Biodiversity and dynamics of meat fermentations: The contribution of molecular methods for a better comprehension of a complex ecosystem

Meat Science 89 (2011) 296–302

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Meat Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m e a t s 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

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Article history: Received 25 February 2011 Received in revised form 8 April 2011 Accepted 12 April 2011 Keywords: Sausages Ecology Molecular methods Culture-dependent methods Culture-independent methods

a b s t r a c t The ecology of fermented sausages is complex and includes different species and strains of bacteria, yeasts and molds. The developments in the field of molecular biology, allowed for new methods to become available, which could be applied to better understand dynamics and diversity of the microorganisms involved in the production 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 approach has highlighted that two main species of lactic acid bacteria, namely Lactobacillus sakei and Lb. curvatus, are involved in the transformation process and that they are accompanied by Staphylococcus xylosus, as representative of the coagulase-negative cocci. These findings were repeatedly confirmed in different regions of the world, mainly in the Mediterranean countries where dry fermented sausages have a long tradition and 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 copresence of multiple strains of the same species. © 2011 Elsevier Ltd. All rights reserved.

Contents 1. 2.

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

1. Introduction 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 materials, 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: [email protected] (L. Cocolin). 0309-1740/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2011.04.011

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subjected to a drying process, are transformed in microbiologically stable final products, characterized by a defined sensory profile. In meat fermentations, different groups of microorganisms, possessing 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.

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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 nitrosomyoglobin. 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 homogeneous 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 fermentation In the last 20 years the advancement in molecular biology has revolutionized the way research is carried out. Today it is possible to

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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 culturedependent 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 cultureindependent and -dependent methods. It is now accepted that such

Fig. 1. Schematic representation of the molecular approaches used in the study of the ecology of fermented sausages.

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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;

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 fermentation, 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

Table 1 Studies exploiting the PCR DGGE as culture-independent methods to investigate the ecology of fermented sausages. Type of product

Region and country

Target group

Type of study

Reference

Fermented sausages Fermented sausages Alheira sausages Fermented sausages (Ciauscolo salami) Naturally fermented ham Fermented sausages (Sopressata) Artisanal dry sausages

Friuli-Venezia-Giulia region, Italy Lombardia region, Italy Portugal Marche region, Italy

LAB, CNC and yeasts LAB and CNC LAB LAB, CNC and yeasts

Throughout fermentation Throughout fermentation Final product Final product

Cocolin et al., 2001; Rantsiou et al., 2005 Cocolin et al., 2009 Albano et al., 2008 Silvestri et al., 2007

Taiwan Campania region, Italy

LAB and CNC LAB and CNC

Throughout fermentation Final product

Tu, Wu, Lock, & Chen, 2010 Villani et al., 2007

Argentina

LAB and CNC

Throughout fermentation Final product

Fontana et al., 2005b Fontana et al., 2005a

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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.

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 fermentationspecific 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 production, 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

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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 techniques 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 characterization 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 straindynamics 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

RAPD–PCR

LAB CNC Bacillus sp. Yeasts LAB LAB CNC LAB CNC Yeasts LAB

Typical Southern Italian sausages (Salame di Senise)

Manufacturing and ripening Final product

Sanz, Hernandez, Ferrus, & Hernandez, 1998 Baruzzi, Matarante, Caputo, & Morea, 2006 Albano et al., 2009 Rantsiou et al., 2006. Rossi et al., 2001 Fontana et al., 2005b Cocolin et al., 2006a Rantsiou et al., 2005

LAB LAB LAB LAB RAPD–PCR and plasmid LAB profiling CNC RAPD–PCR, Sau-PCR CNC and rep-PCR rep-PCR LAB PFGE and RAPD–PCR

CNC

Alheira Portuguese sausages Fermented Greek sausages Artisanal Italian dry sausages Artisanal Argentine fermented dry sausages Fermented Italian sausages Naturally fermented sausages from Greece, Hungary and Italy Naturally fermented sausages from North-central Italy Naturally fermented sausages from Southern Italy Naturally fermented Italian sausages Naturally fermented Italian sausages Low acid Spanish fermented sausages Slightly fermented Spanish sausages Naturally fermented Italian sausages Traditional Ethiopian fermented sausages Fermented Italian sausages (Soppressata)

PFGE and multiplex PCR LAB

Traditional Italian dry fermented sausages

PFGE

Traditional French dry fermented sausages

CNC

Throughout the fermentation

Final product Throughout the fermentation Throughout the fermentation Throughout the fermentation Final product Throughout the fermentation

Cocolin et al., 2009 Bonomo et al., 2008 Urso et al., 2006 Comi et al., 2005 Aymerich et al., 2006 Martin et al., 2006 Iacumin et al., 2006 Bacha, Jonsson, & Ashenafi, 2010 Di Maria, Basso, Santoro, Grazia, & Coppola, 2002 Pennacchia, Vaughan, & Villani, 2006 Corbiere Morot-Bizot, Leroy, & Talon, 2006

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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 provenience. 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 microorganisms 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 investigated. 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 transformation 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.

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