Basil seed gum edible films incorporated with Artemisia sieberi and Achillea santolina essential oils: Physical, antibacterial, and antioxidant properties

Active edible films were prepared by incorporating Artemisia sieberi and Achillea santolina essential oils (EOs) in basil seed gum (BSG) films. The results showed an increase in opacity, contact angel, and water vapor permeability of the films by the addition of EOs, whereas the moisture content, water

(GRAS) for using in human and animal foods and the edible films and coatings manufactured from these biopolymers exhibit considerable mechanical and barriers properties .
Moreover, these film-forming materials can be used as carriers for delivering active ingredients such as flavors, colorants, probiotics, and essential oils on the surface of food products (Khodaei & Hamidi-Esfahani, 2019).
Basil (Ociumum basilicum L.) is an aromatic and the pharmaceutical plant which is widely grown in some regions of Iran and India, and it is broadly used in traditional medicine to heal the colic ulcer, dyspepsia, diarrhea, and inflammations (Khazaei et al., 2014). During the soaking of basil seeds in water, the outer pericarp of the seeds swells and the viscous mass of basil seed gum (BSG) forms. BSG is composed of hydrophobic (glucomannan and highly branched arabinogalactan) and hydrophilic (xylan) fractions (Gahruie et al., 2017;. BSG is a biodegradable, heat resistant, and hydrophilic biopolymer with a considerable rheological properties that makes it an excellent candidate as a natural hydrocolloid in food industry (Hosseini-Parvar et al., 2010;Karimi & Kenari, 2016). The film-forming properties of BSG have been confirmed in previous studies (Gahruie et al., 2017;. Essential oils are natural biocompounds extracted from plants, and they have a wide application in the food industry as a flavoring and antimicrobial agents Tavakolpour et al., 2017). However, the direct incorporation of EOs into food systems due to their possible negative effects on the organoleptic properties of the food or their interactions with the food components and also the low water solubility of EOs is limited (Gahruie et al., 2017). Therefore, some new methods such as encapsulation, emulsion, and using edible films have been proposed to protect the EOs during the processing and storage and also reduce the negative organoleptic effects of these oils on food. Chemical composition, the specific parts of the plant used, methods of extraction, harvesting season are some of the factors influencing the biological activities of essential oils (Vitoratos et al., 2013).
Achillea santolina is one of the 115 known species of Achillea L.
(Asteraceae) genus that it is widely distributed in the northern regions of Europe and Asia (Al-Snafi, 2013;Ebadi, 2006). Antimicrobial, antiinflammatory, antidiabetic, antioxidant, and cardiovascular activities are some of the pharmacological properties attributed to essential oils from A. santolina (Al-Snafi, 2013).
Artemisia sieberi (Artemisia herba-alba) is another medicinal plant from Compositae family growing in Spain, North Africa, and the Middle East. This herbal plant is shown to have considerable antimicrobial activity, and it is commonly used in Iran as a herbal remedies for treating colds, coughing, intestinal disturbances, intestinal parasites infection, and wound healing (Mahboubi & Farzin, 2009).
The effect of incorporation of essential oils on the physicochemical properties of edible films has been reported in various studies.
However, to the authors' knowledge, there are no reports on the active edible films loaded with EOs from A. santolina and A. sieberi.
Thus, the main aim of this research was to formulate active BSGbased films containing different ratios of A. santolina and A. sieberi EOs and evaluate the physical, antioxidant, and antibacterial properties of the prepared films.

| Plant materials
Artemisia sieberi and Achillea santolina (14% MC d.b.) leaves were purchased from a local market in Shiraz city, Fars province, Iran. Basil seeds were purchased from a local market in Fasa city, Fars province, Iran.

| Essential oils extraction
Approximately, 50 g of each plant was hydrodistillated for 3-3.5 hr by an all-glass Clevenger tool. About, 550-ml distilled water was utilized (Pharmacopoeia, 1980). The obtained EOs were dried using anhydrous sodium sulfate and stored in sealed vials at 4°C. The EO yield was determined based on the volume of essential oil relative to the dry weight of the plant. The yield of extraction for A. sieberi and A. santolina EOs were 3.6% and 3.2% (v/w), respectively.

| Essential oil analysis
GC-MS (6890N, Agilent Technologies, Palo Alto, CA, USA) was applied for EOs analysis. An HP-5MS capillary column (30-m length, 0.25-mm internal diameter, and 0.25μm film thickness) with a split ratio of 1:30 and helium as a carrier gas (1.3 ml/min) were used. The injector was set at 265°C, and the EOs were diluted with n-hexane (1:10 v/v). Oven temperature was regulated to 60°C for 6 min, accordingly enhanced to 280°C at 3°C/min. Semi-quantitative data were obtained from FID area percentages without the use of correction factors. Retention indices (RI) were determined by using retention times of n-alkanes (C6-C24) and the compounds were recognized by comparing their mass spectral fragmentation patterns with those of similar compounds from the database (Wiley/NBS library) .

| Extraction of basil seed gum
After sieving, basil seeds (Esfahan variety; 13% MC d.b.) were washed with ethanol (75% w/v) for 10 min, and then ethanol was eliminated from seeds using filtering and drying. Basil seeds were steeped in distilled water (12:1 v/w) at 37°C for 7 hr. Subsequently, seed-water slurry was stirred using a blender at 1,000 rpm for 20 min to separate the gum layer. Final extraction of BSG from the seeds was done by using a cheese cloth filter and the insoluble residue if any was filtered out, exit a concentration of 18% (w/w). The drying process of the extracted gum was carried out by vacuum oven at 50°C ).

| Preparation of film solution
An aqueous hydrocolloid solution containing 6% basil seed gum and proper amount of glycerol (30% w/w) was mixed and heated to 36 ± 1°C under continuous stirring at 500 rpm for 20 min. Then, Tween-20 (15% v/v) was added and the solution mixed for 15 min.
The control film was named "F1" which is BSG film. The EOs-loaded films were prepared by the addition of 3% (v/v) of the oils to the film forming solutions (F2: BSG film incorporated with A. santolina EO; F3: BSG film incorporated with A. sieberi EO; F4: BSG film incorporated with A. santolina EO + A. sieberi EO (1:1 v/v); F5: BSG film incorporated with A. santolina EO + A. sieberi EO (2:1 v/v)); F6: BSG film incorporated with A. santolina EO + A. sieberi EO (1:2 v/v). Afterward, the solutions were degassed by a vacuum pump and the films were cast by pouring the film forming solutions onto petri dishes followed by drying at 35°C for 48 hr. Then, the dried films were peeled off from the petri dishes and conditioned at ambient temperature and 53% RH in chambers containing saturated solutions of Mg (NO 3 ) 2 for 5 days prior the tests.

| Film thickness
The thickness of the films was measured using a digital micrometer (Mitutoyo, Tokyo, Japan). Determinations were made at five different locations for each film.

| Opacity of films
The opacity of films analyzed according to the method by . The films were prepared into rectangular pieces (10 × 40 mm), and the absorbance was read at 550 nm using a Cary 60 UV-VIS spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). The opacity of the films was measured using the equation below where higher opacity values indicates lower transparency (Kanmani & Lim, 2013):

| Swelling index
Approximately, 0.0001 g of the film's cuts left in petri dishes and 35 ml of distilled water was added. Then, they were stored at relative humidity of 50%-55% and 25°C ± 1°C for 24 hr. Afterward, excess water was removed from the films using filter paper and the mass of the film was determined using a microbalance (Mayachiew & Devahastin, 2010). The swelling degree was calculated by the percentage increase in weight of film samples relative to the initial weight of the film.

| Water contact angle measurements
Approximately, 5 µl of water drop were placed on the surface of films (5.0 × 5.0 cm), and the contact angles were recorded using an optical goniometer (Kruss G10, Germany; RH: 30%, 25°C). The angle of the tangent to the basis of the droplet was calculated (Ojagh et al., 2010).

| Moisture content
Moisture content of the films was calculated by determination of weight loss of films in an oven at 90°C for 24 hr ).

| Water vapor permeability, water absorption capacity, and water solubility
The water vapor permeability of the films was recorded gravimetrically using the ASTM E96-00 method with some modification.
The film samples were kept in glass penetration cups with silica gel and the cells were hold in desiccator by means of distilled water at 30°C. The cups were weighed at interval of 1 hr through 24 hr time and water vapor permeability (WVP) of the films were determined (Khodaei, Oltrogge, et al., 2020). The water absorption capacity and solubility of the films in water was measured according to the technique of Sadegh-Hassani and Nafchi (2014).

| Antimicrobial activity
A suspension (0.1 ml of 6 log CFU/ml) of each pathogen was spread on the petri dishes containing Soybean casein digest medium. The films discs (10-mm diameter) were aseptically placed on the center of mediums. The petri dishes were kept at 25°C-37°C and the
diameters of the inhibition zones were calculated using caliper after 24 hr of incubation ).

| Antioxidant assays
2.14.1 | DPPH assay of films Approximately, 3 ml of methanol were added to 25 mg of each film.
Afterward, 2.8 ml of films' extracted solution were added to 0.2 ml of 1-mM DPPH (Sigma-Aldrich) in methanol. After adequate shaking of the solutions in dark condition, the absorbance at 517 nm was read water (Benzie & Strain, 1996).

| Statistical analysis
Statistical analyses were performed with ANOVA and significant differences at p < .05 were performed by Duncan's multiple range tests using SPSS package program. All measurements were made by triplicate and the data are presented as mean ± standard deviation of each treatment.
However, the EO from A. santolina exhibited more complex composition (48 identified components) and as it is shown in

| Physical properties of the BSG-based films containing different EOs
The effect of incorporation of A. sieberi and A. santolina EOs separately or in different combination ratios on the physical properties BSG films are presented in Table 3. The thickness of the films ranged from 0.07 to 0.08 mm the results showed that it was not influenced by the addition of EOs (p ≤ .05). This observation is in consistence with our previous study that the increase in oregano EO in BSG films did not alter the thickness of the films .
Other studies have also confirmed that the incorporation of EOs had no influence on the thickness of gelatin films (Martucci et al., 2015;Moradi et al., 2016).
The transparency is another important factor of edible films since it has a direct effect on the marketability, appearance, and acceptance of the final product by the consumers . As can be seen in

The water contact angel (WCA) is another important index
showing the hydrophobic characterization of the film's surface (Phan et al., 2005 Valderrama et al. (2015) that the incorporation of rosemary and thyme EOs had no effect on the SI of the chitosan edible films.
The moisture content (MC) % of the films represent the possible interaction of EOs and the biopolymers that may influence the affinity of the films to water molecules . As shown in Table 3 The effect of EOs addition on the water absorption capacity (WAC) of the BSG-based films is shown in highest WAC was observed for control film (F1) and the lowest was for F3 treatment.
The water solubility (WS) % of the films is presented in

| Antioxidant activity of films
Antioxidant characterization is one of the most important biological activities of the EOs that it is mainly attributed to the presence of phenolic compounds, terpenoids, and other volatile compounds.
Studies have shown that the EOs can be used as a natural antioxidant agents into the active packaging or films to reduce the oxidation and Similar pattern for ABTS assay was observed ( Figure 2) and the antioxidant activity of the BSG films increased from 0.71 µmol/g film for control film to 45.0 µmol/g film for F6. These results suggest that the incorporation a mixture of the EOs can lead in more active films to scavenging free radicals such as DPPH and ABTS.
FRAP assay has a wide application to study the antioxidant activity in plant materials (Ruiz-Navajas et al., 2013). FRAP assay also confirmed a low ability of BSG film to reduce Fe +3 to Fe +2 (0.04 µmol/g of the film) and incorporation of the EOs significantly increased the antioxidant activity of the films (Figure 3). Similar to DPPH and ABTS assays, films containing both A. santolina and A. sieberi EOs exhibited the highest FRAP values and the highest antioxidant activity was observed for F6 film (4.1 µmol/g of the film).  reported that the addition of oregano EO to BSG films significantly increased the antioxidant activity compared to control films.
Addition of green tea extract to the gelatin films increased the antioxidant activity compared to control film (Dou et al., 2018). Ruiz-Navajas et al. (2013) also observed that the addition of T. moroderi and T. piperella EOs increased the antioxidant activity of chitosan films.

| Antibacterial activity
The antibacterial effects of BSG film containing A. santolina and A.

| CON CLUS ION
In this study, active edible films were prepared by incorporating A.
santolina and A. sieberi essential oils (EOs) into the basil seed gum (BSG) films. The results showed that the presence of EOs had no effect on the thickness and swelling of the films. However, the opacity, water vapor permeability and water contact angle increased. EOs have a great potential to be utilized as a packaging material in order to enhance the storage life of packaged food.

CO N FLI C T O F I NTE R E S T
The authors have declared no conflicts of interest for this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.