Characterization and Antimicrobial Activity of Biodegradable Active Packaging Enriched with Clove and Thyme Essential Oil for Food Packaging Application

Bioactive packaging contains natural antimicrobial agents, which inhibit the growth of microorganisms and increase the food shelf life. Solvent casting method was used to prepare the Poly (lactide)-Poly (butylene adipate-co-terephthalate) (PLA-PBAT) film incorporated with the thyme oil and clove oil in various concentrations (1 wt%, 5 wt% and 10 wt%). The clove oil composite films depicted less green and more yellow as compared to thyme oil composite films. Clove oil composite film has shown an 80% increase in the UV blocking efficiency. The tensile strength (TS) of thyme oil and clove oil composite film decreases from 1.35 MPs (control film) to 0.96 MPa and 0.79, respectively. A complete killing of S. aureus that is a reduction from 6.5 log CFU/mL to 0 log CFU/mL was observed on the 10 wt% clove oil incorporated composite film. Clove oil and thyme oil composite film had inhibited E. coli biofilm by 93.43% and 82.30%, respectively. Clove oil composite film had exhibited UV blocking properties, strong antimicrobial activity and has high potential to be used as an active food packaging.


Introduction
Active packaging is a novel method used to prolong the shelf-life of perishable foods, maintain or improve the quality and safety of prepared foods due to its interaction with the product. Besides, active packaging for consumable goods has the potential to reduce the addition of active compounds into foods, reduction in the movement of particles from film to food and localisation activities could introduce the pathogenic microorganism into the product [1]. Also, the world focus has now shifted to biodegradable and non-biodegradable polymers such as poly-hydroxy alkanoates (PHA), polyethylene (PE), poly (lactide) (PLA), bio-based polyethylene terephthalate (PET), poly (butylene adipate-co-terephthalate) (PBAT) based packaging. Poly(lactide) (PLA) and Poly (butylene adipate-co-terephthalate) (PBAT) are the biodegradable polymers, and various studies were conducted on the blend of PLA and PBAT [2]. clove essential oil from bud, lead and stem. In all essential oils, they also found the major constituent as eugenol in bud (72.08-82.36%), stem (87.52-96.65%) and leaf (75.04-83.58%). Eugenyl acetate was also found in bud (8.61-21.32%), leaf (0-1.45%) and stem (0.07-2.53%). Moreover, β-caryophyllene are found more in leaf (11.65-19.53%), than in bud essential oils (2.76-8.64%) and in stem essential oils (1.66-9.72%) [13].
In the present study, essential oils (thyme oil and clove oil) have been incorporated as an antimicrobial agent in a PLA/PBAT (poly (lactide)/poly (butylene adipate-co-terephthalate) blend film for food packaging application. Effect of thyme oil and clove oil on the structural, functional, mechanical, antimicrobial and biofilm inhibition properties of the composite films was studied. To the best of author's knowledge, this is the first report to incorporate thyme oil and clove oils into the biodegradable polymer (PLA/PBAT) blend film and investigate their potential as an active food packaging.

Preparation of Composite Films
By using a solvent casting method, the essential oils (thyme and clove) were incorporated into biodegradable films of PLA-PBAT [19]. The blending ratio of PLA:PBAT was taken as 98:2 [20]. In 100 mL chloroform various concentrations of essential oil (1 wt%, 5 wt%, and 10 wt% of polymer resin) were pipetted out. To make up the equal volume in all the beaker ethanol was added in the samples accordingly. 4 g of polymer resin (3.92 g of PLA and 0.08 g of PBAT) and 50 wt% of glycerol [21] were added and mixed using a magnetic stirrer at 23 ± 2 • C for 24 h. A control sample of the PLA/PBAT film was prepared using the same method, followed by the addition of only ethanol (without any essential oil).
After the stirring of 24 h the solubilised mixture was poured onto a Teflon coated glass plate (24 cm × 30 cm) and spread evenly with a bend glass rod and allowed to dry at the room temperature for 24 h. The prepared films were peeled off from the glass plates and placed into the incubator at 25 • C and 50% relative humidity (RH) for 48 h. The Control films prepared was named as Poly (lactide)-Poly (butylene adipate-co-terephthalate) (PLA-PBAT) [22]. The thyme containing films were PLA/PBAT-Thyme 1% , PLA/PBAT-Thyme 5% , and PLA/PBAT-Thyme 10% . Clove containing films were named as PLA/PBAT-Clove 1% , PLA/PBAT-Clove 5% , and PLA/PBAT-Clove 10% .

Surface Colour and Optical Properties
ColorQuest XE (Hunter Lab) spectrophotometer was used to determine the surface colour of composite films. The colour values were taken in terms of L (+lightness, −darkness), a (+redness, −greenness), and b (+yellowness, -blueness). White colour plate (L = 93.97, a = −0.88, b = 1.21) was used as a standard background. For each film the readings are taken in triplicate from different locations. The mean value with the standard deviation of the readings was considered. The total colour difference which determines the difference between the colour values of standard tile and film samples (∆E) was considered using an equation: where ∆L, ∆a, and ∆b are the differences between standard colour plate, each colour value and the colour value of the film samples, respectively [22,23].
The optical properties such as the light transmittance spectrum was observed using UV-Vis spectrophotometer. The rectangular films of (3 cm × 7 cm) were cut and mounted between two spectrophotometer magnetic cells. The light transmittance spectrum of the composite film was measured between the wavelength of 200-700 nm. UV-light barrier property was obtained by the percent transmittance at 280 nm (T 280 ) and transparency of the films were determined at 600 nm (T 600 ).
Fourier-transform infrared spectrometer (FTIR) (Thermo Scientific) was used to determine the infrared spectrum of absorption of the composite films. FTIR was operated at the resolution of 4 cm −1 . The film sample of 4 cm × 4 cm were cut and positioned on the ray exposing stage (crystal plate). The spectrum was recorded at wavenumber of 4000-400 cm −1 . The readings were taken in triplicates from the surface. The functional groups present in the composite films were determined by the peak at specific wavenumber.

Thickness and Tensile Properties
The thickness of the composite films was determined by Digital micrometre (Mitutoyo, Japan) having 0.001 mm resolution. Five readings were taken from the random positions. The mean values and standard deviation were considered of these readings.
Mechanical properties such as tensile strength (TS) and elongation at break (EB) of the composite films were measured by using standard ASTM D 882-88 method [24] by Instron Universal Testing (Model 5565, Instron Engineering Corporation, Canton, MA, USA). The composite films were cut into rectangular strips of 3 cm × 15 cm. Grip length of 50 mm and a crosshead speed of 50 mm/min using a 500 N load cell set in Instron Instrument were used to operate at room temperature until the sample broke at a certain point. The Tensile Strength was calculated using the Equation: where F is the maximum force (N) required to separate the sample, and A is the initial cross-sectional area (m 2 ) of the composite films [22].
Percentage elongation at break (EB) is the ratio between changed film length and initial length of the samples [25]. The EB was calculated using the following formula: where X o is the initial grips separation (50 mm) of samples, X f is the film elongation at the moment of failure.

Surface Hydrophobicity
Water contact angle (WCA) determines the interaction of the film surface with the liquid interphase by using a dynamic contact angle analyser (FTA-200 system) [26]. It evaluates the surface is hydrophobic or hydrophilic in nature. Rectangular films of 3 cm × 8 cm placed on the stainless-steel platform having the water contact angle analyser attached. With the help of a micro-syringe a drop of distilled water approximately 10 µL was dropped on the film surface. The interaction of the drop on the surface of the film was observed by taking a picture with a high-speed camera and analysed it by the image processed by computer. Three readings were taken from the different location. Also, the experiment was performed in triplicates.

Antibacterial Activity
The Japanese Industrial Standard (JIS Z 2801:2000) [27] was used to determine the antibacterial efficacy of the composite films incorporated with thyme oil and clove oils against Staphylococcus aureus (ATCC 25923) and Escherichia coli (NCTC 9001). The kinetic study was performed using square pieces of the composite films (5 cm × 5 cm). Each film was sterilised each side using UV light for 20 min. Films were then placed on the aluminium plate (5.5 cm × 5.5 cm) and aseptically transferred into a sterilised petri dish containing filter paper which was wetted with autoclaved water. The initial bacterial inoculum 10 6 CFU/mL was prepared aseptically for the test. 200 µL of the test inoculum was then pipetted onto three pieces of each film, leaving one film as a positive control. Each petri dish containing the sample was covered with a lid and then incubated with the test inoculum at the temperature of 37 • C and relative humidity (RH) of >90%. Films were tested at 0 h, 4 h, 8 h, 12 h and 24 h. One set of inoculated samples was immediately tested after 0 h by placing the samples in a sterilised stomacher bag. 20 mL of Maximum Recovery Diluent (MDR) was poured and mixed gently by the stomacher (AGB Scientific-Lab blender 400) for 40-45 s. For the viable cell counts the samples were taken from the MRD culture, appropriately diluted and plated on Tryptic Soy Agar plates. The above process is repeated to determine the viable cell count after 4 h, 8 h, 12 h and 24 h samples and the control sample after their incubation.

Biofilm Inhibition
To study the biofilm inhibition of the essential oil composite film common food borne pathogen E. coli (NCTC 9001) was used. Three square pieces of 4 cm × 4 cm of each film (one set for positive control and two sets for biofilm inhibition) were cut and sterilised using UV light for 20 min. Films were then placed on the aluminium plate (5 cm × 5 cm) and transferred into a sterilised petri dish containing filter paper which was wetted with autoclaved water. 400 µL of the prepared test inoculum (10 6 CFU/mL) was put on the film and incubated at 37 • C and relative humidity (RH) of >90% for 72 h. After the incubation, each film was rinsed with sterile water three times. The films were then stained with 1% (w/v) crystal violet (500 µL) for 45 min [28]. The test film samples were again washed thrice with the sterile distilled water. Biofilms were quantified by eluting crystal violet with 95% ethanol (500 µL) for 10 min and transferring 100 µL aliquots to 96 well plates. The absorbance was then determined at 600 nm to determine the percent inhibition of E. coli biofilm.

Statistical Analysis
All measurements are determined in triplicates. Statistical differences between multiple sample comparisons were evaluated by analysis of variance (ANOVA) and multiple comparisons (Fischer's least significant difference test) using STATGRAPHICS Centurion XV software (Stat Point Technologies Inc. Warrenton, VA, USA). Differences were considered to be significant if the value of p < 0.05. All results are stated as mean ± standard deviation.

Surface Colour
To make the food product look more attractive, is played by the appearance of the active food packaging. The surface colour and transparency are the major factor to adopt the food packaging. The film appeared to be homogeneous, smooth and flexible. L (+lightness, −darkness), a (+redness, −greenness), and b (+yellowness, −blueness) values of the composite films incorporated with the essential value steadily increased (Tables 1 and 2). With the increase in the concentration of both the essential oils (thyme and clove) the lightness was observed to decrease significantly (p < 0.05). Also, the a-value of the thyme oil composite film significantly decreased by 1.2 folds while, for the clove oil composite film decreased by 2.07 folds as the oil concentration increases from 1 wt% to 10 wt%. The letters (a-d) at each concentration specifies groups that are significantly different (p < 0.05). In addition, the yellowness (b-value) of films increased significantly in the clove oil composite film. It was observed that the yellowness of the thyme composite film increased by 1.4 folds with the increase in the concentration of the oil, whereas, in the clove oil composite oil increased by 3.7 folds. The greenness and yellowness values of the clove oil composite film (PLA/PBAT-clove oil films) are significantly different from each other and depicted the highest a and b values, which means less green and more yellow as compared to PLA/PBAT-thyme oil films. The phenolic compounds present in the clove oil could be a possible reason for the increase in the yellowness in the PLA/PBAT-clove oil films [29]. The thyme oil composite film had shown decrement in the total colour difference (∆E values) while, of PLA/PBAT-clove blend film ∆E values increased as the oil concentration increased (Tables 1 and 2). Similar results have been observed by various studies [30], after the incorporation of the clove oil in polymer film has observed the transformation of colour from bluish to yellowish. Furthermore, Ejaz [31] et al., had observed the decrease in transparency with the increase in the concentration of clove oil in the film matrix due to the presence of the colouring elements in essential oil and heterogenous network of the film.

Optical Properties
Transparency of the active food packaging plays a very important role in the acceptability of film by the consumers. Another optical property which plays a significant role in the food industry is the UV barrier property of the film. The film must be able to prevent the oxidation of the food induced by the UV light and increase the shelf life of the food. The transmission spectrum of light was measured Foods 2020, 9,1117 7 of 16 at 280-700 nm to determine the optical properties of the composite film (Figure 1a,b). The control film (PLA/PBAT) showed the higher transmittance at 300 nm, whereas the essential oil incorporated film showed no or low transmittance at 300 nm.
at 280 nm (T280), which indicates the superior UV barrier property of the film significantly (p < 0.05). The decrease in the transmittance at 280 nm was due to the interruption in the passage of light through the film surface [32,33]. The T280 (for UV light) depicted that for the PLA/PBAT-Thyme 10% UV light blocking property increased by 20% with respect to the control film while, of the PLA/PBAT-Clove 10% increased by 80% (Tables 1 and 2). A very high concentration of the phenolic compound in the clove oil absorbed UV light which results for the best UV blocking properties for the clove oil composite film. These results align with various studies in which a UV barrier property had been observed by the incorporation of clove essential oil in the film matrix. Mulla [33] et al., observed a significant increase in the UV barrier property after the incorporation of clove oil. Similarly, Ejaz [31] et al., had observed the improvement in the UV blocking property of the composite film.  As seen in the Figure 1b, the transmittance of the PLA/PBAT-thyme oil films had shown a remarkable increase above 280 nm whereas, the PLA/PBAT-clove oil films increased above 380 nm. Furthermore, the incorporation of the essential oil (clove and thyme oil) decreased the transmittance at 280 nm (T 280 ), which indicates the superior UV barrier property of the film significantly (p < 0.05). The decrease in the transmittance at 280 nm was due to the interruption in the passage of light through the film surface [32,33]. The T 280 (for UV light) depicted that for the PLA/PBAT-Thyme 10% UV light blocking property increased by 20% with respect to the control film while, of the PLA/PBAT-Clove 10% increased by 80% (Tables 1 and 2). A very high concentration of the phenolic compound in the clove oil absorbed UV light which results for the best UV blocking properties for the clove oil composite film. These results align with various studies in which a UV barrier property had been observed by the incorporation of clove essential oil in the film matrix. Mulla [33] et al., observed a significant increase T 600 (for visible light) depicts the transparency of the composite films. No significant difference was observed between control film and 1 wt% thyme composite film, and between 5 wt% and 10 wt% thyme composite film. The transmittance of light at 600 nm of the control film is observed to be 72.46%. On the incorporation of thyme oil (10 wt%) transmittance value is observed as 81.28% (Table 1) whereas by the incorporation of clove oil (10 wt%) it becomes 65.53% ( Table 2). The value observed suggests that on the incorporation of thyme oil the transparency of the film increased, while clove oil incorporation had decreased the transparency of the film. Teixeria [34] et al., had also observed the decrease in the transparency due to the incorporation of clove oil. Sanuja [35] et al., had also observed the increase in the opaqueness and decrease in the transparency of the packaging film due to the incorporation of the clove oil. The transparency of the PLA/PBAT-clove oil films is observed to decrease due to the presence of phenolic compounds which led to the yellowish tint composite film. Therefore, due to the high property of UV blocking PLA/PBAT-clove films could be used as an active packaging material.

FTIR Analysis
For the qualitative analysis of the chemical properties of the composite film Fourier transform infrared spectroscopy (FTIR) was performed. The FTIR spectra has shown the peaks demonstrating the chemical composition of the composite film (

Thickness of the Films
Digital micrometer was used to measure the thickness of the composite films. It was observed that the thickness of the composite films increases with the increase in the concentration of the essential oil (thyme oil and clove oil). The thickness of the thyme oil increased by 3.08 folds (113.33 μm of PLA/PBAT-Thyme 10% ) with respect to the control PLA/PBAT film (36.71 μm) while, the thickness of the clove oil composite film increased by 2.90 folds (106.67 μm of PLA/PBAT-Clove 10% ) depicting significant difference in the values (Tables 3 and 4). Similar results are seen in studies where the thickness increased with the incorporation of essential oil in the active packaging film [37,38]. This could be due to the distribution pattern of the essential oil in the matrix of the film.

Thickness of the Films
Digital micrometer was used to measure the thickness of the composite films. It was observed that the thickness of the composite films increases with the increase in the concentration of the essential oil (thyme oil and clove oil). The thickness of the thyme oil increased by 3.08 folds (113.33 µm of PLA/PBAT-Thyme 10% ) with respect to the control PLA/PBAT film (36.71 µm) while, the thickness of the clove oil composite film increased by 2.90 folds (106.67 µm of PLA/PBAT-Clove 10% ) depicting significant difference in the values (Tables 3 and 4). Similar results are seen in studies where the thickness increased with the incorporation of essential oil in the active packaging film [37,38]. This could be due to the distribution pattern of the essential oil in the matrix of the film.  The letters (a-d) at each concentration specifies groups that are significantly different (p < 0.05).

Tensile Properties of the Films
Universal Testing Machine (Zwick/Roell-A730271) was used to determine the mechanical properties (tensile strength (TS) and elongation at break (EB)) of the essential oil incorporated composite film. The tensile strength of the composite films was observed to decrease with the incorporation of the essential oils (thyme oil and clove oil). The tensile strength of the control PLA/PBAT film was observed as 1.35 MPa (Tables 3 and 4). No significant difference in the control film, 1 wt% and 5 wt% thyme oil composite film was observed. As the thyme oil in the composite film increases from 1 wt% to 10 wt% the TS decreases from 1.52 MPa to 0.96 MPa, respectively, whereas, the increase in the clove oil concentration in the composite film the TS decreases from 0.94 MPa to 0.79 MPa. This shows a significant difference in the 10 wt% thyme oil composite film and 10 wt% clove oil composite films from other films. The increase in the concentration of the oil in the composite oil could weaken the structure as the stronger polymer-polymer interaction is replaced by a weaker polymer-oil interaction [39,40].
The flexibility of the film is depicted by the elongation at break (EB). The EB is observed to increase with the increase in the concentration (Tables 3 and 4). A significant difference between the 10 wt% thyme oil, 10 wt% clove oil composite film and the control film was observed. The EB of the control film PLA/PBAT was observed to be 5.63%. With the increase in the thyme oil concentration in the composite film (PLA/PBAT-Thyme 10% ) the EB increases to 16.51%, while, for the increase in the clove oil concentration in the composite film (PLA/PBAT-Clove 10% ) the EB increases to 25.67%. The improved flexibility of the composite films is due to a better interaction between PLA and PBAT polymers and a presence of the essential oil.
Many studies show that the incorporation of the essential oil influences the TS depending on the interaction of the oil with the composite film matrix [39]. Bonilla [41] et al., had incorporated basil and thyme essential oil in chitosan film and studied that the film had become more fragile with increased strechability [41]. Furthermore, Chen [42] et al., had studied 14.13% decrease in TS and 26.64% increase in EB after the incorporation of clove oil in poly(vinyl alcohol) active film. Hosseini [43] et al., had also stated that the destabilization process could take place during the drying of the film and could lead to the reduction in TS.

Water Contact Angle (WCA)
Water Contact angle (WCA) is used to determine the surface hydrophobicity. WCA indicates the interaction of the film with the liquids which is important for the application of the film in the food industry [44]. The surface is determined to be hydrophobic if the contact angle higher than 65 • [22,45]. The control PLA/PBAT film has shown WCA as 61.61 • (Tables 3 and 4). No significant difference was observed in the WCA value of the control, 1 wt% and 5 wt% of the essential oil (thyme oil and clove oil) incorporated films, whereas the WCA value of higher concentration (10 wt% of thyme and clove oil) composite film showed significant difference. As the thyme oil concentration in the composite film increased from 1 wt% to 10 wt% the WCA increased from 72.57 to 80.57 • , whereas as the clove oil concentration increased from 1 wt% to 10 wt% in the composite film the WCA increased from 62.24 to 74.74 • . Therefore, the incorporation of the clove oil (10 wt%) and thyme oil (1 wt%, 5 wt% and 10 wt%) had increased the hydrophobicity of the film, which could play a significant role in the increase in the quality of the active packaging.

Antibacterial Activity
Antibacterial active packaging prevents the growth of bacteria on the surface of the food due to direct contact of the packaging material. The antibacterial efficiency of the essential oil (thyme and clove oil) incorporated composite film was determined against E. coli (Gram negative bacteria) and S. aureus (Gram positive bacteria). The incorporation of the clove oil in the composite film had demonstrated significant antibacterial activity. It was also observed in Figure 3c,d that, as the thyme oil composite film doesn't demonstrate antibacterial activity for 24 h. Though 5 wt% thyme oil composite film or the thymol content shows antimicrobial activity for the first 4 h against E. coli whereas, 10 wt% thyme oil composite film shows antibacterial activity for 8 h against S. aureus and 12 h against E. coli. Thyme oil composite film (10 wt%) slightly reduces the growth of E. coli from 6.5 log CFU/mL to 6.32 log CFU/mL in 12 h after that it reaches to 6.84 log CFU/mL. As shown in Figure 3a, the 1 wt% clove oil composite film has shown antibacterial effect against E. coli and S. aureus in the first 4 h, whereas 5 wt% clove oil composite have shown antibacterial activity against E. coli up till 12 h, after that it has not shown antibacterial activity. However, in the presence of 10 wt% clove oil composite film (PLA/PBAT-Clove 10% ) E. coli growth had reduced from 6.5 log CFU/mL to 4.4 log CFU/mL. However, an exceptional antibacterial activity has been observed by 10 wt% clove oil composite film against S. aureus. In presence of 5 wt% clove oil composite film the S. aureus growth had reduced from 6.5 log CFU/mL to 4.5 log CFU/mL. 10 wt% clove oil composite film had shown a complete killing of S. aureus by reducing growth from 6.5 log CFU/mL to 0 log CFU/mL. It has been reported that the incorporation of essential oil including clove oil has higher bacterial inhibition efficacy against Gram positive bacteria [46]. Mulla [33] et al., had also studied strong antibacterial efficacy of clove essential oil against S. typhimurium and L. monocytogenes. Moreover, Mupalla et al., (2014) had observed that the clove oil film had shown promising antibacterial activity. Also, the films were observed to be more active against S. aureus than B. cereus [47].

Biofilm Inhibition
Aggregation of bacteria on the surface led to the formation of the biofilm. Biofilms on the food contact surfaces causes critical problems in the food industry. A biofilm study was observed against E. coli for 72 h. As shown in Figure 4, the control film shows minimal biofilm inhibition (5.27%). The thyme oil composite film (1 wt%) had inhibited biofilm by 55.96%, whereas 1 wt% clove oil composite film had inhibited E. coli growth by 60.31%. As the concentration rises to 5 wt%, thyme oil composite film had inhibited biofilm growth by 71.39% and clove oil composite film had inhibited biofilm growth of E. coli by 75.65%.
Aggregation of bacteria on the surface led to the formation of the biofilm. Biofilms on the food contact surfaces causes critical problems in the food industry. A biofilm study was observed against E. coli for 72 h. As shown in Figure 4, the control film shows minimal biofilm inhibition (5.27%). The thyme oil composite film (1 wt%) had inhibited biofilm by 55.96%, whereas 1 wt% clove oil composite film had inhibited E. coli growth by 60.31%. As the concentration rises to 5 wt%, thyme oil composite film had inhibited biofilm growth by 71.39% and clove oil composite film had inhibited biofilm growth of E. coli by 75.65%.
However, a significant inhibition of E. coli biofilm growth was observed by clove oil composite film (10 wt%). 10 wt% clove oil composite film had inhibited the biofilm by 93.43%. Moreover, clove oil was more effective than thyme oils as they contain higher concentrations of eugenol, 180 mg [48].Therefore, the results signify that the high concentration (10 wt%) of essential oils (thyme oil or clove oil) inhibit the formation of the biofilm (E. coli) on all tested composite films, though the percentage inhibition of clove oil (93.43% inhibition) was higher than thyme oil (82.30% inhibition). Cui [49] et al., had also studied that the incorporation of clove oil-loaded chitosan nanoparticle had inhibited E. coli biofilm up to 99.99%.

Conclusions
Thyme oil and clove oils were incorporated into the biodegradable polymer (PLA/PBAT) blend film and their potential as an active food packaging was investigated. Incorporation of clove oil and thyme oil had a very high influence on the composite film properties such as optical, morphological, mechanical properties, antibacterial efficacy and biofilm inhibition property. The clove oil composite films depicted pale yellow film with less transparency and high UV-light barrier property as compared to thyme oil composite films. Clove oil composite film has shown 80% UV barrier property. Significant decrease in the tensile strength by the incorporation of the essential oil has been observed. The tensile strength decreases from 1.35 (control film) to 0.96 MPa (PLA/PBAT-Thyme 10% ) and 0.79 MPa (PLA/PBAT-Clove 10% ). However, as the concentration of essential oil increases, the water contact angle (WCA) of the composite films has increased significantly. Also, a very high antibacterial property had been shown by clove oil composite film. Clove oil composite film had reduced E. coli growth by 2.1 log CFU/mL. Also, a complete killing of S. aureus that is, a reduction from 6.5 log  However, a significant inhibition of E. coli biofilm growth was observed by clove oil composite film (10 wt%). 10 wt% clove oil composite film had inhibited the biofilm by 93.43%. Moreover, clove oil was more effective than thyme oils as they contain higher concentrations of eugenol, 180 mg [48]. Therefore, the results signify that the high concentration (10 wt%) of essential oils (thyme oil or clove oil) inhibit the formation of the biofilm (E. coli) on all tested composite films, though the percentage inhibition of clove oil (93.43% inhibition) was higher than thyme oil (82.30% inhibition). Cui [49] et al., had also studied that the incorporation of clove oil-loaded chitosan nanoparticle had inhibited E. coli biofilm up to 99.99%.

Conclusions
Thyme oil and clove oils were incorporated into the biodegradable polymer (PLA/PBAT) blend film and their potential as an active food packaging was investigated. Incorporation of clove oil and thyme oil had a very high influence on the composite film properties such as optical, morphological, mechanical properties, antibacterial efficacy and biofilm inhibition property. The clove oil composite films depicted pale yellow film with less transparency and high UV-light barrier property as compared to thyme oil composite films. Clove oil composite film has shown 80% UV barrier property. Significant decrease in the tensile strength by the incorporation of the essential oil has been observed. The tensile strength decreases from 1.35 (control film) to 0.96 MPa (PLA/PBAT-Thyme 10% ) and 0.79 MPa (PLA/PBAT-Clove 10% ). However, as the concentration of essential oil increases, the water contact angle (WCA) of the composite films has increased significantly. Also, a very high antibacterial property had been shown by clove oil composite film. Clove oil composite film had reduced E. coli growth by 2.1 log CFU/mL. Also, a complete killing of S. aureus that is, a reduction from 6.5 log CFU/mL to 0 log CFU/mL was observed by 10 wt% clove oil composite film. The thyme oil composite film (10 wt%) had inhibited biofilm by 82.30% against E. coli whereas, clove oil composite film (10 wt%) had inhibited the E. coli biofilm by 93.43%. Thus, the clove oil composite film has demonstrated strong UV barrier, antibacterial and biofilm inhibition property, which can avoid the undesirable photochemical reaction, adherence and growth of pathogenic bacteria which can increase the shelf life of the packed food. Therefore, clove oil composite film can be used as an active packaging.