Investigation of the chemical properties of Mentha pulegium essential oil and its application in Ocimum basilicum seed mucilage edible coating for extending the quality and shelf life of veal stored in refrigerator (4°C)

Abstract Nowadays, the tendency toward the application of natural preservatives to extent the shelf life of food products has grown. The purpose of the present research was to evaluate the effect of the basil seed mucilage (BSM)‐based edible coating containing different concentrations of Mentha pulegium essential oil (MPEO) on the shelf life of the veal stored at refrigerator temperature. Firstly, the chemical composition and functional groups of MPEO were detected through gas chromatography–mass spectrometry (GC‐MS) and Fourier transform infrared spectroscopy (FTIR). Then, the BSM‐based edible coatings containing 0%, 0.5%, 1%, 1.5%, and 2% MPEO were prepared, and the veal samples were coated with them. The physicochemical, microbial, and sensory properties of the samples were investigated during the 9‐day storage period at 4°C. Twenty‐five compounds were detected in MPEO with limonene being the major one (28.44%). The results revealed that the lightness, hardness, and moisture content of the samples decreased during storage. The coating containing the essential oil could properly restrain the rise in pH, peroxide value (PV), and thiobarbituric acid value (TBA). Based on microbial analyses, the shelf life of the coated sample without the essential oil and those containing 0.5%, 1%, 1.5%, and 2% of the essential oil were, respectively, extended up to 3, 6, 9, 9, and 9 days relative to the control. Moreover, the coating containing the essential oil produced no unfavorable effect on the sensory properties of the meat samples. In conclusion, the BSM‐based edible coating containing different concentrations of MPEO can be applied as a natural preservative to enhance the resistance of meat products against microbial spoilage and fat oxidation.


| INTRODUC TI ON
Owing to its abundant nutrients, red meat is one the major food products consumed by humans. Given the high perishability of meat, the hygiene and shelf life of this product are of great importance to producers and consumers. Up to now, various methods, including smoking, drying, and storage at low temperatures have been utilized to extend the shelf life of meat and its products. Application of edible films and coatings is one of the novel technics for the storage of meat and its products (Dave & Ghaly, 2011).
Anti-microbial edible films and coatings containing natural essential oils and extracts are considered to be a novelty in food packaging (Shen et al., 2010). Characteristics, such as biodegradability, gradual release of the active compounds present in the coating, minimizing the moisture loss of food products, and preventing the release of aromatic compounds are among the benefits of employing this technic of storage (Wang et al., 2010). Utilization of herbal essential oils and extracts with anti-microbial and antioxidative effects in edible coatings can be a suitable solution for inhibiting the microbial and chemical spoilage of food products (Alizadeh Behbahani et al., 2017c).
Mentha pulegium L. belongs to the family of Lamiaceae and the genus of Mentha (Khezri et al., 2020). It can be found in the wet regions of Europe, North Africa, Australia, North America, and Asia (Soilhia et al., 2019). The height of some shrubs of this perennial herbaceous plant reaches one meter. Due to its desirable aroma and properties, Mentha pulegium is used as a seasoning and preservative in the food industry (Ahmed et al., 2018). The aerial segments of this medicinal plant are employed in traditional medicine for the treatment of bronchitis, sinusitis, tuberculosis, indigestion, diarrhea, flatulence, and vomiting (Gülçin et al., 2020). Menthone, pulegone, and isomenthone are the major components of Mentha pulegium (Khezri et al., 2020).
Basil (Ocimum basilicum L.) belongs to the Lamiaceae family. Nowadays, it is cultivated in several regions of the world. At the same time, India and Asia are known as its primary origin (Ahmed et al., 2019). This plant absorbs water through the outer segments of its seeds. Glucomannan (43%), xylan (24%), and uric acid (6%) are the major constituents of BSM (Tantiwatcharothai & Prachayawarakorn, 2019). In the structure of the mucilage, glucomannan and xylan act as the hydrophobic and hydrophilic moieties, respectively. Xylan causes swelling by water absorption (Nourozia & Sayyari, 2019). Biodegradability, low-cost production, easy extraction, high water absorption, temperature resistance, and favorable rheological properties have caused BSM to find its application in the preparation of edible films and coatings (Keisandokht et al., 2018;Majdinasab et al., 2020).
The objective of this study was to produce edible coatings based on BSM and MPEO, as well as investigating the physicochemical, microbial, and sensory properties of the coated veal samples during refrigerated storage for 0, 3, 6, and 9 days.

| Chemicals and reagents
The culture media, including plate count agar, mannitol salt agar, eosin methylene blue, and yeast extract glucose chloramphenicol agar were supplied from Conda pronadisa. Chloroform, acetic acid, potassium iodide, potassium sulfate, TBA indicator, and trichloroacetic acid were purchased from Sigma-Aldrich.

| MPEO preparation
The essential oil was bought from Johareh Tam Mashhad, which had been extracted through water distillation.

| Preparation of basil seed mucilage
BSM was extracted according to the method previously optimized by Razavi et al. (2009). To that end, basil seeds were firstly macerated in deionized water at a ratio of 1:50, 50°C and pH = 7. After 10 min, the mixture was centrifuged at 5000 g, and the mucilage was extracted which was further bleached with ethanol 96% at a ratio of 1:3. It was then freeze-dried (OPERON. FDB-5503) and stored in a package impervious to moisture.

| Detection of the chemical compounds of MPEO
GC-MS was employed to detect the constituents of the essential oil. One microliter of the essential oil was injected into a gas chromatograph (Agilent Technologies 7890 A) equipped with a column, 30 m in length, 0.25 mm in internal diameter, and 0.25 µm in film thickness (Agilent Technologies Inc., HP-5 MS) and integrated with a mass spectrometer (Agilent Technologies 5975). The initial temperature of the column was set at 40°C which was elevated up to 250°C at 2.5°C/min. Helium was used as the carrier gas at a flow rate of 1.1 ml/min, and the ionization energy was 70 eV. The chemical compounds of MPEO were detected using the spectra and retention indices of n-alkanes along with referring to the database of natural compounds (Alizadeh .

| Examination of MPEO functional groups
Fourier transform infrared spectroscopy (FTIR) was employed to identify the functional groups of the MPEO. First, the essential oil powder was mixed with potassium bromide and pressed to be converted into a tablet. The essential oil spectrum was recorded in the wavenumber range of 4000-400 cm −1 using a spectrometer (Avatar 370, Thermo Nicolet; Alizadeh Behbahani, Falah, et al., 2020;Falah et al., 2021).

| Veal chemical composition
The moisture, ash, fat, and protein contents of the veal samples were quantified according to AOAC (2005).

| Edible coating preparation and coating of veal samples
In order to coat the veal samples, 1.5 g of the BSM and 0.52 g Tween 80 (35% of the dry weight of the BSM) were made to volume and blended with 100 ml of sterile distilled water. The solution temperature was set at 45-50°C. MPEO, as an ingredient with antibacterial effect, was added to the BSM solution at 0%, 0.5%, 1%, 1.5%, and 2%. Fresh veal samples were macerated and coated for 1 min in the solution containing the BSM and various concentrations of the essential oil. Four samples coated with the BSM and different concentrations (0.5%, 1%, 1.5%, and 2%) of the essential oil, one sample free of both the essential oil and the BSM, and one sample coated with the BSM without the essential oil were stored at refrigerator temperature for 9 days. The physicochemical, microbial, and sensory properties of the veal samples were investigated at certain time intervals (Heydari et al., 2020).

| pH
In order to determine the pH of the samples, 5 g of each sample was mixed with 45 ml of distilled water and homogenized. The pH values of the samples were measured using a digital pH meter at room temperature (Xiong et al., 2020).

| Peroxide value
In order to extract the lipid content of the samples, 60 ml of methanol was added to 15 g of the veal sample in a separatory funnel.
After homogenization, 30 ml of chloroform was also added. After 5 min, another 30 ml of chloroform was added, and the solution was set aside at ambient temperature for 24 h so the sample lipid content would be extracted. Finally, 36 ml of distilled water was added to the contents of the separatory funnel, and the lower phase was separated after 2 h. The solvent was evaporated using a rotary evaporator, and only the sample lipid remained. For the determination of peroxide value (PV), a certain amount of the extracted lipid, 25 ml of acetic acid-chloroform (chloroform: acetic acid ratio of 2:3), 0.5 ml of saturated potassium iodide, 30 ml of distilled water, and 0.5 ml of starch 1% were mixed in a 250-ml Erlenmeyer flask. The released iodine was titrated with sodium thiosulfate 0.01 N. PV was expressed as meq O 2 /kg (Majdinasab et al., 2020).

| Thiobarbituric acid value
First, 2 g of the veal samples was homogenized with 5 ml of trichloroacetic acid 20% for 2 min. The resulting mixture was filtered using a filter paper. Five milliliter of the filtrate was blended with 5 ml of TBA indicator 10 mM in a test tube. The tube was kept in a water bath at 100°C until color change was observed (about 1 h). After cooling down the tube, the absorbance value of the solution was measured at 532 nm (Lashkari et al., 2020).

| Moisture content
For quantifying the moisture content of the samples, they were heated in a hot-air oven at 105°C until reaching a constant weight (5 h) (Barzegar et al., 2020).

| Texture analysis
A texture analyzer (TA, XT2i, the UK) was used to examine the hardness of the meat samples. During the test, the sample cubes (2*2*2 cm 3 ) were compressed up to 30% of their initial height by the aluminum probe with a diameter of 36 mm (36 p/36). The probe speed was set at 5 mm/s (Heydari et al., 2020).

| Microbiological analysis
The microbial load of the veal samples stored at 4°C was assessed at certain time intervals (days 0, 3, 6, and 9). The stock solution was prepared by mixing 5 g of the veal and 45 ml of sterile physiological serum and homogenized for 1 min. Subsequent sequential dilutions were prepared using the stock solution. In all the steps before dilution, the solutions were homogenized for 1 min. Eventually, 100 ml of each solution was poured onto the culture media and spread with an L-shaped rod. The performed microbial tests were as follows: Total viable count was done using plate count agar and incubation at 37°C for 24 h.
Psychrotrophic count was carried out using plate count agar and incubation at 20°C for 24 h.
Mold, yeast, and fungi count were performed using yeast extract glucose chloramphenicol agar and incubation at 27°C for 72 h.
Staphylococcus aureus count was conducted using mannitol salt agar and incubation at 37°C for 24 h.
Escherichia coli count was done using eosin methylene blue and incubation at 37°C for 24 h (Kiarsi et al., 2020).

| Sensory evaluation
The color, odor, texture, and overall acceptability of the samples were assessed on the test days. The veal samples were placed on the plates coded with random three-digit numbers. Nine-point hedonic scale (1 = the lowest quality and 9 = the highest quality) was applied to determine the quality of the samples. The samples with scores less than 4 were considered unacceptable (Barzegar et al., 2020).

| Statistical analysis
The obtained data were analyzed through one-way analysis of variance (ANOVA) using SPSS version 16. Duncan's multiple range test was employed for mean comparison at 95% confidence level. All the experiments were triplicated.
The FTIR results demonstrated that the band in the wavenumber range of 3400-3000 cm −1 was associated with the stretching vibrations of the O-H groups of water molecules, as well as those of the phenolic and alcoholic compounds of the essential oil like menthol (Alavi et al., 2021). The peak at 2968.83 cm −1 was ascribed to the asymmetric stretch of C-H and CH 3 groups (Petrakis et al., 2009).
In addition, the peaks at 2925.47 cm −1 belonged to the asymmetric stretch of CH 2 and the symmetric stretch of CH 3 . The peak appeared at 1675.14 cm −1 was related to the carbonyl (C=O) groups of piperitone and pulegone, 1453.41 cm −1 to CH 2 scissoring and the asym- Mentha pulegium extract, a broad band peaked at 3402 cm −1 was observed which was believed to be associated with the O-H groups of the phenolic compounds. In our study, the band ranging from 3400 to 3000 cm −1 was related to the O-H groups of the phenolic and alcoholic compounds of the essential oil.

| Veal chemical composition
The chemical composition of the veal used in the present research is summarized in Table 2. AOAC standard methods were utilized to determine the meat chemical composition.

| pH
The effect of pH change on the veal samples is depicted in Figure 2a.
ANOVA showed that the pH of all the samples significantly increased during storage; however, the elevation slope was gentler in the coated samples relative to the control. The effect of coating was observed even on the first day of storage such that the pH of the control was more than that of the other samples, but this difference was not significant at 95% confidence level. The highest and lowest pH values on the 9th day of storage belonged to the coated sample containing 2% of the essential oil and the control, respectively, which were significantly different at p < .05.
Cattle age, sex, and diet in addition to the pre-slaughter stresses are among the most important factors influencing the meat initial pH. It has been cited in the literature that bacteria play a key role in pH increase in such a way that bacterial enzymes give rise to pH by

| Peroxide value
The changes in the PV of the veal samples are depicted in Figure 2b.
The PV of all the samples was equal to 1.36 meq O 2 /kg on the first day. The slope of the PV increase was steeper for the control than for the other samples. In contrast, the PV of the coated sample containing 2% of the essential oil showed the least variations and in-    (Xiong et al., 2020). In addition, water-holding capacity has direct and inverse correlations with pH and hardness, respectively. As a result, it can be claimed that during storage, the pH and water-holding capacity of the samples rose, while their hardness was reduced (Heydari et al., 2020). The findings of the present study are in agreement with those achieved by Barzegar et al. (2020) and Xiong et al. (2020).

| Colorimetry
The changes in the L* of the veal samples during storage are demonstrated in Figure 3a. This color index represents the lightness of the samples. According to the obtained data, the lightness of the control was more than that of the coated samples on the first day; however, this difference was not significant (p > .05). As time went by, the lightness of all the samples significantly lowered which was more pronounced for the control than for the other samples. Meat color is considered a primary attribute attracting consumers' attention and is an indicator of the meat freshness from the consumers' points of view (Chaparro-Hernández et al., 2019). The lower lightness of the coated samples, relative to the control, on the first day is likely related to the darkness of the coating. Many researchers believe that the decrease in the lightness of the samples during storage is owing to oxidation, alteration of protein structures, and increased light scattering (Alizadeh Lashkari et al., 2020).
Moreover, the less pronounced reduction in the lightness of the coated samples, compared with the control, is associated with the antioxidant properties of the MPEO in addition to the inhibition of oxygen penetration by the coating, which controlled oxidation (Vital et al., 2016).
The alterations in the a* of the veal samples during storage are presented in Figure 3b. This color index denotes the redness of the samples. It was significantly reduced in all the samples during storage.
The reduction slope was gentler for the control rather than the coated samples. Meat redness is affected by several factors, including myoglobin content, texture pH, and the presence of antioxidants (Mancini & Hunt, 2005). The decreased redness may be due to the conversion of oxymyoglobin (bright pink) to metmyoglobin (brown). The presence of the coating caused a reduction in oxygen pressure, thus inhibiting the production of oxymyoglobin (Kiarsi et al., 2020;Ruan et al., 2019).
The variations in the b* of the meat samples during storage are summarized in Figure 3c. This color index stands for yellowness.
The b* of all the samples significantly lowered during storage. The changes in the b* of the control were more pronounced compared with the coated samples. In the literature, the smaller reduction in the yellowness of coated samples and the coated ones containing an essential oil has been attributed to the color of the coating and the essential oil (Kiarsi et al., 2020;Lashkari et al., 2020).
The total color change of the samples was evaluated using ΔE which was computed using L*a*b* (Figure 3d). The most and least changes, respectively, belonged to the control and the coated sample containing 2% of the essential oil. The results of this research are consistent with those reported by Khare et al., (2016) and Guerrero et al., (2020) (Figure 4).

| Total viable count
The results of the total viable count of the veal samples during refrigerated storage are shown in Figure 5a. The initial total viable count ranged from 3.66 to 4.06 log CFU/g. There was no significant difference between the samples at p < .05. During storage, total viable count was elevated significantly. The rise slope was steeper for the control relative to the other samples, which increased from 4.06 log CFU/g on the first day up to 10.4 log CFU/g on the 9th day.
Additionally, as the essential oil concentration was raised, this response decreased, so that the total viable count of the sample coated with the BSM-based coating containing the MPEO was always lower than that of the other samples on the same day of storage.
Considering the presented standards, the maximum acceptable and permitted microbial load of fresh meat is equal to 7 log CFU/g (Barzegar et al., 2020). Therefore, the microbial load of the control was under this limit only on the first day of storage. Moreover, the microbial loads of the coated sample without the essential oil and the coated one with 0.5% of the essential oil, respectively, exceeded the permitted limit after the 3rd and 6th days.  According to statistical analyses, the control and the coated samples significantly (p < .05) differed from each other on the 3rd, 6th, and 9th days of storage, confirming the protective role of the coating.

| Escherichia coli count
The changes in the E. coli count of the samples during storage are summarized in Figure 5d. There were no significant differences (p > .05) between the samples on the first day. However, this response increased significantly during storage. The greatest change was associated with the control. The increase in the MPEO concentration restrained the rise in E. coli count. On the 9th day of storage, the coated samples significantly (p < .05) differed from the control in terms of E. coli count. The results also indicated that the microbial load of S. aureus was lower than that of E. coli. The difference in the cell-wall structure of these bacteria is probably the reason behind this observation (Alizadeh Kiarsi et al., 2020;Noshad et al., 2021).

| Staphylococcus aureus count
The

| Sensory evaluation
The results of the sensory evaluation (color) of the veal samples during storage are shown in Figure 6a. The samples with sensory scores of more than 4 were considered acceptable. There were no significant differences between the samples on the first day. During storage, the sensory scores of all the samples were reduced, which was less pronounced in the coated samples, particularly in the coated one containing 2% of the essential oil. The sensory attribute (color) of the control and the BSM-coated sample was not acceptable on the 6th and 9th days. On the other hand, all the coated samples containing the essential oil obtained scores of more than 4 until the 9th day.
The control had the lowest color acceptability from the panelists' viewpoints. This may be due to the more darkness of this sample relative to the others. In the coated samples, the coating prevented the direct contact between oxygen and the sample surfaces, while there was a direct contact between oxygen and the control surface. Although the sensory scores of all the samples decreased, the control experienced the largest reduction. The odor score of the control was more than 4 merely on the first day and was not acceptable thereafter. Likewise, the score of the BSM-coated sample without the essential oil was less than 4 on the 6th and 9th days.
It is possible that the decrease in the odor scores of the samples was caused by the products of hydroperoxides degradation.
Although hydroperoxides are themselves tasteless and odorless but are basically unstable and decompose rapidly. The products of the breakdown of hydroperoxides change into aldehydes and ketones with undesirable odor and taste. The rise in the undesirable odor of the samples during storage may also be owing to the activity of microorganisms like Pseudomonas and Achromobacter (Majdinasab et al., 2020;Rezaeifar et al., 2020).
The results of the sensory evaluation (texture) of the samples during storage at 4°C are depicted in Figure 6c. There were no significant differences (p > .05) between the samples on the first day.
Nevertheless, the differences were raised during storage. The reduction slope was steeper in the control rather than the other samples. Sensory analysis showed that all the coated samples containing the essential oil were acceptable until the 9th day. However, the control and the BSM-coated sample free of the essential oil were not acceptable on the 6th and 9th days.
The decrease in the sensory texture score might be caused by microbial activities. Under aerobic conditions, Pseudomonas and Alcaligenes species produce slime on the meat surface, which has a negative impact on its texture. Furthermore, the growth of molds, in particular their mycelium, makes the meat texture adhesive and fluffy, adversely affecting the sensory texture score (Noshad et al., 2021). The results of the microbial tests also confirmed this fact.
The results of the sensory evaluation (overall acceptability) of the veal samples during refrigerated storage are illustrated in Figure 6d. oil, which were considered acceptable until the 9th day, the control and the coated ones without the essential oil were not acceptable after the 3rd and 6th days, respectively. In conclusion, it could be declared that MPEO and the BSM-based coating did not produce adverse effects on the sensory properties of the veal samples.

| Correlation among physicochemical, microbiological, and sensory properties of veal samples
The correlation coefficients of the physicochemical, microbiological, and sensory properties of the veal samples are presented in Table 3.
This coefficient represents the linear relationship between two variables. If the line between the variables passes through all the real points, the correlation is perfect, and if not, the correlation is imperfect. Correlation coefficient varies from −1 to 1. The coefficients of −1 and 1 represent the perfect linear correlation, whereas the values between them denote the imperfect linear correlation. The correlation coefficients of all the variables were more than 0.74. In addition, the smallest coefficient (0.74) belonged to the correlation between moisture content and ΔE. In contrast, the largest coefficient (1.00) pertained to the correlation between color and sensory texture.

| CON CLUS ION
Meat and meat products are always prone to microbial spoilage and oxidation, due to being composed of abundant nutrients and unsaturated fatty acids. The results of the present study demonstrated that the BSM-based coating containing MPEO could appropriately restrain lipid oxidation, moisture loss, hardness reduction, and drastic color changes in the samples, relative to the control. According to microbial analyses, the shelf-lives of the coated sample free of the essential oil and the coated ones containing the essential oil at 0.5%, 1%, 1.5%, and 2% were, respectively, extended up to 3, 6, 9, 9, and 9 days, compared with the control. Moreover, the coating containing the essential oil had no unfavorable effect on the sensory attributes of the veal samples. In conclusion, the BSM-based coating containing MPEO could be utilized to package and extend the shelf life of meat products as well as other fresh food products.