Effect of Active-Edible Coating and Essential Oils on Lamb Patties Oxidation during Display

The use of natural products to reduce the use of synthetic additives in meat products, reducing the oxidation and improving the shelf life is a current challenge. Meat quality from lamb patties during 10 days of display on modified atmosphere packaging (MAP) and active-edible coating were tested under six treatments: uncoated patties without coating (CON); patties with alginate coating (EC) and patties with coating and 0.1 or 0.05% of essential oils (EOs) from either thyme (TH 0.1; TH 0.05) or oregano (OR 0.1; OR 0.05). Display and treatment significantly modified (P < 0.001) all the studied meat quality variables (pH, color, water holding capacity, weight losses, thiobarbituric acid reactive substances (TBARS), antioxidant activity). Display produced discoloration and lipid oxidation, however, the samples with essential oils presented lower (P < 0.001) lipid oxidation than the CON or EC groups. Coated samples with or without EOs showed better color (lower lightness but higher redness and yellowness) and lower water losses (P < 0.001) than the CON. The addition of thyme EO caused a decrease (P < 0.001) in the consumer’s overall acceptability, whereas no statistical differences appeared between CON, EC and oregano EO addition. Thus, using EOs as natural antioxidants, especially those from oregano at low dosages (0.05%), could be considered a viable strategy to enhance the shelf life and the product quality of lamb meat patties without damaging the sensory acceptability.


Introduction
Shelf life is a basic aspect to consider in meat products due to their perishability. Lipid oxidation and color changes (discoloration) are between the main factors that decrease food quality, especially in meat products [1,2]. In fresh meat, color is an important indicator of wholesomeness that determines purchase or leads to product rejection [3].
With the aim of extending the shelf life of processed food products, synthetic additives are frequently used in the food industry. However, consumers have raised concerns with regard to the health risk of using these kinds of synthetics products. The increase in demand for more natural products encourages researchers to investigate new ways to achieve the challenge of preserving or increasing the shelf life with more natural and acceptable methods [1,4,5].
Among these methods, modified atmosphere packaging (MAP) stands out for keeping a desirable color in meat products frequently used by the meat industry. It can be done using

Animals and Preparation of Lamb Patties with Coating
Meat was obtained from 16 commercial hind limbs of lambs from Rasa Aragonesa breed, selected from the cooperative Grupo Pastores ® among their animals labeled as "Ternasco de Aragón" Protected Geographical Indication Quality Label, slaughtered 24 h prior to the lamb patties elaboration. The average weight of the hind limbs was 1.41kg ± 0.075 kg.
Hind limbs were transported on the day of preparation under refrigerated transport to the Meat Quality Laboratory of Zaragoza (Veterinary Faculty, Spain) where they were deboned and the edible portion (muscle and fat) separated and ground (Gesame ® Mod 9432). The edible portion from all animals was mixed and homogenized. Patties were molded by a hand cutter (1 cm thickness) to weigh 55 g each, and randomly distributed to the different treatments tested, with 2 replicates (experiment) of 3 samples per each day of analyses per treatment.
Edible coating was prepared with sodium alginate 2% (w/v) following methodologies described by Vital et al. [14]. For the active coating, the EOs of thyme (Thymus vulgaris QT linalool) and oregano (Origanum vulgare) from Pranarôm International ® (Ghislenghien -Belgium) were used. They were added at 0.1 or 0.05% (w/w) in their correspondent alginate coating and mixed under magnetic stirring. Chemical composition of EOs was analyzed by an Agilent 6890N gas chromatograph coupled with a 5973N mass spectrometer (Agilent, Santa Clara, CA, USA) and an auto-sampler 7863 (Agilent, Wilmingotn, DE, USA). The oven temperature was programmed following the method of Benelli et al. [20]. Helium (99.99%) was used as the carrier gas with a flow rate of 1 mL/min. Injector and detector temperatures were set to 280 • C. Two µL of the essential oil solution in nhexane (1:100) was injected with a split ratio of 1:50. Peaks were acquired in full scan (EI mode, 70 eV) in the range of 28-400 m/z. α-Pinene, camphene, β-pinene, 1-octen-3-ol, myrcene, α-phellandrene, α-terpinene, p-cymene, limonene, 1,8-cineole, γ-terpinene, terpinolene, linalool, borneol, terpinen-4-ol, α-terpineol, thymol, carvacrol, (E)-caryophyllene, α-humulene and caryophyllene oxide were identified by comparison to the authentic standards (Sigma-Aldrich, Milan, Italy). The remaining compounds were assigned by using the combination of retention indices (RIs), calculated using a mixture of linear C 8 -C 30 alkanes (Supelco, Bellefonte, CA, USA) according to the van den Dool and Kratz formula [21] and the similarity of the mass spectra (MS), with respect to those of the ADAMS, FFNSC2 and NIST17 libraries. Semi-quantitative values of the components were obtained by the peak area without correction factors. The values were the mean of three replicates. Table 1 compiles essential oils composition. Lamb patties were equally and randomly divided into six groups: patties uncoatedcontrol (CON); patties with edible coating (EC); patties with edible coating with 0.1% thyme EO (TH 0.1), patties with edible coating with 0.05% thyme EO (TH 0.05), patties with edible coating with 0.1% oregano EO (OR 0.1) and patties with edible coating with 0.05% oregano EO (OR 0.05).
All samples were packaged in an individual polystyrene tray with a modified atmosphere (70% O 2 and 30% CO 2 ) displayed under refrigeration conditions (between 2 and 4 • C) under light exposure (1200 lux, 12 h/ day), simulating market conditions. Samples of CON, EC, TH 0.1, TH 0.05, OR 0.1 and OR 0.05 were removed at 1, 3, 7 and 10 days of display for quality analysis.
Prior to the patties' elaboration, the ground meat was analyzed and characterized according to several meat quality characteristics.

Proximate Composition
The proximate composition of the ground meat used to elaborate the burgers was analyzed according to the standardized ISO protocols [22][23][24][25] for moisture, protein, fat, and ashes. Samples were taken the sampling day after homogenizing the ground meat, vacuum packaged and frozen (−20 • C). Then, they were transported to the Ingeniería y Servicios Cárnicos S.L. where the analyses were performed.

Fatty Acid Composition
Fatty acid analyses were performed after fat extraction described on Bligh and Dyer [26]. The methyl ester preparation included KOH in methanol, with C23:0 as an internal standard. To identify the methyl esters, a gas chromatograph (HP 6890) with a capillary column (100 m ×0.25 mm × 0.20 mm; SP 2380); was used [27]. The carrier gas was nitrogen. Samples were measured in duplicate and the results were expressed as the percentage of total fatty acids.

pH and Weight Losses
pH was registered each day of analysis on the patties using a pH meter (pH 7 portable pHmeter Lab Process) equipped with a penetration pH electrode.
Weight of each patty was registered on the sampling day (after edible coat arrangement) and each day of the analysis, allowing to calculate the water holding capacity as the exudative losses percentage, during display according to the following equation: initial weight (day 0) − weight (analyses day)/initial weight) × 100.

Total Phenolic Compounds and Antioxidant Activity
Total phenolic compounds (TPCs) and antioxidant activity were measured on the EOs (1:1000 v/v with pure methanol) after their extraction from the patties samples (1:1 w/v with methanol), or the edible coatings (1:3 v/v with methanol). With homogenization and centrifugation (15 min, 4000 rpm) extracts were obtained. In the patties, filtration with filter paper was also performed.

Total Phenolic Compounds (TPCs)
The TPCs were measured as described by Singleton and Rossi [28] with modifications. The sample (125 µL) was mixed with 125 µL of Folin-Ciocalteu reagent and 2250 µL of sodium carbonate (28 g/L). The samples reacted during 30 min in the dark (25 • C) and then the absorbance was read at 725 nm using a spectrophotometer (Onda ® , Model: UV-20, Giogio Bormac Srl, Carpi (MO), Italy). The results were expressed as mg gallic acid equivalent (GAE) g of sample. The standard curve of gallic acid concentrations ranged from 0 to 300 mg/L.

DPPH Radical Scavenging Assay
Protocols modified from Li et al. [29] were used to determined DPPH activity. A methanolic solution (2850 µL) containing DPPH (60 µM) was mixed with the samples (150 µL) during 30 min. Five hundred and fifteen nanometers (515 nm) was the absorbance wavelength used to read samples. Antioxidant activity was calculated as DPPH activity (%) = (1−(Abs t/Abs t=0)) × 100 where: A sample t = 0: absorbance at time zero of the sample; A sample t: absorbance at 30 min of the sample.

ABTS Radical Scavenging Assay
The ABTS activity was evaluated according to Re et al. [30] with modifications. ABTS· was obtained through the reaction between 7 mM ABTS (5 mL) and 140 mM potassium persulfate (88 µL), 16 h was the time necessary to incubate. The ABTS radical was mixed with ethanol (absorbance of 0.70 ± 0.02). Seven hundred and thirty-four nanometers (734 nm) was the wavelength used to record the scavenging activity (%). Samples (40 µL) were mixed with ABTS·+ solution (1960 µL) and absorbance was noted after 6 min of reaction. The scavenging activity (%) was calculated as ABTS activity (%) = (1−(Abs t/Abs t=0)) × 100 where: A sample t = 0: absorbance of the sample at time zero; A sample t: absorbance of the sample at 6 min.

Lipid Oxidation Analysis
Lipid oxidation assays were performed by thiobarbituric acid reactive substances (TBARS), according to Pfalzgraf et al. [31]. Absorbance was measured at 532 nm with ONDA UV−20 spectrophotometer. The malonaldehyde (MDA) content was measured and the results were expressed as mg MDA/kg of meat. Lipid oxidation assays were assessed at 1, 3, 7 and 10 days of display.

Color Measurement
Color was determined by the CIEL*a*b* system at 1, 3, 7 and 10 days of display, using a Minolta CM-2002 (Konica-Minolta Business Solutions S.A., Madrid, Spain) spectrophotometer with a 10 • view angle and a D65 illuminant, obtaining lightness (L*), redness (a*) and yellowness (b*).

Consumer Acceptability
Consumer tests were performed in a private room adequately adapted for sensory analysis at the University of Zaragoza (Spain). Eighty consumers were selected randomly within quotas of gender (46.25% women and 53.75% men) and age ( Eight sessions with ten different consumers were carried out. Each consumer assessed 6 different samples of patties, (one from each treatment evaluated), after 7 days of display. The samples were identified with a three-digit code and they were served in a randomized design, to prevent carry-over and order effects [32].
For culinary preparation, a pre-heated grill (200 • C) (SAMMIC ® , P80-2) was used and each patty was individually cooked. Patties were covered with aluminum foil and cooked until reaching an internal temperature of 75 • C. Each sample was cut in five portions, wrapped in aluminum foil, and kept at 50 • C. Consumers were requested to taste the samples and evaluate the acceptability of different attributes (flavor, tenderness and overall acceptability) using a hedonic scale with 9 points which range from 1 (dislike extremely) to 9 (like extremely) without neutral central point (neither like nor dislike). They were informed to rinse their mouth with water and eat an unsalted tasted bread before evaluating each sample, including the first one.

Statistical Analysis
Meat quality attributes were assessed via the analysis of variance using the general linear model (GLM) procedures with SPSS (version 23.0) (IBM SPSS Statistics, SPSS Inc., Chicago, IL, USA) for Windows. Treatments and display were considered as fixed effects and their interactions were also considered. For consumer acceptability, treatment was the only fixed effect evaluated and the consumer was considered as a random effect. Differences between the means were evaluated using the Tukey test (P ≤ 0.050).
The chemical composition of the O. compactum essential oil was characterized by oxygenated monoterpenes, accounting for 86.2%, whereas monoterpene hydrocarbons represented a minor fraction (11.7%) of the oil. The content of sesquiterpene fraction was negligible (1.4%). Among oxygenated monoterpenes, the phenolics carvacrol (65.4%) and thymol (18.5%) represented the two predominant compounds of the EO, accounting together for 83.9% of the total composition. Among the monoterpene hydrocarbons, the most representative compounds were p-cymene (6.3%) and γ-terpinene (3.9%), the biosynthetic precursors of thymol and carvacrol [33]. The remaining constituents identified in the EO were all in percentages < 1.3%.
The chemical profile of T. vulgaris belonged to the linalool chemotype, which is quite rare in this species. Notably, this chemotype has already been noticed in French populations of T. vulgaris [34]. On the other hand, the thymol and carvacrol chemotypes are characteristics in this species [35]. The O. compactum EO composition detected by us was qualitatively consistent with those previously found by other authors [36], though little quantitative variability can be observed. This can be related to harvesting time, plant processing, as well as genetic and geographic factors [37].
Carvacrol and thymol are two characteristic phenolic monoterpenes in several medicinal and aromatic plants belonging to Lamiaceae and Apiaceae families including the genera Thymus, Origanum, Satureja, Ocimum, Thymbra, Trachyspermum and Oliveria [38][39][40]. These compounds are reported as the most active EO components against bacterial and fungal growth.

Proximate Composition and Fatty Acids Analysis of Meat Matrix
The proximate composition (g/100 g) of the original mixed meat matrix presented 69.95% moisture, 19.0% protein, 6.5% fat, 0.78% carbohydrates and 1.05% ashes, having an energetic value of 164 kcal/100 g. Current results are comparable to those from the same Protected Geographical Indication 'Ternasco de Aragón' lamb legs characterized by [41]. However, they differ in terms of fat percentage, which also produces slighter variations on the fatty acid (FA) profile of the commercial legs between studies. The fatty acid profile of meat matrix was: 43.85% of SFA (saturated fatty acids), 46.61% of MUFA (monounsaturated FA) and 5.84% of PUFA (polyunsaturated FA). n-6 PUFA accounted 4.74% and n-3 PUFA 1.02% of total fatty acids. These results are typical of animals mainly fed on concentrates and slaughtered before three months of age, which is how most lambs are reared in Spain [42].

pH and Weight Loss of Lamb Patties
The pH and weight loss of patties are presented in (Tables 2 and 3), respectively. There was an interaction (P < 0.001) between the treatment and display for pH. Until the third day, no differences were observed between treatments (P > 0.05). However, after this, the pH of the CON and EC treatments presented higher values than the other treatments with active coatings (with EO). This increase might be linked to meat spoilage, which results in a switch from a glycolytic to an amino acid-degrading microbial metabolism [43].
The coating decreased weight losses in the lamb patties during display (P < 0.001), and an interaction between the treatments and display time was also observed (P < 0.001). Although all treatments showed an increase in weight loss, this was more pronounced in CON treatments during all days evaluated. The coating acted as a barrier [44], keeping the water in the system, and thus, little or no exudate was released. This has also been observed in beef [14]. The formation of a gelatinous layer from the edible coating around the meat adhered after cooking [14] might help to reduce water losses. Other coatings have proved also to be effective in reducing water losses, such as whey proteins and monoacylglycerols [45] or chitosan with unsaturated fatty acids [46].

Antioxidant Activity of Essential Oils and Lamb Patties
In this study, TPC and antioxidant activity (DPPH and ABTS radical scavenging) were measured. The TPC value was 236.07 mg GAE/g for oregano EO and 22.5 mg GAE/g for thyme EO. The EO had a DPPH radical scavenging of 25.49 and 6.88% for oregano and thyme, respectively. The ABTS scavenging ability for oregano was 63.90 and 6.62% for thyme. Thus, thyme EO had lower TPC antioxidant activity (P < 0.001) than oregano EO. This result was also observed in the respective coatings with patties. Patties with coating + EO had higher antioxidant activity (P < 0.001) than control samples and those coated with oregano presented the highest antioxidant activity in all assessments. Related to ABTS, OR 0.1 presented an average of 28.69%, OR 0.05 of 25.87%, TH 0.1 of 24.40% and TH 0.05 23.70%. For DPPH, the values were 12.85% for OR 0.1, 12.28% for OR 0.05, 11.95% for TH 0.1 and 11.92% for TH 0.05. CON and EC had similar antioxidant activity (21.24% and 21.85% for DPPH, respectively; 11.46 and 11.49% for ABTS). Natural products with notable antioxidant activity have a good potential to be applied in meat industry, due to the content in active compounds that can reduce food deterioration during storage [47,48].

Lipid Oxidation of Lamb Patties
This assay measured the secondary products of oxidation, specially related to rancidity. The effect of coatings on the lipid oxidation of patties was evaluated throughout the display. The inclusion of EO in the coating and display time significantly influenced the TBARS values, and an interaction between these variables was observed (P < 0.001) ( Tables 2 and 4). Patties with coating containing EOs showed a lower value for oxidation, and coating + oregano EO was more efficient in reducing the lipid oxidation than coating with thyme EO. No difference was observed in relation to the EO concentration (0.05 or 0.1%). This difference between oregano and thyme EO might be associated to differences in their composition [49]. In oregano EO, 83.89% of the compounds were constituted by carvacrol and thymol (phenols), both of which have strong antioxidant activity while the essential oil of thyme has as a main compound the monoterpene linalool (54.53%), a major component with lower antioxidant activity than phenols. In this study, it was also possible to observe that the modified atmosphere (CON) was more effective to delay the lipid oxidation when compared with EC (modified atmosphere + coating) without EO. Oxidation is among the main factors in the deterioration of foods, leading to the rejection by the consumer and a decrease in quality. Thus, an edible coating with a natural antioxidant can enhance the shelf-life of meat products, through the prevention of lipid oxidation. Table 4. Lipid oxidation evolution (mg malonaldehyde/kg meat) during the display period of the lamb patties with the edible coating and essential oils (mean ± standard error). A, B, C: different upper letter means statistical differences between the treatments within the display (P < 0.05). a, b, c: different lower letter means statistical differences between displays within the treatment (P < 0.05). CON: patties uncoated; EC: patties with edible coating; TH 0.05: patties with edible coating with 0.05% thyme EO; TH 0.1: patties with edible coating with 0.1% thyme EO; OR 0.05: patties with edible coating with 0.05% oregano EO; OR 0.1: patties with edible coating with 0.1% oregano EO.

Color of Lamb Patties
The color can influence the consumer purchasing decisions [11], being one of the main factors at the time of purchase. In this way, it is important to verify the influence of coating on the color of hamburgers L*, a* and b* values are presented in (Tables 2 and 5). L* values (lightness) decreased until day 3 for all treatments and then remained stable. CON showed higher L*, and this behavior may be associated with the highly oxidizing conditions, compared to samples with EO and coating, such as conformational changes in protein.
The oxidative process may result in the rupture of peptides, protein-protein interactions and modification in amino acid chains. These changes may alter protein structure and function, leading to modifications of food attributes as color, texture and flavor. In addition, related to EC and compared to control, the presence of exudates in the coated patties (effect of coating) darkens the color. This behavior was observed in each coated treatment. Vital et al. [14] also observed an increase in L* when the coating was compared with high oxygen concentration (control sample). Related to a* (redness), CON presented a significant decrease (P < 0.001) over the storage and the coating decreased (P < 0.05) the color losses (compared to CON). In addition, at day 10, the a* values of the coated hamburger was >10, indicating a bright red color. The meat pigment, without oxygen, is in the form of deoxyMb (purple-red color). With air (O 2 ), the pigment oxygenates (MbO 2 ), with a bright red color. The oxygenation process was slowed down by the coating, and the coated treatments reached the maximum a* value between 7 and 10 days. b* value of CON was significantly different (P < 0.001) from the coated hamburgers, decreasing during storage in CON, while in the coated hamburger this parameter was not altered. Additionally, the coated samples exhibited the highest b* values, associated with the yellowish color of the coating. Coated treatments did not present a significant difference related to b*. Coating with EO can reduce color deterioration over the display time, extending the shelf-life of the meat products and making it more attractive to consumers.

Consumer Test
EOs have strong aromatic compounds whose presence can determine the specific aroma of plants and the flavor of condiments, one of their main functions also being to develop desirable flavors and aroma. The effect of treatment on consumer acceptability is compiled in (Table 6). Statistical differences (P < 0.001) were reported by consumers in terms of flavor and overall acceptability. No differences (P > 0.05) were pointed out with respect to tenderness acceptability, which confirms, as in other food products covered with alginate edible coating (beef steaks or fish fillets), that the presence of the coating did not decrease the texture acceptability [11,14,50]. Flavor acceptability of thyme EOs, especially at higher dosages (0.1%) was significantly lower than those reported in the CON group. The addition of oregano EOs decrease the acceptability score (P < 0.001). However, the differences were small related to CON and EC treatments.
Samples from TH 0.1 presented significantly lower overall acceptability that those from CON, EC or OR 0.1, treatments which did not statistically differ between them and obtained the highest overall acceptability scores.
Oregano EO had better acceptability than thyme for lamb patties. Higher dosages of oregano (0.1 vs. 0.05%) are slightly preferred by consumers.
The choice of the EOs added and their concentration in a specific type of food is important because a small amount can cause sensory alterations, positive or negative, depending on both factors. The strong aroma of EOs can modify the food organoleptic properties [9]. Presumably the current results were more affected by the aromatic effect than by the antioxidant effect of EOs used, since the time of display (7 days) does not let strong undesirable off-flavors (rancidity) develop, which would be lower in EO samples with respect to CON or EC, as shown in the TBARS results (Table 4).

Conclusions
Edible coating (alginate-based) decreases the weight losses and discoloration of lamb patties. In addition, alginate coating is effective against lipid oxidation, an effect that is potentiated when EOs are added, which increase the antioxidant activity. Both concentrations of oregano EOs tested (0.1 and 0.05%) showed a higher antioxidant activity and lower lipid oxidation than those from the thyme EO.
Regarding consumer acceptability, patties with oregano were well accepted, as the CON and EC, while the patties with the highest concentration of thyme received lower notes.
Thus, the combination of packaging (MAP) and alginate-based coatings with EO (considering the concentration and the type of EOs added) could be used in diverse meat products (such as lamb patties) in order to maintain or improve their shelf life, without adding undesirable sensorial characteristics to the product depending on the EO.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data presented in this study are available on request from the corresponding author.