Evaluation of the Antioxidant and Antimicrobial Activity of Rosemary Essential Oils as Gelatin Edible Film Component

Abstract

The aim of this work was to evaluate the antioxidant activity and antimicrobial activity of gelatin edible films incorporated in rosemary essential oil (REO). The essential oil of two varieties of rosemary leaves (Rosmarinus officinalis L. var. Typicus and Troglodytarum) was extracted by hydro-distillation method. The antioxidant activity of REO was investigated using DPPH and β-carotene bleaching assays, and REO compounds were identified and quantified using GC-MS. REO has been incorporated into a biopolymer with gelatin and the antibacterial activity against Escherichia coli was determined using disc diffusion method for both REO and edible film with REO. REO extracted from Typicus variety was characterized with higher antioxidant activity, β-carotene and antimicrobial activity, than Troglodytarum variety. However, when the REO was incorporated in gelatin film lower antimicrobial activity were observed in comparison to rosemary essential oils.

Introduction

An edible film or coating can be defined as a thin layer of material from plant or animal sources, which can be eaten with a food product (Galus and Kadzińska, 2015; Mannozzi et al., 2018) or can be used as an additional layer of packaging (Nowacka et al., 2017, 2018; Ramos et al., 2016). They provide a barrier to mass transfer of moisture, gases (oxygen, carbon dioxide), lipid, flavours, aroma, etc., and protect food from the surrounding environment (Galus, 2018; Nowacka et al., 2017). The use of edible films and coatings can enhance food safety, improve the quality of food and extend its shelf-life. One of the most popular biopolymer, which is frequently employed in the preparation of films and coatings is gelatin. It is a natural, water-soluble protein derived from hydrolysis of the insoluble fibrous collagen, which is produced from wastes form slaughtering and fish processing (Alfaro et al., 2014). The film made of this biopolymer has good mechanical and optical properties with sensory acceptability. A gelatin-based edible film constitutes a barrier against oxygen and light, prevents from lipid oxidation and protect food from dryness (Ramos et al., 2016).

Essential oils are natural compounds extracted from plants, and have antimicrobial and antioxidant properties. Due to this fact, they are applied as additives to edible films in order to release antimicrobial constituents in food packagings (Galus and Kadzińska, 2015). Research in this topic was focused on investigating the properties of the enriched edible gelatin films with essential oils from medicinal and aromatic plants, such as oregano, garlic, clove, cinnamon, lemon, fennel, cypress, laurel, lavender, thyme, pine, rosemary, etc. (Gómez-Estaca et al., 2010; Rodríguez et al., 2007; Seydim and Sarikus, 2006). The incorporation of this natural additive can extend the shelf life of some food product during storage (Campos et al., 2011). The bioactive packaging with essential oils is a promising technology to preserve foodstuff, setting a new trend and direction in packaging development (Gómez-Estaca et al., 2010).

Nowadays, the consumers interest in natural food products is constantly growing. The natural products from aromatic and medicinal plants, herbs and spices are added to packaging materials in order to minimize the synthetic additive in foodstuff. Rosemary essential oil is one of materials which can be incorporated into an edible coating (Gómez-Estaca et al., 2010). Recently, the interest in extracts and essential oils from rosemary has aroused due to the content of 1,8-cineole and camphor compounds, which have antimicrobial and antifungal properties (Gómez-Estaca et al., 2010). Gómez-Estaca et al. (2010) demonstrated that rosemary essential oil can provide a considerable growth inhibition of some bacterial strains such as Lactobacillus acidophilus, Listeria monocytogenes, Escherichia coli, Pseudomonas fluorescens, Photobacterium phosphoreum and Shewanella putrefaciens. However, the literature on the impact of rosemary essential oils in film-forming solutions and edible coating on microorganism inhibition is not consistent. For instance, Ponce et al. (2008) have shown that rosemary essential oil incorporated into chitosan edible film did not produce a significant antimicrobial effect, however prevention of browning reactions in butternut squash slices, coated with chitosan edible film incorporated into rosemary essential oil, was observed. Similarly, Seydim and Sarikus (2006) stated that a whey protein edible film enriched with rosemary essential oil from Turkey did not exhibit any antimicrobial activity. In contrast, Abdollahi et al. (2012) noticed good antibacterial activity after adding rosemary essential oil (from Iran) of higher than 1% concentration into chitosan. These differences can be related to composition of principal constituents of essential oils from rosemary of various origin. The Tunisian rosemary essential oil is characterized by the higher amount of 1,8-cineole (47.2–27.5%) and camphor (12.9–27.9%) in comparison to other essential oils from different regions of the world (Zaouali et al., 2010): rosemary essential oils from Spain, France and Turkey contain 19.0–21.8, 5.3–24.8, 2.64% of 1,8-cineole respectively and 16.3–18.9, 3.01–7.5, 1.68% of camphor (Özcan and Chalchat, 2008).

In Tunisia two varieties of rosemary can be found: Rosmarinus officinalis L. var. Typicus which grows in the northern parts of the country, and R. officinalis L. var. Troglodytorum which grows in the southern parts. Due to the different bioclimatic areas in the north and south parts of Tunisia, different properties and chemical composition of the rosemary essential oils are observed. Among natural antimicrobial agents, the Tunisian rosemary (R. officinalis L.) essential oil has been characterized as exhibiting a powerful antibacterial activity (Zaouali et al., 2010) and health benefits as hepatoprotective potential (Rašković et al., 2014), anti-inflammatory and antinociceptive (Takaki et al., 2008), DNA-protective (Slameňová et al., 2011) and anticancer effects (Wang et al., 2012).

The aim of the study was to investigate the difference in antioxidant and antimicrobial activities of the rosemary essential oil between two varieties of rosemary leaves from northern (var. Typicus) and southern (var. Troglodytarum) Tunisia. Moreover, a gelatin edible film incorporated with two rosemary essential oil was evaluated in terms of mechanical properties and antioxidant activity.

Materials and Methods

Material    Fresh aerial parts of wild rosemary (R. officinalis L.) var. Typicus and var. Troglodytarum were collected in winter (January, 2018) from Zaghouan (northern Tunisia) and Chaab Tweel (Matmata, southern Tunisia), respectively. Table 1 presents the characteristics of the places where the Tunisian rosemary plants (R. officinalis L.) grow (Zaouali and Boussaid, 2008; Zaouali et al., 2010). The herbarium specimen was confirmed by the botanist in The Biotechnology Center of Borj-Cedria (Tunisia). The rosemary leaves were dried at room temperature (31 ± 6 °C) without applying any heat treatment to prevent deterioration of active compounds in essential oils. Such material was used for further analyses and preparation of edible films.

Table 1. Characteristics of places where the Tunisian rosemary plant (R. officinalis L.) grows

Variety	Region	Altitude [m]	Rainfall[mm/year]	Bioclimatic zone
Typicus	Zaghouan	970	500–600	Sub-humid
	(northern Tunisia)
Troglodytarum	Chaab Tweel (Matmata)	350	100–150	Upper arid
	(southern Tunisia)

Processing

Rosemary essential oil (REO) extraction procedure    A dried plant material was immediately subjected to hydro-distillation using Clevenger apparatus according to the Clevenger method (Clevenger, 1928). 100 g of rosemary leaves were extracted with 2 L of water for 3 h. The essential oils were collected and dried under anhydrous sodium sulphate. The rosemary essential oils (REO) were stored at 4 °C until ulterior analysis.

Film preparation    Film-forming solutions were prepared by casting procedure in line with the method described by Jridi et al. (2013) and Abdollahi et al. (2012) with a slight modification. 4 g of gelatin from bovine skin (Type B, Sigma Aldrich) was dissolved in 100 g of distilled water using magnetically stirring and thermostatic heating at a temperature of 60 °C for 30 min. Then 15% (w/v) of glycerol (Sigma Aldrich) was added to 4% of gelatin solution as plasticizer. After mixing, gelatin solution was cooled down to 40 °C in a water bath (SONOREX DIGIPLUS, Berlin) and the rosemary essential oil was added at concentration of 2% (w/v). For microbial analysis, two more concentration of rosemary essential oil was used 0.5 and 1%. The control sample did not have any addition of rosemary essential oil. As a next step, 0.5% (w/v) of Tween 80 was added to all solutions as an emulsifier. All the mixtures were homogenized with Ultra Turrax (IKA T25-Digital Ultra Turrax, Staufen, Germany) at 10 000 rpm for 3 min, and afterwards subjected to ultrasonic bath (SONOREX DIGIPLUS, Berlin) in 40 °C for 15 min. The 15 g of a gelatin film-forming solution with two types or rosemary essential oils (REO-Z and REO-CT) and the control sample (without REO) were poured onto a series of Petri dishes glass NORMAX of a diameter of 6 cm and dried in heating chamber at a temperature of 25 °C for 60 h. Dried gelatin films with and without REO were peeled-off and conditioned at 50 ± 5% RH and 2 5 ± 1 °C for 48 h prior to testing. The experiment was conducted in triplicate.

Chemical analysis

Bioactive compounds quantification in rosemary essential oils    The rosemary essential oils' composition was determined by GC-FID and GC-MS analysis according to Kasmi et al. (2017) with a slight modification. The analysis was carried out in triplicate for each sample type.

Gas chromatography analysis (GC-FID)    REOs composition was analyzed using the Hewlett-Packard 6890 chromatograph, which was equipped with a flame ionization detector. An electronic pressure control injector and a polyethylene glycol capillary column (HP Innowax: 30 m × 0.25 mm; 0.25 µm film thickness) were used. N₂ (flow of 1.6 mL/min, split ratio of 1:60) was used as a gas carrier. The column temperature was set at 35 °C for 10 min, then heated up to 205 °C with a rate of 2 °C/min, and afterwards kept constant at 205 °C for 10 min. The injector and detector temperatures were held at 250 °C and 300 °C, respectively.

Gas chromatography/mass spectrometry analysis (GC-MS)    GC-MS analysis was performed on the Agilent Gas Chromatograph (model 7890A) equipped with a mass spectrometer (Agilent 5975C inert XL MSD) and electron impact ionization (70 eV). An apolar HP-5MS capillary column (30 m × 0.25 mm coated with 5% phenyl methyl silicone, 95% dimethylpolysiloxane, 0.25 µm film thickness) was used. Oven temperature was programmed at 40 °C for 1 min, then with a rate of 8 °C/min heated up to 100 °C and kept constant at 100 °C for 5 min. Afterwards, the temperature was heated to 200 °C with a rate of 10 °C/min and kept constant at 200 °C for 3 min and the final temperature was set up at 300 °Cwith a rate of 2 °C/min. The injector temperature was set at 250 °C. The heating temperature program of the GC-MS oven was optimized for better separation of volatile compounds of rosemary essential oils. As a carrier gas was used helium (He, 1 mL/min flow, split ratio of 100:1). Scan time equaled 1 s and mass range varied between50 and 550 m/z. The volatile components were identified by comparison of their retention indices (RI) relative to (C₈-C₄₀) n-alkanes with the ones available in the literature or with those of authentic compounds available in the authors' laboratory. Further identification was made by matching their recorded mass spectra with the ones stored in the mass spectral library (Wiley 09 NIST 2011) of the GC-MS data system.

Antioxidant activity of rosemary essential oils    The antioxidant activity was assessed by DPPH (Zaouali et al., 2010) and b-carotene bleaching assays with a slight modification. The antioxidant activity analysis was carried out in three independent repetitions. DPPH free radical-scavenging activity of rosemary essential oils were express as concentration providing 50% inhibition (IC₅₀), which was calculated from the regression equation of the graph of inhibition percentage and extract concentration of the REOs. Moreover, the synthetic antioxidant BHT (butylated hydroxytoluene) was used as a positive control sample. The percentage of DPPH scavenging activity was expressed as mean IC50 value ± SD (mg/mL). To measure the ability of rosemary essential oils incorporated in gelatin films to prevent the bleaching of β-carotene the following procedure was applied according Jridi et al. (2013).

Antimicrobial activity of REOs and gelatin films incorporated with REOs    The antimicrobial activity of rosemary essential oils (REO-Z and REO-CT) and gelatin films enriched with the rosemary essential oils were carried out by disc diffusion method according to Gómez-Estaca et al. (2010) with a slight modification. Antimicrobial activity was tested over gram negative strains E. coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and gram positive strain Enterococcus faecalis (ATCC 29212). The surface of Mueller-Hinton Agar plates (Merck) were inoculated with 0.1 mL of culture bacterial suspension of E. coli, P. aeruginosa and E. faecalis (10⁵ CFU/mL). Afterwards, the sterile filter paper discs of 6 mm diameter were placed on the plates surface and 10 µL of rosemary essential oils (REO-Z and REO-CT) was added on the discs. In order to assess the antimicrobial activity of edible gelatin films incorporated with rosemary essential oils at concentration of 2% (REO-Z and REO-CT), the 6 mm discs were cut and subjected to decontamination under ultraviolet light for 25 min. Sterile edible gelatin films were placed on the surface of the Mueller-Hinton Agar plates (Merck). All the plates were incubated for 24 h at temperature 37 °C.

The diameter of the inhibition zone (mm) was measured taking into account the initial diameter of the films. The gelatin film without essential oil was used as a negative control and streptomycin antibiotic (10 µL/disc) was used as a positive control. The tests were carried out in triplicate.

Physical properties of gelatin films

Thickness    The thickness was measured with a micrometer (ProGage Thickness Tester, Thwing-Albert, and ISO 534). Three random locations distinct in each film sample were used for thickness determination. The measurement was repeated ten times for one type of an edible film.

Mechanical properties    Mechanical properties of edible gelatin films incorporated with rosemary essential oils were measured after conditioning process carried out for 48 h (at 25 °C and 50 ± 5% relative humidity).

Elongation at break (EAB) and tensile strength (TS) were determined as described by Iwata et al. (2000) using the Universal Testing Machine Lloyd Instruments LRX (Chicago, USA) with NEXYGEN software. Ten strings (2 cm × 4 cm) of film samples were used for testing. The length of the initial grip of was about 3 cm. The samples of gelatin film with rosemary essential oils were clamped and deformed until the samples were broken. There were used tensile test with a 100 N load cell and the cross head speed of 30 mm/min. The maximum load and the final extension at break were applied to calculate TS and EAB, respectively. The measurement was repeated ten times for one type of an edible film.

Statistical analysis    Obtained results were examined by the analysis of variance (ANOVA, the equality of variances was checked through Levene's test) using STATISTICA 12 (StatSoft Inc., USA). Homogenous groups were identified using Tukey's HSD method with a probability of 0.05.

Results and Discussion

Rosemary essential oil characteristics    The Table 2 shows the chemical composition of rosemary essential oil extracted from rosemary leave belonging the northern and the southern Tunisian areas determined by GC-MS. A total of 31 compounds corresponding to 96.23 ± 1.03 and 97.55 ± 1.09% of the total essential oil content were identified in REOs obtained by hydro-distillation from rosemary leaves (R. officinalis L.) collected in winter from Zaghouan (northern, var. Typicus) and Chaab Tweel (Matmata, southern, var. Troglodytarum), respectively.

Table 2. Chemical composition of REOs from two varieties: Typicus (REO-Z) and Troglodytarum (REO-CT)

RI (HP5-MS)	Compound	REO-Z [%]	REO-CT [%]
931	α-pirene	10.48 ± 0.08b	12.85 ± 0.14a
953	camphene	2.59 ± 0.03b	12.20 ± 0.13a
971	β-pirene	1.75 ± 0.02a	1.75 ± 0.02a
988	β-myrcene	0.75 ± 0.01a	0.48 ± 0.01b
1014	α-phellandrene	0.14 ± 0.01	-
1018	3-carene	0.32 ± 0.01a	0.46 ± 0.01b
1026	α-terpinene	0.14 ± 0.01a	0.12 ± 0.01b
1033	p-cymene	1.51 ± 0.02a	1.13 ± 0.01b
1036	d-limonene	1.81 ± 0.02b	1.93 ± 0.02a
1062	γ-terpinene	0.43 ± 0.01a	0.77 ± 0.01b
1084	α-terpinolene	0.20 ± 0.01a	0.23 ± 0.01b
	Total monoterpene hydrocarbons	20.12 ± 0.18a	31.92 ± 0.35b
1024	1,8-cineole	49.43 ± 0.55a	26.11 ± 0.30b
1068	Linalool	0.08 ± 0.01a	0.09 ± 0.01a
1070	(E)-sabinene hydrate	0.07 ± 0.01a	0.07 ± 0.01a
1088	(Z)-sabinene hydrate	0.60 ± 0.01	-
1123	d-fenchyl alcohol	0.08 ± 0.01a	0.07 ± 0.01b
1130	(E)-pinocarveol	-	0.08 ± 0.01
1139	α-campholenal	0.03 ± 0.01	-
1143	Camphor	14.24 ± 0.16b	29.35 ± 0.33a
1165	Borneol	6.21 ± 0.07a	3.05 ± 0.01b
1291	bornyl acetate	-	2.63 ± 0.03
1189	methyl eugenol	4.08 ± 0.05a	2.19 ± 0.03b
1410	α-terpineol	-	0.26 ± 0.01
	Total oxygenated monoterpene	74.83 ± 0.83a	63.91 ± 0.71b
1391	α-copaene	0.05 ± 0.01a	0.06 ± 0.01a
1439	(E)-caryophyllene	0.04 ± 0.01	-
1454	Muurolene	0.95 ± 0.01a	0.45 ± 0.01b
1499	Aromadendrene	0.05 ± 0.01a	0.06 ± 0.01a
1530	delta-cadinene	-	0.12 ± 0.01
	Total sesquiterpene hydrocarbons	1.13 ± 0.01a	0.68 ± 0.02b
1485	α-amorphene	0.03 ± 0.01	-
1606	Caryophyllene oxide	0.14 ± 0.01a	0.07 ± 0.01b
1652	α-eudesmol	-	0.98 ± 0.01
	Total oxygenated sesquiterpene	0.14 ± 0.01a	1.04 ± 0.02b
	Total identified	96.23 ± 1.03a	97.55 ± 1.09a

Different letters in rows indicate a different homogenous group (α = 0.05).

The main compounds in the REO from rosemary leaves from Zaghouan (northern, var. Typicus) and Chaab Tweel (Matmata, southern, var. Troglodytarum) had the same major compounds but different quantity in total content. The main compounds in the REO-Z were: 1,8-cineole, camphor and α-pinene and contained also considerable amounts of borneol, α-terpineol and camphene. The chemical analysis of the REO-CT demonstrated that the main compounds were: camphor, 1,8-cineole, α-pinene and camphene, and there was present borneol and α-terpineol at considerable amounts. These results were in line with the literature which enlists the major components of the rosemary essential oil as 1,8-cineole (27.23%), α-pinene (19.43%), camphor (14.26%), camphene (11.52%) and β-pinene (6.71%) (Pintore et al., 2002; Wang et al., 2018). The varieties used and bioclimatic zone caused changes in the composition of REOs. For example, in the Tunisian REOs of two varieties Typicus and Troglodytarum the main compounds were: 1,8-cineole in the range from 26 to 51.2%, and camphor in the range from 4.9 to 29.7% (Zaouali et al., 2010).

The chemical analysis of rosemary essential oil revealed that the two main volatile compounds 1,8-cineole and camphor were correlated with the variety of rosemary, which corresponds to the northern (var. Typicus) and southern (var. Troglodytarum) of Tunisian bioclimatic zones, respectively. The abovementioned results are in agreement with the data obtained by Zaouali et al. (2010). In addition, the high amount of these two main volatile compounds of the REO might be connected with the period of harvest. For the purpose of this research, the leaves were collected in winter, however the ones used by Zaouali et al. (2010) were collected during the vegetative stage (spring). The latter contained lower values of 1,8-cineole (47.2%) and camphor (27.9%), what can be explained by the plant acclimation to the hard stress condition (water stress), the climatic change and the harvest time during the year, which can influence the essential oil composition (Atti-Santos et al., 2005). 1,8-cineol compound is known to have antioxidant, antimicrobial (Wang et al., 2012) and anti-inflammatory properties (Juergens et al., 2003). Moreover, 1,8-cineol, α-pinene and β-pinene are used in medicine as gastroprotective and anti-tumoral agents (Wang et al., 2012). It is noteworthy that rosemary essential oil from northern part of Tunisia (REO-Z) exhibits a higher amount of 1,8-cineole in comparison to the rosemary essential oil from other countries. For example, REO from Morocco (Lahsen et al., 2015) and Turkey (Özcan and Chalchat, 2008) were characterized by a lower amount of 1,8-cineole (42.24 and 2.64%, respectively). Therefore, the rosemary essential oil from Zaghouan area can be used as an active compound for packaging, what was also confirmed by the antimicrobial test for gelatin film (Figure 1).

Fig. 1.

Inhibition diameter of REOs and gelatin films incorporated with REOs of two varieties: Typicus (REO-Z) and Troglodytarum (REO-CT) against E. coli, P. aeruginosa and E. faecalis, different letters indicate a different homogenous group for each microorganism (α = 0.05).

The antioxidant activity of rosemary was investigated by the DPPH free radical scavenging and the β-Carotene bleaching assays (Table 3). According to the results given by the DPPH free radical scavenging assay, the REO obtained from rosemary leaves collected from northern Tunisia (REO-Z; var. Typicus) exhibited a slightly higher (not significant, p > 0.05) antioxidant activity (3.96 ± 0.56 µL/mL) in comparison to the rosemary essential oil from southern Tunisia (REO-CT, var. Troglodytarum). Both rosemary essential oils (REO-Z and REO-CT) were characterized by lower antioxidant activity in comparison to the synthetic antioxidant BHT (1.75 ± 0.27 µL/mL). However, Zaouali et al. (2010) obtained higher antioxidant activity of the rosemary essential oil of Troglodytarum variety in comparison to rosemary essential oil var. Typicus, what might be linked to harvest time and stress condition, which the plant was exposed to (Atti-Santos et al., 2005). Compared to other essential oils extracted from leaves collected in different countries e.g. eastern Morocco, Tunisian rosemary essential oils presents a higher antioxidant activity. Moroccan rosemary essential oils were characterized by IC₅₀ equal to 28.97 ± 0.86 (R. tournefortii, wild plant) and 20.17 ± 1.04 µL/mL (R. Tournefortii, domesticated plant) and a lower antioxidant activity of R. Tournefortii variety was connected with lower concentration of 1,8-cineole (Tahri et al., 2015).

Table 3. Antioxidant activity (DPPH reducing ability and β-carotene bleaching method) of REOs from two varieties: Typicus (REO-Z) and Troglodytarum (REO-CT)

Rosemary essential oil	DPPH IC50[µg/mL]	β-carotene [%]
REO-Z	3.96 ± 0.56 a	20.99 ± 0.23 b
REO-CT	4.09 ± 0.58 a	10.0 ± 0.12 a
BHT	1.75 ± 0.27 b	75.40 ± 0.97 c

Different letters in columns indicate a different homogenous group (α = 0.05).

The antioxidant activity of the Tunisian rosemary essential oils obtained by the β-carotene bleaching assay showed that rosemary essential oil form northern Tunisia demonstrated higher antioxidant activity when compared to those from southern. Inhibition percentage of beta-carotene bleaching for REO-Z and REO-CT was 20.99 ± 0.23 and 10.0 ± 0.12%, respectively. The synthetic antioxidant (BHT) was characterized by a higher inhibition percentage which was about 75.40 ± 0.97%.

Antimicrobial activity of rosemary essential oils and gelatin films incorporated with REOs    Rosemary essential oils are considered as natural antimicrobial agents. The power of antimicrobial activity is correlated with chemical composition of rosemary essential oils, as aforementioned. The variations of rosemary oils' composition depend on, among others, stage of plant development, region where a plant grows, agronomic and environmental conditions (Zaouali et al., 2010). In this research rosemary essential oils came from different locations and bioclimatic zones i.e. from the northern part of Tunisia (var. Typicus - REO-Z) and southern (var. Troglodytarum - REO-CT). These two varieties were characterized by different antibacterial activity. The results of antimicrobial activity estimated by the diameter of inhibition for E. coli, P. aeruginosa and E. faecalis bacteria strains are presented in Figure 1. The higher antibacterial activity was observed for the REO from Zaghouan - northern Tunisia (REO-Z, var. Typicus) for E. coli and E. faecalis strains, which was related to a higher content of 1,8-cineole and α-terpineol (Table 2) than the REO form southern Tunisia (REO-CT, var. Troglodytarum). 1,8-cineole has a strong antibacterial activity connected with its hydrophobicity and α-terpineol shows very good antibacterial activity against E. coli, causing cell wall and cell membrane rupturing (Li et al., 2014). Moreover, the content of monoterpenes in essential oils determines their antimicrobial activity due to their lipophilic character, which cause disruption of microbial cytoplasmic membrane integrity (Campos et al., 2011). However, for P. aeruginosa strains higher antibacterial activity were obtained for REO from southern Tunisia (REO-CT, var. Troglodytarum) than for the REO form southern Tunisia (REO-CT, var. Troglodytarum).

In our study, the inhibition zone for REO-Z was equal to 16.3 ± 0.6 mm, 12.3 ± 0.6 mm and 23.7 ± 0.6 mm for E. coli, P. aeruginosa and E. faecalis, respectively. A significantly lower antibacterial activity was obtained for REO from Chaab Tweel - Matmata (REO-CT, var. Troglodytarum) for E. coli with inhibition zone of 14.3 ± 0.6 mm and for E. faecalis (17.7 ± 0.6 mm). However, for P. aeruginosa the inhibition zone for REO-CT was higher (23.33 ± 1.2 mm) in comparison to REO-Z. Zaouali et al. (2010) reported higher antimicrobial activity of REOs from Matmata region, yet similar to the inhibition zone for REO from Zaghouan, however, they collected the rosemary leaves in different period (spring, April), which could have influence on chemical composition of REO. Moreover, Zaouali et al. (2010) found that rosemary essential oils show antimicrobial activity against other gram-negative (Klebsiella pneumoniae) and gram-positive (Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Staphylococcus epidermis, Streptococcus feacalis) bacteria (Zaouali et al., 2010). Furthermore, the Turkey rosemary essential oils displayed antifungal activity (Özcan and Chalchat, 2008). Pintore et al. (2002) stated that rosemary essential oils from Sardinia and Corsica were characterized by moderate antibacterial activity. Both tested rosemary essential oils from Tunisia (REO-Z, REO-CT) were characterized by similar or significantly higher antibacterial inhibition as streptomycin antibiotic.

The inhibition diameter of rosemary essential oils (REO-Z, REO-CT) incorporated into gelatin film against E. coli, P. aeruginosa and E. faecalis was characterized by lower value in comparison to alone REO, which is connected to the concentration of the REOs in edible coating and the composition of rosemary essential oil. With the use of a higher concentration of rosemary essential oil (0.5, 1 and 2%) in the gelatin film, a higher microbiological activity was obtained for all tested strains (Figure 1). However, the gelatin film limited the antimicrobial effect of REOs. Similarly, rosemary oleoresin showed meaningful antimicrobial activity against L. monocytogenes, however chitosan film-solutions containing 1% of rosemary oleoresin exhibited limited antimicrobial effects (Ponce et al., 2008).

Not only REO composition but also the composition of edible film has impact on the antimicrobial activity of REOs. For example, whey protein isolate-based films enriched with Turkish rosemary essential oil at a concentration from 1 to 4% were not effective against several microorganisms (Seydim and Sarikus, 2006). In our investigation, gelatin film without REO exhibited the inhibition diameter equal to 6.0 ± 0.1 mm for all tested strains. When gelatin film with REO-Z and REO-CT were tested the inhibition zone was larger with higher REO concentration. The inhibition zone for gelatin film incorporated with REO-Z was in the range from 7.3 ± 0.6 to 10.7 ± 1.2 mm, from 6.0 ± 0.1 to 9.7 ± 0.6 mm, and from 10.7 ± 0.6 to 14.7 ± 0.6 mm for E.coli, P. aeruginosa and E. faecalis, respectively. The gelatin film with REO-CT was characterized by lower inhibition zone for E.coli and E. faecalis strains in comparison to the gelatin film with REO-Z. However, a different tendency was observed for P. aeruginosa strains, which the larger inhibition zone was noticed for gelatin film with REO-CT than for gelatin film with REO-Z component. Moreover, the values of inhibition zone for E.coli and P. aeruginosa, even when the gelatin film was incorporated with the highest concentration (2%) of REOs, were significantly lower when compared to streptomycin antibiotic. This means that gelatin films enriched with REOs have antimicrobial activity against E.coli and P. aeruginosa, however the ability to inhibit bacterial growth is not so strong as for antibiotics. What is worth to mention, the antimicrobial activity was similar to streptomycin antibiotic for E. faecalis strains, when the gelatin film was incorporated with REOs with a concentration above 1% for REO-Z and 2% for REO-CT. These results shows meaningful antimicrobial activity against E. faecalis for gelatin films with REOs.

Film characteristics    The thickness and mechanical properties (tensile strength - TS and elongation at break - EAB) of gelatin films with or without the addition of REOs of different varieties: Typicus (REO-Z) and Troglodytarum (REO-CT) are presented in Table 4. An ideal edible film should be characterized by high mechanical strength (tensile strength TS and elongation at break EAB) (Du et al., 2011).

Table 4. Thickness and mechanical properties (tensile strength - TS and elongation at break - EAB) of gelatin films incorporated with REOs (2%) of two varieties: Typicus (REO-Z) and Troglodytarum (REO-CT)

Type of film	Thickness [µm]	TS [MPa]	EAB [%]
Gelatin film (control)	45.2 ± 1.6 a	9.3 ± 1.3 a	21.3 ± 3.8 a
GF_REO-Z 2%	62.8 ± 2.3 c	12.3 ± 1.1 b	28.6 ± 4.1 b
GF_REO-CT 2%	55.6 ± 4.4 b	10.4 ± 1.5 ab	24.2 ± 3.1 ab

Different letters in columns indicate a different homogenous group (α = 0.05).

Gelatin film was characterized by TS and EAB equal to 9.3 ± 1.3 MPa and 21.3 ± 3.8%, respectively. The incorporation of gelatin film in REO changed the film into more flexible. Tensile strength values increased to 10.4 and 12.3 MPa for gelatin films incorporated with REO-CT and REO-Z (GF_REO-CT and GF_REO-Z), respectively. It is noteworthy that the mechanical properties of both GF_REO-Z and GF_REO-CT enriched with the same concentration of REO (2%) exhibited a higher tensile strength (TS) and elongation at break (EAB) when compared to the control film. However, only for GF_REO-Z the changes of mechanical properties were statistically significant (p < 0.05). This effect was linked to thickness of obtained edible gelatin films with REOs, which were characterized by significantly higher thickness. Similar effect was observed by Abdollahi et al. (2012) for chitosan film incorporated with rosemary essential oil, who noted that TS and EAB increased by about 7 and 40%, respectively. The authors explained these changes by some interaction in polymer chain, which led to increase of the interchain forces in the chitosan matrix. However, for gelatin film incorporated with cinnamon essential oil, with the concentration of about 2%, decrease of the tensile strength and elongation at break was observed (Wu et al., 2017). Furthermore, the reduction of elongation at break and tensile strength have been reported by other authors (Galus and Kadzińska, 2015).

In our research, the growth of TS and EAB in gelatin films incorporated with REOs also might be linked with the increase of the interaction between gelatin monomers and might have induced the increase of the polymer chain-to-chain interactions. Moreover, Irissin-Mangata et al. (2001) stated that lower temperature of heating during preparation of enriched gelatin film with the rosemary essential oils can boost the physical strength (TS and EAB) of these films (GF_REO-Z and GF_REO-CT).

Conclusions

It can be concluded that rosemary essential oils from different varieties and bioclimatic zones were characterized by diverse chemical composition, which was responsible for their antimicrobial and antioxidant activity.

Rosemary essential oils were incorporated into gelatin film with success. Gelatin film enriched with rosemary essential oils from northern part of Tunisia (var. Typicus) was characterized by a higher antimicrobial activity against Escherichia coli and Enterococcus faecalis in comparison to Troglodytarum. Moreover, films containing rosemary essential oils of variety Typicus demonstrated better mechanical properties.

The films containing rosemary essential oils, especially var. Typicus from the north of Tunisia, showed potential to be used as an active film in food preservation. However, more research is required for better understanding the antimicrobial behaviour of single compounds of rosemary essential oils and combination of diverse natural compounds to obtain synergistic effects and higher antimicrobial activity in edible gelatin films.

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