Physicochemical properties and antibacterial activity of cornstarch based films incorporated with lemongrass (Cymbopogon citratus) essential oil

Philippine e-Journal for Applied Research and Development 15(2025), 51-61 ISSN 2449-3694 (Online) PURL: https://pejard.slu.edu.ph/vol.15/2025.11.21.pdf DOI: https://doi.org/10.5281/zenodo.17694787 Physicochemical properties and antibacterial activity of cornstarchbased films incorporated with lemongrass (Cymbopogon citratus) essential oil Betina Dannah M. Villanueva1, Adeliza A. Dorado1*, Ma. Josie V. Sumague1, and Romel M. Felismino1 1Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippine Los Baños *[email protected] Received, 02 May 2025; Accepted, 13 November 2025; Published, 21 November 2025 Copyright @ 2025 B.D.M. Villanueva, A.A. Dorado, MJ.V. Sumague, and R.M. Felismino. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Bioplastic films prepared from cornstarch incorporated with lemongrass essential oil (LEO) were evaluated in terms of their physicochemical and antibacterial properties. The films were prepared via solution casting to contain different amounts of LEO (0%, 0.5%, 1%, and 2% w/w). The films were characterized for thickness, color properties, moisture content, tensile strength, and elongation at break. Antibacterial activity against Staphylococcus aureus (BIOTECH 1582) and Salmonella typhimurium (BIOTECH 1826) was determined via disk diffusion using 6 mm film discs on Mueller–Hinton agar. Significant improvement in elongation at break (25.06 ± 7.39%) at 0.5% LEO, and increase in film thickness and moisture content with LEO addition were observed. All films containing LEO displayed strong antibacterial activity. Interestingly, the control film (0% LEO) exhibited notable baseline antibacterial activity (inhibition zones >29 mm) probably due to the vinegar and glycerol in its formulation. Considerable increase in antimicrobial activity occurs with 2% LEO against S. aureus. Overall, films prepared with 0.5% LEO showed the best possible balance between mechanical flexibility and antimicrobial activity, suggesting that this formulation may be used in preparing active food packaging materials. Keywords: antibacterial packaging, elongation at break, microbial zone of inhibition, tensile strength Philippine e-Journal for Applied Research and Development Website: pejard.slu.edu.ph ISSN 2449-3694 (Online) Introduction Food packaging plays a crucial role as it protects food ingredients and products from potential damage and deterioration (Petkoska et al., 2021; Shanbhag et al., 2023). Conventional food packaging includes paper, glass, plastics, and metals. Although over 20% of paper and paperboard are recycled, various plastics are recycled at a very low rate (< 20%) (Jeevahan & Chandrasekaran, 2019). Thus, the common practice of discarding packaging materials especially conventional plastics poses a serious environmental problem, as these are widely used and are non-biodegradable. Moreover, conventional plastics are derived from nonrenewable petroleum reserves and generate carbon dioxide and toxic substances when incinerated (Petkoska et al., 2021). Due to pressing issues surrounding the use of conventional plastics, the food industry now considers the development of biodegradable, active, and edible packaging materials among its top priorities. These alternative packaging G.G.L.-Co Yu Kang, T.J. Parreñas, and N.P. Nolasco 52 B.D.M. Villanueva, A.A. Dorado, MJ.V. Sumague, and R.M. Felismino materials include films that are prepared from edible materials such as lipids, proteins, and polysaccharides. These bioplastic films may be formed into wraps, pouches, bags, capsules, and casings (Jeevahan & Chandrasekaran, 2019). In recent years, starch-based films have attracted considerable attention due to their inherent biodegradability, high availability, and low cost (Dorado et al., 2017). Starch is a natural polymer that may be formed into colorless, odorless films with low oxygen permeability (Cano et al., 2014; Garcia et al., 2020). However, starch-based films suffer from high water vapor permeability because of the abundance of hydroxyl groups in starch, and likewise exhibit poor mechanical properties (Dorado et al., 2017; Garcia et al., 2020). To overcome these drawbacks, a plasticizer is added to the film matrix to improve flexibility and mechanical properties (Youssef & El-Sayed, 2018). Low-molecular weight polyols are often used as plasticizers, with glycerol being the most studied polyol. Molecular weight is associated with the efficacy of a plasticizer: the smaller the molecule, the greater the plasticizing effect. Aside from plasticizers, crosslinking agents such as vinegar can be added to improve the film’s strength. Vinegar is an acetic acid solution that releases acetate and hydrogen ions that, in turn, react with starch polymers so that they become disordered. This disorder arising from the ionization of water and acetic acid renders the film more homogenous (Shanbhag et al., 2023). To obtain unique functionality, additives such as antimicrobial agents, colors, and flavors are incorporated into the starch matrix depending on its intended use (Bilal, Zhao & Iqbal, 2020). For example, to prevent microbial growth, starch-based films can be infused with antimicrobial agents such as essential oils (EOs). When incorporated into the film matrix, EOs can be released into the food upon contact with the film surface (do Evangelo, 2019). Furthermore, EOs serve to reduce the water vapor permeability of starch-based films (Garcia et al., 2020). EOs have an oily and volatile nature which influence the structural characteristics and degree of hydrophobicity of polymeric films, thus altering their mechanical and barrier properties (do Evangelo et al., 2019; Atares & Chiralt, 2016). EOs are the best replacement to petroleumderived additives in food packaging materials because they are naturally abundant, ecofriendly, and possess superior antimicrobial and antioxidant attributes (Zubair et al., 2022). However, although the antimicrobial properties of several EOs in biodegradable films have been widely studied, there are very few reports on their effects on film properties (do Evangelo et al., 2019). Some such reports include works on starch/chitosan films infused with lemongrass essential oil (LEO) wherein antimicrobial activity was evaluated along with water vapor permeability, water solubility, and mechanical properties (Perdana et al. 2021); on cassava starch/chitosan films incorporated with LEO where antimicrobial activity and mechanical properties were evaluated against storage time (Perdana et al., 2022); and, on cornstarch films containing Zanthoxylum bungeanum essential oil (ZYO) wherein antimicrobial activity and physical characteristics (morphology, optics, mechanical and barrier properties) were reported (Wang et al., 2020). In this study, we will report a combination of microbiological (efficacy against Gram-positive and Gram-negative bacteria) and physicochemical (surface color, transparency, moisture content, tensile strength, elongation) evaluations of LEO-infused films prepared from cornstarch, glycerol, and vinegar, with focus on the effects of varying LEO concentrations (0.5– 2% w/w LEO). As such, this study centers on the potential of cornstarch matrices combined with commonly available plasticizer and acid to help address the dual challenge of environmental pollution from synthetic packaging and food spoilage due to microbial contamination. Furthermore, this study provides insights into optimizing film composition for mechanical performance and antimicrobial efficacy. Methodology Preparation of films Commercial cornstarch (Cream pure cornstarch), distilled water (Absolute), and 5% acidity vinegar (Heinz distilled vinegar) were purchased from a local supermarket. Laboratorygrade glycerol (Scharlau A.R.) and LEO were procured from Chemline Scientific Corporation and Young Living Philippines, respectively. 53 Philippine e-Journal for Applied Research and Development Website: pejard.slu.edu.ph ISSN 2449-3694 (Online) Philippine e-Journal for Applied Research and Development 15(2025), 51-61 The cornstarch-based films were prepared via solution casting according to the methods of Resianingrum et al. (2016), do Evangelo et al. (2019) and Wang et al. (2020) with some modifications. The starch-based mixture consisted of distilled water, cornstarch, vinegar, and glycerol to which LEO was added at different amounts. Films were prepared by mixing cornstarch (7.67% w/w), glycerol (7.67% w/w), and vinegar (7.67% w/w) in water (77% w/w) and heated at 80–85 °C using a covered double-boiler steamer for 15 min to prevent volatilization of oil. LEO was added directly to the solution at 0%, 0.5%, 1%, and 2% w/w of total solution weight (Table 1) and stirred thoroughly. The mixture (Figure 1a) was cast onto wax-paper-lined trays (380 × 280 mm) and dried at 40 °C for 72 h. Dried films were stored in desiccators until analysis. The preparation of packaging films was done in triplicates for each treatment. Film thickness The thickness of the films was measured as the average of ten measurements taken at the center and periphery of the film using a micrometer caliper. Tensile strength and elongation at break The tensile strength (TS) and percent elongation at break (%E) of the sheets were determined according to the ASTM D882 (Standard Test Method for Tensile Properties of Thin Plastic Sheeting) using an Instron Universal Testing Machine (Model 5585H). All Table 1 Formulation of the cornstarch-based films. Components % Lemongrass essential oil (LEO) 0 0.5 1.0 2.0 Cornstarch (g) 10 10 10 10 Distilled water (g) 100 99.35 98.7 97.4 Glycerol (g) 10 10 10 10 Vinegar(g) 10 10 10 10 LEO (g) 0 0.65 1.3 2.6 Total (g) 130 130 130 130 film samples were pre-conditioned at 23 ± 2°C and 50 ± 5% relative humidity for at least 48 h prior to mechanical and color testing in accordance with ASTM D618 (Standard Practice for Conditioning Plastics for Testing). For every formulation, ten replicates were tested. To account for batch-tobatch variation, these replicates were taken from each of the three independently prepared film sheets for each treatment. The TS was calculated using Eq. 1 where Lp is the peak load (N) and a is the cross-sectional area of the strips (m2) while the %E was determined using Eq. 2 where ∆l is the change in length at breaking point (mm) and l is the original length (mm) of the film strip. 3 grade glycerol (Scharlau A.R.) and LEO were procured from Chemline Scientific Corporation 91 and Young Living Philippines, respectively. 92 The cornstarch-based films were prepared via solution casting according to the methods 93 of Resianingrum et al. (2016), do Evangelo et al. (2019) and Wang et al. (2020) with some 94 modifications. The starch-based mixture consisted of distilled water, cornstarch, vinegar, and 95 glycerol to which LEO was added at different amounts. Films were prepared by mixing 96 cornstarch (7.67% w/w), glycerol (7.67% w/w), and vinegar (7.67% w/w) in water (77% w/w) 97 and heated at 80–85 °C using a covered double-boiler steamer for 15 min to prevent 98 volatilization of oil. LEO was added directly to the solution at 0%, 0.5%, 1%, and 2% w/w of 99 total solution weight (Table 1) and stirred thoroughly. The mixture (Figure 1a) was cast onto 100 wax-paper-lined trays (380 × 280 mm) and dried at 40 °C for 72 h. Dried films were stored in 101 desiccators until analysis. The preparation of packaging films was done in triplicates for each 102 treatment. 103 Table 1. Formulation of the cornstarch-based films. 104 Components % Lemongrass essential oil (LEO) 0 0.5 1.0 2.0 Cornstarch (g) 10 10 10 10 Distilled water (g) 100 99.35 98.7 97.4 Glycerol (g) 10 10 10 10 Vinegar(g) 10 10 10 10 LEO (g) 0 0.65 1.3 2.6 Total (g) 130 130 130 130 105 106 Film thickness 107 108 The thickness of the films was measured as the average of ten measurements taken at 109 the center and periphery of the film using a micrometer caliper. 110 111 Tensile strength and elongation at break 112 113 The tensile strength (TS) and percent elongation at break (%E) of the sheets were 114 determined according to the ASTM D882 (Standard Test Method for Tensile Properties of Thin 115 Plastic Sheeting) using an Instron Universal Testing Machine (Model 5585H). All film samples 116 were pre-conditioned at 23 ± 2°C and 50 ± 5% relative humidity for at least 48 h prior to 117 mechanical and color testing in accordance with ASTM D618 (Standard Practice for 118 Conditioning Plastics for Testing). For every formulation, ten replicates were tested. To 119 account for batch-to-batch variation, these replicates were taken from each of the three 120 independently prepared film sheets for each treatment. The TS was calculated using Eq. 1 121 where 𝐿𝐿𝐿𝐿𝑝𝑝𝑝𝑝 is the peak load (N) and 𝑎𝑎𝑎𝑎 is the cross-sectional area of the strips (m2) while the %E 122 was determined using Eq. 2 where ∆𝑙𝑙𝑙𝑙 is the change in length at breaking point (mm) and 𝑙𝑙𝑙𝑙 is 123 the original length (mm) of the film strip. 124 𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 = 𝐿𝐿𝐿𝐿 𝑝𝑝𝑝𝑝 𝑎𝑎𝑎𝑎 Eq. 1 %𝐸𝐸𝐸𝐸= � ∆𝑙𝑙𝑙𝑙 𝑙𝑙𝑙𝑙 � × 100 Eq. 2 Eq. 1 Eq. 2 Color properties The color of the cornstarch-based films was measured using the CIELAB coordinates (L*, a*, and b*) obtained from Chroma Meter CR-400 (Minolta, Tokyo, Japan). Prior to measurements, the instrument was calibrated using the conventional two-point method – zero calibration (black standard) and white calibration using the white calibration tile provided by the manufacturer. Films from each formulation was tested in three runs, with three replicates for 54 B.D.M. Villanueva, A.A. Dorado, MJ.V. Sumague, and R.M. Felismino each run. The whiteness index (WI) that acts as an indirect indicator of changes in the visual characteristics of the film (Garcia et al., 2020; Wang et al., 2020) was computed using Eq. 3. 4 Color properties 125 The color of the cornstarch-based films was measured using the CIELAB coordinates 126 (L*, a*, and b*) obtained from Chroma Meter CR-400 (Minolta, Tokyo, Japan). Prior to 127 measurements, the instrument was calibrated using the conventional two-point method – zero 128 calibration (black standard) and white calibration using the white calibration tile provided by 129 the manufacturer. Films from each formulation was tested in three runs, with three replicates 130 for each run. The whiteness index (WI) that acts as an indirect indicator of changes in the visual 131 characteristics of the film (Garcia et al., 2020; Wang et al., 2020) was computed using Eq. 3. 132 𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊 =100 −�(100 −𝐿𝐿𝐿𝐿 ∗ ) + 𝑎𝑎𝑎𝑎 ∗2 +𝑏𝑏𝑏𝑏 ∗2 Eq. 3 133 Moisture Content 134 135 The moisture content of the films was obtained according to the methods of Wang et 136 al. (2020) with some modifications. The samples were first conditioned at 60 ± 5% RH for 48 137 h. Approximately 1 g of a film sample was cut into tiny pieces and placed in a tared crucible. 138 The crucible containing the sample was placed in an oven at 100 ± 5 oC for 24 h, and then 139 allowed to equilibrate at room temperature in a desiccator before re-weighing. Thereafter, the 140 crucible was returned to the oven for 30 min, and then cooled and reweighed. These re-drying 141 and reweighing steps were repeated until the weight of the two previous readings did not differ 142 by more than 0.001 g. The moisture content (MC) of the film was calculated using Eq. 4. 143 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 = 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑤𝑤𝑤𝑤𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑖𝑖𝑖𝑖ℎ𝑐𝑐𝑐𝑐−𝑓𝑓𝑓𝑓𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑎𝑎𝑎𝑎𝑓𝑓𝑓𝑓 𝑤𝑤𝑤𝑤𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑖𝑖𝑖𝑖ℎ𝑐𝑐𝑐𝑐 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑤𝑤𝑤𝑤𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑖𝑖𝑖𝑖ℎ𝑐𝑐𝑐𝑐 × 100 Eq. 4 144 Microbial activity of the films 145 146 Prior to the conduct of the microbial activity test, Gram-positive Staphylococcus aureus 147 (BIOTECH 1582) and Gram-negative Salmonella typhimurium (BIOTECH 1826) obtained 148 from the University of the Philippines Los Baños - National Institute of Biology and 149 Biotechnology (UPLB-BIOTECH), were grown at room temperature generating 9.63 × 108 150 CFU mL-1 and 3.3 × 109 CFU mL-1 of S. aureus and S. typhimurium, respectively. 151 The antimicrobial activity of the films were evaluated by disk diffusion assay according 152 to the Kirby-Bauer method (Christenson et al., 2017) with modifications. The films were cut 153 into 6-mm diameter discs and placed on the surface of the Mueller-Hinton agar (MHA) 154 previously inoculated with 0.1 mL of the bacteria culture through spread plating (Wang et al., 155 2020). The turbidity of the inoculant was adjusted with sterile broth prior to being spread onto 156 the agar plates to an approximate cell density of 1.5 × 108 CFU mL-1. The antimicrobial 157 effects of cornstarch-based films containing different concentrations of LEO against S. aureus 158 and S. typhimurium were carried out using a zone of inhibition assay on MHA. The plates were 159 then incubated at 37 °C for 24 h. After incubation, the inhibition zone was measured on the 160 Eq. 3 Moisture Content The moisture content of the films was obtained according to the methods of Wang et al. (2020) with some modifications. The samples were first conditioned at 60 ± 5% RH for 48 h. Approximately 1 g of a film sample was cut into tiny pieces and placed in a tared crucible. The crucible containing the sample was placed in an oven at 100 ± 5 oC for 24 h, and then allowed to equilibrate at room temperature in a desiccator before re-weighing. Thereafter, the crucible was returned to the oven for 30 min, and then cooled and reweighed. These re-drying and reweighing steps were repeated until the weight of the two previous readings did not differ by more than 0.001 g. The moisture content (MC) of the film was calculated using Eq. 4. 4 Color properties 125 The color of the cornstarch-based films was measured using the CIELAB coordinates 126 (L*, a*, and b*) obtained from Chroma Meter CR-400 (Minolta, Tokyo, Japan). Prior to 127 measurements, the instrument was calibrated using the conventional two-point method – zero 128 calibration (black standard) and white calibration using the white calibration tile provided by 129 the manufacturer. Films from each formulation was tested in three runs, with three replicates 130 for each run. The whiteness index (WI) that acts as an indirect indicator of changes in the visual 131 characteristics of the film (Garcia et al., 2020; Wang et al., 2020) was computed using Eq. 3. 132 𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊 =100 −�(100 −𝐿𝐿𝐿𝐿 ∗ ) + 𝑎𝑎𝑎𝑎 ∗2 +𝑏𝑏𝑏𝑏 ∗2 Eq. 3 133 Moisture Content 134 135 The moisture content of the films was obtained according to the methods of Wang et 136 al. (2020) with some modifications. The samples were first conditioned at 60 ± 5% RH for 48 137 h. Approximately 1 g of a film sample was cut into tiny pieces and placed in a tared crucible. 138 The crucible containing the sample was placed in an oven at 100 ± 5 oC for 24 h, and then 139 allowed to equilibrate at room temperature in a desiccator before re-weighing. Thereafter, the 140 crucible was returned to the oven for 30 min, and then cooled and reweighed. These re-drying 141 and reweighing steps were repeated until the weight of the two previous readings did not differ 142 by more than 0.001 g. The moisture content (MC) of the film was calculated using Eq. 4. 143 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 = 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑤𝑤𝑤𝑤𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑖𝑖𝑖𝑖ℎ𝑐𝑐𝑐𝑐−𝑓𝑓𝑓𝑓𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑎𝑎𝑎𝑎𝑓𝑓𝑓𝑓 𝑤𝑤𝑤𝑤𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑖𝑖𝑖𝑖ℎ𝑐𝑐𝑐𝑐 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑤𝑤𝑤𝑤𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑖𝑖𝑖𝑖ℎ𝑐𝑐𝑐𝑐 × 100 Eq. 4 144 Microbial activity of the films 145 146 Prior to the conduct of the microbial activity test, Gram-positive Staphylococcus aureus 147 (BIOTECH 1582) and Gram-negative Salmonella typhimurium (BIOTECH 1826) obtained 148 from the University of the Philippines Los Baños - National Institute of Biology and 149 Biotechnology (UPLB-BIOTECH), were grown at room temperature generating 9.63 × 108 150 CFU mL-1 and 3.3 × 109 CFU mL-1 of S. aureus and S. typhimurium, respectively. 151 The antimicrobial activity of the films were evaluated by disk diffusion assay according 152 to the Kirby-Bauer method (Christenson et al., 2017) with modifications. The films were cut 153 into 6-mm diameter discs and placed on the surface of the Mueller-Hinton agar (MHA) 154 previously inoculated with 0.1 mL of the bacteria culture through spread plating (Wang et al., 155 2020). The turbidity of the inoculant was adjusted with sterile broth prior to being spread onto 156 the agar plates to an approximate cell density of 1.5 × 108 CFU mL-1. The antimicrobial 157 effects of cornstarch-based films containing different concentrations of LEO against S. aureus 158 and S. typhimurium were carried out using a zone of inhibition assay on MHA. The plates were 159 then incubated at 37 °C for 24 h. After incubation, the inhibition zone was measured on the 160 Eg. 4 Microbial activity of the films Prior to the conduct of the microbial activity test, Gram-positive Staphylococcus aureus (BIOTECH 1582) and Gram-negative Salmonella typhimurium (BIOTECH 1826) obtained from the University of the Philippines Los Baños - National Institute of Biology and Biotechnology (UPLB-BIOTECH), were grown at room temperature of around 30oC generating 9.63 x 108 CFU mL-1 and 3.3 x 109 CFU mL-1 of S. aureus and S. typhimurium, respectively. The antimicrobial activity of the films were evaluated by disk diffusion assay according to the Kirby-Bauer method (Christenson et al., 2017) with modifications. The films were cut into 6-mm diameter discs and placed on the surface of the Mueller-Hinton agar (MHA) previously inoculated with 0.1 mL of the bacteria culture through spread plating (Wang et al., 2020). The turbidity of the inoculant was adjusted with sterile broth prior to being spread onto the agar plates to an approximate cell density of 1.5 x 108 CFU mL-1. The antimicrobial effects of cornstarchbased films containing different concentrations of LEO against S. aureus and S. typhimurium were carried out using a zone of inhibition assay on MHA. The plates were then incubated at 37 °C for 24 h. After incubation, the inhibition zone was measured on the film disks (Manab et al., 2011). The film with no EO (0% LEO) served as the untreated control. Three replicates from each of the three runs of each treatment were tested. Statistical Analysis All means and standard deviations were calculated and analyzed statistically using Microsoft Excel. Significant differences between the samples were determined for all characteristics measured using analysis of variance (ANOVA) at a 95% confidence level and Tukey pairwise comparison. Results All films were macroscopically homogeneous without visible oil droplets or exudates. Figure 1a shows a sample of heated film solution on tray while Figure 1b shows samples of dried films. Tables 2 and 3 list the physiochemical characteristics of the films. Films with 0.5% LEO exhibited the highest tensile strength (0.966 ± 0.252 MPa) and elongation at break (25.06 ± 7.39%), outperforming both untreated films and films treated with higher amounts of LEO. It appears that disruption of the polymer network occurred at higher amounts of LEO, resulting in weaker mechanical performance. Loss of transparency with incorporation of LEO suggests its entrapment in microdroplets, consistent with literature reports on EO–polymer films. Also, incorporation of LEO significantly increased film thickness, consistent with literature reports. The films exhibit antibacterial activity as indicated by the presence of circular zones of inhibition in disk diffusion assays for S. 55 Philippine e-Journal for Applied Research and Development Website: pejard.slu.edu.ph ISSN 2449-3694 (Online) Philippine e-Journal for Applied Research and Development 15(2025), 51-61 Table 2 Thickness, tensile strength, and elongation at break of cornstarch-based films incorporated with lemongrass essential oil (LEO). Treatment (% LEO) Thickness (mm) Tensile Strength (MPa) Percentage Elongation at Break 0% (Control) 0.120 ± 0.034b 0.860 ± 0.303ab 14.072 ± 3.101b 0.5% 0.177 ± 0.051a0.966 ± 0.252a25.060 ± 7.390a 1% 0.161 ± 0.041ab 0.594 ± 0.171bc 14.500 ± 2.866b 2% 0.186 ±0.029a0.475 ±0.166c12.930 ±3.880b Each data point represents the mean ± standard deviation of 10 replicates obtained from each formulation. The multiple comparisons were determined by Tukey at p < 0.05. Means in the same column with different letters are significantly different. Table 3 Color values, whiteness index(WI), and moisture content of cornstarch-based films incorporated with lemongrass essential oil (LEO). Treatment (% LEO) L* a* b* WI Moisture Content (%) 0% (Control) 92.249± 0.610 a -0.049 ± 0.043a3.690 ± 0.218b91.412 ± 0.597a22.195 ± 0.331c 0.5% 91.456 ± 0.490a -0.303 ± 0.068b4.131 ± 0.311b90.496 ± 0.409a24.920 ± 0.364b 1% 91.949 ± 0.963a -0.421 ± 0.086c4.950 ± 0.566a90.531 ± 1.040a28.839 ± 0.778a 2% 92.082 ± 1.253a -0.410 ± 0.103c4.919 ± 0.530a90.661 ± 1.297a24.145 ± 0.634b Each data point represents the mean ± standard deviation of triplicates obtained from triplicate preparations of films. The multiple comparisons were determined by Tukey at p < 0.05. Means in the same column with different letters are significantly different. L*, a*, b* are the coordinates of the CIELAB color space diagram. L* (0 to 50 is considered dark while 51 to 100 is considered light), a* (positive values indicate redness while negative values indicate greenness), b* (positive values indicate yellowness while negative values indicate blueness). Figure 1 (a) a tray of film solution, and (b) samples of dried LEO-treated cornstarch films. 56 B.D.M. Villanueva, A.A. Dorado, MJ.V. Sumague, and R.M. Felismino typhimurium (Figure 2a) and S. aureus (Figure 2b). The diameter of these zones (Figure 2c) was measured and summarized in Table 4. While Tukey’s HSD test identifies differences between treatments, the biological significance was determined based on the classification of inhibition zone diameters. All samples demonstrated very strong inhibition (>20 mm), indicating effective antibacterial activity. Figure 2 (a) disk diffusion assays for S. typhimurium (L-R: 0%, 0.5%, 1%, 2%), (b) S. aureus (L-R: 0%, 0.5%, 1%, 2%), and (c) the method for estimating diameters of microbial inhibition zones. Table 4 Zone of inhibition of antibacterial activity of cornstarch-based films incorporated with lemongrass essential oil (LEO). Treatment (% LEO) Diameter of Zone of Inhibition (mm) S. aureus S. typhimurium 0% (Control) 32.78±4.60 ab 29.33±1.94 bc 0.5% 32.22±2.91 abc 28.44±1.24 c 1% 31.89±3.18 abc 28.33±2.50 c 2% 34.22±1.72a 30.67±1.80 abc Each data point represents the mean ± standard deviation of triplicates obtained from triplicate preparations of films. The multiple comparisons were determined by Tukey at p < 0.05. Means that do not share a letter are significantly different. Inhibition zone diameters ≥20 mm are indicative of very strong antibacterial activity (Davis and Stout, 1971). 57 Philippine e-Journal for Applied Research and Development Website: pejard.slu.edu.ph ISSN 2449-3694 (Online) Philippine e-Journal for Applied Research and Development 15(2025), 51-61 Discussion Thickness of films Incorporating LEO significantly increased the thickness of the cornstarch-based films (Table 2). This result is in agreement with findings from starch-based films incorporated with Zanthoxylum bungeanum essential oil (Wang et al., 2020), cornstarch films with orange essential oil (do Evangelho et al., 2019), carboxymethyl xylan films with licorice essential oil (Luis et al., 2019), and cassava starch-based edible films with lemongrass oil (Resianingrum et al., 2016). In their study, Luis et al. (2019) attributed this behavior to the entrapment of microdroplets of licorice essential oil into the film matrix, which gives rise to less dense and thicker films. In this study, the film with the highest amount of LEO (2% w/w) showed the highest mean thickness. However, the effects of varying amounts of LEO on the thickness of the films are not significantly different from each other. Meanwhile, although the thickness of films prepared with 0%, 0.5% and 2% LEO are significantly different, the thickness of films prepared with 0% and 1% LEO are not significantly different. Nevertheless, the ideal thickness for films which is < 0.25 mm (Hatmi et al., 2020) was achieved in this study. In the development of packaging materials, the thickness of the film is measured for the evaluation of other properties – structural, mechanical, thermal, and barrier properties. For example, in the case of barrier properties, thicker films are desired since this would mean a longer path for water vapor to traverse to penetrate the film. Tensile strength (TS) The mechanical properties of the films were significantly influenced by the addition of LEO (Table 2). The mean TS of the film with 0.5% LEO was the highest (0.966 ± 0.252 MPa); however, it did not differ statistically from that of the control film (0.860 ± 0.303 MPa). In their study on cassava starch-based edible films, Resianingrum et al. (2016) explained that LEO contains solid components that lessen the intercellular gaps in edible films which results in more compact and firmer film matrices. This may explain the observed increase in TS in films prepared with 0.5% LEO. At higher amounts of LEO (1% and 2%), the TS decreased significantly relative to the control. Similar results were obtained for cornstarch films containing orange essential oil (do Evangelho et al., 2019); tapioca-based edible films incorporated with cinnamon essential oil (Utami et al., 2019); and chitosan-based films incorporated with thyme, clove, and cinnamon essential oils (Hossenei et al., 2009). The decrease in TS can be attributed to essential oil causing the formation of a heterogeneous film structure (Utami et al., 2019) or the breakup of the polymer film network (Hossenei et al., 2009). However, other factors must be considered when evaluating the TS of films. The concentration and composition of the starch (i.e., amylose and amylopectin) greatly affect the tensile strength (Hatmi et al., 2020). Elongation at break Compared with the other formulations, the film prepared with 0.5% LEO showed a significantly improved elongation at break (25.060 ± 7.390%), which means that a plasticizing effect occurs at this formulation (Table 2), similar to observations in films with low amounts of essential oil (do Evangelho et al., 2019). Plasticizer molecules disrupt the starch’s cohesiveness, decrease intermolecular connections, and increase polymer mobility (Resianingrum et al., 2016). Meanwhile, the decrease in elongation at break at higher amounts of LEO was similarly observed in the study of Hossenei et al. (2009), wherein both the TS and elongation at break decreased in 1% thyme essential oil and 1.5% clove essential oil formulations of chitosan-based films; and in the study of Utami et al. (2019) wherein both the TS and elongation at break decreased when 1% of cinnamon essential oil was added in tapioca-based edible films. According to Utami et al. (2019), essential oils create compact film structures, thereby improving continuity in polysaccharide networks, which leads to a decrease in elongation. Moreover, Hosseini et al. (2009) explains that a cross-linking effect can be induced by strong interactions between polymers and essential oils that reduces the free volume and molecular mobility of polymers, leading to a 58 B.D.M. Villanueva, A.A. Dorado, MJ.V. Sumague, and R.M. Felismino decrease in elongation at break. In this study, it may be proposed that maximum polymer (i.e., starch) mobility occurs at 0.5% LEO incorporation, which resulted in a maximum film elongation of about 25%. Films made from polysaccharides have elongation values from 1 to 80% (Hatmi et al., 2020), which puts the elongation at break of the LEO-infused cornstarch-based film in this study at the lower spectrum of the range of values. This means that the addition of LEO does not significantly influence the elongation at break. The elongation at break might be influenced more remarkably by changes in the amount of plasticizer (i.e., glycerol) relative to cornstarch which unfortunately was not within the scope of this study. Surface color properties Table 3 shows the mean of the color parameters L* (0 to 50 is considered dark while 51 to 100 is considered light), a* (positive values indicate redness while negative values indicate greenness), b* (positive values indicate yellowness while negative values indicate blueness), and WI of films prepared with different LEO treatments. There were no significant differences in brightness (L*) and light transmittance in terms of WI values of the films. The control (0% LEO) films were brighter and more transparent than all the treated films. At the lowest LEO treatment (0.5% LEO), a significant difference in the color of the films with regard to their greenish hue was observed. At 1% LEO, the yellowish hue was significantly different from the control. These changes in color property of LEO-treated films may be attributed to light scattering caused by lipid droplets in the film network (Wang et al., 2020). All in all, compared with the control, and despite the differences in color, the LEOtreated films meet consumer requirements for food packaging materials. Color is a key factor considered in food packaging films, as this can influence consumer perception and acceptance of food products. Moisture Content The incorporation of LEO showed a significant increase in the moisture content of LEO-treated films (Table 3). Similar results in EO-treated films were reported by Wang et al. (2020), Utami et al. (2019), Hossenei et al. (2009), Resianingrum et al. (2016), and do Evangelo et al. (2019). The increase in moisture content of films treated with EO can be attributed to the formation of porous structures that facilitate the entry of water molecules between polymer chains (do Evangelo et al., 2019). This allows water molecules to accumulate and deposit between polymer chains via hydrogen bonding. The wide difference in molecular weight of plasticizer and starch can also cause an increase in moisture content, as greater gaps between molecular weights make for bigger space between molecules that can be occupied by water molecules. The moisture content of films is an important parameter that must be measured and reduced to a minimum, as this influences the growth of microorganisms and the shelf life of food products. Antibacterial activity of films In this study, the disk diffusion assay was prepared to evaluate the antimicrobial activity of cornstarch-based films incorporated with LEO against S. aureus and S. typhimurium (Figure 2). The microbial inhibition effect of the films is indicated by the clear zone formed around the films, with a wider clear zone suggesting a stronger antimicrobial activity. Davis and Stout (1971), as cited by Resianingrum et al. (2016), categorized the inhibition power criteria in terms of the diameter of inhibition zone as follows: very strong inhibition (≥ 20 mm), strong (10-20 mm), medium (5-10 mm), and weak (≤ 5 mm). Based on these criteria, the control and all LEO-treated films exhibit very strong antibacterial activity against both S. aureus and S. typhimurium, where every inhibition zone is more than 20 mm (Table 4). One important finding is that the control film possesses strong inherent antimicrobial property as indicated by sizable inhibition zones (32.78 ± 4.60 mm for S. aureus; 29.33 ± 1.94 mm for S. typhimurium). This outcome may be attributed to the glycerol and vinegar in the film formulation. Nevertheless, the inhibition zones of the control and LEO-treated films do not differ statistically from each other. It may be noted, however, that the inhibition zone diameter of films with 2% LEO against S. aureus is considerably higher than those of the other films. 59 Philippine e-Journal for Applied Research and Development Website: pejard.slu.edu.ph ISSN 2449-3694 (Online) Philippine e-Journal for Applied Research and Development 15(2025), 51-61 Both glycerol and vinegar possess antimicrobial properties. In Schlievert and Peterson’s (2012) study on the antibacterial activity of glycerol monolaurate in broth and biofilm cultures, it was reported that glycerol monolaurate has potent activity against Grampositive bacteria including S. aureus, and is a bactericidal for a wide range of potential bacterial pathogens. In a study wherein glycerol is a component of the film formulation, Utami et al. (2019) reported antimicrobial activity in tapiocabased edible films when cinnamon essential oil was not incorporated in the film. On the other hand, vinegar is rich in phenolic compounds and is well known to have antioxidant and antimicrobial potential. Bakir et al. (2017) tested the antimicrobial activity of 18 vinegar samples against Gram-negative S. typhimurium, E. coli and S. aureus, and reported that all samples exhibited antibacterial activity against these bacteria with zones of inhibition ranging from 8.58 to 15.81 mm. The LEO-treated films tended to have a slightly larger zone of inhibition against the Gram-positive bacteria (S. Aureus) compared with the Gram-negative bacteria (S. typhimurium). A similar outcome was observed by Wang et al. (2020), wherein the inhibitory zones of films incorporated with Zanthoxylum bungeanum essential oil against Gram-positive bacteria (S. aureus and L. monocytogenes) were larger than those against Gram-negative bacteria (E. coli). The antibacterial activity of LEO is due to an interaction between the main oil constituents and the bacterial cell membrane. LEO contains lipophilic terpenes that can change the fluidity and permeability of microbial membrane or change the intracellular pH and ATP concentrations, which result in cell rupture that inhibits microbial growth (Majewska et al., 2019). The outer membrane of Gram-negative bacteria contains lipopolysaccharide molecules that limit the diffusion of hydrophobic compounds. In contrast, Gram-positive bacteria have a thick peptidoglycan layer, which can act as preventive barrier to certain essential oils (Burt, 2004, as cited by Wang et al., 2020). Limitations and future directions In this study, the transparency of film was indirectly measured by determining the whiteness index (WI). WI was used to measure the color shift and departure from whiteness brought on by the addition of the yellowish LEO. More accurate measurements of transparency can be done by measuring luminous transmittance and haze, typically with ASTM D1003. Other than the determination of film transparency, the following activities are suggested for future study: measurement of barrier properties (i.e., water vapor and oxygen transmission rate); morphological characterization via Scanning Electron Microscopy (SEM) to visualize oil droplet dispersion; quantitative antimicrobial assessment using broth microdilution (MIC) assays; sensory evaluation of the film’s impact on packaged food products; and, assessment of the film’s biodegradability. Conclusion Lemongrass essential oil (LEO) was successfully incorporated into cornstarch-based films. The effect of LEO on the mechanical and physical properties of the films depends on the amount incorporated in the film, e.g., tensile strength and flexibility increased at 0.5% LEO but decreased at higher LEO treatments. The base film exhibits potent inherent antimicrobial activity due to glycerol and vinegar in its formulation. This already strong activity was only slightly enhanced by the addition of LEO. This outcome was probably influenced by the disk diffusion assay’s limitations for hydrophobic compounds. References ASTM D882 (Standard Test Method for Tensile Properties of Thin Plastic Sheeting). (2010). ASTM International. West Conshohocken, PA. ASTM D618 (Standard Practice for Conditioning Plastics for Testing). (2021). ASTM International. West Conshohocken, PA. Atares, L. & Chiralt, A. (2016). Essential oils as