International Journal of Food Microbiology 157 (2012) 130–141

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Review

Spoilage microbiota associated to the storage of raw meat in different conditions Agapi I. Doulgeraki a, Danilo Ercolini b,⁎, Francesco Villani b, George-John E. Nychas a a b

Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Technology, Agricultural University of Athens, Athens GR‐11855, Greece Dipartimento di Scienza degli Alimenti, Università degli Studi di Napoli Federico II, Via Università, 100-80055 Portici (NA), Italy

a r t i c l e

i n f o

Article history: Received 6 February 2012 Received in revised form 21 May 2012 Accepted 22 May 2012 Available online 25 May 2012 Keywords: Meat microbiota Meat spoilage Meat storage Molecular methods

a b s t r a c t The spoilage of raw meat is mainly due to undesired microbial development in meat during storage. The type of bacteria and their loads depend on the initial meat contamination and on the specific storage conditions that can influence the development of different spoilage-related microbial populations thus affecting the type and rate of the spoilage process. This review focuses on the composition of raw meat spoilage microbiota and the influence of storage conditions such as temperature, packaging atmosphere and use of different preservatives on the bacterial diversity developing in raw meat. In addition, the most recent tools used for the detection and identification of meat microbiota are also reviewed. © 2012 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . Detection and identification of microbial communities in meat 2.1. PCR-based molecular typing . . . . . . . . . . . . . 2.2. Typing by non PCR-based methods . . . . . . . . . . 2.3. Identification and typing by PCR and/or sequencing . . 2.4. Identification by SDS-PAGE profiling . . . . . . . . . 2.5. Culture-independent methods . . . . . . . . . . . . 3. Spoilage associated microbial populations and storage conditions 3.1. Influence of packaging atmosphere . . . . . . . . . . 3.2. Influence of temperature . . . . . . . . . . . . . . . 3.3. Influence of natural antimicrobials and other “hurdles” 4. Conclusions and future perspectives . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Meat is recognized as one of the most perishable foods. This is due to its chemical composition that favours microbial growth to unacceptable levels contributing significantly to meat deterioration or spoilage. When large numbers of microorganisms are present in raw meat, there will be changes such that it becomes unappealing and unsuitable for human consumption (Fung, 2010; Gram et al., 2002). The initial microbial load of meat depends on the physiological status of the

⁎ Corresponding author. Tel.: + 39 081 2539449; fax: + 39 081 2539407. E-mail address: [email protected] (D. Ercolini). 0168-1605/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2012.05.020

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animal at slaughter, the spread of contamination into slaughterhouses and during processing, while temperature and other conditions of storage during distribution can also influence the rate of spoilage (Nychas et al., 2008). The different microbial groups that will potentially contribute to meat spoilage depend on (i) the storage conditions applied and (ii) their competition. The development of such microbial association is reported to significantly affect the type of spoilage. For example two distinct situations are possible: (i) one when facultative anaerobic or anaerobic Gram-positive microbiota determines the changes in the ecosystem (e.g. meat stored under low oxygen availability with or without the presence of antimicrobial gases) and (ii) another where aerobic or facultative anaerobic Gram-negative bacteria (meat stored aerobically and/or under high oxygen tension) dominate the microbial succession.

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The physicochemical spoilage progress of meat and meat products has been extensively reviewed. However, the succession of microbial association is not always described and many reports are based on the determination of viable counts of microorganisms during storage (Borch et al., 1996; Dainty, 1996; Ingram and Dainty, 1971; Labadie, 1999; Nychas et al., 2008; Nychas and Skandamis, 2005; Skandamis and Nychas, 2002; Stanbridge and Davies, 1998). Microbial analysis alone might not be enough to understand the actual shifts of the microbial ecology of raw meat in response to different storage conditions. Studies have established that spoilage is caused only by a fraction of species and strains of the initial microbial association (Nychas and Skandamis, 2005). Despite the fact that this concept has contributed significantly to our understanding of meat spoilage, there is still uncertainty of how and when these bacterial species or strains are influenced by the specific storage conditions and, as a consequence, the influence that they can have on the type and rate of spoilage development is to be further investigated. The concept of ‘succession’ of spoilage-related microbial groups i.e. Ephemeral/Specific Spoilage Organisms (E/SSO), was only recently taken into consideration (Chenoll et al., 2007; Ercolini et al., 2006a, 2011; Gram et al., 2002; Nychas et al., 2008; Pennacchia et al., 2011). Recently, in fact, several studies have focused on meat with the aim of describing the diversity of the spoilage associated microbial populations in response to different storage conditions (Brightwell et al., 2009; Diez et al., 2008; Doulgeraki et al., 2010, 2011; Ercolini et al., 2006a, 2009, 2010a, 2011; Fontana et al., 2006; Jiang et al., 2010; Pennacchia et al., 2011; Sakala et al., 2002a; Xu et al., 2010). The storage temperatures as well as the different applied treatments such as addition of preservatives, vacuum pack (VP) and modified atmosphere packaging (MAP) were found to affect the microbial association such as ESOs of the product and consequently the spoilage process (Ercolini et al., 2010a, 2011; Nychas et al., 2008; Pennacchia et al., 2011; Stanbridge and Davies, 1998). The characterization of the spoilage microbiota not only at species but also at strain level is also an important issue that has been increasingly taken into account recently. This approach could potentially play a pivotal role in the understanding of meat spoilage as different strains of the same species may have different spoilage activities or can be differently affected by storage conditions (Casaburi et al., 2011; Doulgeraki et al., 2010, 2011; Ercolini et al., 2010b). Hence, the study of bacterial physiology could be of help in understanding the spoilage occurrence and dynamics. The basic knowledge of the microbial populations developing during meat storage has been acquired in the past by using traditional microbiology approaches. In the recent years, the development and application of powerful molecular techniques have contributed to produce reliable data on the microbial species and strains occurring during meat storage. In this review, a description will be provided of the bacterial communities developing during storage of meat under different conditions (temperature, packaging, addition of preservatives). The methods recently applied for the detection and identification of meat microbiota will also be presented and discussed. 2. Detection and identification of microbial communities in meat The application of molecular techniques in microbial ecology of food has changed the way of studying the microbial diversity in complex food ecosystems (Cocolin and Ercolini, 2008). The molecular methods allow rapid and reliable identification and typing of microorganisms providing a clear picture of the microbial community in a particular environment. Studies of muscle (e.g. meat and fish) microbial ecology and more specific microbial meat spoilage have recently benefited by the use of such tools and the most recent results described in the literature are based on the application of both culture-dependent and culture-independent molecular methods

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(Broekaert et al., 2011; Diez et al., 2008; Ercolini et al., 2006a, 2010a, 2011). Several molecular techniques that have been developed during the last decades are applied for both identification and typing of bacteria at species or strain level; they are basically employed to overcome the drawbacks of conventional phenotypic methods (Cocolin et al., 2011; Doulgeraki et al., 2010; O'Sullivan, 2000; Rantsiou and Cocolin, 2006; Schillinger and Holzapfel, 2006; Temmerman et al., 2004). A list of the above techniques and their most recent applications is reported in Table 1. In general, identification and characterization by classical phenotypic methods, do not always allow efficient characterization of the microbiota at species level (Holzapfel, 1998; Stanbridge and Davies, 1998). These methods commonly rely on the detection of DNA polymorphisms between species or strains and differ in their dynamic range of taxonomic discriminatory power and reproducibility (Ben Amor et al., 2007). 2.1. PCR-based molecular typing The polymerase chain reaction (PCR) is a fast, accurate, sensitive and easy operating technique. The PCR based method was reported to allow differentiation at the species (Welsh and McClelland, 1992) and intra-species level (Seal et al., 1992) depending on the stringency of the PCR conditions. The specificity of this technique is directly associated with the primer selection and the amplification conditions. The differences between strains can be detected by exploiting primers that are annealing in various regions of the genome thereby producing a band pattern mainly by randomly amplified polymorphic DNA‐PCR (RAPD-PCR) and repetitive extragenic palindromic-PCR (rep-PCR) (Cocolin et al., 2008; Ercolini et al., 2010b; Versalovic et al., 1991; Welsh and McClelland, 1992). RAPD‐PCR has been intensively used in studies of genomic diversity among bacterial species (Byun et al., 2001; Yost and Nattress, 2002) and to characterize mesophilic and psychrotrophic mesophilic bacteria isolated from meat (Ercolini et al., 2009; Casaburi et al., 2011). Similarly, rep-PCR has been used to differentiate between closely related bacterial strains (Gevers et al., 2001) and spoilage related microbiota of artisan-type cooked ham (Vasilopoulos et al., 2008, 2010). The main drawback of random PCR methods like RAPD‐PCR is their low reproducibility; however the profiles obtained from rep-PCR analysis are specific for a species and they are also more reproducible (Cocolin et al., 2008). 2.2. Typing by non PCR-based methods Restriction enzyme analysis coupled with pulsed-field gel electrophoresis (REA–PFGE) is a powerful method for strain characterization. Genomic DNA from different strains is digested by specific restriction enzymes and then subjected to PFGE; the result obtained is a strain-specific band pattern (Cocolin et al., 2008). PFGE has been used to differentiate genomic restriction patterns of strains as well as to monitor the bacterial strain succession during storage of meat (Doulgeraki et al., 2010, 2011; Jones, 2004), and is regarded as a very reliable technique to study the succession of bacteria in meat at strain level. The aforementioned studies concluded that different LAB and enterobacteria strains within the same species were found depending on the storage conditions, which strengthen the opinion that one population may replace another without an observable decline in total counts. Additionally, the importance of monitoring the strain succession by such technique was highlighted. 2.3. Identification and typing by PCR and/or sequencing A valuable tool in bacterial taxonomy for determining relationships between bacterial groups and identify species is the sequencing of 16S rRNA gene (Baylis, 2006; Ercolini, 2004; Hugenholtz et al., 1998; Maukonen and Saarela, 2009; Nocker et al., 2007). This is because the 16S rRNA gene has conserved as well as variable regions and can allow

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Table 1 Techniques applied recently for monitoring the bacterial communities on meat, poultry and meat products. Technique

Product

Target

Reference

Species-specific PCR

Cooked, MA packaged poultry Beef, different packaging temperature conditions Beef, pork and poultry Vacuum packaged beef Beef MA packed cooked meat product Peroxyacetic acid treated vacuum packaged beef Beef, different packaging conditions

Carnobacterium spp., Lactococcus spp., Lactobacillussakei Pseudomonas spp. Lactobacillus, Leuconostoc spp. Brochothrix thermosphacta Lactic acid bacteria Bacteria Pseudomonads, entrobacteria, lactic acid bacteria, Brochothrix Lactic acid bacteria, enterobacteria Bacteria Lactic acid bacteria, Bacteria Pseudomonads, B. thermosphacta, lactic acid bacteria, enterobacteria Lactic acid bacteria Bacteria Pseudomonads Enterobacteria Bacteria Mesophilic, psychrotrophic bacteria Lactic acid bacteria Pseudomonas fragi Bacteria Brochothrix spp. Lactic acid bacteria Bacteria Psychrophilic and psychrotrophic clostridia Cold-tolerant clostridia Lactic acid bacteria Enterobacteria Lactic acid bacteria

Barakat et al. (2000) Doulgeraki et al. (2010) Ercolini et al. (2007) Fontana et al. (2006) Pennacchia et al. (2009) Audenaert et al. (2010) Brightwell et al. (2009) Ercolini et al. (2006a)

PCR-DGGE

Beef stored in nisin activated packaging Vacuum packaged beef Vacuum packed-pork Tray-packed pork Beef (air, vacuum)

T-RFLP Real-time PCR SDS-PAGE RAPD-PCR rep-PCR

RFLP

PFGE

MA packed artisan-type cooked ham MA packed meat Tray-packed pork Beef, different packaging temperature conditions Vacuum packaged beef Beef Vacuum-packaged beef Beef MA packed artisan-type cooked ham Meat, poultry MA packaged, marinated broiler legs Peroxyacetic acidtreated vacuum packaged beef ‘Blown pack’ spoilage of vacuumpacked meats Raw vacuum-packed chill-stored meat Beef, different packaging temperature conditions Beef, different packaging temperature conditions Chilled stored Vacuum packed beef

designing primers targeting all bacteria and spanning regions with species-specific sequences (Ercolini, 2004). More than 30 million full and partial sequences can be found in public databases, while the sequence databases for other genes contain only limited number of sequences limiting their use in microbial ecological studies (Maukonen and Saarela, 2009; Nocker et al., 2007). Nevertheless, limited heterogeneity of the 16S rRNA gene of Pseudomonas and members of Enterobacteriaceae did not allow their identification to species level in different cases (Baylis, 2006; Ercolini et al., 2006a). In fact, it has been recognized that identity of 16S rRNA sequences is not necessarily enough to guarantee species identity (Fox et al., 1992). Therefore, although 16S rRNA sequences can be used routinely to distinguish and establish relationships between genera and well-resolved species, in some cases alternative target genes are to be taken into account. Besides 16S rRNA gene, other housekeeping or functional genes might be used as targets for PCR. The multi-locus sequencing typing (MLST) has become fundamental for the characterization of food-borne microorganisms and several genes can be used depending on the phyla of interest. The MLST is based on PCR amplification and sequencing of several housekeeping genes; after sequence comparison, even one difference in the sequence is considered enough to define a different biotype. Among the core bacterial genes, the gene for the RNA polymerase beta subunit, rpoB has emerged as one of the few potential candidates for identification of bacteria, especially when studying closely related isolates (Mollet et al., 1997; Adekambi et al., 2008). Additionally, the internal transcribed spacer (ITS) has been reported as a widely used tool to differentiate closely related strains at species and/or strain level (Khan et al., 2005; Xu et al., 2010). A further alternative for identification without post PCR sequencing is the use of species-specific PCR assays.In the case of pseudomonads, the sequence heterogeneity of the carA gene has been found useful for simultaneous detection of Ps. fragi, Ps. lundensis and Ps. putida by multiplex species-specific PCR (Ercolini et al., 2007). By contrast, the sequences of the genes recA, gyrB, fliC, and rpoD may be only supportive for Pseudomonas species

Ercolini et al. (2010a, 2010b) Fontana et al. (2006) Jiang et al. (2010) Jiang et al. (2011) Pennacchia et al. (2011) Vasilopoulos et al. (2010) Nieminen et al. (2011) Jiang et al. (2011) Doulgeraki et al. (2011) Sakala et al. (2002a) Ercolini et al. (2009) Yost and Nattress (2002) Ercolini et al. (2010b) Vasilopoulos et al. (2010) Xu et al. (2010) Bjorkroth et al. (2005) Brightwell et al. (2009) Broda et al. (2000) Cavill et al. (2011) Doulgeraki et al. (2010) Doulgeraki et al. (2011) Jones (2004)

differentiation (Bellingham et al., 2001; Hilario et al., 2004; Yamamoto et al., 2000). A species-specific real-time PCR assay was developed for the identification of Brochothrix thermosphacta in meat without cultivation although the real-time assay did not prove useful for a quantitative estimation of the microorganism in meat (Pennacchia et al., 2009). In another study, species specific primers were described for the differentiation of Carnobacterium and Lactococcus isolated from cooked, modified atmosphere packaged, refrigerated, poultry meat (Barakat et al., 2000). Similarly, multiplex PCRs using 16S rRNA-specific primers allowed differentiation between Leuconostoc species, while speciesspecific PCR was used to confirm the presence of Lactobacillus sakei and Lb. curvatus in VP beef (Fontana et al., 2006). On the other hand, a widely used PCR based method allowing strain grouping is RFLP analysis of the 16S rRNA gene. In RFLP, the differences in the DNA sequence are identified by using a restriction endonuclease cutting the DNA in specific restriction sites, giving a specific pattern for a given species. This method has been applied in the field of fermented sausages (Cocolin et al., 2008) as well as meat for the characterization of LAB (Bjorkroth et al., 2005), spoilage microbiota (Brightwell et al., 2009) and Clostridia (Broda et al., 2000; Cavill et al., 2011). 2.4. Identification by SDS-PAGE profiling The comparison of whole-cell protein patterns obtained by highly standardised sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) has also been successfully applied for microbial identification at species or subspecies level, even of closely related species (Pot et al., 1994). The high taxonomic resolution of this technique, regarding inter- and intra-species divergence, which is often the case in the Enterobacteriaceae family, has been shown (Coenye et al., 2001; Hantula et al., 1990; Holmes et al., 1991). SDS-PAGE has been used to monitor the changes of the microbiota in VP beef during storage (Sakala et al., 2002a) and determine the species diversity of Enterobacteriaceae community of minced beef (Doulgeraki et al.,

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Table 2 Examples of microbial species occurring during chilled storage of raw meat in different conditions. Microorganisma

Storage conditions Aerobic

Aeromonas spp. A. baumannii A. salmonicida Acinetobacter spp. Achromobacter spp. Alcaligenes spp. Alteromonas spp. Arthrobacter spp. Bacillus spp. Bradyrhizobium spp. Brochothrix thermosphacta Buttiauxella agrestis B. gaviniae B. noackiae Carnobacterium divergens C. maltaromaticum Chromobacterium spp. Citrobacter freundii Clostridium spp. Cl. algidicarnis Cl. estertheticum Cl. frigidicarnis Cl. gasigenes Cl. putrefaciens Enterobacter cloacae E. agglomerans Hafnia alvei Klebsiella spp. Kluyvera spp. Kocuria spp. Kurthia spp. Lactococcus spp. L. piscium Lactobacillus spp. Lb. algidus Lb. curvatus Lb. sakei Lb. kimchii Lb. graminis Lb. oligofermentans Leuconostoc spp. L. carnosum L. gasicomitatum L. gelidum L. mesenteroides L. pseudomesenteroides L. kimchii Limnobacter spp. Listeria spp. Microbacterium spp. Micrococcus spp. Moraxella spp. Paenibacillus spp. Pantoea spp. P. agglomerans P. anantis Photobacterium spp. P. kishitaniiclade Proteus vulgaris Providencia spp. Pseudomonas spp. Ps. fluorescens Ps. fragi Ps. lundensis Ps. migulae Ps. putida Ps. syringae Psychrobacter spp. Rahnella spp. R. aquatilis Ralstonia spp. Rudaea cellulosilytica Serratia spp.

Modified atmosphere packaging

Vacuum packaging

x x x x x x

x

x x x x

x

x

x x x x

x x

x

x x x

Antimicrobial active packagingb

x x x x x x x x x

x x

x x

x x x x x x x

x x x x x x x

x x

x

x

x

x x x

x x

x

x x

x x x

x

x x x x

x x

x x x

x x

x x

x x x

x x x x x x x

x x x

x

x x

x x x x x x x x x x x x

x x

x

x x

x

x x

x

x

x

x x x

x x x x

x

x x

x (continued on next page)

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Table 2 (continued) Microorganisma

S. grimesii S. liquefaciens S. marcescens S. proteamaculans Shewanella putrefaciens Staphylococcus spp. Staph. pasteuri Staph. saprophiticus Staph. xylosus Stenotrophomonas maltophilia Streptococcus spp. Strep. parauberis Weissella spp. Yersinia spp.

Storage conditions Aerobic

Modified atmosphere packaging

Vacuum packaging

Antimicrobial active packagingb

x x x x x x

x x

x x x x

x

x x x x x

x

x

x x

x

x x x

x

x x x

x indicates the occurrence of the microorganism in the specific storage condition. a The species are indicated as occurring in the different storage conditions if, according to the literature, they were identified at least once in the specific conditions regardless of the time of storage. b The antimicrobial packaging refers to nisin activated antimicrobial packaging (Ercolini et al., 2010a, 2011); in other works on antimicrobial packaging, species identifications were not performed.

2011). Although in both studies a clear discrimination was supplied between the species, the remarkable intra-species differentiation capacity of this technique was demonstrated with a further subdivision of S. proteamaculans into 2 sub-clusters (Doulgeraki et al., 2011).

2.5. Culture-independent methods It has been reported that only a small fraction of microorganisms is analyzed by conventional methods, and the isolated strains often do not represent the real spectrum of microorganisms and their genes active in the habitat of choice (Ampe et al., 1999; Engelen et al., 1998; Ward et al., 1990). On the other hand, culture independent methods are believed to overcome problems associated with selective cultivation such as (i) inability to detect some bacteria on the known media, (ii) lack of knowledge of the real conditions under which most of bacteria are growing in their natural habitat and (iii) difficulty to develop media for cultivation accurately resembling these conditions. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of ribosomal RNA genes, that has been proposed for studies of microbial ecology by Muyzer et al. (1993), is perhaps the most commonly used among the culture-independent fingerprinting techniques (Ercolini, 2004). Nowadays, PCR-DGGE is widely applied to analyze the DNA extracted from meat and meat products (Cocolin et al., 2007, 2011; Ercolini et al., 2006a, 2010a; Fontana et al., 2005; Pennacchia et al., 2011; Rantsiou et al., 2005; Villani et al., 2007). The advantage of using PCR-DGGE is the possibility to have a species related profile of meat samples during storage. Briefly, each meat sample in a particular storage condition will be associated to a specific microbial profile where the identification of the bands will provide the exact species composition of that particular sample without the need for cultivation. A series of PCR-DGGE profiles from meat during storage will allow changes in microbial composition to be detected at only one glance. In addition, the possibility to analyze bulk cells from selective culture media also by PCR-DGGE (Ercolini et al., 2001, 2006a, 2010a, 2011) can be also extremely important in order to achieve a rapid and reliable combination of culture-dependent and culture-independent data on meat microbiota. Despite the success of PCR-DGGE to provide a rapid survey of the bacterial community, the technique has several acknowledged drawbacks that have been reviewed in the past (Ercolini, 2004). The latter include selective DNA extraction, preferential PCR amplification (Reysenbach et al., 1992), amplification efficiency when working with difficult templates (Nocker et al., 2007) and presence of multiple rRNA copies with overestimation of the diversity (Nubel et al., 1996). The use of PCR-DGGE for screening communities can further be limited by the small fragment size of the PCR products;

amplification of 300–400 bp might not contain enough information for a precise taxonomic classification (Nocker et al., 2007; Ovreas, 2000). In alternative, culture-independent terminal restriction fragment length polymorphism (T-RFLP) has shown reproducibility and potential for a higher throughput than PCR-DGGE (Nieminen et al., 2011). T-RFLP has been applied to bacterial composition and succession in food like fish (Reynisson et al., 2009; Tanaka et al., 2010), milk (Rasalofo et al., 2011) and meat (Nieminen et al., 2011). It has to be noted that the taxonomic resolution level of the T-RFLP method when applied for the characterization of psychrotrophic bacterial communities in MAP minced meat was in between genus and species within the investigated LAB isolates and within family and genus within the investigated Gram-negative isolates (Nieminen et al., 2011). Finally, high throughput sequencing tools such as direct pyrosequencing of PCR amplicons after culture-independent DNA extraction, despite the cost still associated to the approach, can be an extremely valuable tool for the evaluation of microbial diversity in meat and meat products. Very recently, it was shown that direct pyrosequencing of 16S rRNA gene amplicons from meat stored in different packaging conditions was much more powerful than PCR-DGGE for the detection of microbial succession during storage of meat (Ercolini et al., 2011). In fact, a wider number of species could be identified by pyrosequencing compared to PCR-DGGE and, moreover, the pyrosequencing approach can give a quantitative estimation of the abundance of a single taxon in each sample based on the number of reads of its particular 16S rRNA gene sequence. 3. Spoilage associated microbial populations and storage conditions According to the literature, genera occurring on freshly cut meat frequently are Acinetobacter, Pseudomonas, Brochothrix, Flavobacterium, Psychrobacter, Moraxella, Staphylococcus, Micrococcus, lactic acid bacteria (LAB) and different genera of the family of Enterobacteriaceae (Blickstad et al., 1981; Blickstad and Molin, 1983; Dainty and Mackey, 1992; Dainty et al., 1983; Enfors et al., 1979; Erichsen and Molin, 1981). The environment then enforces a selection pressure on the bacterial community, and those groups of bacteria best adapted to the environment will outgrow the others, become dominant, and reach high numbers. Thus, the survival, growth and succession of specific spoilage bacteria can be affected by a diversity of ecophysiological factors in the physical and chemical environment. These factors, including meat constituents, temperature, pH, oxygen or carbon dioxide (packaging atmosphere) and competing microbiota are important in maintaining meat quality over time (Koutsoumanis et al., 2006; Lambert et al., 1991). A list of species that can be found in raw meat during chill storage in different conditions is reported in Table 2.

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3.1. Influence of packaging atmosphere Survival and growth of spoilage organisms are greatly affected by the gaseous composition of the atmosphere surrounding the meat. It is well known that the aerobic storage can accelerate spoilage due to the fast growing of the pseudomonads while vacuum (VP) and MAP can favour the dominance of a facultative anaerobic population including LAB and B. thermosphacta (Enfors et al., 1979; Lambropoulou et al., 1996; Newton and Gill, 1978; Nychas and Skandamis, 2005). Similarly, psychrophilic and psychrotrophic Clostridium spp. have been confirmed as causative agents of ‘blown pack’ spoilage of VP chilled meats (Broda et al., 1996a; Dainty et al., 1989; Kalchayanand et al., 1989). On the other hand, limited information was available in the past decades about the influence of the packaging on the specific bacteria at species or strain level (Samelis, 2006). Nowadays, the spoilage microbiota as well as the dynamics of the specific bacteria during spoilage of meat is studied. Both storage time and packaging have a strong effect on the bacterial communities in chilled beef (Doulgeraki et al., 2010, 2011; Ercolini et al., 2006a, 2009, 2010a, 2011; Pennacchia et al., 2011), and pork (Jiang et al., 2010; Li et al., 2006); this is also highlighted in Table 2. Members of the Ps. fluorescens group, along with the psychrotrophic Ps. fragi, Ps. lundensis and Ps. putida, are often isolated from aerobically spoiled meat even during storage at low temperatures (Ercolini et al., 2007, 2010b; Labadie, 1999; Stanbridge and Davies, 1998). Similarly, phenotypic and molecular characterization of the psychrotrophs isolated from fresh and spoiled meat revealed the presence of three major species of Pseudomonas (Ps. fragi, Ps. fluorescens and Ps. lundensis) (Liao, 2006). It has been observed by a number of investigators that Ps. fluorescens is more abundant on fresh meats than Ps. fragi but that the latter becomes dominant over time (Lebert et al., 1998). Ps. fragi was reported to be the most frequently dominating species, followed by the Ps. lundensis and Ps. fluorescens (Banks and Board, 1983; Dainty and Mackey, 1992; Erichsen and Molin, 1981; Molin and Ternstrom, 1982; Molin et al., 1986; Shaw and Latty, 1982, 1984; Stanbridge and Davies, 1998). High concentrations of CO2 (up to 10%) have been found to inhibit the growth of Ps. fluorescens and Ps. fragi in red meat (Gill and Tan, 1980), and Ps. fragi was inhibited more than the other pseudomonads like Ps. fluorescens and Ps. lundensis (Stanbridge and Davies, 1998). Using a species-specific assay, it was proved that Ps. fragi occurs almost in all the samples during storage of beef in various conditions including air, MAP and VP (Ercolini et al., 2007, 2010a, 2011; Pennacchia et al., 2011). On the other hand, Olofsson et al. (2007) have found several Pseudomonas species other than Ps. fragi as contaminating population of refrigerated beef. However, due to its strong occurrence in fresh and spoiled meat, Ps. fragi plays a significant role in the spoilage of meat (Gill, 2003; Lebert et al., 1998; Stanbridge and Davies, 1998), and it is supposed that meat can be considered its ecological niche (Ercolini et al., 2010b; Labadie, 1999). Overall, it can be concluded that while Ps. fragi appears the dominant Pseudomonas regardless of the packaging conditions, all the other Pseudomonas species mainly occur during air storage of raw meat. Shewanella, a genus closely related to Pseudomonas has been also found in a wide range of muscle foods (Gram and Daglaard, 2002; McMeekin, 1982; Molin and Ternstrom, 1982; Nychas et al., 2007), and in combination with Pseudomonas species has been found to contribute significantly in the spoilage of animal origin foods. Shewanella putrefaciens strains are characterised by their ability to produce malodorous compounds e.g. Hydrogen sulphide combines with muscle pigment to give a green discoloration, contributing in this way to the development of spoilage odours and deterioration of meat. Specifically, Sh. putrefaciens has been considered the primary cause of spoilage of chill-stored VP meat (Molin and Ternstrom, 1982; Nychas et al., 2008) and high pH VP meat (Borch et al., 1996). Several authors have detected many members of the Enterobacteriaceae on raw beef, lamb, pork, and poultry products, as well as on offal meats (Garcia-Lopez et al., 1998). However, the

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genera Serratia, Enterobacter, Pantoea, Klebsiella, Proteus and Hafnia, often contribute to meat spoilage (Borch et al., 1996; Nychas et al., 1998). With regard to their meat spoilage potential, the most important Enterobacteriaceae are the species S. liquefaciens, Hafnia alvei and Enterobacter (Pantoea) agglomerans (Samelis, 2006). Among the Enterobacteriaceae, Serratia spp. is the most commonly found genus in meat. S. grimesii and S. proteamaculans occur in meat stored in air, MAP and VP storage; although S. grimesii is often found at late stages of storage (Ercolini et al., 2006a, 2010a; Pennacchia et al., 2011). S. proteamaculans was recovered from fresh meat, while Citrobacter freundii and Proteus vulgaris were recovered from minced beef stored aerobically and under MAP, respectively (Doulgeraki et al., 2011). S. liquefaciens has been found to be the most common member of the Enterobacteriaceae in meat stored in different atmospheres (Doulgeraki et al., 2011; Lee et al., 1985; Patterson and Gibbs, 1977; Stanbridge and Davies, 1998), while H. alvei is very frequently encountered in minced beef stored under MAP or VP (Borch et al., 1996; Doulgeraki et al., 2011; Drosinos and Board, 1995; Lee et al., 1985; Nychas et al., 1998; Stanbridge and Davies, 1998). This could be associated with the fact that H. alvei did not compete well in the high oxygen atmosphere, where pseudomonads tended to be prevalent (Stanbridge and Davies, 1998). Additionally, Rahnella spp. has been shown to potentially play an important role in the spoilage of meat and was found as the dominant enterobacterium in the late phases of refrigerated storage of beef in MAP and VP (Ercolini et al., 2006a; Pennacchia et al., 2011). LAB are recognized as important competitors of the other spoilage related microbial groups under VP/MAP conditions (Castellano et al., 2004; Gill, 1996; Nychas and Skandamis, 2005; Stanbridge and Davies, 1998; Tsigarida et al., 2000). Particularly, Lactobacillus spp., Carnobacterium spp. and Leuconostoc spp. are associated to the spoilage of refrigerated raw meat (Labadie, 1999), while they can also become dominant throughout storage in reduced O2 availability (Lambert et al., 1991). LAB populations in VP beef were mainly represented by Lb. curvatus, Lb. sakei, and Leuconostoc spp. (Fontana et al., 2006; Pennacchia et al., 2011; Yost and Nattress, 2002). Similarly, Lactobacillus was the major component of the microbiota in chilled pork for VP (Blixt and Borch, 2002; Borch et al., 1996; Greer et al., 1994; Jiang et al., 2010; Li et al., 2006). More specifically, Lb. sakei has been associated with fresh meat (Champomier-Verges et al., 2001) as well as spoilage of meat both under VP and MAP (Borch and Molin, 1988; Chenoll et al., 2007; Doulgeraki et al., 2010; Ercolini et al., 2006a, 2009) and it is known to be among the most common psychrotrophic lactobacilli detected. Such dominance of Lb. sakei would deserve in depth investigations in order to work out the reasons, beyond the psychrotrophic nature, of its selective development during meat storage. Similarly, it has been reported that only psychrotrophic LAB like Lb. sakei, Lb. curvatus, Lb. fuchuensis, C. divergens, C. maltaromaticum and Leuconostoc sp. attained high cell numbers in MAP and chill-stored VP meat (Nieminen et al., 2011; Sakala et al., 2002a, 2002b; Schillinger and Lucke, 1986; Shaw and Harding, 1984). More species of lactobacilli can be found during storage under vacuum at 4 °C including Lb. algidus beyond Lb. sakei. In addition, at 1 °C Lactobacillus spp., Weissella spp. and L. mesenteroides can occur indicating an influence of the temperature in the development of different species under the same packaging conditions. Holzapfel (1998) reported that more rarely Lb. plantarum and Lb. casei are associated with meat systems and in lower frequency and numbers than Lb. curvatus and Lb. sakei. Additionally, leuconostocs have been identified as predominant organisms in beef stored aerobically (Doulgeraki et al., 2010) and under VP/MAP (Stanbridge and Davies, 1998; Yost and Nattress, 2002) while their presence in the initial mesophilic bacterial microbiota is very frequent (Borch et al., 1996; Doulgeraki et al., 2010). In a latter study it has been reported that L. gasicomitatum may respire while growing on high-oxygen MAP meats thus dominating the spoilage LAB population due to effective growth and improved stress resistance (Johansson et al., 2011). Moreover, the dominance of leuconostoc could be explained by the fact that among the LAB isolated

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during meat spoilage only these exhibited AI-2‐like activity, whereas their percentage in the recovered final population depended on storage time and temperature. The results provide a basis for further research concerning the effect of storage temperature on the expression of genes encoding AI-2 activity and on the diversity of the ephemeral bacterial population (Blana et al., 2011). The two species of carnobacteria that can be found in meat during storage are C. divergens and C. maltaromaticum. C. divergens is recognised as a predominant species on raw meat regardless of the packaging conditions (Leisner et al., 2007), it has been found in meat stored both in air and VP (Ercolini et al., 2010a, 2011; Pennacchia et al., 2011), and is commonly associated with the spoilage of meat (Axelsson, 2008; Ercolini et al., 2009; Jones, 2004; Laursen et al., 2005; Sakala et al., 2002a; Stanbridge and Davies, 1998). By contrast, C. maltaromaticum, whose complete genome has been recently described (Leisner et al., 2012), has been also investigated as possible spoilage agent in fresh meat (Casaburi et al., 2011; Ercolini et al., 2009) and it has been suggested that its actual role in the sensory spoilage of meat can be considered negligible at least in absence of other spoilage bacteria (Casaburi et al., 2011). The psychrotrophic B. thermosphacta is an important meat spoilage bacterium and commonly associated with the spoilage of fresh meats (Cantoni et al., 2000; Ercolini et al., 2006a; Rattanasomboon et al., 1999; Russo et al., 2006; Samelis, 2006; Stanbridge and Davies, 1998). The capability of B. thermosphacta to grow on meat during both aerobic and vacuum storage makes it a significant meat colonizer and an important portion of the spoilage microbiota (Dainty et al., 1983; Pin et al., 2002; Labadie, 1999), and it can be occasionally the dominant organism (Borch et al., 1996). It has been reported that Brochothrix sp./B. thermosphacta was detectable in the middle of the storage of VP pork but was difficult to be observed at the last sampling point (Jiang et al., 2010), which confirmed that these spoilage bacteria were unable to compete against LAB in chill-stored meat under anaerobic conditions (Russo et al., 2006; Sakala et al., 2002a). However, B. thermosphacta was always found in beef stored in MAP (Ercolini et al., 2006a), in air, and VP (Ercolini et al., 2010a, 2011; Pennacchia et al., 2011). Psychrothophic clostridia such as Cl. algidicarnis, Cl. algidixylanolyticum, Cl. estertheticum, Cl. frigidicarnis, Cl. gasigenes and Cl. putrefaciens have been identified as the causative agents of ‘blown pack’ spoilage of VP chilled meat (Adam et al., 2010; Brightwell et al., 2007; Broda et al., 1996a, 1999, 2000; Dainty et al., 1989; Kalchayanand et al., 1989; Lawson et al., 1994; Silva et al., 2011). Cl. frigidicarnis is associated with spoilage of chilled red meat and was originally isolated from spoiled, VP, temperature abused beef (Adam et al., 2011). On the other hand, Cl. estertheticum seems to be the species more frequently associated with ‘blown pack’ spoilage (Adam et al., 2010; Cavill et al., 2011). Similarly, Cl. gasigenes has been identified as the causing agent of ‘blown pack’ spoilage in New Zealand (Broda et al., 2002), while Cl. algidicarnis has been mainly associated with deep-tissue spoilage (Broda et al., 1996b; De Lacy et al., 1998). In another study, the involvement of the two aforementioned species in ‘blown pack’ spoilage of VP has been described, and it was suggested that more than one species of psychrotrophic clostridia may be associated with this kind of spoilage (Silva et al., 2011). “Bone taint” is a spoilage typically characterized by putrid odours coming from the internal part of the muscle in contact with the bone. The taints can be mainly due to the development of clostridia that are capable of growing without oxygen. Cl. algidicarnis and Cl. putrefaciens have been implicated in cases of “bone taint” in chilled VP lamb and pork (Broda et al., 1996a, 2000, 2003, 2009; Cavill et al., 2011; Ross, 1965). 3.2. Influence of temperature Storage temperature is considered the most important factor affecting meat spoilage by affecting the lag phase duration, the maximum specific growth rate and the final cell numbers (Labuza and Fu, 1993;

Mataragas et al., 2006). Although most countries have established regulation with maximum temperature limits for refrigeration storage, it has been shown that temperature conditions higher than 10 °C are not unusual (Koutsoumanis et al., 2006). Thus, a shift of the microbial populations has been observed under different storage temperatures. Psychrotrophic bacteria which belong to microbial genera of both Gram positive, such as LAB, and Gram negative bacteria, such as Pseudomonas spp. and Enterobacteriaceae could be developing in meat at chill temperatures (Gill and Newton, 1978; Holzapfel, 1998). More accurately, species of Pseudomonas are particularly involved in the spoilage of meat stored at chill temperatures (Ercolini et al., 2007, 2010b; Labadie, 1999). On the other hand, the microbiota of VP chillstored meat is characterized in most cases by psychrotrophic LAB (Borch et al., 1996; Hitchener et al., 1982; Nychas et al., 1998; Shaw and Harding, 1984) and psychrophilic and psychrotrophic Clostridium spp. (Adam et al., 2010; Silva et al., 2011). In the case of temperature abuse, it has been reported that Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. were predominant in the spoilage microbiota at 30 °C (Gill and Newton, 1978). It has been noted that the storage temperature can affect the spoilage potential of different bacteria (Stanbridge and Davies, 1998) and species belonging to the same bacterial group do not necessarily grow at the same temperature (Doulgeraki et al., 2010). Thus, differences on bacterial species/strains were observed for beef (Doulgeraki et al., 2010, 2011; Fontana et al., 2006) during storage at different temperatures. Furthermore, the microbiota recovered from the fresh beef was markedly different from that at the end of storage at chill temperatures, while an increased diversity at strain level occurred at low temperatures combined with MAP (Doulgeraki et al., 2010). Similarly, within the LAB population obtained from minced beef, Lb. sakei and Leuconostoc spp. were identified as significant members of the microbiota at chill and abuse temperatures, respectively (Doulgeraki et al., 2010). S. liquefaciens represented the dominant isolate of Enterobacteriaceae during storage of minced beef (Doulgeraki et al., 2011) and pork (Blickstad et al., 1981) at chill and abuse temperatures especially under aerobic conditions. On the other hand, H. alvei was the dominant member of Enterobacteriaceae on beef steaks stored in MAP at 5 °C (Stanbridge and Davies, 1998) and on minced beef at 10 °C (Doulgeraki et al., 2011). H. alvei has been shown as a major spoilage microorganism in meat, in particular due to its psychrotolerant character which gives an adaptation advantage over other microorganisms (Borch et al., 1996). The observation that different species/strains of Enterobacteriaceae occurred at different temperatures could be explained by temperature-induced differences in adaptation and competitiveness within this group of spoilage organisms (Doulgeraki et al., 2011). Within the same species, it was also demonstrated that the growth and spoilage related activities are also influenced by the temperature and that in isolates from meat, the main difference between mesophilic and psychrotrophic bacteria is the fact that the latter are disadvantaged in the growth at 30 °C (Ercolini et al., 2009). 3.3. Influence of natural antimicrobials and other “hurdles” Numerous methods are available to control spoilage and thus extend the shelf life of meat. Novel preservation technologies include the application of the concept of bioprotective cultures and natural antimicrobial compounds such as essential oils or enzymes and bacteriocins, while all preservation treatments may also be used in combination to make use of synergistic effects (Schillinger and Holzapfel, 2006). In the case of natural antimicrobial compounds, it has been reported that the bacterial counts were significantly suppressed in meat stored aerobically in the presence of oregano essential oil (Tsigarida et al., 2000). Moreover, oregano essential oil, as a potential ‘hurdle’, was found to affect the contribution of spoilage microorganisms to the microbial association as well as to the physico-chemical changes of the minced meat (Burt, 2004; Skandamis and Nychas, 2001). The volatile compounds

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of oregano essential oil also are capable of affecting both growth and metabolic activity of microbial populations in meat stored in MAP (Skandamis and Nychas, 2002). It has been reported that the growth of meat spoilage bacteria in vitro such as Lb. sakei, Lb. curvatus and C. maltaromaticum, B. thermosphacta, Ps. fluorescens and S. liquefaciens was affected by the presence of essential oils (Outtara et al., 1997). On the other hand, differences in microbial metabolites produced with the addition of oregano essential oil, have been associated with changes in the metabolic pathways of some members of the microbial association (homofermentative or heterofermentative pathway of glucose metabolism by LAB) or a selection of homofermentative LAB due to the essential oil (Nychas et al., 1998; Skandamis and Nychas, 2001). Indeed, Axelsson (2004) concluded that the metabolism of LAB could be influenced, while a shift in the microbial metabolism of LAB in meat stored under VP/MAP conditions has also been reported (Borch and Agerhem, 1992). Similarly, volatile compounds of essential oil suppressed the leuconostocs and seemed to have provided Lb. sakei with an ecological advantage as it has found to dominate the final stage of storage (Doulgeraki et al., 2010). In the latter case, it has also to be noted that the presence of essential oil affected strain diversity as it was observed that different Lb. sakei strains dominated the final stage of storage compared to the untreated samples. Additionally, in a similar study the essential oil reduced the number of different recovered S. liquefaciens and P. vulgaris strains, indicating an inhibitory effect on the diversity of these species at strain level, while an advance in H. alvei diversity was observed (Doulgeraki et al., 2011). The use of bacteriocins is of great interest, although nisin and pediocin PA-1are the only commercially available (Cotter et al., 2005). Bacteriocins may be added as biopreservatives to improve the microbial stability and safety of chill-stored fresh meat (Samelis, 2006). Advantages of nisin are its broad antimicrobial spectrum and bactericidal mode of action (Stiles and Hastings, 1991). However, nisin is more active against Grampositive than Gram-negative organisms and an improved effect against Gram negatives can be obtained by the combined use of chelators such as EDTA (Boziaris and Adams, 1999). Although the effect of these compounds on the total spoilage microbiota is well established, limited information is available about their effect on specific spoilage bacteria at species or strain level and how the bacteriocin can influence the spoilage dynamics during meat storage. Several studies have been performed on bacteriocins for improving the safety of foods (Deegan et al., 2006). It has been reported that variations in strain sensitivities and development of strain resistance/adaptation are also of major concern for the application of bacteriocins in food (Galvez et al., 2007). In a meat model system, nisin reduced viable counts of E. coli, reduced growth of Staph. aureus, and suppressed slime-producing bacteria (Garriga et al., 2002). Similarly, a synergistic activity of nisin and lysozyme against Gram-positive bacteria, including spoilage lactobacilli and Staph. aureus was observed (Chung and Hancock, 2000; Nattress and Baker, 2003).In the case of bacteriocins produced from Carnobacterium, they proved to be effective mainly towards microorganisms such as other LAB, Enterococcus and L. monocytogenes (Stoffels et al., 1992; McMullen and Stiles, 1996). Indeed, antibacterial spectrum of activity has been reported for antilisterial bacteriocin from C. maltaromaticum (formely C. piscicola) and for Piscicolin 126 (Jack et al., 1996). For other carnobacteriocins the antagonistic activity reported has been reported more restricted to Listeria species (Ahn and Stiles, 1990; Stoffels et al., 1993), while a limited number of C. divergens and C. maltaromaticum isolates possessed a biopreservative potential due to their production of bacteriocins with a wide spectrum of inhibition (Laursen et al., 2005). This would indicate that strains of the same Carnobacterium species may produce different bacteriocins (McMullen and Stiles, 1996), or that the production rate for the same bacteriocin differs among strains (Stoffels et al., 1993), thus variations in the antibacterial spectra can be expected within the same species (Schobitz et al., 1999). Bacteriocins can be also used to develop active packaging devices (Mauriello et al., 2004; Mauriello and Villani, 2011) by coating their

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solutions on the surface of plastic films to be used for meat packaging. The contact between activated surface and meat was proved to be very important for an effective antimicrobial effect (Ercolini et al., 2006b). In addition, the thickness of the coating and the type of bacteriocin solution to be employed can influence the coating homogeneity and antimicrobial activity (La Storia et al., 2008). A nisin-containing cellophane coating was found to reduce viable counts of total aerobic bacteria in fresh veal meat stored at 8 °C (Guerra et al., 2005). Similarly, an overall reduction of the viable counts in meat stored in nisinactivated packaging was observed during storage of meat under vacuum although the species diversity showed a variation during time that was strongly dependent on the sample and method of detection (Ercolini et al., 2010a, 2011). The injection of meat and meat products with brine containing salt, sugar, organic acids and herbs or spices is increasingly applied in Northern Europe. This treatment is mainly performed to obtain desired organoleptic properties, but it has been also reported that the marination introduces or selects for LAB in the product (Bjorkroth, 2005). Additionally, soy sauce and wine based marinades were found to decrease significantly the total viable counts and also to inhibit Pseudomonas spp. in beef stored at chill and abuse temperatures (Kargiotou et al., 2011). However, limited research has been done to describe the development and final composition of the bacterial flora in marinated meat (Schirmer et al., 2009). LAB such as Carnobacterium, Lactobacillus and Leuconostoc have been the main genera associated with spoiled, marinated broiler meat strips stored under MAP (Bjorkroth et al., 2000; Susiluoto et al., 2003). Similarly, in marinated, skin containing broiler legs stored in MAP the spoilage LAB population has been found to be dominated by carnobacteria that overgrew Enterococcus species occurring in the initial fresh meat (Bjorkroth et al., 2005). In another study, L. gasicomitatum was found to be associated with the spoilage of high-oxygen MAP beef steaks from two different manufactures (Vihavainen and Bjorkroth, 2007). In addition, the development of the microbiota in marinated pork products was found to be affected by the plant, as two different bacteria, L. carnosum and Lb. algidus occurred in samples from two different production sites (Schirmer et al., 2009). A new species, Lb. oligofermentans was also found to be associated with spoilage of MAP marinated poultry meat (Koort et al., 2005). 4. Conclusions and future perspectives Although several studies on muscle foods have clearly shown that storage temperature combined with packaging conditions as well as the application of different treatments/antimicrobials induced a selection of the spoilage microbiota even at strain level, still further research is needed in this area. Interesting insights could come from the simultaneous detection and localization of bacteria in meat as it was done in other foods (Ercolini et al., 2003). Indeed, the differential distribution of species could suggest specific ecological reasons for the establishment of sites of actual microbial growth in the meat. Similar studies have reported the distribution of bacteria in meat, cheese and olives (Ercolini et al., 2003; Gram et al., 2002; Nychas et al., 2002; Wilson et al., 2002). This approach, which has been mainly applied to fermented foods, could be valuable to highlight the sites of metabolite release in meat and consequently affect the choice and/or development of specific storage conditions. Such findings can also possibly contribute to the explanation of why, regardless of the species diversity, different metabolic compounds can be detected in meat during storage (Ammor et al., 2009; Argyri et al., 2010, 2011; Casaburi et al., 2011; Ercolini et al., 2009, 2010b, 2011; Ingram and Dainty, 1971; Nychas et al., 2008; Skandamis and Nychas, 2001, 2002). The above observations can be fundamental in understanding the spoilage process and in explaining the presence of different products or by-products that occur in different storage conditions. Thus, further studies are needed in order to monitor the succession of strains during meat storage as the comparison of various strains of the same species

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may reveal specific characteristics of the spoilage-associated biotypes that can help their establishment in the meat ecological niche. A significant amount of data has been filed in the recent years thanks to the application of molecular techniques to identify and monitor the microbiota developing in raw meat. However, the principal limitation of a molecular approach is the fact that it cannot be directly related to the spoilage development because no information is provided (i.e. metabolic activities) beyond identification. In addition, it is worth to underline as a conclusion of this review that although the molecular techniques offer advantages in reliability and speed of identification, and have contributed to the production of a significant amount of ecological data, their use has so far not changed much the acknowledged structure of the spoilage-associated microbiota occurring in meat. The current generation approaches of genome sequencing together with the culture-independent analyses of microbial communities by direct sequencing and metagenomics are going to deepen the structure of meat microbiota as well as other microbial ecosystems associated to foods. The above approaches will be hopefully complemented with transcriptomics and metatranscriptomic analyses of meat microbiota in order to further contribute to the advance in the knowledge of such a complex event as meat spoilage. Acknowledgements This study was supported by a EU project (SYMBIOSIS-EU) within the 7th Framework Programme (ref. grant agreement no. 211638). The information in this article reflects only the authors' views and the community is not liable for any use that may be made of the information contained herein. References Adam, K.H., Flint, S.H., Brightwell, G., 2010. Psychrophilic and psychrotrophic clostridia: sporulation and germination processes and their role in the spoilage of chilled, vacuum-packaged beef, lamb and venison. International Journal of Food Science and Technology 45, 1539–1544. Adam, K.H., Brunt, J., Brightwell, G., Flint, S.H., Peck, M.W., 2011. Spore germination of the psychrotolerant, red meat spoiler, Clostridium frigidicarnis. Letters in Applied Microbiology 53, 92–97. Adekambi, T., Drancourt, M., Raoult, D., 2008. The rpoB gene as a tool for clinical microbiologists. Trends in Microbiology 17, 37–45. Ahn, C., Stiles, M.E., 1990. Plasmid-associated bacteriocin production by a strain of Carnobacterium piscicola from meat. Applied and Environmental Microbiology 56, 2503–2510. Ammor, M.S., Argyri, A., Nychas, G.-J.E., 2009. Rapid monitoring of the spoilage of minced beef stored under conventionally and active packaging conditions using Fourier transform infrared spectroscopy in tandem with chemometrics. Meat Science 81, 507–514. Ampe, F., Ben Omar, N., Moizan, C., Wacher, C., Guyot, J.-P., 1999. Polyphasic study of the spatial distribution of microorganisms in mexican pozol, a fermented maize dough, demonstrates the need for cultivation-independent methods to investigate traditional fermentations. Applied and Environmental Microbiology 65, 5464–5473. Argyri, A.A., Panagou, E.Z., Tarantilis, P.A., Polysiou, M., Nychas, G.-J.E., 2010. Rapid qualitative and quantitative detection of beef fillets spoilage based on Fourier transform infrared spectroscopy data and artificial neural networks. Sensors and Actuators B: Chemical 145, 146–154. Argyri, A.A., Doulgeraki, A.I., Blana, V.A., Panagou, E.Z., Nychas, G.J.E., 2011. Potential of a simple HPLC based approach to quantify spoilage of minced beef stored at different temperatures and packaging systems. International Journal of Food Microbiology 150, 25–33. Audenaert, K., D'Haene, K., Messens, K., Ruyssen, T., Vandamme, P., Huys, G., 2010. Diversity of lactic acid bacteria from modified atmosphere packaged sliced cooked meat products at sell-by date assessed by PCR-denaturing gradient gel electrophoresis. Food Microbiology 27, 12–18. Axelsson, L.T., 2004. Lactic acid bacteria: classification and physiology, In: Salminen, S., Von Wright, A., Ouwehand, A. (Eds.), Lactic Acid Bacteria Microbiology and Functional Aspects, 3rd edition. Marcel Dekker, New York, pp. 1–66. Axelsson, L.T., 2008. Lactic acid bacteria: classification and physiology. In: Salminen, S., Von Wright, A., Ouwehand, A. (Eds.), Lactic Acid Bacteria Microbiology and Functional Aspects. Marcel Dekker, New York, pp. 19–66. Banks, J.G., Board, R.G., 1983. The classification of pseudomonads and other obligately aerobic Gram-negative bacteria from British pork sausage and ingredients. Systematic and Applied Microbiology 4, 424–438. Barakat, R.K., Griffiths, M.W., Harris, L.J., 2000. Isolation and characterization of Carnobacterium, Lactococcus, and Enterococcus spp. from cooked, modified atmosphere packaged, refrigerated, poultry meat. International Journal of Food Microbiology 62,

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