Chitosan and Essential Oils Combined for Beef Meat Protection against the Oviposition of Calliphora vomitoria, Water Loss, Lipid Peroxidation, and Colour Changes

Meat production has a higher economic and ecological impact than other commodities. The reduction in meat loss and waste throughout the whole supply chain is a demanding challenge. In recent years, the interest in the food-grade polysaccharide chitosan (CH) and essential oils (EOs) employed as allies in meat protection has increased. In this work, we selected five EOs obtained from plants traditionally used as spices, and after their chemical characterisation, a trained panel of expert sensorial analysts determined that, among them, Laurus nobilis (Lauraceae) and Piper nigrum (Piperaceae) EOs were the most suitable to season meat. Therefore, the effect of CH, the L. nobilis and P. nigrum EOs, and EOs-enriched CH solutions on meat was tested to assess how they deter the oviposition behaviour of the blowfly Calliphora vomitoria (Diptera: Calliphoridae) and against water loss, lipid peroxidation, and colour changes. All the applied treatments, compared to the control, did not accelerate meat dehydration but increased colour lightness, an attractive feature for consumers, and discouraged the blowfly’s oviposition. In particular, the P. nigrum EO-enriched CH was the most active in repelling C. vomitoria without negatively affecting the organoleptic qualities and shelf-life of meat.


Introduction
According to the Food and Agriculture Organization [1], global meat production in 2020 accounts for about 337.3 million tonnes, and around 4% of the worldwide food loss and waste is exactly represented by this commodity [2]. Even if this percentage is lower than that of cereals, fruits, and vegetables, meat production has a higher economic and ecological impact. Indeed, it leads to the highest greenhouse gas emissions among all foodstuffs [3]. In less developed countries, loss and waste are localised at the production and storage levels due to inadequate infrastructures and technologies [2]; in industrialised regions, such as Europe, they occur during distribution, as well as at the retail and home consumption stages [4].
Meat products have a relatively short shelf-life and require undisrupted cold temperatures along the transport network to avoid spoilage [5]. Lipids, pigments, proteins, and vitamin oxidation are critical processes that also negatively affect meat quality [6].
The meat supply chain is also threatened by the Diptera Calliphoridae flies, commonly known as blowflies. In slaughterhouses, industries, and stores, if hygienic conditions are not optimal, blowflies target meat for their oviposition. The resulting maggots feed on the infested products causing their rotting and spoilage [7,8]. Moreover, adult blowflies can act as mechanical vectors of pathogenic bacteria and protozoa [9,10] as they come into contact solutions, 0.5% (v/v) of vegetal glycerol (A.C.E.F. s.p.a., Fiorenzuola d'Arda, Italy), 0.6% (v/v) of Tween ® 80 (Sigma-Aldrich), and 0.1 or 1.0% (v/v) of the five selected EOs were added to the previously dissolved CH. The EOs concentration was adjusted based on the quantity of the solution employed in the different trials, as explained in Sections 2.4 and 2.7. Successively, the EOs-enriched CH solutions were homogenised on a hot plate stirrer at 18 • C and 28× g for 4 min. Glycerol is a plasticiser that improves the CH mechanical properties, and Tween ® 80 is a surfactant used to ensure wettability [21]. The obtained solutions were stored at 4 • C for no longer than 7 days and heated to 18 • C before use. We prepared the solutions for the sensory analysis and colour assessment of meat during the pre-screening, oviposition deterrence trial with C. vomitoria, and meat preservation and quality analysis during storage.

Selection and Training of Assessors
The selection and training of assessors were performed according to the Department of Agriculture, Food and Environment (DAFE) of the University of Pisa internal procedure, which is based on a normalised technical procedure reported in the literature [22], with some modifications.
All the potential new assessors have been involved in a multi-step training period arranged every year to select a sub-group of future panellists, characterised by the necessary motivation during the whole activity (attendance at more than 75% of training sessions), together with the minimum sensory skills required for food tasting and description (including visual, aroma, and taste attributes).
This multi-step general training is arranged over a period of three months as follows: 1.
Theoretical introduction to the principles of human physiology of sight, smell, and taste.

2.
Arrangement of preliminary training tests, mainly based on the utilisation of model standard solutions, to collect information about the tasting capacity of each assessor (i.e., sensory acuity, odour and flavour memory, term use and recall, scoring consistency). 3.
As the discrimination relies as much on odour memory (that accumulates with experience) as on sensory acuity, ten tasting sessions were carried out in the morning, in a well-ventilated quiet room and in a relaxed atmosphere to evaluate different commercial foods. A sub-group of panellists (eleven people, three males and eight females, ranging from 26 to 65 years old) was selected, starting from the assessors already included in the official panel of the DAFE. All the assessors had previous experience in the food and EOs sensory descriptive analysis and were provided with a specifically developed sensory sheet consisting of a non-structured, parametric, and descriptive scoring chart. Furthermore, all the assessors were also asked to provide a list of some specific olfactory descriptors freely chosen to describe the olfactory profiles of the different samples tested.

Meat, Chitosan, Essential Oils, and Essential Oils-Enriched Chitosan Solutions Sensory Analysis
Samples for sensory analysis were prepared as described below: 1.
1.0 mL of 2.0% CH solution containing 0.1% of one of the five EOs (corresponding to 1.0 µL EO sample −1 ) on the glass Petri dish; 5.
Raw beef mince (3 g + 600 µL of water) with 100 µL of 1.0% EtOH solutions of one of the five EOs (corresponding to 1.0 µL EO sample −1 ) in the embedding mould; 6.
Raw beef mince (3 g + 600 µL of water) with 1.0 mL of 2.0% plain CH solution in the embedding mould;

7.
Raw beef mince (3 g + 600 µL of water) with 1.0 mL of 2.0% CH solution containing 0.1% of one of the five EOs (corresponding to 1.0 µL EO sample −1 ) in the embedding mould.
In order to obtain the same quantity of EO in the pertinent samples (2, 4, 5, and 7), the used concentration of EO (0.1 or 1.0%) was adjusted based on the quantity of the employed solution (100.0 µL or 1.0 mL).
The trained panel of the DAFE of the University of Pisa evaluated the smell profiles of all the samples following the sensory wheel reported in Figure 1.  For the determination of the chromatic characteristics of raw beef mince, an Eoptis CLM-196 colourimeter (Eoptis S.r.l., Trento, Italy) was used. The instrument interfaces through the USB port to a PC with a Microsoft Windows operating system. The acquired colour values are expressed using the native CIE (Commission Internationale de l'Éclairage) coordinates L*, a*, and b* (CIELAB), according to the official method OIV-MA-AS2-11. L* defines the colour lightness (with L* = 0 black and L* = 100 white); a* is the position between red and green (−a* = green and +a* = red); b* is the position between yellow and blue (−b* = blue and +b* = yellow) [23]. The identification of colours in the CIELAB space can also be performed using the so-called cylindrical coordinates: h* and C*. h* defines the psychometric hue, while C* defines the psychometric chroma; they are related, respectively, to the perceptual terms of hue and saturation [23].
The Chroma value C* was calculated by the relation: The colour difference among samples was expressed as ∆E * ab :

Calliphora vomitoria Rearing
The whole rearing procedure was carried out according to Farina et al. [16], with minor changes. C. vomitoria mature larvae were purchased from the commercial supplier Altomare (Vittoria Apuana, Italy) and reared in a plastic box (27 × 21 × 12 cm) with a netted lid for ventilation. Larvae were fed with raw beef mince and kept under laboratory conditions (temperature 23 • C, RH 60-70%, natural photoperiod) until pupation. Adult blowflies (Figure 2), after the species identification [24], were reared in a 75.0 × 75.0 × 115.0 cm polyester and knitted mesh tent (BugDorm-2400 Insect Rearing Tent, MegaView Science Co., Ltd., Taichung, Taiwan) under the same laboratory conditions. Adults were fed a solid diet (sucrose and yeast extract 4:1) and water ad libitum. Yeast was proven to be necessary to provide the proteins needed to stimulate oviposition in Diptera [25].

Calliphora vomitoria Oviposition Deterrence Trial
For the oviposition deterrence assays, adults of C. vomitoria were moved into 47.50 × 47.50 × 93.0 cm nylon and knitted mesh cages (BugDorm-4M4590DH, MegaView Science Co., Ltd., Taichung, Taiwan). Each cage contained one hundred and fifty unsexed blowflies (sex ratio 1:1), 10-20 days old, fed a solid diet (sucrose and yeast extract 4:1) and water ad libitum. Cages were also furnished with a beaker covered by cotton gauze containing 500 mL of water to maintain humidity and were kept under fluorescent lamps (14,000 lux) to provide even lighting during the whole duration of the trials, at 23 • C and RH 60-70%. The methodology was adapted from Bedini et al. [13][14][15] with minor changes.
Firstly, the protection against C. vomitoria oviposition given by the L. nobilis and P. nigrum EOs was evaluated. In each cage, a total of sixteen cubic embedding moulds (2.1 cm side) were positioned; they were filled with 5 g of raw beef mince with 9% of fat and added with 1.0 mL of water to avoid dehydration. The meat surface was flattened and treated with 100 µL of 0.0 (control, CTR), 0.5, 1.0, and 2.0% EtOH solutions of one of the two EOs (corresponding to 0.0, 0.5, 1.0, and 2.0 µL EO sample −1 ). Four moulds, each one containing one of the different EO concentrations, were positioned in correspondence with the four inner corners of the cage, at about 5 cm from the edges, as schematised in Figure 3. The bases of the moulds were glued with double-sided tape to a circular lid (10.0 cm diameter) to avoid overturning. The test lasted 24 h, during which the female blowflies were free to lay their eggs in the preferred sample. . Schematic representation of how the moulds containing the beef meat were arranged in the oviposition deterrence trial. The grey square represents the cage seen from above; the green, blue, purple, and yellow squares represent the moulds containing the meat samples (one control and three different treatments); the white circles represent the supports to which the moulds are glued.
Afterward, following the same protocol and scheme (Figure 3), the protection given to the meat samples by 1.0 mL of 0.5, 1.0, and 2.0% plain CH solutions were tested, compared with an untreated meat CTR.
By taking into consideration the previously obtained results, the need to use the lowest concentration possible of EOs to propose an economically advantageous treatment, and the ease of application of the treatments based on their fluidity, the 1.0% EtOH EO and 1.0% plain CH solutions were selected. Therefore, the protective effect of 100 µL of the 1.0% EtOH solution of L. nobilis or P. nigrum EOs (corresponding to 1.0 µL EO sample −1 ) was compared to 1.0 mL of the 1.0% CH solution, 1.0 mL of 1.0% CH solution containing 0.1% of one of the two EOs (corresponding to 1.0 µL EO sample −1 ), and an untreated CTR ( Figure 3). In order to obtain the same quantity of EO in the pertinent samples, the used concentration of EO (0.1 or 1.0%) was adjusted based on the quantity of solution employed (100.0 µL or 1.0 mL).
All the experiments were replicated three times, applying the same methodology. The laid eggs were counted 24 h from the beginning of the assays, using the piece counter function of an analytical balance (KERN ABS-N, Kern & Sohn, Balingen, Germany). The protection of the different treatments against C. vomitoria was assessed as the percentage of oviposition according to the following formula: NT ÷ NCG × 100, where NT is the number of eggs laid on the specific treatment, and NCG is the total number of eggs laid in the cage.

Meat Preservation and Quality Analysis
The effect of the CH edible coatings, with or without the EOs enrichment, was tested on the shelf-life of raw beef mince with 9% of fat. All the CH solutions were prepared as reported in Section 2.2. Meat patties (10.43 ± 0.07 g weight, 3.5 cm diameter) were manually made and treated with 1.0 mL of the 1.0% plain CH solution, 0.1% of the L. nobilis or P. nigrum EOs, and 1.0% CH solution enriched with 0.1% of EO (L. nobilis or P. nigrum). The 1.0 mL treatments were applied to the patties by spraying them, and the coated samples were stored at 5 • C in plastic Petri dishes (8.5 cm diameter), simulating home storage conditions. Treated beef patties were compared to control (CTR) and untreated patties, and the analysis was performed after the coatings solidified on the beef surface (day 0).
The weight loss percentage and colour determination (L*, a*, and b*-CIELAB) were assessed on days 0, 4, and 7 (n = 6 for each group and time). Meat patties were further analysed to evaluate the lipid peroxidation status by measuring the concentration of the thiobarbituric acid reactive substances (TBARS) at each time point, using a pro-UV-vis spectrophotometer (Amersham Biosciences Ltd., Amersham, UK). In detail, samples were homogenised in 5% trichloroacetic acid (TCA, 1 g:10 mL w/v) and centrifuged at 10,000× g for 20 min at 4 • C; after that, the supernatant was collected. The extract (200 µL) was added to 1.0 mL of either TBA (thiobarbituric acid) solution (15% TCA and 0.01% butylated hydroxytoluene) or + TBA solution (15% TCA, 0.375% TBA, 0.01% butylated hydroxytoluene). Samples were then shaken and boiled at 100 • C within a block heater for 20 min. Before analysis, samples were let to cool down in an ice bath, and the absorbance was then read at 532, 440 and 600 nm. The results were expressed as nmol of malondialdehyde (MDA) equivalent g− 1 FW [26,27].

Data Analysis
The results of the sensory analysis were processed by the Big Sensory Soft 2.0 software (version 2018, Centro Studi Assaggiatori, Brescia, Italy). Sensory data were analysed by two-way ANOVA with panellists and samples taken as main factors [28].
Differences in the oviposition of C. vomitoria among treatments were assessed by oneway ANOVA, with the percentage of laid eggs as the dependent variable and the treatment as the main factor. Means were separated by Tukey HSD post hoc test. Oviposition percentage data were transformed into arcsine values before statistical analysis. Data were processed by SPSS 22.0 software (SPSS Inc., Chicago, IL, USA).
One-way ANOVA and Tukey HSD post hoc test was also applied to assess weight loss, lipid peroxidation, and colour changes during the storage of raw beef patties, with the treatment as the main factor. In the case of colour determination, the effect of the time of storage was also checked for each treatment.

Chemical Composition of the Essential Oils
The complete composition of all the analysed EOs is reported in Table 1.    In the A. sativum EO, 19 compounds (86.1% of the total composition) were detected, all belonging to the non-terpene sulphur derivatives chemical class, of which the most abundant were diallyl tetrasulphide (27.3%) and di-2-propenyl trisulfide 18.3%.
Twenty-nine compounds were identified in the S. rosmarinus EO (100% of the total composition). Over 60% were represented by oxygenated monoterpenes, of which 1,8-cineole accounted for up to 41.1%.

Meat, Chitosan, Essential Oils, and Essential Oils-Enriched Chitosan Solutions Sensory Profiles
The EOs selected for the treatment of meat, chosen among the spices traditionally used to season meat dishes [30], were A. sativum, L. nobilis, O. basilicum, P. nigrum, and S. rosmarinus. Figure 4 shows the overall descriptors used to define their profiles before their utilisation on meat samples. In order to complete the analysis, panellists were asked to list some specific descriptors when necessary (Table 2). According to the compositions shown in Table 1, the best smell profiles were attributed to the L. nobilis and P. nigrum EOs, with high scores on the floral, fruity, and spicy descriptors ( Table 2). On the contrary, given the presence of several compounds with aromatic sulphur notes (Table 1), the A. sativum EOs were characterised by a high number of unpleasant aromas (Table 2), together with the highest smell intensity and persistency (Figure 4).  Figure 5 shows the overall pleasantness attributed to all the EOs EtOH solutions together with data related to the meat samples treated with the various EOs, CH, and EOs enriched CH solutions. Among the selected five EOs, the P. nigrum showed the highest score for overall pleasantness, closely followed by L. nobilis, while the lowest score was attributed to the A. sativum EO. The latter was below the acceptability limit, generally fixed at 5 when 9 is the maximum score value.
Without treatment, the smell of plain meat was described as rancid and cadaverinelike, while that of plain CH was described as acetic and acetone-like due to the use of glacial acetic acid for its preparation (Section 2.2). Nevertheless, the presence of CH reduced the smell intensity of the treated meat, thus reducing the off-flavour detection and improving the global pleasantness score.  Overall, the presence of CH did not significantly affect the sensorial profile of the meat treated with the EOs. When the meat was treated with the EOs or EOs-enriched CH, the best sensorial profiles were obtained with the L. nobilis EOs and P. nigrum, while the worst ones were associated with the A. sativum EO. Furthermore, meat samples treated with the O. basilicum and S. rosmarinus EOs were close to the limit of acceptability, regardless of the presence of CH.

Colourimetric Determination
Soon after treatment, the visual appearance of the meat samples treated with all the combinations of EOs and EOs-enriched CH solutions was deeply affected by the treatment. Table 3 shows that, when meat was treated with the EOs-enriched CH solutions, the colour was generally less vivid. When using the A. sativum EO, the shade changed from red/brown to yellow/brown or greenish/brown. Table 3. Colour coordinates (L*, a*, b*) of beef meat samples in cubic embedding moulds. In each column, different letters (a-g) indicate statistically significant differences.

Calliphora vomitoria Oviposition Deterrence Activity
The oviposition deterrence assays indicated that both the EOs and plain CH could strongly affect the oviposition behaviour of C. vomitoria females. By using the plain CH solutions alone, the C. vomitoria oviposition was reduced up to eleven times (F 3,11 = 18.887, p = 0.001), but with no significant differences among CH concentrations (0.5, 1.0, and 2.0%) (Tukey HSD, p > 0.05).
Similarly, a clear repellent effect was observed for the EO-treated samples, with significant differences both for the P. nigrum (F 3,11 = 36.332, p < 0.001) and L. nobilis EOs (F 3,11 = 45.011, p < 0.001). However, while no significant differences were detected among different concentrations of the P. nigrum EO (0.5, 1.0, and 2.0% in EtOH) (Tukey HSD, p > 0.05), the effect of the L. nobilis EO was dose-dependent with significant differences among the concentrations. In detail, for the L. nobilis EO, the post hoc test indicated a significant difference between the 0.5 and 1.0% EO concentrations ( Figure 6). As previously explained in the Materials and Methods section (Section 2.7), we decided to use the treatments with 1.0% CH, 1.0% EOs, and 1.0% CH solution containing 0.1% of the EOs to be compared. In this case, the ANOVA showed significant differences among the treatments both for the P. nigrum (F 3,11 = 43.676, p < 0.001) and L. nobilis (F 3,11 = 248.649, p < 0.001) EOs. In detail, the post hoc test indicated that among the P. nigrum treated samples, the most effective treatment was the EO-enriched CH solution, whose effect was significantly stronger than that of the plain CH and CTR. On the contrary, among the L. nobilis treated samples, significant differences were shown only among the CH, EO, and the EO-enriched CH solution with the EO and the CTR (Figure 7).

Meat Characterisation during Storage
Given the results above discussed the sensorial characterisation of the proposed treatments and the protection they gave against the C. vomitoria oviposition, we also evaluated meat preservation for 7 days by treating the samples with the L. nobilis or P. nigrum EOs, CH, and the corresponding EOs-enriched CH solutions.

Weight Loss
Weight loss (%) was calculated in comparison to the initial weight of each sample (day 0). Significant changes in the weight loss percentage (Figure 8) were observed after 4 (F 5,30 = 8.103; p < 0.0001) and 7 days (F 5,30 = 4.1342; p < 0.01). All the treatments showed a similar trend in comparison with the CTR samples. However, after 4 days, the L. nobilis EO significantly differed from the CH treatments, both plain and enriched. Moreover, after both 4 and 7 days, the enriched CH solutions performed better than the respective EOs alone.

Colour Modifications
During storage, the colour was assessed by measuring the L*, a*, and b* parameters according to the CIELAB system ( Table 4). The difference among treatments was compared to verify how the application of an edible coating may influence the attractiveness compared to the CTR beef patties. The lightness index L* was affected by the treatments at day 0 (F 5,30 = 5.522; p = 0.001) and after 7 days (F 5,30 = 7.111; p < 0.001). It is interesting to note that, at the beginning of the storage, samples coated with the CH enriched with the L. nobilis (+10.6%) and P. nigrum (+9.6%) EOs but also with plain CH (+10.5%) displayed a higher lightness compared to the CTR group. After 7 days, the enriched CH solutions (+10.5 % and +9.0% for L. nobilis and P. nigrum, respectively) and plain CH (+9.2%) still conferred higher lightness values to meat if compared to CTR.
The a* coordinate was significantly affected by the treatments after 4 (F 5,30 = 2.874; p < 0.050) and 7 days (F 5,30 = 4.246; p < 0.010). In the first case, the only significant difference was found between the CTR and CH enriched with the L. nobilis EO, with the latter having a lower value of about −11%, while the other treatments had similar values to the CTR. After 7 days, all the treatments showed a lower a* compared to the CTR (−10.2%, −13.4%, −11.6%, −11.5% for CH, CH+L. nobilis EO, L. nobilis EO, and P. nigrum EO, respectively), except for the CH enriched with the P. nigrum EO, which was similar to the CTR.
The b* coordinate was significantly affected by the treatments applied only at day 0 (F 5,30 = 4.778; p < 0.010). In particular, all the treatments showed no difference with the CTR group, but the CH-treated samples displayed a lower value if compared with the EOs treatments (−29.7% and −34.6%, for the L. nobilis and P. nigrum EOs, respectively) and CH enriched with the P. nigrum EO (−33.2%).
Besides the evaluation of the differences induced by the different coatings, the changes in the colour indexes occurring during storage were checked for each treatment (Table 4). While the greater changes in all the parameters (L*, a*, and b*) utilised to measure the meat's colour were already evident after the first 24 h, regardless of the treatment, some further indications can be highlighted and discussed even during the 7 days storage.
In particular, for the plain CH, CH enriched with the L. nobilis or P. nigrum EOs, and L. nobilis EO, no changes for any of the coordinates investigated were found during the observation time. Both the CTR and CH samples showed a significant increase in a* after 4 and 7 days. Specifically, in CTR samples, a* was 14.4% and 20.7% higher (F 5,30 = 11.419; p ≤ 0.001) after 4 and 7 days, respectively, as compared to the beginning of storage. The CH samples displayed a similar trend, with an increase in a* (F 5,30 = 5.792; p < 0.050) of +13.1% at 4 days and +12.8% at 7 days compared to day 0. For samples treated with the P. nigrum EO only, L* underwent a little increase of +5.9 % (F 5,30 = 4.290; p < 0.050) after 4 days of storage.
The total colour differences (∆E ab ) compared to the initial values (0 days) were calculated at 4 and 7 days of storage for each group (Table 4). During the whole observation period, CTR samples displayed the highest colour change, regardless of the preserving solution adopted.
Moreover, when the total colour differences (∆E ab ) were calculated among samples on each day of storage (Table 5a-c), the higher ∆E ab values were detected when chitosan was added to the meat, regardless of the storing time considered.

Lipid Peroxidation Index
The presence of secondary products of lipid oxidation (Figure 9) was evaluated at 0, 4, and 7 days of cold storage. After 4 days, the treatments applied on the beef patties' surface caused some significant differences in this parameter (F 5,12 = 6.030; p < 0.010). Indeed, CH and EOs, both the L. nobilis and P. nigrum, produced a reduction in the lipid peroxides concentration (−40%, −49%, and −44%, respectively) when compared to the CTR group, while the EOs-enriched CH had similar concentrations to the CTR and the other treatments. The lipid peroxidation index was also significantly affected by the treatments at the end of the storage (F 5,12 = 6.718; p < 0.010). In particular, no significant differences were found for all the treatments applied compared to the CTR group, even if a trend towards a lower lipid peroxidation index can be appreciated in the case of the CH and CH enriched with the L. nobilis EO; however, some differences emerged among the coating treatments. Specifically, the treatment with the two EOs alone, both the L. nobilis and P. nigrum, caused an increase in the TBARS concentration compared to the CH (+57% and +56%, respectively) and CH enriched with the L. nobilis EO (+46% and 45%, respectively).

Discussion
Meat protection, preventing the loss and waste of this commodity with a particularly negative ecological impact, is a demanding challenge that must be addressed. In recent years, the interest in innovative and sustainable packaging able to improve the shelf-life of meat has increased. In this study, we assessed beef meat protection against oviposition by the blowfly C. vomitoria and its preservation using a CH edible coating mixed with two EOs (L. nobilis and P. nigrum) selected by expert sensorial analysts based on their suitability for meat.
The Moroccan L. nobilis EO used by Nafis et al. [33] revealed a composition similar to that reported in the present study, with 1,8-cineole (eucalyptol) as the main constituent (40.85%), followed by α-terpinyl acetate (12.64%) and methyl eugenol (8.72%). Two laurel EOs, one extracted from a Greek accession and one from a Georgian one, exhibited 1,8-cineole (30.8 and 29.2%, respectively) and α-terpinyl acetate (14.9 and 22.6%, respectively) as major components, as shown by the results of the present work. The EO from Greece also contained 8.0% of α-terpineol and 6.0% of terpinen-4-ol; the EO from Georgia was composed of 12.2% of sabinene and 8.1% of methyl eugenol [34].
The O. basilicum EO characterised in this paper was a methyl chavicol-chemotype (76.3%), as stated by the manufacturer. This chemotype was found in Turkey (city of Zonguldak), as reported by Telci et al. [35], and in Mississippi (United States), according to a study on 38 basil genotypes [36].
β-Caryophyllene is commonly reported as the main compound of P. nigrum EO: it accounted for up to 51.12% in a black pepper EO used by Andriana et al. [37], and a similar percentage (47.14-50.88%) was reported by Rmili et al. [38].
The five EOs proposed for meat preservation were initially selected based on their traditional use in meat seasoning [30]. Among them, the P. nigrum and L. nobilis EOs showed the best sensorial profile both in pure solution and in combination with meat, regardless of the presence of CH. On the contrary, the A. sativum EO showed the lowest overall pleasantness in all the conditions tested (EtOH solution, EO + meat, EO + CH + meat). With the only exception of the A. sativum, the addition of EOs significantly improved the sensorial profile of meat samples, regardless of the presence of CH. Furthermore, when the A. sativum EO was utilised, the colour of meat samples was also negatively affected.
The oviposition deterrence on C. vomitoria was already evaluated using several EOs extracted from culinary herbs. Complete meat protection was achieved using A. sativum EO at the concentration of 1.25 µL EO cm −2 [8] and Artemisia dracunculus L. (Asteraceae) EO at a substantially lower concentration of 0.05 µL EO cm −2 [13]. Three EOs from distinct Origanum vulgare L. (Lamiaceae) chemotypes offered different levels of protection. At the concentration of 0.32 µL EO cm −2 , the thymol/γ-terpinene oregano chemotype EO avoided almost 90% of the oviposition, and the thymol/p-cymene and carvacrol chemotypes EOs more than 60% [15]. Similarly, our results show that, at the concentration of 0.48 µL EO cm −2 , the L. nobilis and P. nigrum EOs exert protection of 89 and 93%, respectively.
To the best of our knowledge, none of the EOs used in this work has been used to control C. vomitoria before, but they were applied as repellents against other insect pests as well as insecticides. In a repellence assay on stored products pests, an L. nobilis EO at 78.63 nL EO/cm 2 proved to be highly repellent (more than 80%) towards Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae) after 24 h of exposure [41]. A 3.0% L. nobilis EO formulated with olive oil protected for 52.3 min from Culex pipiens molestus Forskål (Diptera: Culicidae) bites [42]. Erler et al. [43] tested the repellence of an L. nobilis EO against C. pipiens female mosquitoes in a Y-tube olfactometer, reporting a more than 80% repellent effect with 10 µL of EO in an exposure time of 255 s.
Sticking to the repellence, Chaubey [44] found that a P. nigrum EO was 100% repellent on filter paper in Petri dishes starting from the concentration of 0.8% in acetone against Sitophilus zeamais (Motsch.) (Coleoptera: Curculionidae) and 97.5 ± 0.5% repellent from the concentration of 0.0125% in acetone against Sitophilus oryzae (L.) [45]. A different accession of a P. nigrum EO induced reduction in the oviposition and eggs hatching, a delay in the transformation of larvae into pupae, and a decrease in the final number of adults in Callosobruchus chinensis L. (Coleoptera: Bruchidae) [46]. CH finds various applications in insect pest control, both as a repellent and insecticide. Different CH concentrations (from 0.5 to 5%) were successfully used on paper and wood to inhibit the activity of the termites Reticulitermes flavipes (Kollar), Reticulitermes virginicus Banks, and Coptotermes curvignathus (Holmgren) (Isoptera: Rhinotermitidae) [47,48]. Moreover, several EOs have been added to CH matrixes to enhance their efficacy and persistence. Melissa officinalis L. (Lamiaceae) nanoencapsulated EO in CH (from 0.06 to 0.30 mL EO in 1.5% CH) showed antifeedant activity and toxicity by fumigation on Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) [49]. Cymbopogon spp. (Poaceae) EO adsorbed on a CH and silica gel matrix successfully repelled adults of the mosquito Aedes aegypti L. (Diptera: Culicidae) for up to 4 h [50]. CH enriched with Ferulago campestris (Besser) Grecescu (Apiaceae) EO (from 10 to 25% EO in 2.0% CH) hindered the reproductive activity of Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae) females on the common bean Phaseolus vulgaris L. (Fabaceae) [51].
Concerning meat dehydration during the 7 days of storage at cold temperatures, all the treatments had no effects if compared with the CTR group at any time point. However, EOs alone generally caused a higher loss compared to the CH enriched with EOs. Based on our results, CH might be able to mitigate the negative effects that EOs can have on the dehydration of food products, improving the water barrier properties. Similarly to our observation, Ummarat and Seraypheap [52], studying the post-harvest effects of EOs on rambutan fruits (Nephelium lappaceum Linn.-Sapindaceae), found that Cymbopogon nardus L. (Poaceae) EO at concentrations higher than 0.04% enhanced the weight loss compared to their CTR.
Another crucial attribute of meat products is the aesthetical quality in terms of surface colour. In our study, the application of plain CH or CH enriched with the EOs increased the lightness compared to the CTR beef: this could be an important feature for the consumers' acceptability. The higher L* coordinate values at the beginning and after 7 days of storage were likely due to the coating itself. Indeed, Jo et al. [53] and Giatrakou et al. [54] found a similar effect in their studies regarding different CH treatments for meat preservation. Conversely, Lekjing [55] studied CH coatings with or without the addition of Syzygium aromaticum (L.) Merr. and Perry (Myrtaceae) EO applied on cooked pork sausages and found a decrease in this parameter when comparing treatments with CTR samples.
However, the coordinate a*, indicating redness, was similar to the CTR values until 4 days of storage, except for the CH enriched with the L. nobilis EO. At the end of the storage period, the lower a* induced by all treatments, except for the CH enriched with the P. nigrum EO, resulted in a less bright red colour, according to the instrument, which might suggest the likely ongoing oxidation processes.
As the different redness could be caused by the CH coatings or EOs themselves, we also compared the time-course modifications for each treatment. That, indeed, revealed no significant changes in the patties coated with the EOs-enriched CH solutions, but a little time-dependent increase for the CTR and CH samples occurred. Myoglobin is the principal protein responsible for meat colour, and its oxygenation causes the conversion of this molecule into oxymyoglobin, which gives a bright red colour [56]. Then, over time, deoxyand oxymyoglobin forms are further oxidised to metmyoglobin, causing the production of the brown colour of meat [57]. Therefore, it can be hypothesised that an oxygenation process of myoglobin in the CTR and CH samples was likely ongoing during the storage of beef patties, while the other treatments slowed down the oxygenation reactions. Even the increase at 4 days of storage of the L* coordinate of beef patties treated with the P. nigrum EO likely suggests changes in the protein structure caused by oxidation, as indicated by MacDougall [58]. Finally, the differences in total colour (∆E) calculated for each group at both times of storage in respect of 0 days pointed out that untreated beef changed greatly and significantly compared to the EO-enriched and CH-treated samples. On the other hand, at each storage time, the higher ∆E ab values were detected when chitosan was added to the meat, while the distance between the chromatic coordinates (∆E ab ) showed how all the meat samples treated with different preserving solutions could not be distinguishably discriminated (∆E ab < 6) in colour if compared with each other and control [59].
The results related to the TBARS concentration indicated that the lipid peroxidation status was affected by the treatments differently according to the different times of storage. Indeed, at 4 days, a positive influence of CH and the EOs alone was noticed compared to the untreated beef, confirming their ability to slow down the oxidative reactions occurring within the biological matrix. Similarly, Vital et al. [60] found a reduction in lipid peroxidation in beef treated with rosemary and oregano EOs compared to untreated beef. Moreover, CH is known for its antioxidant properties, as reported in other studies on beef [61,62]. After 7 days, the lipid peroxidation was similar to the CTR group for all treatments, meaning that the protective effect of the EOs noted at the previous time point faded. Indeed, the treatments with the EOs alone caused an increase in this lipid peroxidation index in comparison with the CH treatments. In particular, the L. nobilis EO, when added to the CH solution, performed better. This might indicate that, after 7 days, the EOs might have undergone a natural auto-oxidation of some lipid components that, instead, was prevented by the EO addition into the CH solution. This auto-oxidation was likely able to set off other oxidative reactions within the food matrix. Indeed, CH acts as a selective gas barrier, i.e., towards oxygen, as demonstrated by several other studies [63,64], the property might have protected the EOs included in it.

Conclusions
The results presented in this work show that edible coatings made of CH and selected EOs can be promising, innovative allies in beef meat protection. Regarding the smell profiles, the application of the L. nobilis or P. nigrum EOs, alone or mixed with CH, enhances the odour pleasantness of raw meat, masking the usual cadaverine-like smell. Interestingly, the P. nigrum EO enriched CH is significantly active in repelling the blowfly C. vomitoria, avoiding its oviposition on meat. That feature could be successfully exploited for the implementation of EOs-enriched CH sprayable coatings able to reduce meat loss and waste due to the Calliphoridae flies in slaughterhouses, industries, and stores where the hygienic conditions are not optimal. All the treatments proposed, compared to the control, do not accelerate meat dehydration and lipid peroxidation after 7 days of storage, preserving its organoleptic qualities and shelf-life. Interestingly, in earlier days, a pronounced antioxidant effect against lipid peroxidation was achieved with the EOs treatments, but this protection was transient and faded later. Furthermore, the treatments increase the colour lightness of meat, an attractive feature for consumers.