Use of Essential Oils to Increase the Safety and the Quality of Marinated Pork Loin

This study aimed at evaluating the effects of the addition of an oil/beer/lemon marinade solution with or without the inclusion of oregano, rosemary and juniper essential oils on the quality, the technological properties as well as the shelf-life and safety of vacuum-packed pork loin meat. The results obtained suggested that, aside from the addition of essential oils, the marination process allowed to reduce meat pH, thus improving its water holding capacity. Instrumental and sensorial tests showed that the marination also enhanced the tenderness of meat samples, with those marinated with essential oils being the most positively perceived by the panelists. In addition, microbiological data indicated that the marinated samples showed a lower microbial load of the main spoiling microorganisms compared to the control samples, from the 6th to the 13th day of storage, regardless of the addition of essential oils. Marination also allowed to inhibit the pathogens Salmonella enteritidis, Listeria monocytogenes and Staphylococcus aureus, thus increasing the microbiological safety of the product. Overall outcomes suggest that the oil/beer/lemon marinade solution added with essential oils might represent a promising strategy to improve both qualitative and sensory characteristics as well as the safety of meat products.


Introduction
In the past decade, global consumer demand for marinated meat products has significantly increased [1,2]. The reasons behind this scenario are mainly related to the nutritional characteristics, the extended shelf-life as well as the improvement of sensorial and textural traits of this kind of commodity [2,3]. In addition, marination technology allows to diversify meat products and, conferring them peculiar sensorial traits, to offer a broader choice to the consumers [4]. Marination is a widely used process in the meat industry consisting in the injection or immersion of meat cuts into aqueous solutions containing a wide range of ingredients such as water, salt, vinegar, lemon juice, wine, soy sauce, brine, essential oils, tenderizers, herbs, spices and organic acids [5,6]. Depending on the selected ingredients, a huge variety of marinade solutions, either alkaline or acid, exists. The firsts contain phosphates, while the seconds are usually prepared with the addition of organic acids or their salts [7,8]. Another type of marinade solution are the water/oil emulsions. Overall, the addition of marinade Table 1. Marinade solutions tested in the preliminary trials.
Subsequently, in order to select the combination allowing to obtain the best organoleptic properties of the product without altering its flavor, the addition of essential oils to the marinade solutions was tested. The essential oils considered during the preliminary tests were thyme, rosemary, oregano, and juniper, in different combinations and concentrations (0.02, 0.04, 0.06 and 0.08% on the final product). The evaluation was done by an untrained panel of 20 panellist taking into consideration the sensory parameters such as color, odour, overall accettability before and after cooking.
On the basis of preliminary results (data not shown), the marinade solution selected for the main experiment was composed by olive oil/beer/lemon juice (1:2:1, 10% w/w) with a mixture of oregano (0.02%), rosemary (0.03%) and juniper (0.03%) essential oils.

Ingredients and Microorganisms Used
The pork loin slices used in this work were obtained from a local retailer the same day of the trial and kept at refrigerated temperatures (4 ± 1 • C) until the analyses. The marinade solution was composed as follows: the bock style beer Moretti la rossa (7.2% ABV) (Heineken Italia S.p.A., Pollein, AO, Italy), extra virgin olive oil (Monini, Spoleto, PG, Italy) and concentrated lemon juice (LIMMI, Perugia, PG, Italy). The essential oils used in this experimentation were oregano, rosemary, and juniper (Flora, Pisa, PI, Italy).
The strains used in the challenge test trial, Listeria monocytogenes Scott A, Salmonella enteritidis E5 and Staphylococcus aureus SR41 belonged to the Department of Agricultural and Food Sciences of Bologna University. The strains were maintained at −80 • C before experiments and before inoculation they were cultured twice in Brain Heart Infusion broth (BHI, Oxoid Ltd. Basingstoke, UK) at 37 • C for 24 h.

Preparation of the Samples and Shelf-Life Trials
The experiment was carried out on a total of 81 slices of pork loin (having an average weight of 60 g), divided into 3 groups (27 slices/group) as follows:
As previously mentioned, the marinade solution was realized by mixing bock style beer, concentrated lemon juice and extra virgin olive oil at a 2:1:1 ratio using an Ultraturrax (IKA-WERKE, Labortechnik, Staufen, Germany) (13,000 rpm, 30 s, in ice). Part of this solution was used for samples belonging to the experimental group M, while the remaining was added of a mixture of essential oils (0.08% of the final weight) consisting of oregano (0.02%), rosemary (0.03%) and juniper (0.03%) and included in the samples M + E. Each pork loin slice (about 60 g), was added of 1% NaCl, calculated on the final weight of the marinated product. Subsequently, the samples were placed in heat-resistant plastic bags, in which the marinating solution was directly added, with the only exception of the samples belonging to the control group to which only 1% NaCl was included. The amount of marinade solution added to the samples corresponded to 10% (w/w) of the final product. The slices were then vacuum packaged (99.9%) and placed in a small-scale tumbler (model MHG-20, VakonaQualitat, Lienen, Germany) under vacuum conditions (−0.95 bar) and at a temperature of 2-4 • C. Tumbling was performed in 60 min at a speed of 20 rpm including two working cycles (25 min per cycle) and a 10 min pause cycle. The vacuum-tumbled loin slices were then stored at 4 • C and used for analytical determinations after 3, 9 and 15 days of storage.

pH
The pH of the samples was determined by taking an aliquot of meat (avoiding fat and connective tissue) according to Jeacocke [24]. About 2.5 g of finely chopped meat were homogenized for 30 s by Ultraturrax in 25 mL of a solution 5 mM of sodium iodoacetate and 150 mM of KCl at pH 7.0. The pH was determined by pH meter (mod. Jenway 3510; Electrode 924001, Cole-Parmer, Stone, UK) previously calibrated. The pH determination was performed after 3, 6 and 15 days of refrigerated storage on raw meat samples.

Color
Color was assessed by a Minolta ® CR-400 colorimeter (Milan, Italy), previously calibrated using a standard white ceramic tile, in standardized illuminant (C) and observation angle (0 • with respect to an area of 8 mm in diameter) conditions. The CIELAB system [25] was utilized and the parameter of lightness (L*), redness (a*) and yellowness (b*) were used to objectively define color. The color determination was performed, for each group, after 3, 9 and 15 days of refrigerated storage on raw meat samples.

Marinade Uptake
Marinade uptake (i.e., the ability of meat to bind the saline solution added) was calculated by the difference in weight of the samples before and after the marination process. The amount of marinade solution absorbed was calculated as a percentage of the initial weight of the meat sample, according to the formula: Marinade uptake (%) = [(Weight after marination − Initial weight)/Initial weight] × 100 Shear force was assessed by a texture analyzer TA-HDi 500 (Stable Micro System, Godalming, Surrey, UK) equipped with a 5-kg load cell and a Warner-Bratzler shear probe. From each cooked sample, sub-samples (having the dimension of 4 × 1 × 0.5 cm) were excised and placed inside the load cell. The resulting shear force was expressed as kg/cm 2 .

Sensory Analysis
Panel tests were performed after 3, 9 and 15 days of refrigerated storage on cooked samples in order to test their visual appearance, olfactory acceptability and taste. The analysis was carried out by 20 untrained panelists who evaluated on a 1 to 5 scale the following parameters: meat odor intensity, spicy odor intensity, color intensity, flavor intensity, tenderness, overall assessment and finally favorite sample.

Microbiological Analysis
During storage at 4 • C, the cell count over time of lactic acid bacteria, yeasts, total aerobic mesophilic bacteria, total aerobic psychrotrophic bacteria, Pseudomonas spp. and To perform microbiological analyses, 10 g of meat sample were diluted into 90 mL of physiological solution (0.9% (w/v) NaCl), homogenized by a BagMixer 400 P (Interscience, St Nom la Bretèche, France), followed by serial dilution in physiological solution. The MRS agar plates were incubated 24 h at 37 • C, the PCA plates for the detection of psychrotrophic bacteria were incubated at 10 • C for 7 days, all the other agar media were incubated at 30 • C for 24-48 h.

Challenge-Test Trials
The preparation of marinated pork loin was done similarly to what reported in paragraph 2.3. The experiment was carried out on a total of 60 slices of pork loin (having an average weight of 60 g), divided into 3 groups (20 slices/group). Three groups of samples were obtained:

Listeria monocytogenes
Scott A, Salmonella enteritidis E5 and Staphylococcus aureus SR231, used in the challenge test belongs to the Department of Agricultural and Food Sciences (DISTAL, University of Bologna) collection. The bacterial strains were cultured overnight two times in Brain Heart Infusion (Oxoid Ltd., Basigstone, UK) at 37 • C. The pathogens were directly inoculated on the loin slices through 0.5 mL of physiological solution for the control group, while for the groups M + P and M + E + P were added to the marinating solution before the addition to the product. The inoculum was done in order to have an initial cell load of the pathogens, on the product, of approximately 4.0 log CFU/g. After the addition of the marinating solution the product was packaged and churned as reported in paragraph 2.3. The samples were stored at 4 • C and used for microbiological analyses immediately after the marinating and after 3, 6, 9, 13 and 15 days.

Microbiological Analysis
During the storage microbiological analyses were performed in order to detect the cell loads of the inoculated L. monocytogenes, S. enteritidis and S. aureus. Specifically, the entire slice of loin (about 60 g) was placed in sterile bags and added with sterile physiological solution in a 1:2 (w/w) ratio and then homogenized for 2 min by a BagMixer 400 P (Interscience, St Nom la Bretèche, France) followed by serial dilution in physiological solution. L. monocytogenes, S. enteritidis and S. aureus were detected in specific selective agar media. Listeria Selective Agar (LSA, Oxoid Ltd., Basingstoke, UK) supplemented with Listeria selective supplement (SR0140, Oxoid Ltd., Basingstoke, UK) for the enumeration of L. monocytogenes; Bismuth Sulphite Agar (BSA, Oxoid Ltd., Basingstoke, UK) for the detection of S. enteritidis, while Baird-Parker Agar base (BPA, Oxoid Ltd., Basingstoke, UK) added with Egg Yolk Tellurite Emulsion (SR0054, Oxoid Ltd., Basingstoke, UK) for the enumeration of S. aureus. The agar plates were then incubated at 37 • C for 24 h.

Statistical Analysis
Data were analyzed using the one-way ANOVA option of Statistica software (version 8.0; StatSoft., Tulsa, Oklahoma, USA) in order to test the effect of the addition of a marinade solution (with or without the inclusion of essential oils) at each sampling time (3, 9 and 15 days). Following, mean values were separated through Tukey honest significant difference (HSD) test, by considering a significance level of p < 0.05.

pH and Color
As reported in Figure 1, at each sampling time, control samples showed significantly higher pH than the marinated ones (p < 0.05) which, in their turn, exhibited similar values. A slight decrease in pH following refrigerated storage was observed for all the experimental groups, with C samples showing the greatest pH decline. In more detail, control samples exhibited an average pH decrease of 0.32 units, while M and M + E decreased of 0.19 and 0.17, respectively. As reported in Figure 1, at each sampling time, control samples showed significantly higher pH than the marinated ones (p < 0.05) which, in their turn, exhibited similar values. A slight decrease in pH following refrigerated storage was observed for all the experimental groups, with C samples showing the greatest pH decline. In more detail, control samples exhibited an average pH decrease of 0.32 units, while M and M + E decreased of 0.19 and 0.17, respectively.  Results concerning the evolution of color parameters (lightness-L*, redness-a*, yellowness-b*) during the refrigerated storage are reported in Figure 2. Overall, regardless the storage time, no significant differences were found either in L* or a* values among the experimental groups. Although these differences were not statistically significant, non-marinated samples showed noticeably higher a* values at both 9 and 15 days of storage. On the contrary, marination treatment exploited a remarkable effect on yellowness (b*): at each storage time, both M and M + E exhibited significantly higher b* values if compared to the control (p < 0.05).

Marinade Uptake and Cooking Loss
Data concerning the marinade uptake during the refrigerated storage are shown in Figure 3. Albeit any difference has been detected among the experimental groups at 9 and 15 days of storage, at day 3, a significantly (p < 0.05) higher marinade uptake has been observed in M + E samples in comparison to M (7.8 vs. 7.3%, respectively).

Marinade Uptake and Cooking Loss
Data concerning the marinade uptake during the refrigerated storage are shown in Figure 3. Albeit any difference has been detected among the experimental groups at 9 and 15 days of storage, at day 3, a significantly (p < 0.05) higher marinade uptake has been observed in M + E samples in comparison to M (7.8 vs. 7.3%, respectively).

Marinade Uptake and Cooking Loss
Data concerning the marinade uptake during the refrigerated storage are shown in Figure 3. Albeit any difference has been detected among the experimental groups at 9 and 15 days of storage, at day 3, a significantly (p < 0.05) higher marinade uptake has been observed in M + E samples in comparison to M (7.8 vs. 7.3%, respectively).

Shear Force
Results concerning the shear force of cooked pork loin samples after 3, 9 and 15 days of refrigerated storage are displayed in Figure 5. After 3 days of refrigerated storage, non-marinated samples showed significantly higher shear forces than the marinated ones (M and M + E) (p < 0.05), with M + E group exhibiting the lowest values. Albeit no statistical difference has been detected at 9 and 15 days likely due to the high variability of data, M + E samples showed the lowest shear force values, thus suggesting that the effect of essential oils on improving meat tenderness is considerable in particular in the first days of storage.

Shear Force
Results concerning the shear force of cooked pork loin samples after 3, 9 and 15 days of refrigerated storage are displayed in Figure 5. After 3 days of refrigerated storage, non-marinated samples showed significantly higher shear forces than the marinated ones (M and M + E) (p < 0.05), with M + E group exhibiting the lowest values. Albeit no statistical difference has been detected at 9 and 15 days likely due to the high variability of data, M + E samples showed the lowest shear force values, thus suggesting that the effect of essential oils on improving meat tenderness is considerable in particular in the first days of storage.  Results concerning the shear force of cooked pork loin samples after 3, 9 and 15 days of refrigerated storage are displayed in Figure 5. After 3 days of refrigerated storage, non-marinated samples showed significantly higher shear forces than the marinated ones (M and M + E) (p < 0.05), with M + E group exhibiting the lowest values. Albeit no statistical difference has been detected at 9 and 15 days likely due to the high variability of data, M + E samples showed the lowest shear force values, thus suggesting that the effect of essential oils on improving meat tenderness is considerable in particular in the first days of storage. The results showed that, regardless the sampling time, the marinated meat, and especially that with essential oils (M + E), exhibited better scores compared to the non-marinated one, with the only exception of meat flavor intensity parameter. In addition, the marinated samples showed a greater The results showed that, regardless the sampling time, the marinated meat, and especially that with essential oils (M + E), exhibited better scores compared to the non-marinated one, with the only exception of meat flavor intensity parameter. In addition, the marinated samples showed a greater intensity of flavor and taste, positively perceived by the panelists. In particular, marinated meat slices showed higher tenderness, color, flavor and taste intensities for the whole storage period, resulting in an overall improved acceptability compared to the controls. Considering the effect of essential oils, no differences between M and M + E samples were observed after 3 days of storage. However, starting from the second panel test (day 9), M + E samples showed higher scores for spicy flavor and taste intensity compared to M samples. The differences among M and M + E samples intensified at the end of storage (day 15), when the M + E group showed the highest scores for overall acceptability, thus being the preferred sample for over 60% of panelists.

Microbiological Analysis
The microbiological analyses were aimed to detect various microbiological groups frequently associated with the spoilage of processed meat products. In particular, during the refrigerated storage of the samples, the cell loads of total aerobic mesophilic and psychotropic bacteria, lactic acid bacteria, yeasts, Pseudomonas spp., total coliforms and Brochotrix thermosphacta were detected.
In Figure 7, the cell loads of mesophilic aerobic bacteria, lactic acid bacteria, yeasts, Pseudomonas spp., total coliforms and Brochotrix thermosphacta are reported. intensity of flavor and taste, positively perceived by the panelists. In particular, marinated meat slices showed higher tenderness, color, flavor and taste intensities for the whole storage period, resulting in an overall improved acceptability compared to the controls. Considering the effect of essential oils, no differences between M and M + E samples were observed after 3 days of storage. However, starting from the second panel test (day 9), M + E samples showed higher scores for spicy flavor and taste intensity compared to M samples. The differences among M and M + E samples intensified at the end of storage (day 15), when the M + E group showed the highest scores for overall acceptability, thus being the preferred sample for over 60% of panelists.

Microbiological Analysis
The microbiological analyses were aimed to detect various microbiological groups frequently associated with the spoilage of processed meat products. In particular, during the refrigerated storage of the samples, the cell loads of total aerobic mesophilic and psychotropic bacteria, lactic acid bacteria, yeasts, Pseudomonas spp., total coliforms and Brochotrix thermosphacta were detected.
In Figure 7, the cell loads of mesophilic aerobic bacteria, lactic acid bacteria, yeasts, Pseudomonas spp., total coliforms and Brochotrix thermosphacta are reported. The data obtained indicated a satisfactory microbiological quality of the raw meat. In fact, the initial cell load of the main spoiling microorganisms was below 3.0 log CFU/g, independently on the use of marinade solution or the addition of essential oils. As expected, the mesophilic bacteria represented the main microbial spoiling group. In fact, a fast increase of the cell load of this group was observed in all the samples starting from the sixth day of refrigerated storage. However, from day 6 of storage, samples M and M + E showed significant lower cell loads compared to C, while no differences were observed between M and M + E samples. The C samples were the only ones found to exceed 8.0 log CFU/g after 15 d of storage. The same trend was observed for psychotropic aerobic bacteria. In general, the highest inhibition due to marination and the addition of essential oils was observed against the Gram-negative bacteria Pseudomonas spp. and total coliforms. Otherwise, minor differences were observed considering B. thermosphacta since no significant differences were observed starting from day 9 of storage. However, depending on the sample, this microorganism reached a cell load ranging between 4.4 and 4.8 log CFU/g.
A different trend was observed for yeasts and lactic acid bacteria. In fact, starting from day 6 of storage, yeasts resulted significantly higher in samples M and M + E compared to the control. However, yeasts never exceed 5.0 log CFU/g for the whole period of storage. In case of lactic acid bacteria, no significant differences were detected at the end of the storage among the samples.

Challenge Test
In order to evaluate the effects of the marinade solution with or without essential oils on the safety of vacuum packed pork loin slices, a challenge test inoculating Listeria monocytogenes Scott A, Salmonella enteritidis E5 and Staphylococcus aures SR31 was performed. Figure 8a-c shows the cell loads of the pathogen microorganisms during the refrigerated storage. The data obtained indicated a satisfactory microbiological quality of the raw meat. In fact, the initial cell load of the main spoiling microorganisms was below 3.0 log CFU/g, independently on the use of marinade solution or the addition of essential oils. As expected, the mesophilic bacteria represented the main microbial spoiling group. In fact, a fast increase of the cell load of this group was observed in all the samples starting from the sixth day of refrigerated storage. However, from day 6 of storage, samples M and M + E showed significant lower cell loads compared to C, while no differences were observed between M and M + E samples. The C samples were the only ones found to exceed 8.0 log CFU/g after 15 d of storage. The same trend was observed for psychotropic aerobic bacteria.
A similar tendency was observed for Pseudomonas spp. Starting from day 3 of storage C samples showed significant higher cell loads compared to M and M + E samples. No significant differences regarding the cell load of Pseudomonas spp. were observed between M and M + E samples, with the only exception of day 3. At the end of the storage Pseudomonas spp. resulted 6.67, 5.61 and 5.88 log CFU/g respectively in C, M and M + E samples. Total coliforms resulted significantly lower in M and M + E samples compared to C ones, excepted at day 3 of storage. The greatest differences were observed at day 15 when coliforms were 5.25, 4.18 and 4.22 log CFU/g respectively in samples C, M and M + E. In general, the highest inhibition due to marination and the addition of essential oils was observed against the Gram-negative bacteria Pseudomonas spp. and total coliforms. Otherwise, minor differences were observed considering B. thermosphacta since no significant differences were observed starting from day 9 of storage. However, depending on the sample, this microorganism reached a cell load ranging between 4.4 and 4.8 log CFU/g.
A different trend was observed for yeasts and lactic acid bacteria. In fact, starting from day 6 of storage, yeasts resulted significantly higher in samples M and M + E compared to the control. However, yeasts never exceed 5.0 log CFU/g for the whole period of storage. In case of lactic acid bacteria, no significant differences were detected at the end of the storage among the samples.

Challenge Test
In order to evaluate the effects of the marinade solution with or without essential oils on the safety of vacuum packed pork loin slices, a challenge test inoculating Listeria monocytogenes Scott A, Salmonella enteritidis E5 and Staphylococcus aures SR31 was performed. Figure 8a-c shows the cell loads of the pathogen microorganisms during the refrigerated storage. It is noteworthy to mention that marination allowed a significant (p < 0.05) reduction of the initial microbial cell load of all the pathogens, regardless of the presence or absence of essential oils. The highest initial cell load reduction, compared to control samples, was observed for S. aureus, and ranged between 0.7 and 1.0 log CFU/g, followed by S. enteritidis (0.7-0.8 log CFU/g) and L. monocytogenes (0.5-0.6 log CFU/g). In all cases, the differences in the pathogen levels between marinated and not marinated samples increased during the storage period. At the end of the storage, M and M + E samples showed cell loads lower than 2.0 logarithmic cycles for L. monocytogenes and S. aureus and lower than 1.5 logarithmic cycles in the case of S. enteritidis. On the contrary, an increase of the level of all the pathogens in C samples, greater in the case of L. monocytogenes, was observed during storage. Contrarily, a decrease of the pathogen loads in the marinated products was observed during the storage but without allowing their complete inactivation. Considering the effect of the addition of essential oils, no significant differences were found between the samples M and M + E for S. enteritis and S. aureus while in the case of L. monocytogenes the samples M + E showed a significantly It is noteworthy to mention that marination allowed a significant (p < 0.05) reduction of the initial microbial cell load of all the pathogens, regardless of the presence or absence of essential oils. The highest initial cell load reduction, compared to control samples, was observed for S. aureus, and ranged between 0.7 and 1.0 log CFU/g, followed by S. enteritidis (0.7-0.8 log CFU/g) and L. monocytogenes (0.5-0.6 log CFU/g). In all cases, the differences in the pathogen levels between marinated and not marinated samples increased during the storage period. At the end of the storage, M and M + E samples showed cell loads lower than 2.0 logarithmic cycles for L. monocytogenes and S. aureus and lower than 1.5 logarithmic cycles in the case of S. enteritidis. On the contrary, an increase of the level of all the pathogens in C samples, greater in the case of L. monocytogenes, was observed during storage. Contrarily, a decrease of the pathogen loads in the marinated products was observed during the storage but without allowing their complete inactivation. Considering the effect of the addition of essential oils, no significant differences were found between the samples M and M + E for S. enteritis and S. aureus while in the case of L. monocytogenes the samples M + E showed a significantly lower cell load with respect to samples M starting from day 13 of storage. The greatest antimicrobial effect from marinating was observed against S. aureus. In fact, a reduction of more than 3.5 log CFU/g at the end of storage compared to the initial load of C samples was observed.

Discussion
The marinade solution prepared with extra virgin olive oil, beer, concentrated lemon juice and a mixture of essential oils used within this study was selected based on the findings of preliminary trials. Considering that offering a marinated product including typical ingredients and flavors belonging to the Mediterranean diet may represent an added value to product itself, all the marinade ingredients and essential oils chosen in this work derive from plants commonly used in the traditional recipes of this area. The selected marinade solution was then tested with the aim of exploring its effect on the shelf-life, safety and quality traits of pork loin slices during refrigerated storage.
Aside from the inclusion of essential oils, the addition of the marinade solution significantly reduced the pH of vacuum-packed pork loin. These outcomes might be ascribed to the addition of an acid marinade solution in which the inclusion of beer (pH = 3.96) and concentrated lemon juice (pH = 2.26) results in a remarkable reduction in pH. This might be desirable for several reasons. First, meat pH exerts a direct effect on its water holding capacity (WHC), since it is generally held that the ability of meat to retain water progressively improves above and below pH values corresponding to the isoelectric point of meat proteins (i.e., 5.5 in the case of pork meat) [26]. Furthermore, processed meat products with a low pH are less likely to develop pathogen microbial growth and off-odors, thus having an improved safety and shelf-life [27,28]. Lastly, reduced pH values might also be advantageous to facilitate the action of collagenases and other proteolytic enzymes responsible for meat tenderization during the refrigerated storage [29].
The addition of the marinade solution, regardless of the use of essential oils, also exerted a significant effect on the yellowness (b*) of meat samples, while lightness (L*) and redness (a*) were not affected. The higher b* values detected for marinated samples might be likely due to the presence of coloring compounds in the solution itself (i.e., extra virgin olive oil, beer and concentrated lemon juice) which might have increased the yellowness of samples. However, the increase in b* values did not negatively affect the sensory evaluation by panelists who associated to the marinated samples in general, and to those including essential oils in particular, a better color retention if compared to the control.
Beside all, the marinating process is a widely used procedure at industrial level implemented with the aim to improve not only the sensory and eating qualities of meat products but also their technological properties, with a special reference to WHC [30,31]. Accordingly, satisfactory marinade uptakes (of more than 7%) were observed for both marinated pork loin groups after 3 days of storage. Albeit little literature is available concerning the effects of essential oils to improve the technological properties of meat, the remarkable improvement in marinade uptakes might be ascribed to the acid pH of the marinade solution. Indeed, as lemon juice contains citric acid, this ingredient is often included within the marinade solution to improve meat WHC by lowering its pH [32]. These outcomes are in agreement with those reported by other authors that observed a marinade uptake ranging between 4.6 and 9.7% in acidic marinated Longissimus dorsi muscles [33]. However, it is noteworthy to remember that the marinade uptake is strongly related to the meat type, marination technique as well as the duration of the process [34].
The marination process allowed to remarkably reduce the cooking losses compared to control samples after 3 days of refrigerated storage. This trend is in agreement with what reported by Gao et al. [35] who assessed the effect of marination on the main quality aspects of vacuum-packed pork loin meat. However, after both 9 and 15 days of storage, marinated meat (either M or M + E) exhibited slightly higher cooking losses if compared to the control group. This trend might be likely due to the greater marinade uptake measured during the storage period, which might have resulted in a higher loss of fluids during cooking. Therefore, it is reasonable that raw meat, added with salt without the inclusion of marinade solution, presented reduced cooking losses after a week of refrigerated storage.
Several authors have reported an increase in tenderness of marinated poultry, pork and beef [11,32,35]. Accordingly, the addition of marinade solution with or without essential oils allowed to reduce the shear forces of pork loin meat of about 40% and 22.8%, respectively, just after 3 days of refrigerated storage. These outcomes suggest the effectiveness of an acidic marinade solution to improve the tenderness of meat samples, as previously reported by Miller [36]. Accordingly, several studies have reported that acidic substances in the marinating solution (including lemon juice) can play a crucial role in the tenderization of marinated meat, leading to meat fibers swelling and enhancing proteolysis [37,38].
The sensory analysis data, according to the available literature, suggested that the marinated samples, and in particular those in which essential oils were added to the marinade, were tender and characterized by better color, flavor and taste intensity compared to the control samples. On the other hand, the positive effect of acidic marinade solutions on tenderness and other quality characteristics of different types of meat is widely reported in the literature [2,39]. The addition of essential oils strongly increased the overall acceptability of the samples, especially at the end of the storage, resulting in the preference of the consumers. Recently, many studies have reported an improvement of the sensory qualities and an extended shelf life of meat and meat products supplemented with different essential oils including, rosemary, thyme, oregano, basil, coriander, ginger, garlic, clove, juniper and fennel, used alone or in combination [40,41]. In addition, essential oils are widely reported as characterized by a strong antioxidant activity [42,43]. A wide literature reports a reduction of the lipid oxidation of meat and meat products added with essential oils during storage [40,44,45]. A better sensory quality and a longer shelf-life is normally associated to the reduction of lipid oxidation [45,46].
The predominant spoiling bacteria associated to refrigerated pork and beef, are Pseudomonas spp. during storage in aerobic conditions and lactic acid bacteria belonging to the genus Lactobacillus spp., Leuconostoc spp. and Carnobacterium spp. but also Brochothrix thermosphacta, Enterobacteriaceae and psychrophilic Clostridium spp. in case of anaerobic conditions [47,48]. Meat defects due to off-odors and off-flavors normally linked to a discoloration, gas production and acidification are generally associated to the growth of these microorganisms [49][50][51]. Our results indicate a satisfactory initial microbiological quality of the pork loin used in the present study. In fact, for all the main microbiological spoilage agents considered, the cell load was lower than 3.0 log CFU/g. During storage, an increase of the total viable mesophilic and psychotropic bacteria, Pseudomonas spp. lactic acid bacteria and B. thermosphacta was observed. The enumeration of total viable mesophilic and psychotropic microorganisms represents one of the most widely used and recognized criteria for evaluating the microbiological quality of meat [52]. Generally, the product is considered acceptable when the cell load of these microorganisms is lower than 7.0 log CFU/g [53] and this level is generally taken at industrial level as the upper threshold to determine the product expiry date. Our results showed that marinated samples overcome this limit only after 15 days of storage while control samples exceeded the limit after 9 days of refrigerated storage. The marination, regardless the addition of essential oils, showed the highest inhibition against the Gram-negative bacteria Pseudomonas spp. and total coliforms. Several literature data showed that species belonging to Pseudomonas and other psychotropic microorganisms are the predominant cause of alteration of fresh packaged meat [54]. Several Pseudomonas spp. are responsible for the formation of superficial patinas and off-flavor when their concentration reaches levels between 7-8 log CFU/g in chilled meat products [55].
Currently, foodborne outbreaks caused by foodborne pathogens transmitted from meat product still represent a significant public health challenge [56]. Considering the last 10-15 years the most important foodborne bacterial pathogens associated to meat belong to Salmonella spp., Escherichia coli, Campylobacter jejuni and Staphylococcus aureus [57][58][59]. Our results showed a clear inhibitory effect of the tested marinades on the growth kinetic of Listeria monocytogenes, Salmonella enteritidis and Staphylococcus aureus resulting in an increased safety of the product. In particular, the tested marinating solution proved an immediate inhibitory effect against all the pathogens. In addition, an increase of pathogens cell load during storage was observed in control samples, while the marinated products induced a more or less marked decrease of the pathogens load without allowing their complete deactivation. Regarding the addition of essential oils, a significant additional antimicrobial effect, compared to marinated samples, was observed only against Listeria monocytogenes. The antimicrobial activities of essential oils and their bioactive components are well known and reviewed in a wide literature even if strongly affected by microbial species, strains, and physico-chemical and process variables [17,18,21,60,61]. Although strain dependent and affected by application conditions, the greatest resistance of Gram-negative bacteria, due to the presence of the outer membrane acting as a barrier to hydrophobic molecules, to many essential oils is well known [62]. Among the Gram-positive bacteria, the very high resistance of Staphylococcus aureus to many stress factors and antimicrobials including essential oils and their components is well documented [62,63]. Also the action mechanisms of several essential oil components against many microorganisms, including the target microorganisms taken into consideration in the present research, have been clarified by molecular tools [64][65][66][67]. The limited antimicrobial effects of the essential oils in the present work is probably due to the masking effect of ethanol and its synergistic effects with low pH values and NaCl of marinade. In fact, as shown by Lanciotti et al. [68] studying the boundary between the growth and no growth of Salmonella enteritidis, Bacillus cereus and Staphylococcus aureus in the presence of different growth controlling factors through probabilistic models, the effects of ethanol on the limitation of growth of the considered species was significant also at concentration of about 1% and not merely additive with temperature and NaCl concentration. Also, the presence of organic acids and the pH reduction by marinade contribute to mask the effects of the essential oils on the target microorganisms considered [69].
Several authors have reported the antimicrobial effect of marinating solution components [10,12]. In particular the antimicrobial effect of some acidic marinade solutions containing alcoholic drinks is associated to the presence of ethanol but also to phenolic derivatives and organic acids, contributing the last to the reduction of the pH of the product [10,70,71]. In addition, the combination of organic acids, ethanol and sodium chloride can strongly inhibit several microorganisms including pathogens like Salmonella, Listeria monocytogenes, Escherichia coli and Staphylococcus aureus [72,73].

Conclusions
The results of the present study highlighted that the marination of pork loin slices using a solution (formulated with typical ingredients from Mediterranean area) with a mix of extra virgin olive oil, beer and lemon juice (in the presence/absence of essential oils) allows to obtain an overall improvement of the technological and sensory properties of meat. In particular, panel test results suggest a clear preference for marinated products with the addition of essential oils. Furthermore, the tested marinade solution exerted a remarkable meat pH reduction and significant antimicrobial activity both towards the common spoiling microflora normally present on the product and on pathogenic microorganisms deliberately inoculated, improving product safety and shelf-life. The use of marinade allowed the extension of the shelf-life of six days. In addition, offering a marinated product formulated with typical ingredients and flavors belonging to the Mediterranean diet may represent an added value to product itself. However, the addition of essential oils did not lead to a further increase of the antimicrobial activity exerted by the marinade solution. Though, the results obtained in this study suggest that an optimization of the concentration and type of essential oils used for the marination of pork loin could further increase its antimicrobial activity.