*2.1. Antimicrobial Films*

Lysozyme-Lactoferrin (LZ-LF) water solutions were coated together onto PET: the formulation included gelatin as the main biopolymer network, glycerol as plasticizer, and a lipid phase (a monoglyceride acetylate) as slipping agent, in order to avoid blocking of the film coils during the unwinding operations on the reels. The total dry matter was 19.1 wt %, where LZ and LF accounted for 3 wt % (on the total weight). The procedure has been up-scaled in a pilot plant coating machine at a speed of 10 m/min. The removal of the solvent (water) was achieved by the combined effect of IR lamps and a flux of mild air. Right before the coating deposition, the plastic web underwent an in-line corona treatment to improve surface wettability.

Carvacrol (4.8%) was instead incorporated in a coextruded multilayer film 73 μm thick, made up of two external layers of polypropylene, two tie layers, and an internal layer of ethylene–vinyl alcohol copolymer with a 29% ethylene molar content (EVOH-29), prepared as reported elsewhere [11]. Briefly, films were obtained by extrusion processes using a flat sheet die 500 mm wide and three single screw extruders (Dr. Collin GmbH, Edersberg, Germany) with 30 mm of screw diameter and *L*/*D* ratio of 30. Die, feeding block, and transfer lines were maintained at 250 ˝C. Temperature of chill roll was set at 45 ˝C and an additional airknife was used. Line speed was 9.5 m/min.

#### *2.2. Sample Preparation*

Fresh salmon fillets cut to cubes (*ca.* 120 g) were packaged in laminated material made up of polyethylene/polyamide/polyethylene in Bremerhaven (Germany) and then transported on ice to the University of Milan, where they arrived the next day (Figure 1). Fillets were repackaged into pouches (20 cm ˆ 12 cm) made from a high barrier multilayer film after being wrapped in sheets of the antimicrobial films. The transfer of the samples from the original package to the experimental packages was made in sterile conditions under a safety cabinet. Samples were all packaged under vacuum. Reference samples were also prepared in an identical way, without wrapping in antimicrobial films.

**Figure 1.** Sample of salmon fillets, cut into cubes, employed in shelf-life trials.

After packaging, samples were all stored at 0 ˝C and analyses were performed at days 0, 1 and 4. At day 1, some samples were moved to 5 ˝C, where they were left until the microbiological analysis.

#### *2.3. Microbiological Analysis*

Microbial analyses were performed at days 0, 1 and 4 after packaging. Samples were taken from the surface of the samples (2–3 mm) and transferred aseptically and weighed in a sterile Stomacher bag, diluted with peptone water (PW) (Scharlab, Barcelona, Spain) and blended in Stomacher (IUL S.L., Barcelona, Spain) for 6 min. Ten-fold dilution series in PW of the obtained suspensions were made and plated on selective solid media: TSA (Merck, Germany) for mesophiles and psychrotrophs, MEA (Sigma Aldrich, St. Louis, MO, USA) for yeasts and fungi, *Pseudomonas* agar base (Himedia, India) for *Pseudomonas* spp., VRBLA (Violet Red Bile Agar, Merck, Germany) for coliforms, MRS agar (Scharlab, Barcelona, Spain) for lactic acid bacteria, and Lyngby Iron agar (Oxoid, UK) for H2S-producing bacteria. Colonies were counted after incubation at 30 ˝C for 24 h for mesophiles, 10 ˝C for 10 days for psychrotrophs, 30 ˝C for five days for yeasts and fungi, 25 ˝C for 24 h for *Pseudomonas*, 37 ˝C for 24 h for coliforms, 25 ˝C for five days for lactic acid bacteria and 20 ˝C for three days for H2S-producing bacteria. Counts were performed in triplicate and reported as logarithms of the number of colony forming units (log CFU/g salmon), and means and standard deviations were calculated.

#### **3. Results and Discussion**

Aerobic mesophiles and psychrotrophs as well as H2S-producing bacteria were the prevalent population in the salmon at time of packaging (3.3–3.6 log CFU/g), followed by LAB (2.2 log CFU/g and *Enterobacteriaceae* (1.5 log CFU/g) (Table 1). According to the International Commission on Microbiological Specifications for Foods, most aquatic animals at the time of harvest have microbial counts in the range of 2 to 5 log CFU/g [12]. In the present study the initial value of microbial load was in the same range, as also reported by other authors [13]. After one day of storage at 0 ˝C, the psychrotroph population increased up to 5.5 log CFU/g in control samples. Salmon packed in carvacrol active films was characterized by a reduced psychrotroph population (3.9 log CFU/g), while LZ-LF–coated films were ineffective (5.8 log CFU/g) (Table 2). Gram-negative psychrotrophic bacteria are the major group of microorganisms responsible for the spoilage of fresh fish at chilled temperatures [2]. In this study, mesophiles in the first 24 h remained in the range 3.9–4.1 log CFU/g, without significant difference between the control sample and those stored in the active packages.


**Table 1.** Microbial population (log CFU/g) present in salmon at time of packaging.

**Table 2.** Microbial population (log CFU/g) in salmon packed in two different antimicrobial materials and in one reference material (control) after storage for one day at 0 ˝C.


At day 1 some samples were moved to 5 ˝C while, in parallel, other samples were kept at 0 ˝C. All samples were then stored for up to four days, one day more than the normal shelf-life suggested by the company.

After four days at 0 ˝C, salmon packed in carvacrol films maintained psychrotroph and mesophile populations at low levels, *i.e.*, around 4.0 log CFU/g, compared to 5.0 log CFU/g in control samples (Table 3). Also, LZ-LF–coated films showed an interesting performance, reducing the populations up to 3.0 and 3.7 log CFU/g, respectively. Note also, at this storage temperature, the efficacy of the two active films in reducing the *Pseudomonas* population of approximately 1 log cycle compared to control, from 3.2 to 2.2–2.3 log CFU/g.

Salmon samples stored for four days at 5 ˝C without active packaging were characterized by mesophile and psychrotroph population of 5.3 log CFU/g. Carvacrol active films were effective in maintaining only the mesophile population at a low level of 4.6 log CFU/g, while LZ-LF–coated PET reduced both the populations down to 4.5 and 3.8 log CFU/g, respectively. It must be noted, also, that in this last case there was a reduction of H2S-producing bacteria to 2.7 instead of 4.7 log CFU/g as seen in the control samples. This positive observation that LZ/LF–coated PET films could prevent growth of H2S-producing bacteria is of actual relevance since this type of microorganism has been identified as the most potent in causing rejection of fresh salmon fillets, due to the production of off-odors during growth [4].


**Table 3.** Microbial population (log CFU/g) in salmon packed in two different antimicrobial materials and in one reference material (control) after storage for four days at 0 ˝C and 5 ˝C, comparatively.

*Enterobacteriaceae* were also found to be members of the microbial association implicated in the spoilage of fresh sliced salmon during refrigerated storage. This finding is in agreement with results reported for different fish species, including fresh Atlantic salmon [14] as well as rainbow trout [15], in which *Enterobacteriaceae* were determined as a part of the microbial population at the end of the product shelf-life under refrigerated storage. In this study, *Enterobacteriaceae* were always found in low levels, less than 3 log CFU/g; although *Enterobacteriaceae* can grow at low temperatures, their proliferation was slow during refrigerated storage, possibly because their growth rate is lower than that of other Gram-negative psychrotrophic spoilers [16].

The LAB population was always found lower than 3.2 log CFU/g, and was not influenced by the active packaging employed. The low LAB count in this study was expected since they tend to grow slowly at refrigeration temperatures [13].

The obtained data satisfied the recommended microbiological limits for fresh and frozen fish reported in [12], which are defined only for aerobic plate counts performed at 20–25 ˝C (5.5 to 7 log CFU/g) and for *E. coli* (1 to 2.7 log CFU/g). The first data reflect handling practices in the fish industry, from shipboard to market delivery, while the second parameter is considered an indicator of contamination and, when present in high numbers, suggests temperature abuse in product handling.

The present research was aimed at investigating the efficacy of two antimicrobial food packaging materials on microbial spoilage of fresh salmon fillets. Incorporation of natural antimicrobials into food packaging materials to control the growth of spoilage and pathogenic organisms has been researched for the last decades. As regards fish products and shelf-life, the vast majority of data relates to the possibility of applying antimicrobial edible films in contact with the fish surface. Jasour *et al.* [17] evaluated the effect of an edible coating based on chitosan coated with the lactoperoxidase system (LPS) on the quality and shelf-life extension of rainbow trout during refrigerated storage at 4 ˝C. Results indicated that antimicrobial coating was found efficient in reducing *Shewanella putrefaciens*, *Pseudomonas fluorescens* as well as psychrotrophic and mesophilic bacterial populations compared to the control sample.

Few authors have investigated the performance of an antimicrobial package prepared by applying a coating procedure. Gomez Estaca *et al.* [18] produced a complex gelatin-chitosan film incorporating clove essential oil which was applied to fish during chilled storage: the growth of microorganisms was drastically reduced in Gram-negative bacteria, especially *Enterobacteriaceae*, while LAB remained constant for much of the storage period. Neetoo and Mahomoodally [19] compared the antimicrobial efficacy against *Listeria monocytogenes* in smoked salmon fillets of films or direct coatings incorporating nisin (Nis) and sodiumlactate (SL), sodiumdiacetate (SD), potassiumsorbate (PS), and/or sodium benzoate (SB) in binary or ternary combinations on cold smoked salmon. Surface treatments incorporating Nis (25,000 IU/mL) in combination with PS (0.3%) and SB (0.1%) had the highest inhibitory activity, reducing the population of *L. monocytogenes* by a maximum of 3.3 log CFU/cm2 (films) and 2.9 log CFU/cm<sup>2</sup> (coatings) relative to control samples after 10 days of storage at 21 ˝C. During refrigerated storage, coatings were more effective in inhibiting growth of *L. monocytogenes* than their film counterparts. Cellulose-based coatings incorporating Nis, PS, and SB reduced the population of *L. monocytogenes*, and anaerobic and aerobic spoilage microbiota by a maximum of 4.2, 4.8, and 4.9 log CFU/cm2, respectively, after four weeks of refrigerated storage.

In the present study the carvacrol-incorporated multilayer film was found effective in preventing mesophiles and psychrotrophs at shorter storage time and at lower temperature, while lysozyme-lactoferrin–coated PET was mostly efficient in decreasing H2S-producing bacteria at longer storage time and higher temperature. Even if it is not intended as a way to "clean" a contaminated food product, an antimicrobial package solution can indeed contribute to reducing the microbial population in food items, thus lowering the decay of organoleptic features and increasing shelf life.
