1. Introduction
European tradition has a strong connection with traditional food products, which are a result of a long history, cultural diversity, and varied climates, making them an indispensable component of the local economy [
1]. Regulation 1151/2012 oversees the classification of these products, denoted as PDO (protected designation of origin) and PGI (protected geographical indication), throughout Europe. A publicly accessible register lists these designated products. Notably, in Italy, dry fermented sausages, including salami, enjoy protection even in the absence of PDO or PGI status. The production of salami is, in fact, widespread, and each territory has its own in Italy [
2]. Salame Napoli (recognized by the Italian Ministry of Agriculture as a traditional agri-food product) is manufactured according to traditional technologies in the Campania region. This product is among the most highly favored choices among consumers, and as a result, it is now produced throughout Italy [
3].
The fermentation is one of the best methods to preserve meat, ensuring a product’s stability from both a microbiological and organoleptic perspective, consequently providing a long shelf life [
4]. Fermented meat products are produced with finely or coarsely ground lean and fat meat cuts (depending on the type of product to be obtained). Ingredients such as salt, spices, and authorized additives are added to the mix. Subsequently, the homogenate is obtained, the dough is stuffed into casings (natural, such as pig gut, or artificial) [
5]. The autochthonous bacterial flora in meat aids in fermentation, resulting in the desired sensory characteristics of the finished products [
6]. This is primarily achieved through two main phenomena: the drop in pH and water activity (a
w) levels. However, in many production processes, these processes can also be accelerated by adding starter bacteria [
7]. Fermentation starts during the maturing phase of the products under controlled humidity and temperature conditions, and the physical, chemical, and microbiological phenomena that take place are closely interconnected [
8]. The loss of water is the principal event, occurring due to the evaporation of the water from the product, leading to a progressive drop in pH and water activity. These processes are initially related to the activity of lactic bacteria; later, the activity of surface molds becomes prominent [
9].
The use of starter cultures is widespread in the production processes of many traditional Italian agri-food products, such as Salame Napoli. For its preparation, a specific number and type of selected starter cultures are established and can be added to the recipe to enhance the characteristics of the product [
2]. Individual or mixed microbial cultures, also called starters, are used in known concentrations to promote and conduct the fermentation in meat products. Furthermore, they may be useful to speed up the ripening times, contributing to increased safety and standardizing the fermented products [
10]. Selected bacteria, molds, and yeasts are the microorganisms used as starter cultures in fermented products. Among bacteria, lactic acid bacteria (LAB) are the most prominent group, followed by other bacterial groups, including Gram-positive, catalase-positive cocci, primarily coagulase-negative staphylococci (CNS), and Micrococcaceae [
7].
Lactobacillus genus strains, including
L. sakei,
L. plantarum, and
L. casei, are commonly used in the production of fermented foods. They have an important role in the production of organic acids (lactic and acetic acids), aromatic compounds, protein hydrolysis, and the production of specific bacteriocins and bactericidal peptides that inhibit microbial growth [
11]. LABs are usually combined in starter cultures with non-pathogenic coagulase-negative Staphylococci, including species such as
Staphylococcus xylosus and
Staphylococcus carnosus. Their role is to ensure the organoleptic properties typical of the fermented meat products through the reduction of nitrates, which ensures the brightness of the red color, and through lipolytic and proteolytic activities, contributing to the improvement of texture and flavor [
12]. The combination of the activities carried out by the LAB and the CNS results in the product experiencing a reduction in pH and a
w, the initiation of proteolytic and lipolytic activities, as well as the production of molecular compounds. All these factors ensure the inhibition of unwanted microorganisms, consequently enhancing the safety of the product and improving the sensory properties of the food product [
13].
The aim of this study was to evaluate the effects of two commercial starter cultures featuring different technologies and acidifying profiles during the process (acidification, appearance, and weight loss) and on the quality (physical–chemical, microbiological, rheological, and sensory characteristics) of typical Italian dry-cured Salame Napoli.
2. Materials and Methods
2.1. Starter Cultures Selected
Two commercial starter cultures were used in the production of Salame Napoli: Euroferment Medium (SM) and Euroferment Rapid (SR), both produced by Europrodotti S.p.a. (Concorezzo, Italy). Euroferment Medium consisted of a combination of Staphylococcus xylosus and Lactobacillus plantarum strains in ratio 1:1, while Euroferment Rapid was a blend of Lactobacillus sakei and Micrococci (Staphylococcus carnosus and S. xylosus) strains in ratio 1:1.
2.2. Sample Preparation and Ripening Process
Salame Napoli of the study were manufactured according to industrial practices by a local company. In particular, 70% lean pork meat (shoulder, thigh, neck, and loin), 14% pork underbelly, and 12% lard were used. The meat and lard were minced with a 6 mm plate to obtain the mixture, and sodium chloride (30 g kg−1), pepper (3 g kg−1), and ascorbic acid (2 g kg−1) were added, along with a commercial mixture for salamis, including pepper, ascorbic acid, smoke aroma, sodium nitrite and potassium nitrate, dextrose, lactose, sucrose, and garlic.
The lyophilized starter cultures were rehydrated [15 g of Euroferment Medium (SM) and 20 g of Euroferment Rapid (SR) dissolved in 100 mL of chlorine-free water for processing 100 kg of meat] for four hours, after which they were added to each meat batter. The meat batter was then divided into two batches. The rapid starter culture was added to one batch (SR), while the medium starter culture was added to the other batch (SM). The meat batter was stuffed into the artificial casing to obtain salami of the same size (60 cm in length and 6 cm in diameter). The obtained salamis were placed on two separate trolleys and placed in a room for 24 h at 4 ± 3 °C (cooling phase). On the second day (dripping phase), the salamis were transferred to a new room and maintained at 23 ± 1 °C and 96 ± 2% relative humidity (RH) to facilitate water removal through dripping. Subsequently, both salamis were dried for 5 days (drying phase) and then ripened for 28 days (ripening phase) under controlled temperature and relative humidity, as reported in
Table 1.
2.3. Experimemental Design
During the process, at intervals of every 24 h until the end of the drying phase and every 48 h until the end of the ripening phase, a sample of both SM and SR batch was collected to monitor the pH and temperature values (each measurement was performed in triplicate, taking the mean value as the result). The change in water content throughout the ripening process of the salami was assessed by tracking weight loss. Three salamis per batch were weighted at the beginning, after the drying phase, and at the end of ripening phase. The findings are expressed as a percentage of the initial weight. During the process, a visual evaluation was performed to assess the uniformity and the potential presence of macroscopic defects, such as irregularities in color, texture, or the presence of any physical abnormalities, such as air pockets, atypical molds, or discoloration.
After the ripening, the surface molds on the salamis were scrubbed away using hot water jets. Microbiological, physicochemical, texture, and color analysis were conducted on the edible portion of six samples (three for each salami type, SM and SR) after removing the casing aseptically. The sensory analysis was carried out on slices of three salamis per batch by a quantitative descriptive sensory panel of 12 assessors.
2.4. Microbiological Analysis
To isolate and enumerate the microorganisms listed below, ten grams were taken representative of each sample (SM and SR) and placed in sterile stomacher bag. Ninety milliliters of sterilized peptone water (PW; Oxoid, Madrid, Spain) were added to each bag (1:10,
w/
v), and then, the content was homogenized for three minutes at 230 rpm using a peristaltic homogenizer (BagMixer
®400 P, Interscience, Saint Nom, France). Subsequently, ten-fold serial dilutions were prepared, and microorganisms were cultured as follows: (i) Total mesophilic aerobic bacterial counts (TAB 30 °C) were determined using plate count agar (PCA; Oxoid, Madrid, Spain) and incubated at 30 °C for 48–72 h [
14]; (ii) β-glucuron-positive
Escherichia coli were isolated on Tryptone Bile x-Glucuronide (TBX; CM0945, Oxoid, Basingstoke, Hampshire, UK) and incubated at 44 °C for 24–48 h [
15]; (iii)
Pseudomonas spp. were cultured on CFC agar (Cetrimide-Fucidin-Cephalothin agar with a modified CFC selective supplement, SR0103E; Oxoid, Basingstoke, UK) and incubated aerobically at 25 °C for 48 h [
16]; (iv) mesophilic lactic acid bacteria (LAB) were enumerated using De Man, Rogosa, and Sharpe agar (MRS, CM0361; Oxoid, Hampshire, UK) and incubated aerobically for 72 h at 30 °C [
17]; (v) yeasts and molds were detected on Dicloran Rose-Bengal Chloramphenicol Agar (DRBC; Oxoid, Madrid, Spain) incubated at 25 °C for 120–168 h [
18]. After incubation and counting, the data were expressed as logarithms of the number of colony-forming units (Log CFU g
−1), and the means and standard deviation were calculated.
Research of pathogenic bacteria was conducted to detect
Listeria monocytogenes,
Salmonella spp.,
Clostridium perfringens, and
Bacillus cereus. To detect
Listeria monocytogenes, 25 g of salami samples (SM and SR) were homogenized in 225 mL (1:10,
w/
v) of half Fraser broth (HFB, CM1053, Oxoid), spread on ALOA petri dishes (Agar Listeria according to Ottaviani and Agosti), and then incubated at 37 °C for 24 h [
19]. For
Salmonella spp., homogenization of 25 g of sample in 225 mL (1:10,
w/
v) of buffered peptone water (BPW; Oxoid, Madrid, Spain) was required, followed by transfer to Rappaport–Vassiliadis broth (RVS) and incubation at 41.5 °C for 24 h. Subsequently, the samples were plated on xylose lysine deoxycholate (XLD; Oxoid, Hampshire, UK) agar petri dishes and incubated at 37 °C for 24 h [
20]. For
Clostridium perfringens [
21] and
Bacillus cereus [
22], 1 mL of the solution prepared with peptone water (PW; Oxoid, Madrid, Spain) was inoculated on petri dishes and then incubated anaerobically at 37 °C for 18–24 h for both bacteria.
2.5. Physical–Chemical Analysis
During the ripening process, pH of salami was evaluated using a portable pH meter (HI9025, Hanna Co., Villafranca Padovana, Italy) equipped with a puncture electrode, which was inserted into three small incisions. The final product’s (SM and SR) pH and aw values were determined using a pH meter (Crison-Micro TT2022, Crison Instruments, Barcelona, Spain) and a hygrometer (Aqualab Decagon series 4 TEV), respectively. The analyses were performed in triplicate.
Moisture, protein, salt, and fat content were determined in triplicate on the finished products (SM and SR) using standard procedures [
23]. Lipid oxidation was assessed by measuring the thiobarbituric acid reactive substances (TBAR
s) using the methods described by Di Paolo et al., 2023 [
24]. The lipolysis of ripened salami was evaluated by measuring the free fatty acids (FFA
s), expressed as the percentage (%) of oleic acid present in the sample [
25].
2.6. Texture and Color Analysis
Texture profile analysis (TPA) and color evaluation (CIEL*a*b*) were performed on 3 cm thick slices of SM and SR salami. According to Ambrosio et al., 2021 [
26], the colorimetric evaluation required a Konica Minolta CR300 colorimeter (Minolta, Osaka, Japan). Color measurements were performed on both the external and internal surfaces (three measurements for each surface). For texture profile analysis (TPA), the Shimadzu EZ-Test texturometer (Shimaduz Corporation, Kyoto, Japan) was employed, following the procedures described by Ambrosio et al., 2021 [
26]. The following parameters were evaluated from the TPA curve: adhesiveness (N × mm), cohesiveness (N), hardness (N), cohesion (N), gumminess (N), chewiness (N), resilience. Three slices of each salami were used to obtain an average of 7–10 repetitions, resulting in approximately 3–4 repetitions per slice. The average values of repetitions obtained from the analysis of six salamis (three from SM and three from SR) were used for the statistical analysis.
2.7. Sensory Analyses
The panel test was carried out on 8 mm thick slices of salami by a quantitative descriptive sensory panel of 12 assessors according to ISO 8586 [
27]. Panelists underwent training in preliminary training sessions using various samples of commercial salami to establish a shared vocabulary for describing sensory attributes. After training, the samples were evaluated three times per batch, resulting in a total of six tested slices (three of SM salami and three of SR salami) for each panelist. Each attribute term was meticulously described and explained to eliminate any ambiguity about its relevant meaning. A total of 8 attributes were chosen (frequency of citation > 60%) to characterize the salami: color intensity (appearance), odor intensity, ripened odor (flavor sensory profile), salt, pepper, ripened flavor (gustatory sensory profile), hard, and gummy (tactile profile). Using a structured scale, panelists rated the intensity of each attribute, assigning scores between 0 (absence of the sensation) and 7 (extremely intense). To mitigate external bias elements, each of the tasters evaluated two slices of salami, commonly referred to as “test 1” and “test 2”. The average score given for each sample (SM and SR) and session (three session for each sample) was recorded and utilized in the subsequent statistical analysis.
2.8. Statistical Analysis
Statistical analyses were conducted using IBM SPSS Statistics, version 28 (IBM Analytics, Armonk, NY, USA). Microbiological parameters (TAB 30 °C, Escherichia coli, mesophilic lactobacilli, Bacillus cereus, Clostridium perfringens, Pseudomonas spp., yeast, and molds) as well as physicochemical parameters (chemical composition, lipolysis and oxidation index, and rheological and sensorial parameters) were statistically analyzed with a one-way analysis of variance (ANOVA). A significance level of less than 0.05 (p < 0.05) was considered statistically significant. The analyses were performed in triplicate (n = 3), and all data were presented as mean ± standard deviation.