioned above, underlining the
need to better follow strain dynamics during the transformation process. While there is an important number
of papers dealing with bacterial ecology by using molecular methods, studies on mycobiota of fermented
sausages are just a few. This review reports on how the application of molecular methods made possible a better
comprehension of the sausage fermentations, opening up new fields of research that in the near future will
allow to unravel the connection between sensory properties and co- presence of multiple strains of the same
species.
© 2011 Elsevier Ltd. All rights reserved.

Contents

1. Introduction ...................................................................................................................................................................................................................... 296

2. Molecular approaches in sausage fermentation ................................................................................................................................................................297
2.1. Culture-independent methods: Denaturing gradient gel electrophoresis .............................................................................................................. 298
2.2. Culture-dependent methods: The molecular characterization of the isolates ...................................................................................................... 300
3. Conclusions and future challenges .................................................................................................................................................................................... 301
References .................................................................................................................................................................................................................................. 302

1. Introduction subjected to a drying process, are transformed in microbiologically

Meat fermentations are challenging microbial ecosystems in
which bacteria, yeasts and molds coexist. A great diversity can be
revealed, including not only several species belonging to different
genera, but also strains of the same species, that participate in the
fermentation process. Through fermentation, perishable raw mate-
rials, such as meat and fat, supplemented with sodium chloride and
⁎ Corresponding author at: via Leonardo da Vinci 44, 10095 Grugliasco, Torino,
Italy. Tel.: + 39 011 670 8553; fax: + 39 011 670 8549.
E-mail address: lucasimone.cocolin@unito.it (L. Cocolin).
stable final products, characterized by a defined sensory profile.
In meat fermentations, different groups of microorganisms, posses-
sing different biochemical potentials, contribute for the formation of the
sensory profile of the final product. The acidification process, in which
sugars, such as glucose, lactose and sucrose, are transformed in lactic acid,
thereby reducing the pH of the meat and creating a hostile environment
for pathogenic bacteria, is the main activity of lactic acid bacteria (LAB)
(Ammor & Mayo, 2007). These microorganisms may also be able to
produce proteinaceous compounds, called bacteriocins (De Vuyst & Leroy,
2007), active against closely related bacteria, including pathogens such as
Listeria monocytogenes, thereby increasing the competitiveness of the
producer cells and the safety of the final products.

0309-1740/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2011.04.011
 L. Cocolin et al. / Meat Science 89 (2011) 296–302
Other bacteria relevant for the transformation process are the
coagulase-negative cocci (CNC). They participate in the development
and stability of a generally appreciated red color through nitrate
reductase activity that eventually leads to the formation of nitroso-
myoglobin. Furthermore, nitrate reduction produces nitrite that can
limit lipid oxidation (Talon, Walter, Chartier, Barriere, & Montel, 1999).
LAB and CNC are also responsible for flavor generation through
proteolysis and lipolysis, although it should be pointed out that the
process involved in aroma formation is very complex and it has been
recently reviewed in great detail elsewere (Toldrà, 2008).
Lypolytic and proteolytic activities are also associated to yeasts and
molds. In fermented sausages, yeasts are preferably found internally,
while molds are only present on the surface where there is availability of
oxygen. Yeasts have been described as powerful proteolytic agents
(Santos et al., 2001), and their contribution to the volatile compound
production in salchichón was recently reported (Andrade, Córdoba,
Casado, Córdoba, & Rodríguez, 2010). Molds participate in the
transformation process thanks to their ability to produce lipases and
proteases, thereby enhancing the final organoleptic characteristics.
Moreover due to their capability to create micro-pores on the casing,
they facilitate the dehydration process. Lastly, growing as an homoge-
neous layer on the surface of the sausage, they also protect lipids from
oxidation in the presence of light (Incze, 2004).
The microbial activity and interaction are a key factor for the final
quality characteristics of fermented sausages. As described by Lücke
(2000), LAB are the main responsible for the production of semi-dried
products sold after less than 2 weeks ripening, which are characterized
by an acid flavor. When longer ripening times are employed, there is a
greater diversity and activity of microorganisms, which lead to higher
levels of volatile compounds with low sensory thresholds, resulting in
products with more rich organoleptic profile.
In this review, the most recent advancements in the identification,
characterization and dynamics of the microorganisms involved in the
fermentation of sausages, as unraveled by molecular methods, will be
presented. Understanding the microbial biodiversity and ecology can allow
a better control of the transformation process, resulting in products with
high quality and safety and unique sensory characteristics.
2. Molecular approaches in sausage ferThentation
In the last 20 years the advancement in molecular biology has
revolutionized the way research is carried out. Today it is possible to
Fig. 1. Schematic representation of the molecular approaches used in the study of the ecology of fermented sausages.
297
detect, identify and quantify microorganisms by targeting their nucleic
acids. Moreover, there are methods that can be implemented in
research laboratories, able to differentiate strains belonging to the same
species, opening up new area of interest in which specific strain
dynamics during fermentation can be studied. This last aspect is
receiving much attention in the field of food fermentations because it
allows to understand if a strain inoculated as starter culture is able to
dominate the transformation process. In the case of naturally fermented
sausages, strain(s) capable to drive the fermentation can be identified,
therefore facilitating development of autochthonous starters.
Nucleic acids can be analyzed either from isolates obtained from
the food matrix by traditional microbiological methods, or more
recently their direct extraction from the food sample has been
proposed. In the first case, the approach is considered culture-
dependent while the second case is culture-independent. There is a
scientific consensus on the fact that culture-dependent methods are
not able to properly describe the diversity of complex ecosystems
(Hugenholtz, Goebel, & Pace, 1998): populations that are present in
low numbers or that are in a stressed or injured state will most
probably be unintentionally excluded from consideration if traditional
microbiological methods are used. Moreover, cells that are in a viable
but not culturable (VNBC) state will not be detected, because of their
incapability to form colonies on microbiological media (Cocolin, Dolci,
& Rantsiou, 2008). The use of culture-dependent methods, relying on
the cultivation of the microorganisms can produce a picture of the
ecology of fermented sausages, which is not totally correct, because of
the biases inherent to the different capabilities of the microorganisms
to grow on the synthetic media used in the laboratory. An example of
the use of culture-dependent and -independent molecular methods in
the study of the microbiota of fermented sausages is presented in Fig.
1. After the homogenization of the fermented sausage sample,
researchers can either approach the study of its microbiota by
applying traditional microbiological methods, that often include
cultivation on synthetic media and after growth, isolation of
individual colonies, or a direct extraction of the nucleic acids. It
should be pointed out that also culture-dependent methods can
exploit molecular approaches, however these are used to characterize
isolates that have grown on agar media. In the last 10 years, a number
of evidences have been produced highlighting that often, there are
significant differences between the results obtained with culture-
independent and -dependent methods. It is now accepted that such
298 L. Cocolin et al. / Meat Science 89 (2011) 296–302
differences are partly due to biases introduced by cultivation (Cocolin
et al., 2004). However, as reported below, also culture-independent
methods possess some limitations due to generally high limits of
detection, thereby minor populations are not taken into
consideration.
When culture-independent approaches are employed for the study
of ecology and biodiversity in food fermentations, the target molecules
considered are DNA and RNA. The significance of the results that can be
obtained using one or the other nucleic acid has to be properly evaluated
since these two molecules have different properties and meaning. DNA
is a very stable molecule and it is long present also after the cell has died.
On the contrary RNA, and especially messenger RNA (mRNA), can have
very short life. For these reasons, studying the DNA of a microbial
ecosystem will allow definition of the microbial ecology and diversity,
while the RNA analysis will highlight more properly the microbial
populations that are metabolically active, thereby contributing to the
fermentation process (Cocolin et al., 2008).
2.1. Culture-independent methods: Denaturing gradient gel electrophoresis
The culture-independent method that has become more popular
and has been applied extensively in sausage fermentation is
represented by denaturing gradient gel electrophoresis (DGGE)
(Rantsiou & Cocolin, 2008) and examples of studies exploiting such
technique are reported in Table 1. The technique is based on the
electrophoretic separation of PCR-generated double stranded DNA in
a polyacrylamide gel containing a gradient of chemical denaturants
(urea and formamide). As the DNA molecule encounters an
appropriate denaturant concentration, a sequence-dependent, partial
denaturation of the double strand occurs. This change in the
conformation of the DNA structure causes a reduced migration rate
of the molecule. In the temperature gradient gel electrophoresis
(TGGE), the temperature is the main denaturing agent. When the
method is used for microbial profiling, DNA and/or RNA are subjected
to PCR and/or Reverse Transcription (RT)-PCR with universal primers,
able to prime amplification for all the microbes present in the sample.
After this step, the complex mixture of the DNA molecules obtained
can be differentiated and characterized if separated in denaturing
gradient gels. Every single band that is visible in D/TGGE gels
represents a component of the microbiota. The more bands are
visible, the more complex is the ecosystem. Bands can be excised from
the gels and after re-amplification can be sequenced in order to
obtained the corresponding microbial species. By using these
methods, it is possible not only to profile the microbial populations,
but also to follow their dynamics during time. Modern image analysis
systems have proven to be of value for the analysis of DGGE bands and
their associated patterns. For instance, pairwise matching of DGGE
bands in separate gel lanes has facilitated the calculation of similarity
coefficients to describe relationships between microbial communities
(van der Gucht et al., 2001). It should be noted that these methods are
not quantitative (Rantsiou & Cocolin, 2006). DGGE analysis has been
applied mainly to Italian fermented sausages (Aquilanti et al., 2007;
Table 1
Studies exploiting the PCR DGGE as culture-independent methods to investigate the ecology of fermented sausages.
Type of product Region and country
Fermented sausages Friuli-Venezia-Giulia region, Italy
Fermented sausages Lombardia region, Italy
Alheira sausages Portugal
Fermented sausages Marche region, Italy
(Ciauscolo salami)
Naturally fermented ham Taiwan
Fermented sausages Campania region, Italy
(Sopressata)
Artisanal dry sausages Argentina
Cocolin et al., 2009; Cocolin, Manzano, Cantoni, & Comi, 2001;
Rantsiou et al., 2005; Silvestri et al., 2007; Villani et al., 2007), but
studies on the fermentation dynamics of Argentinean (Fontana,
Cocconcelli, & Vignolo, 2005a; Fontana, Vignolo, & Cocconcelli, 2005b)
and Portuguese (Albano, Henriques, Correira, Hogg, & Teixeira, 2008)
sausages are available as well.
The first work published (Cocolin et al., 2001) regarding direct
DGGE analysis, was focused on the bacterial dynamics, throughout 45
days of fermentation and ripening, of a traditional fermented sausage
produced in the Friuli-Venezia-Giulia region of Italy. In DNA and RNA
DGGE gels, multiple bands were visible for the first 3 days of
fermentation, when different species, most of them related to
Staphylococcus spp., were identified. From the 10th day of ripening only
the LAB bands were present. The LAB population was characterized
by Lactobacillus sakei and Lb. curvatus throughout the process. Lb.
plantarum was only detected on the first day in the DNA gel and the
band could have been generated from dead cells. Staphylococcus species
were found only in the meat mixture before sausages were filled and for
the first 3 days. The only Staphylococcus species represented in the
DGGE gel after 3 days was S. xylosus, which produced a specific band in
the gel until the end of fermentation. The corresponding band in the
RNA gel was only present at day 0 and day 3 and then disappeared.This
study highlighted how fermentation of sausages is a highly competitive
process, in which wide species diversity can be found in the very first
days of fermentation. After 3 days, only a few populations were able to
dominate becoming the relevant actors of the transformation. This
evidence was afterwards confirmed by Cocolin et al. (2007), where both
raw meats and fermented sausages were examined by DGGE. The higher
complexity of the patterns in raw materials when compared to the end
fermentation product was highlighted. In a later study, focusing on
traditional fermented sausages from the same region of Italy, the goal
was to follow the fermentation in three different plants (C, L and U), in
which no starters are being used, and producing sausages with ripening
times of respectively, 120, 45 and 28 days (Rantsiou et al., 2005). At each
sampling point, PCR–DGGE was employed and the resulting bands
were identified by sequencing (Fig. 2). A general consideration that
resulted from this study is that the main differences detected in the
ecology, between the three sausages, were not represented by the
species of microorganisms identified by band sequencing, but by their
relative distribution between the fermentations. In all three
fermentations, a stable signal from the beginning of the period studied
was visible for Lb. curvatus and Lb. sakei, that remained constant
throughout the transformation. In one fermen- tation, a band that
corresponded to Lb. paracasei was present for part of the period while
Lactococcus garviae was detected in two of the three sausages. No Lb.
plantarum was detected in these sausages. As seen from the DGGE
profiles, important contribution to the microbial ecology was given by
Staphylococcus species. S. equorum or S. succinus were present in all the
fermentations, while S. xylosus was mainly present in one of them. When
the DGGE gels were digitalized and subjected to cluster analysis, two
main outcomes could be observed: samples collected at
Target group Type of study Reference
LAB, CNC and yeasts Throughout fermentation Cocolin et al., 2001; Rantsiou et al., 2005
LAB and CNC Throughout fermentation Cocolin et al., 2009
LAB Final product Albano et al., 2008
LAB, CNC and yeasts Final product Silvestri et al., 2007
LAB and CNC Throughout fermentation Tu, Wu, Lock, & Chen, 2010
LAB and CNC Final product Villani et al., 2007
LAB and CNC Throughout fermentation Fontana et al., 2005b
Final product Fontana et al., 2005a
 L. Cocolin et al. / Meat Science 89 (2011) 296–302
Fig. 2. DGGE profiles of the bacterial ecology of three naturally fermented sausages from the North of Italy during fermentation and ripening. The numbers on the lanes indicate the
sampling days and the most relevant species found after sequencing of the bands are indicated on the right side of the gels (modified from Rantsiou et al., 2005).
the beginning of the fermentation were characterized by unique DGGE
patterns and they represented single-sample clusters, the dendrogram
obtained showed 4 main clusters that were grouping samples from the
same producer, highlighting the diversity of the products considered in
the study (Rantsiou et al., 2005).
PCR–DGGE was also applied to profile the bacterial community of
artisanal Argentinean sausages (Fontana et al., 2005b). Targeting the
V3 region of 16S rRNA coding gene, a highly complex fingerprint was
obtained at day 0 of fermentation that was characterized by the presence
of Lb. plantarum, Pediococcus acidilactici, Lb. sakei, Lb. curvatus, S.
equorum (identified by co-migration with control strains) and
Corynebacterium variabilis (identified by sequencing). A more basic
fingerprint was obtained at day 5 and day 14 (last day of ripening) with
the presence of Lb. plantarum, Lb. sakei and Lb. curvatus while S.
saprophyticus represented the Staphylococcus group. Similar results
were obtained by studying a sausage from Tucumán, Argentina
(Fontana et al., 2005a) and comparing it to a ripened sausage from
Córdoba region. A high microbial diversity was observed at day 0 with
the presence of Lb. plantarum, Lb. sakei, S. saprophyticus, Co. variabilis
and when the two sausages were compared, a common band, associated
with Lb. sakei was detected.
By applying PCR–DGGE to the traditional Italian salami ‘Ciauscolo’
from the Marche region, in Central Italy, overall, 5 LAB species were
detected: Lb. plantarum, Lb. curvatus, Lb. sakei, P. acidilactici and
L. lactis. No Staphylococcus bands were observed in the DGGE profiles,
either because the populations were below the detection limit or due to
the higher number of LAB that may create a masking effect. The most
frequently detected species were, once again, Lb. curvatus and Lb.
sakei.
Knowledge produced at the beginning of the 0Xs, has been
confirmed by studies published in the last 5 years. In a study of the
ecology of a fermented sausage from southern Italy, Soppressata,
targeting the V3 and V1 regions, it was possible to identify S. xylosus,
S. succinus and S. equorum among the staphylococci and Lb. sakei and
Lb. curvatus within the lactobacilli (Villani et al., 2007). Lastly, in
Alheira sausages, from Portugal (Albano, Henriques, Correia, Hogg, &
Teixeira, 2008), as well as in fermented sausages from Northern Italy
(Cocolin et al., 2009), the application of PCR–DGGE was allowed to
highlight the important role that LAB have in their production.
Culture-independent methods to study the ecology of yeasts
during sausage fermentation have been used at lesser extend when
compared to bacteria. Some examples of these studies will be
described below.
299
The yeast ecology has been described by targeting the 26S rRNA
conding gene for Italian fermented sausages (Cocolin, Urso, Rantsiou,
Cantoni, & Comi, 2006a; Rantsiou et al., 2005; Silvestri et al., 2007)
and overall, the DGGE profiles were less complex and oftentimes,
characteristic of the products. In particular, when a cluster analysis of
the yeast DGGE profiles was carried out, from the samples collected
during production of three fermented sausages, a fermentation-
specific distribution was obtained, with clusters containing samples
from the same fermentation (Rantsiou et al., 2005). D. hansenii, a
proteolytic yeast that is associated with fermented sausage produc-
tion, was dominant in one of the three fermentations, but was not
seen in the other two productions. Candida krisii and Willopsis
saturnus and C. sake or austromarina characterized respectively the
other two fermentations.
The predominance of D. hansenii was also confirmed by a later study
in Italian sausages fermented at low temperatures, probably due to its
physiological characteristics and it should therefore be considered a well
adapted, to the specific environment, yeast species (Flores, Durà, Marco,
& Toldrà, 2004). Throughout the 60 days of fermentation a stable band,
at both DNA and RNA level, identified as
D. hansenii, was visible in the DGGE gels (Cocolin et al., 2006a).
In the ‘ciauscolo’ salami of the Marche region, the most frequently
detected species was D. hansenii, while C. physchrophila and
Saccharomyces barnettii were occasionally found (Silvestri et al.,
2007).
The application of PCR–DGGE in the field of fermented sausages
offers a better understanding of the biodiversity and dynamics of the
populations involved in the transformation. However it should be
mentioned that pitfalls, associated with sampling, DNA extraction,
DNA purity, PCR conditions, formation of heteroduplex and chimeric
molecules, may still exist, thereby the results obtained need to be
verified and validated (Ercolini, 2004). One important aspect that has
to be taken into consideration when applying DGGE in food
fermentation is the sensitivity limit. It has been demonstrated that
populations that are below 103–104 colony forming units (cfu)/g will
not be detected (Cocolin et al., 2001). This is especially valid when in
the same ecosystem two populations, one at high and the other at low
counts, exist, as it usually happens in sausage fermentations.
Moreover, due to the extensive application of sequencing, databases
have seen tremendous growth of the sequences deposited. Often,
these entries are classified as ‘unculturable microorganism’, since they
have been detected only by culture-independent methods and no
significant similarity to available sequences was obtained. This aspect
300 L. Cocolin et al. / Meat Science 89 (2011) 296–302

introduces potential difficulties in the understanding of the ecology of
fermented foods and at the same time underlines the need to improve
the traditional cultivation methods.
2.2. Culture-dependent methods: The molecular characterization of the
isolates
In the 90s, molecular techniques for the identification and
characterization of microorganisms isolated from fermented meat
products, started to be used side by side or to substitute morphological,
phenotypical and biochemical tests. Although the two different
approaches, in most of the cases, arrived at similar results, molecular
techniques immediately showed a higher level of reproducibility,
automatism and speed (Cocolin et al., 2008).
The increasing availability of the sequences of the 16S rRNA gene
and the intergenic region between 16S rRNA and 23S rRNA genes
allowed the development of different methods for the identification of
microbial species of interest in the field of sausage fermentation. Probes
targeting ribosomal RNA, species specific PCR primers, restriction
fragment length polymorphism (RFLP) analysis of the 16S rRNA gene,
multiplex PCR, TGGE and DGGE coupled with DNA sequencing have
been applied for the identification of LAB and CNC isolated from
fermentation of sausages (Rantsiou & Cocolin, 2008).
Isolated strains can also be characterized by molecular methods,
such as randomly amplified polymorphic DNA (RAPD)–PCR analysis,
ribotyping and PCR amplification of repetitive bacterial DNA elements
(rep-PCR), in order to understand the dynamics and diversity within
the same species. Studies applying molecular methods for the
characterization of the microbiota of fermented sausages are
indicated in Table 2. These approaches are supposed to be used on
isolates that have been previously identified with molecular tech-
niques and in this way intra-species differences are highlighted. Using
appropriate software, fingerprints from gels can be digitalized and
analyzed in order to produce similarity dendrograms, where clusters
can be identified (Fig. 3). Based on the composition of the clusters
important information of strain biodiversity and dynamics can be
achieved.
While the application of molecular strategies has become a routine
step for identification purposes, only a few studies in the field of
fermented sausages have been published focusing on the characteriza-
tion of LAB and CNC. Some examples will be reported below.
In fermented products, the possibility to detect strains able to carry
out fermentation processes is extremely important in the selection of
those that could be used as starters. Rossi, Tofalo, Torriani, and Suzzi
(2001) compared and combined RAPD profiles, from S. xylosus strains
isolated from dry sausages and underlined the suitability of RAPD–PCR
analysis to discriminate strains with technologically relevant
activities.
Cocolin, Urso, Rantsiou, Cantoni, and Comi (2006b) exploited
molecular characterization of the LAB and CNC isolates to follow the
development of an inoculated commercial starter in fermented
sausages. RAPD characterization was carried out on isolates from the
starter used and from LAB and CNC isolated during the same
production. The analysis of the profiles of the starter isolates revealed 3
Lb. plantarum RAPD biotypes in the starter, of which only one was able
to conduct the fermentation based on the similarity of the RAPD
fingerprints obtained from the isolates during the fermentation.
S. xylosus strains from the starter culture were able to predominate only
in the latter stages of fermentation. The different behavior of Lb.
plantarum and S. xylosus could be explained considering the fact that
starter culture was dissolved in white wine, thus inhibiting the initial
development of S. xylosus, not able to overcome the ethanol stress.
Considering natural fermentations, several studies have exploited
the potentials of molecular characterization to understand strain-
dynamics and dominance. Fontana et al. (2005b) used this approach
to demonstrate that the ripening process of Argentinean artisanal
fermented sausages was driven by a limited number of Lactobacillus
and Staphylococcus strains selected from environmental microbiota
for the ability to best compete under the prevailing conditions of the
ecological niche. Comi et al. (2005), studying three fermented
sausages from the Northeast of Italy highlighted, by RAPD–PCR, that
the lactobacilli population was distributed in a fermentation-specific
way and showed a higher degree of heterogeneity for Lb. sakei
compared to Lb. curvatus. The same approach was followed by Urso,

Table 2
Literature reporting on molecular characterization of microorganisms isolated from fermented sausages.

Method Target group Product Type of study Reference

RFLP LAB Dry-cured sausages Final product Sanz, Hernandez, Ferrus,

& Hernandez, 1998
RAPD–PCR LAB CNC Bacillus sp. Typical Southern Italian sausages (Salame di Senise) Manufacturing and Baruzzi, Matarante, Caputo,
Yeasts ripening & Morea, 2006

LAB Alheira Portuguese sausages Final product Albano et al., 2009

LAB Fermented Greek sausages Rantsiou et al., 2006.
CNC Artisanal Italian dry sausages Throughout the Rossi et al., 2001
LAB CNC Artisanal Argentine fermented dry sausages fermentation Fontana et al., 2005b

Yeasts Fermented Italian sausages Cocolin et al., 2006a

LAB Naturally fermented sausages from Greece, Hungary and Rantsiou et al., 2005
Italy

LAB Naturally fermented sausages from North-central Italy Cocolin et al., 2009
LAB Naturally fermented sausages from Southern Italy Bonomo et al., 2008
LAB Naturally fermented Italian sausages Urso et al., 2006
LAB Naturally fermented Italian sausages Comi et al., 2005
RAPD–PCR and plasmid LAB Low acid Spanish fermented sausages Final product Aymerich et al., 2006
profiling CNC Slightly fermented Spanish sausages Martin et al., 2006

RAPD–PCR, Sau-PCR CNC Naturally fermented Italian sausages Throughout the Iacumin et al., 2006
and rep-PCR fermentation

rep-PCR LAB Traditional Ethiopian fermented sausages Throughout the fermentation

fermentation
PFGE and RAPD–PCR CNC Fermented Italian sausages (Soppressata) Throughout the
Bacha, Jonsson, & Ashenafi, 2010 L. Cocolin et al. / Meat Science 89 (2011) 296–302 301

Di Maria, Basso, Santoro, Grazia, & Coppola, 2002

PFGE and multiplex PCR LAB Traditional Italian dry fermented sausages Final product Pennacchia, Vaughan, & Villani,
2006
PFGE CNC Traditional French dry fermented sausages Throughout the Corbiere Morot-Bizot, Leroy,
fermentation & Talon, 2006
302 L. Cocolin et al. / Meat Science 89 (2011) 296–302
Fig. 3. Cluster analysis of Lactobacillus curvatus from different sausages in the North of Italy by Rep-PCR obtained by using the Bionumerics software (Applied Maths). Using a
coefficient of similarity of 70%, 4 groups could be differentiated that included isolates coming from different origins (unpublished results).
Comi, and Cocolin (2006), Bonomo, Ricciardi, Zotta, Parente, and
Salzano (2008) and Cocolin et al. (2009) for Italian fermented sausages
and Albano et al. (2009), for traditional fermented sausages produced
in Portugal confirming the plant specific distribution.
Considering CNC, Iacumin, Comi, Cantoni, and Cocolin (2006)
compared RAPD–PCR and rep-PCR, together with Sau-PCR to
characterize S. xylosus strains isolated from naturally fermented
sausages from different areas of Italy, in order to detect a possible strain
differentiation depending on their specific geographic prove- nience.
The authors confirmed the theory that, depending on temperature,
humidity and ingredients of each production plant, there is a selection
of microorganisms, which influences the characteristics of the final
products. More specifically, all the strains isolated from one plant, show
the capability to grow at 10 °C and differentiate from the other CNC. The
specific plant was the only one using low temperature ripening, thereby
selecting a population of CNC able to grow under these environmental
conditions (Iacumin et al., 2006). The results presented in this study
were further confirmed by a study carried out by Leroy, Giammarinaro,
Chacornac, Lebert, and Talon (2010), in which the ecology of CNC in
small-scale sausage production units in France was studied. After
pulsed field gel electrophoresis (PFGE) analysis it was determined that
the processing plant can contribute to the establishment of the
fermented sausages microbiota by selecting persistent strains, most
probably previously introduced via raw meats, and able to adapt to the
environmental conditions.
As described for the culture-independent methods, also molecular
characterization of yeast isolates was carried out less frequently if
compared to the available studies focusing on bacteria. RAPD–PCR
analysis with primer M13 was used by Cocolin et al. (2006a) to
characterize D. hansenii strains, from Italian fermented sausages. The
authors noticed a shift in D. hansenii population from the beginning to
the end of sausage maturation; in fact, strains present during the early
stages of the fermentation were grouped in clusters that differed from
those defined in the final phases of the maturation.
3. Conclusions and future challenges
Understanding the dynamics, diversity and behavior of microor-
ganisms during sausage fermentations is a very interesting and
challenging task. As underlined above, due to their metabolic
activities, microorganisms are able to transform the raw materials
into a final product that possesses totally different properties, in terms
of safety, shelf life and sensory profile. Thereby the comprehension of
the ecology of the fermentation process can help the producers to
reach high quality of their products.
The last 20 years have seen incredible advancements in the field of
microbiological analyses. The discovery of the PCR revolutionized the
way microorganisms are detected and characterized and allowed
development of new approaches that allow the study of the sausage
microbiota without any cultivation. The information collected so far by
applying molecular methods in this field confirms the finding that
were produced in the 50s, when the first studies on the ecology of
sausages were published. However, a deeper level of comprehension is
now achieved. Using DGGE, it is possible to compare different products
and understand how similar they are, based on the profile of the
populations that are detected, moreover by molecular characterization
of isolates during the transformation process is possible to understand
species and strains biodiversity.
While it can be admitted that the knowledge that has been achieved
for the study of the microbiota of fermented sausages is substantial,
strain dynamics and successions are still under investi- gated. The
outcomes from the first studies on this specific subject, underline the
complexity of the ecology at strain level, during sausage fermentation.
Biotypes of the same species are coming from the raw materials and the
processing plants and their ability to grow and dominate depends on a
lot of parameters, one of which is the fermentation conditions. The
possibility to follow specific strains nowadays is possible through the
application of molecular methods for the characterization, however
cultivation and isolation are still necessary, until when protocols for
direct characterization, using total DNA extracted from the sample, will
become available. Since the overall quality of these products is strictly
connected to the populations that are able to develop and to carry out
the transforma- tion process, and more specifically to certain biotypes
within a species, the availability of more accurate methods to
understand their dynamics is indispensable. An aid may come from
modern sequencing techniques, which are be able to properly profile
complex microbial ecosystems. If regions of the DNA able to
differentiate between strains of the same species are properly selected,
then pyrosequencing should be able to point out their diversity in terms
of abundance of a specific sequence compared to others. Lastly,
targeting the RNA also, important insights of specific activities of the
different biotypes can be obtained. This aspect is extremely relevant,
especially when the differences at the sensory profile between sausages,
produced with the same ingredients but in plants of different
geographical areas, are taken into consideration. Ecology studies
conducted so far underlined that the species that are more involved in
the fermentation process are Lb. sakei and Lb. curvatus for the LAB,
and S. xylosus for CNC. Molecular characterization showed that a high
level of heterogeneity exists within these species, which can partially
explain the different sensory profiles. However, a clear indication of the
role of the different biotypes will be clarified only when it will be possible
to understand their behavior in terms of contribution to the aroma
profile, through gene expression analysis, by the means of
transcriptomics and proteomics.
 L. Cocolin et al. / Meat Science 89 (2011) 296–302
References
Albano, H., Henriques, I., Correia, A., Hogg, T., & Teixeira, P. (2008). Characterization of
microbial population of ‘alheira’ (a traditional portuguese fermented sausage) by PCR–
DGGE and traditional cultural microbiological methods. Journal of Applied
Microbiology, 105, 2187–2194.
Albano, H., van Reenen, C. A., Todorov, S. D., Cruz, D., Fraga, L., Hogg, T., et al. (2009).
Phenotypic and genetic heterogeneity of lactic acid bacteria isolated from “alheira”, a
traditional fermented sausage produced in portugal. Meat Science, 82, 389–398.
Ammor, M. S., & Mayo, B. (2007). Selection criteria for lactic acid bacteria to be used as
functional starter cultures in dry sausage production. An update. Meat Science, 76,
138–146.
Andrade, M. J., Córdoba, J. J., Casado, E. M., Córdoba, M. G., & Rodríguez, M. (2010). Effect of
selected strains of Debaryomyces hansenii on the volatile compounds production of
dry fermented sausage “salchichón”. Meat Science, 85, 256–264.
Aquilanti, L., Santarelli, S., Silvestri, G., Osimani, A., Petruzzelli, A., & Clementi, F. (2007).
The microbial ecology of a typical italian salami during its natural fermentation.
International Journal of Food Microbiology, 120, 136–145.
Aymerich, T., Martin, B., Garrica, M., Vidal-Carou, M. C., Bover-Cid, S., & Hugas, M.
(2006). Safety properties and molecular strain typing of lactic acid bacteria from
slightly fermented sausages. Journal of Applied Microbiology, 100, 40–49.
Bacha, K., Jonsson, H., & Ashenafi, M. (2010). Microbial dynamics during the fermentation
of wakalim, a traditional Ethiopian fermented sausage. Journal of Food Quality, 33,
370–390.
Baruzzi, F., Matarante, A., Caputo, L., & Morea, M. (2006). Molecular and physiological
characterization of natural microbial communities isolated from a traditional
Southern Italian processed sausages. Meat Science, 72, 261–269.
Bonomo, M. G., Ricciardi, A., Zotta, T., Parente, E., & Salzano, G. (2008). Molecular and
technological characterization of lactic acid bacteria from traditional fermented
sausages of Basilicata region (Southern Italy). Meat Science, 80, 1238–1248.
Cocolin, L., Diez, A., Urso, R., Rantsiou, K., Comi, G., Bergmaier, I., et al. (2007).
Optimization of conditions for profiling bacterial populations in food by culture-
independent methods. International Journal of Food Microbiology, 120, 100–109.
Cocolin, L., Dolci, P., & Rantsiou, K. (2008). Molecular methods for identification of
microorganisms in traditional meat products. In F. Toldrà (Ed.), Meat Biotechnology
(pp. 91–127). New York: Springer.
Cocolin, L., Dolci, P., Rantsiou, K., Urso, R., Cantoni, C., & Comi, G. (2009). Lactic acid
bacteria ecology of three traditional fermented sausages produced in the north of Italy
as determined by molecular methods. Meat Science, 82, 125–132.
Cocolin, L., Manzano, M., Cantoni, C., & Comi, G. (2001). Denaturing gradient gel
electrophoresis analysis of the 16S rRNA gene V1 region to monitor dynamic
changes in the bacterial population during fermentation of Italian sausages. Applied
and Environmental Microbiology, 67, 5113–5121.
Cocolin, L., Rantsiou, K., Iacumin, L., Urso, R., Cantoni, C., & Comi, G. (2004). Study of the
ecology of fresh sausages and characterization of populations of lactic acid bacteria by
molecular methods. Applied and Environmental Microbiology, 70, 1883–1894.
Cocolin, L., Urso, R., Rantsiou, K., Cantoni, C., & Comi, G. (2006a). Dynamics and
characterization of yeasts during natural fermentation of Italian sausages. FEMS Yeast
Research, 6, 692–701.
Cocolin, L., Urso, R., Rantsiou, K., Cantoni, C., & Comi, G. (2006b). Multiphasic approach
to study the bacterial ecology of fermented sausages inoculated with a commercial
starter culture. Applied and Environmental Microbiology, 72, 942–945.
Comi, G., Urso, R., Iacumin, L., Rantsiou, K., Cattaneo, P., Cantoni, C., et al. (2005).
Characterization of naturally fermented sausages produced in the North East of Italy.
Meat Science, 69, 381–392.
Corbiere Morot-Bizot, S., Leroy, S., & Talon, R. (2006). Staphylococcal community of a
small unit manufacturing traditional dry fermented sausages. International Journal of
Food Microbiology, 108, 210–217.
De Vuyst, L., & Leroy, F. (2007). Bacteriocins from lactic acid bacteria: Production, purification,
and food applications. Journal of Molecular Microbiology and Biotechnology, 13, 194–199.
Di Maria, S., Basso, A. L., Santoro, E., Grazia, L., & Coppola, R. (2002). Monitoring of
Staphylococcus xylosus DSM 20266 added as starter during fermentation and ripening
of soppressata molisana, a typical Italian sausage. Journal of Applied Microbiology,
92, 158–164.
Ercolini, D. (2004). PCR–DGGE fingerprinting: Novel strategies for detection of microbes
in food. Journal of Microbiological Methods, 56, 297–314.
Flores, M., Durà, M. -A., Marco, A., & Toldrà, F. (2004). Effect of Debaryomyces spp. on
aroma formation and sensory quality of dry-fermented sausages. Meat Science, 68,
439–446.
Fontana, C., Cocconcelli, P. S., & Vignolo, G. (2005a). Monitoring the bacterial population
dynamics during fermentation of artisanal Argentinean sausages. International
Journal of Food Microbiology, 103, 131–142.
303
Fontana, C., Vignolo, G., & Cocconcelli, P. S. (2005b). PCR–DGGE analysis for the
identification of microbial populations from Argentinean dry fermented sausages.
Journal of Microbiological Methods, 63, 254–263.
Hugenholtz, P., Goebel, B. M., & Pace, N. R. (1998). Impact of culture-independent studies
on the emerging phylogenetic view of bacterial diversity. Journal of Bacteriology, 180,
4765–4774.
Iacumin, L., Comi, G., Cantoni, C., & Cocolin, L. (2006). Molecular and technological
characterization of Staphylococcus xylosus isolated from Italian naturally fermented
sausages by RAPD, Rep-PCR and Sau-PCR analysis. Meat Science, 74, 281–288.
Incze, K. (2004). Mould ripened sausages. In Y. H. Hui, L. Meunier-Goddik, Ä. S. Hansen,
J. Josephsen, W. -K. Nip, P. S. Stanfield, & F. Toldra (Eds.), Handbook of Food and
Beverage Fermentation Technology (pp. 480–491). Boca Raton, Fl: CRC Press.
Leroy, S., Giammarinaro, P., Chacornac, J. -P., Lebert, I., & Talon, R. (2010). Biodiversity of
indigenous staphylococci of natural fermented dry sausages and manufacturing
environments of small-scale processing units. Food Microbiology, 27, 294–301.
Lücke, F. K. (2000). Utilization of microbes to process and preserve meat. Meat Science,
56, 105–115.
Martin, B., Garriga, M., Hugas, M., Bover-Cid, S., Veciana-Nogues, M. T., & Aymerich, T.
(2006). Molecular, technological and safety characterization of Gram-positive catalase-
positive cocci from slightly fermented sausages. International Journal of Food
Microbiology, 107, 148–158.
Pennacchia, C., Vaughan, E. E., & Villani, F. (2006). Potential probiotic Lactobacillus
strains from fermented sausages: Further investigations on their probiotic properties.
Meat Science, 73, 90–101.
Rantsiou, K., & Cocolin, L. (2006). New developments in the study of the microbiota of
naturally fermented sausages as determined by molecular methods: A review.
International Journal of Food Microbiology, 108, 255–267.
Rantsiou, K., & Cocolin, L. (2008). Fermented meat products. In L. Cocolin, & D. Ercolini
(Eds.), Molecular Methods and Microbial Ecology of Fermented Foods (pp. 91–118).
New York: Springer.
Rantsiou, K., Drosinos, E. H., Gialitaki, M., Metaxopoulos, I., Comi, G., & Cocolin, L.
(2006). Use of molecular tools to characterize Lactobacillus spp. isolated from Greek
traditional fermented sausages. International Journal of Food Microbiology, 112, 215–
222.
Rantsiou, K., Urso, R., Iacumin, L., Cantoni, C., Cattaneo, P., Comi, G., et al. (2005). Culture
dependent and independent methods to investigate the microbial ecology of Italian
fermented sausages. Applied and Environmental Microbiology, 71, 1977–1986.
Rossi, F., Tofalo, R., Torriani, S., & Suzzi, G. (2001). Identification by 16S–23S rDNA
intergenic region amplification, genotypic and phenotypic clustering of Staphylococ-
cus xylosus strains from dry sausages. Journal of Applied Microbiology, 90, 365–371.
Santos, N. N., Santos-Mendonça, R. C., Sanz, Y., Bolumar, T., Aristoy, M. -C., & Toldrá, F.
(2001). Hydrolysis of pork muscle sarcoplasmatic proteins by Debaryomyces hansenii.
International Journal of Food Microbiology, 68, 199–206.
Sanz, Y., Hernandez, M., Ferrus, M. A., & Hernandez, J. (1998). Characterization of
Lactobacillus sake isolates from dry-cured sausage by restriction fragment length
polymorphism analysis of the 16S rRNA gene. Journal of Applied Microbiology, 84,
600–6006.
Silvestri, G., Santarelli, S., Aquilanti, L., Beccaceci, A., Osimani, A., Tonucci, F., et al. (2007).
Investigation of the microbial ecology of Ciauscolo, a traditional Italian salami, by
culture-dependent techniques and PCR–DGGE. Meat Science, 77, 413–423.
Talon, R., Walter, D., Chartier, S., Barriere, C., & Montel, M. C. (1999). Effect of nitrate and
incubation conditions on the production of catalase and nitrate reductase by
staphylococci. International Journal of Food Microbiology, 52, 47–56.
Toldrà, F. (2008). Biotechnology of flavor generation in fermented meats. In F. Toldrà
(Ed.), Meat Biotechnology (pp. 199–215). New York: Springer.
Tu, R. -J., Wu, H. -Y., Lock, Y. -S., & Chen, M. -J. (2010). Evaluation of microbial dynamics
during the ripening of traditional Taiwanese naturally fermented ham. Food
Microbiology, 27, 460–467.
Urso, R., Comi, G., & Cocolin, L. (2006). Ecology of lactic acid bacteria in Italian
fermented sausages: Isolation, identification and molecular characterization.
Systematic and Applied Microbiology, 29, 671–680.
van der Gucht, K., Sabbe, K., de Meester, L., Vloemens, N., Zwart, G., Gillis, M., et al.
(2001). Contrasting bacterioplankton community composition and seasonal
dynamics in two neighboring hypertrophic freshwater lakes. Environmental
Microbiology, 3, 680–690.
Villani, F., Casaburi, A., Pennacchia, C., Filosa, L., Russo, F., & Ercolini, D. (2007). The
microbial ecology of the Soppressata of Vallo di Diano, a traditional dry fermented
sausage from southern Italy, and in vitro and in situ selection of autochthonous starter
cultures. Applied and Environmental Microbiology, 73, 5453–5463.