Listeria Monocytogenes in Soft Spreadable Salami: Study of the Pathogen Behavior and Growth Prediction During Manufacturing Process and Shelf Life

Recently, particular attention has been addressed to the control of Listeria monocytogenes in ready-to-eat meat products, such as fermented salami, as a consequence of several listeriosis outbreaks associated with the consumption of these types of products. A short-ripened spreadable salami, typically produced in the Umbria region (Central Italy), was challenged with L. monocytogenes aiming to evaluate the pathogen’s growth dynamics and to define its growth potential during processing and storage time. The pathogen counts were stable in the inoculum level (2 Log CFU/g) during the production process and up to 30 days of storage time, decreasing thereafter. The growth potentials registered for process phase and storage time were 0.40 and −1.28, showing that the application of the hurdles technology principle successfully creates an unfavorable environment for L. monocytogenes growth.


Introduction
Listeria monocytogenes is an ubiquitous Gram-positive bacterium able to contaminate a wide variety of foodstuffs; particularly, it is considered a relevant foodborne pathogen commonly associated with ready-to-eat (RTE) products [1,2]. The microorganism is responsible for numerous outbreaks of listeriosis worldwide [3][4][5][6] with high mortality rates in specific population categories such as immunocompromised subjects, elderly and pregnant women [7][8][9]. L. monocytogenes is also considered a real concern in the meat industry. Once the microorganism is introduced into a meat processing plant through contaminated unprocessed raw materials, and due to its distinct characteristics of resistance to environmental conditions, it can survive through the manufacturing process [10][11][12]. Furthermore L. monocytogenes is able to adhere to surfaces in the food-processing environments and, once attached, it may produce biofilms that are resistant to disinfection and from which cells can become detached and constantly contaminate food products [13,14]. As a consequence, in spite of food producers' efforts, L. monocytogenes may not be completely eliminated during the production of dry-fermented meat products [15], suggesting that further measures must be taken to avoid the growth of this pathogen [16,17]. In the literature, it is reported that L. monocytogenes growth during fermentation and drying of meat products can decrease due to the combined action of several hurdles such as the reduction in water activity (a w ) and pH or the employ of additives, such as nitrite and nitrate salts [18,19]. However, in some cases, the physicochemical parameters of fermented salami can be permissive for Listeria growth [20,21], particularly for those products with short ripening [22]. In Central Italy, there are several RTE spreadable fermented meat products with short ripening times and high fat contents [23][24][25][26]. The characteristics of these RTE salami products together with the short maturation could increase the chance that L. monocytogenes survives and proliferates during the manufacturing process. For RTE foods that are able to support the growth of L. monocytogenes, food business operators (FBO) can benefit from scientific studies in order to demonstrate that the product complies with the regulation's criteria throughout the shelf-life [27][28][29]. Nevertheless, few studies and practices are found in the literature that could be applied by FBO to their own products, especially for artisanal and niche ones [30]. The aim of the present investigation was to evaluate the growth dynamics of L. monocytogenes and to define its growth potential during the processing and storage time of an artisanal spreadable "Monti Sibillini" salami manufactured by a producer in the Umbria region (Central Italy), and characterized by a short ripening time and a soft consistency. Furthermore, this study also aimed to propose a valuable scientific tool enabling food manufacturers who produce salami with similar characteristics to prove whether their product is supportive of L. monocytogenes growth, according to EC Regulation No. 2073/2005 [28].

Bacterial Cultures and Inoculum Preparation
A multi-strain mix of L. monocytogenes was used in the study to carry out a challenge test. The mix consisted of an authenticated reference strain (WDCM 00021) and two strains from Istituto Zooprofilattico Sperimentale dell'Umbria and Marche "Togo Rosati" (IZSUM) collection isolated from food matrices (Lm15011/14 from salami and Lm36206/14 from dry sausage); each strain was lyophilized and kept at +4 • C in the IZSUM collection. The inoculum to be used in the challenge study of salami was prepared according to the guidelines of the European Union Reference Laboratory for Listeria monocytogenes [31]. Briefly, the bacterial cultures used in the study were regenerated into Brain Heart Infusion (BHI; Oxoid, Basingstoke, UK) by incubation at 37 • C for 24 h. Aliquots of each activated culture (0.1 ml) were transferred into tubes containing BHI and incubated for 72 h at 10 ± 1 • C, a temperature close to the storage condition of the product. At the end of the incubation time, the cultures were equally combined to form the multi-strain cocktail, which was subsequently centrifuged at 2178 g at 10 • C for 5 min, the supernatant was discarded and the pellet resuspended in 10 mL of sterile physiological solution. Counts were performed by serial decimal dilution and inoculation in Agar Listeria Ottaviani Agosti (ALOA Selective Supplement, ALOA Enrichment Supplement; Biolife, Italy), incubated at 37 • C for 24−48 h and the multi-strain suspension was diluted with sterile physiological solution to obtain an inoculum able to determine a final concentration in meat batter of approximately 2 Log Colony Forming Units (CFU)/g. Generally, a microbiological challenge test assessing the Listeria monocytogenes growth potential (δ) is performed only on final meat products, starting at the day of product contamination and finishing at the end of the shelf life [31]. In this study, the contamination was performed at the beginning of the production process (into meat batter), taking into account the most probable conditions of natural contamination of the product and the specific packaging choices of the manufacturer (the salami is commercialized whole and under vacuum). Therefore, the evaluation of the pathogen's behavior, as well as all the other analytical determinations, were performed during both the production process and the shelf life.

Salami Production
The soft spreadable salami investigated here is a typical product of the Umbria region, in Central Italy. Its soft consistency is attributable to the high level of fat, the fine mincing of meat and the specific processing techniques. Meat batter was prepared using the following swine cuts: shoulder 30%, jowl and belly (70%). Moreover, salt 2.7%, ground black pepper 0.4%, ground garlic 0.1%, white wine (10 mL/kg), L-ascorbic acid (E301), potassium nitrate (E252) and sodium nitrate (E250) were also added in compliance with EC regulation N • 1333/2008 including subsequent amendments and additions [32]; no starter cultures were employed. For each batch, a total of 15 kg of meat batter was processed. The meat batter was minced twice with a 2.5 mm plate and then stuffed into casings of approximately 500 g each. Subsequently, the products underwent a drying process in a controlled environment for about 5 days, with decreasing temperature (T) and relative humidity (RH), as detailed below: T 24 • C RH 65% for 9 h, T 23 • C RH 65% for 3 h, T 22 • C RH 65% for 24 h, T 21 • C RH 60% for 20 h, T 21 • C RH 55% for 18 h, T 20 • C RH 50% for 14 h, T 19 • C and RH 45% for 28 h. The ripening phase took place in a controlled environment for another 5 days, with a T of 14-16 • C and an RH around 70%. Each salami was vacuum packaged and stored at +4 • C for 60 days.

Preparation of Samples
To conduct the challenge test, three batches of salami were produced, each batch consisted of 3 experimental groups of 6 products sized 500 g.
The first group (LM) was produced at the pilot plant of the IZSUM by experimentally contaminating the meat batter with a suspension of multi-strain mix of L. monocytogenes at a final concentration of approximatively 2 Log CFU/g. These sample units were used to evaluate the behavior of L. monocytogenes during salami production and ripening.
The second group (CTR) was produced at the pilot plant of the IZSUM by adding an amount of sterile saline solution, instead of the contaminating suspension, equivalent to the inoculum (50ml). These sample units were employed to determine the physicochemical characteristics of salami (considering also the potential influences of saline solution), to detect and/or enumerate the eventual L. monocytogenes occurring naturally in the products and to ensure that the same ripening condition and product evolution occurred in both the manufacturing process and the IZSUM. A third group (CTRFBO) was produced and ripened by the manufacturer in order to assure that the products that are routinely produced by the processor are equivalent to those submitted to experimental contamination.

Storage Conditions and Sampling
LM and CTR samples were ripened at the IZSUM according to the maturation profile (time/T/RH) applied by the producer and subsequently packaged under vacuum.
The shelf life defined by the manufacturer for the salami was equivalent to 60 days (starting from the end of ripening phase) stored at +4 • C therefore according to the EURL reference document [31], after ripening, the products were stored at +8 • C for 7 days and at +12 • C for the remaining storage time (53 days).
Microbiological and physicochemical analyses were performed at 0 (day of production), 1, 2, 5, 10 (maturation time), 40 and 70 days (storage time). All analyses were carried out on two replicates of salami and three replicates for each batch (N = 3 batch; n = 3 replicates per sampling time); at time 0 the sample was constituted by meat batter (approximately 100g), while at the following time points the sampling unit was a whole salami unpacked up until day 10, vacuum packed at 40 and 70 days.

Enumeration and Isolation of Viable Lactic Acid Bacteria
The enumeration of lactic acid bacteria (LAB) was conducted at 0, 1, 2, 5 and 10 days of maturation and at 40 and 70 days of storage time, in CTR samples of each batch according to the method reported by Ortenzi et al. [4]. The analyses were conducted using the Tempo®automated system (Tempo LAB®, bioMérieux, Mercy Etoile, France) that has been proved to achieve performance levels similar to the standard NF ISO 15214, 1998 [33,34]. Counting results were reported in terms of Log CFU/ g.

Detection and Enumeration of Listeria Monocytogenes
The detection and enumeration of L. monocytogenes were conducted following the previously reported sampling times in all sample units of each batch according to Annex I of Regulation No. 2073/2005 [28]. The validated method AFNOR BIO 12/11-03/04 [35] for the detection of Listeria monocytogenes in human food products was employed. Briefly, a pre-enrichment was performed in half-Fraser broth (Biolife, Italy), incubated for 24 h at 30±1 • C and then sub-cultured in Fraser broth (Biolife, Italy) and incubated for 24 h at 37±1 • C. Subsequently the VIDAS LMO2 test (VIDAS®, bioMérieux, Mercy Etoile, France) was performed. Samples found to be positive to the mentioned test are confirmed through isolation on ALOA growth medium. The limit of detection of the method is of 0.4 CFU in 25 g. For bacterial enumeration, the EN ISO 11290-2 [36] reference method was used with a limit of detection of 10 CFU/g. Counting results were reported in terms of Log CFU/g.

Physicochemical Determination
The physicochemical determinations were carried out at 0, 1, 2, 5 and 10 days of ripening and at 40 and 70 days during storage time in the sample units of each batch belonging to the CTR groups.
The pH measurements were performed through a puncture electrode probe connected to a portable pH meter (Mettler Toledo Inc., Columbus, OH, USA). The salt (NaCl) content was determined by the Volhard method according to AOAC (method n 935.43) [37]. Water activity (aw) was measured at 25 • C with the a w recorder AquaLab, series 3, Model TE (Decagon Devices Inc., Pullman, WA, USA).

Statistical Analysis
Bacterial counts were converted to Log CFU per g or mL. The individual means and standard deviations of the microbiological and physicochemical results were determined on the basis of the average of two samples. The data were statistically analyzed using SAS version 2001 (SAS institute inc., Cary, NC, USA).
Differences between mean values were detected through the Post-hoc Tukey's test and evaluations were based on a confidence interval of 95%. The growth potential (δ) of L. monocytogenes is estimated as the difference between the median of results at the end of the challenge test and the median of results at the beginning of the challenge test, in three replicates, for three batches [31].
During the production process and shelf life of the product, the fitting of L. monocytogenes growth curves was performed using the free software program DMFit (https://www.combase.cc/index.php/en/ DMFit) to measure growth parameters such as maximum specific growth rate (µmax, 1/h) and Lag time (λ, 1/h) using the model of Baranyi and Roberts [38].

Results and Discussion
In total, 54 salami (3 batches/3 groups/6 salamis) of 500 g each were produced. Three samples from each salami were submitted to analytical determinations during the testing period. Table 1 shows the results of the physicochemical determinations and LAB enumeration performed on CTR and CTRFBO salamis during ripening and storage. No significant differences (p < 0.05) were detected among the two groups, therefore the discussion took into account the characteristics of the spreadable salami produced by the processor.
The results highlight that the acidification process had taken place since the early stages of ripening, reaching the lowest value at day 10 (5.27). During the storage time the pH tended to show a slight increase reaching a final value (day 70) of 5.44, as reported elsewhere in different types of salami [39].
A reduction in a w values for salami occurred gradually and constantly throughout maturation and storage, from an initial value of 0.961 for the batter to a final value (day 70) of 0.931 (Table 1). The NaCl value, as expected, increased with ripening and storage time from an average value of 2.47% at day 0 to a final mean value (day 70) of 3.39% (Table 1). Different letters in the same row (v, w, x, y, z) indicate differences between mean values during sampling times (p ≤ 0.05). No differences were registered within the same column for each parameter considered. CTR, produced by the food business operator and ripened/stored at the Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche "Togo Rosati"; CTRFBO, samples produced and ripened/stored by the manufacturer in order to compare the products submitted to challenge testing to the products that are routinely produced by the processor. SEM, standard error mean.
The physicochemical properties of the salami investigated in this study are in agreement with those obtained by other authors in similar products [23,24,40,41], although a w values registered were higher than the threshold of 0.90 proposed for the microbial stability in order to prolong the shelf life of salami [19].
To evaluate the background microbiota, the lactic acid flora during the ripening and shelf life time was investigated ( Table 1). The results of the behavior of LAB during ripening and storage showed no difference among batches.
A significant growth (p < 0.05) of LAB was observed until the end of the manufacturing process with counts reaching 8.94 Log CFU/g at day 10 (LAB increase in 4 Log CFU/g from day 0). At the end of storage time (70 days) the LAB decreased to a value of 7.92 Log CFU/g ( Table 1).
The comparison of the results obtained from the analysis of the three batches showed no intrabatch nor interbatch variability of the physicochemical properties or of the background microbiota, confirming that the production process of the studied salami is fully standardized, differing from what was reported by other studies of analogous products [30]. This outcome also confirms the importance of considering multiple batches and various replicates per batch in order to establish the inherent variability in physicochemical characteristics and growth potential linked to the product and its processing/storage conditions, as recommended by Annex II on challenge testing of EC Regulation 2073/2005 [28].
As mentioned above, in the present study, the contamination was performed on meat dough, and the evaluation on the pathogen evolution and the definition of the growth potential (δ) was performed during both the production process and the shelf life.
The behavior of L. monocytogenes during spreadable salami ripening and storage time, considering the three batches of LM samples, is shown in Figure 1; the pathogen was undetectable in control samples. L. monocytogenes artificially inoculated in salami was able to survive throughout the ripening time (2.43 ± 0.19 Log CFU/g at 10 days) while a decrease in the pathogen concentration was observed during storage (1.07 ± 0.11 Log CFU/g at 70 days; Figure 1). At the end of the storage period (day 70) for batch 1 the L. monocytogenes was undetectable (<10 CFU/g) (data not shown).
Appl. Sci. 2020, 10, x FOR PEER REVIEW 1 of 15 The behavior of L. monocytogenes during spreadable salami ripening and storage time, considering the three batches of LM samples, is shown in Figure 1; the pathogen was undetectable in control samples. L. monocytogenes artificially inoculated in salami was able to survive throughout the ripening time (2.43 ± 0.19 Log CFU/g at 10 days) while a decrease in the pathogen concentration was observed during storage (1.07 ± 0.11 Log CFU/g at 70 days; Figure 1). At the end of the storage period (day 70) for batch 1 the L. monocytogenes was undetectable (<10 CFU/g) (data not shown). Concerning δ definition, as reported in Table 2A, at day 0 (beginning of production process) the median concentrations of L. monocytogenes were 2.15 Log CFU/g, 2.23 Log CFU/g and 2.10 Log CFU/g for batch 1, 2 and 3, respectively. The calculated growth potential of L. monocytogenes for the targeted spreadable salami was 0.13 Log CFU /g in batch 1 and 0.40 Log CFU /g in batch 2 and batch 3. The highest δ value obtained is retained among all tested batches, therefore, for the production process the δ is 0.40 Log CFU/g [31]. The calculated δ is ≤ 0.5 Log CFU/g, therefore, according to reference guidelines, it is assumed that during this time frame the salami is not able to support the growth of Concerning δ definition, as reported in Table 2A, at day 0 (beginning of production process) the median concentrations of L. monocytogenes were 2.15 Log CFU/g, 2.23 Log CFU/g and 2.10 Log CFU/g for batch 1, 2 and 3, respectively. The calculated growth potential of L. monocytogenes for the targeted spreadable salami was 0.13 Log CFU /g in batch 1 and 0.40 Log CFU /g in batch 2 and batch 3. The highest δ value obtained is retained among all tested batches, therefore, for the production process the δ is 0.40 Log CFU/g [31]. The calculated δ is ≤ 0.5 Log CFU/g, therefore, according to reference guidelines, it is assumed that during this time frame the salami is not able to support the growth of L. monocytogenes [31]. This evidence corroborates the results previously reported for the pathogen behavior ( Figure 1); indeed, no increase in L. monocytogenes loads was observed at any time during the process, whereas, a decreasing trend of the pathogen concentration was detected during the storage time (from day 10 to day 70). At day 0 of storage (that corresponds to the sampling day 10) the registered median concentrations of L. monocytogenes were 2.28 Log CFU /g for batch 1, 2.63 Log CFU /g for batch 2 and 2.50 Log CFU /g for batch 3 (Table 2B). The growth potential of L. monocytogenes was −1.28 Log CFU /g for batch 1 and 3 and −1.63 Log CFU /g for batch 2, therefore, concerning the storage time of spreadable salami, the δ is −1.28 Log CFU/g. As reported for the production process, in the conservation time the calculated δ is also lower than 0.5 Log CFU/g, confirming that the product is unsupportive of Listeria growth [31]. Furthermore, in this phase, not only is the calculated δ below the limit set by the reference document, but it is also represented by a negative value (< 0), suggesting, therefore, that during the conservation time the hurdles applied to the spreadable salami are able to exert their action against the growth of Listeria, decreasing the pathogen concentration ( Figure 1 and Table 2B).
The growth curves of L. monocytogenes in experimentally produced spreadable salami contaminated at the beginning of the production process and stored according to the abovementioned conditions were obtained by fitting the observed growth data using the DMFit program ( Figure 2). According to the Combase Predictor tool (part of the Modelling Toolbox, www.combase.cc) [42] in culture media with equivalent growth conditions of salami, a maximum L. monocytogenes population of 8.51 log CFU/g was reached in about 625 h (26 days, data not shown). However, the fitted growth curves show that the pathogen concentration reached a maximum level of 2.43 log CFU/g and then the growth was suppressed (Figure 2), corroborating the results reported above concerning the definition of Listeria growth potential. The DMFit program was also used to calculate growth parameters for each condition tested ( Table 3). The maximum specific growth rates (μmax, 1/h) ranged from −0.0019 to −0.0013, while the latency phase (Lag) ranged from 776 to 1032 hours. The lower asymptote was not reached at the end of the considered shelf life, therefore the final values for the three batches were not determined. Table 3. Output parameters estimated by the DMFit program for each growth curve in three batches of experimentally produced spreadable salami. The results of the fitting of observed growth data corroborate the outcomes of the challenge test. According to the Combase Predictor tool (part of the Modelling Toolbox, www.combase.cc) [42] in culture media with equivalent growth conditions of salami, a maximum L. monocytogenes population of 8.51 log CFU/g was reached in about 625 h (26 days, data not shown). However, the fitted growth curves show that the pathogen concentration reached a maximum level of 2.43 log CFU/g and then the growth was suppressed (Figure 2), corroborating the results reported above concerning the definition of Listeria growth potential. The DMFit program was also used to calculate growth parameters for each condition tested ( Table 3). The maximum specific growth rates (µmax, 1/h) ranged from −0.0019 to −0.0013, while the latency phase (Lag) ranged from 776 to 1032 h. The lower asymptote was not reached at the end of the considered shelf life, therefore the final values for the three batches were not determined.

Batch µmax (Log CFU/h) Lag (h) SE
The results of the fitting of observed growth data corroborate the outcomes of the challenge test. For each of the three growth curves, in fact, the value of µ max was negative, confirming that the growth of the targeted pathogen was suppressed in the food environment considered.
As shown in Table 3, the latency phase (Lag) of the three fitted curves ranges from a minimum value of 32 days (780 h) to a maximum value of 42 days (1032 h) for batch 2 and 1, respectively. The values registered in this study are remarkably higher than the value obtained through the growth prediction performed with the Combase Predictor tool [42] that was of 0.72 days (17.3 h, data not shown). As reported in the literature, the duration of the latency phase is characterized by inherent variability and by partially defined physiological and molecular processes [43], however, recent findings suggest that changes in the lag-time in order to develop tolerance can be considered the first change made by bacteria in response to stressor factors [44]. The optimization and elongation of Lag may therefore represent a strategy adopted by bacterial communities to tolerate environmental stress [43][44][45], that may correspond, in this study, to the hurdles present in the food matrix. These results demonstrate the inhibitory effect of the process and the product characteristics on L. monocytogenes growth, even though the studied spreadable salami showed a pH and a w values compatible with the growth of the microorganism [28]. In fact, similarly to what has been reported by other authors, the physicochemical properties of spreadable salami are not sufficient to justify the pathogen behavior [41], indeed, it has already been highlighted that L. monocytogenes is able to survive in environments with pH and a w values lower than those found in this study [46].
The factors that could affect the behavior of L. monocytogenes in RTE meat products are numerous, such as reductions in pH [47] and a w [48], the use of additives [49], and the presence of indigenous microflora [50]. The presence of NaCl is considered a method to control the pathogen growth, however, the concentration used in the present experiment is not demonstrated to limit L. monocytogenes growth [51]. Similarly, the temperature applied during the ripening phase and the storage period could not have negatively influenced the growth capacity of L. monocytogenes since the strains used for experimental contamination had undergone a process of thermal adaptation. The main hurdle in L. monocytogenes growth, as shown in the literature, is probably represented by lactic acid flora [50]. Several studies have shown, in fact, that the presence of LAB in foods can hinder the growth of pathogens by means of competition for nutrients, antimicrobial metabolite production and more generally through microbial antagonism [52,53].
Specifically, as reported in the literature, the reduction in L. monocytogenes count in some foodstuffs may have been attributable to the inhibitory effects of various metabolic products of LAB such as carbon dioxide, short-chain organic acids, hydrogen peroxide, diacetyl, and antimicrobial peptides [54].
The inhibitory activity of LAB towards sensitive food spoilage or pathogenic bacteria, such as L. monocytogenes, has been reported for various food products [52,55,56]. Particularly, Al-Zeyara et al. [57] and Cornu et al. [58] state that the lactic flora present in fermented products, such as salami, exerts a remarkable inhibitory effect on L. monocytogenes growth especially when the aforementioned microflora is present in concentrations higher than 4.5 Log CFU/g by creating an unfavorable environmental condition for the pathogen.
Studies of the LAB population of spreadable salami produced in Central Italy revealed that the most frequently detected lactic species were Lactobacillus sakei, Lactobacillus plantarum, Lactobacillus curvatus and Pediococcus pentosaceus [25,40,59,60].
Some authors claim that the anti-listerial potential of bacteriocinogenic L. sakei and L. curvatus represents an efficient tool to counteract the development of pathogens such as L. monocytogenes in food, even at low temperatures [61][62][63][64]. Furthermore, evidences have also shown that non bacteriocin-producing strains of L. sakei are able to retard spoilage and to reduce the number of pathogens by developing dominant populations in vacuum packaged fresh meats [65]. The inhibitory potential of non bacteriocinogenic L. sakei strains might be attributable to the high specialization of the ecological niche represented by meat products, enabling this microorganism to be an efficient competitor for nutrients and to have a better colonizing capability [66].
Furthermore, some L. plantarum strains have been characterized as bacteriocin producers [67,68], highlighting their potential application as bio-preservatives in meat and meat products [69]. Indeed, some studies have suggested that certain strains of L. plantarum are able to produce class IIb bacteriocins with synergistic antibacterial effects [70]. Barbosa et al. [69] have also highlighted the remarkable antilisterial activity of L. plantarum isolated from a fermented salami and able to produce a two-peptide lantibiotic plantaricin.
Similarly to what was previously mentioned for L. sakei and L. plantarum, it has been reported in literature that P. pentosaceus effectively inhibits the growth of L. monocytogenes in pork fermented sausage, mainly due to the production of a non-lantibiotic class IIa bacteriocin [71]. It is likely that the inhibition of L. monocytogenes in the targeted spreadable salami was determined by the combination of the physicochemical parameters associated with the inhibitory effect of the lactic flora exerted mainly through the production of antimicrobial peptides, as reported by others authors [16,39,57]. Furthermore, it has been reported elsewhere that in presence of nitrate and nitrite additives, the fermentation process is able to limit and even suppress the growth of L. monocytogenes in fermented sausages [18]. In this study, the food matrix was challenged with Listeria monocytohenes during both ripening and storage by performing the contamination on meat batter at the beginning of the production process. This innovative approach enabled the thorough assessment of pathogen behavior in this peculiar meat production compared to challenge study only during shelf life [20,39]. Finally, the results of the present study are in agreement with the hurdle technology concept [72,73], which demonstrates that the combination and interaction of all the major preservative factors for foods are pivotal for optimal food preservation ensuring food stability and safety.

Conclusions
The dry-fermented process of salami, characterized by a short ripening time, could be a source of risk for consumers caused by Listeria monocytogenes. A useful strategy aiming to limit the presence and the growth of such pathogen is to favor the development of the native lactic flora that, in combination with other preservative factors such as additives, can act as a hurdle against Listeria creating an unfavorable environment for its survival.