Fresh tapioca was procured from the local market in Coimbatore, Tamil Nadu, India, for starch extraction. Basil leaves were collected from nearby farmsin and around Coimbatore.

Glycerol, carboxy methyl cellulose (CMC; with sodium salt), and food-grade citric acid (CA; 95%−97% purity) were sourced from Sigma-Aldrich in Coimbatore for the formulationof edible films. Glycerol was used as the plasticizer, CMC as filler and CA as crosslinker to enhance the functional properties of the tapioca film as active edible film.

Tapioca starch was extracted using a wet extraction technique according to the procedure demonstrated by Shao et al. (2023) and Amaraweera et al. (2021). The fresh tapioca was peeled, chopped into pieces and ground in industrial wet blender. Then, washed with 10 times of water to the quantity of pulp, stirred it for 5 min and then, filtered through triple-layered sieve (0.03 mm mesh). Again, the filtrate was allowed to settle down by adding five times of water (to the quantity of filtrate), and the white mass was collected carefully by decanting. The collected starch was dried at 65°C for 5 h in hot air oven (Technico) and the moisture content was below 12%.

Basil leaves were cleaned, shade-dried, and pulverized in a blender. Powdered basil leaves (50 g) were soaked overnight in 250 ml ethanol (99.9% analytical grade ethanol, China Make) in a beaker. Extraction was performed using a Soxhlet apparatus with ethanol as the solvent. The extraction process was conducted at 80°C for 2 h and repeated four times. The extracts were collected and allowed to stand at room temperature to facilitate the evaporation of the residual solvent. The beaker was then weighed to determine the final weight of the extract. The extract was filtered through Whatman No. 1 filter paper, and the filtrate was stored in dark bottles at 4°C for preservation (Romano et al., 2022). The extracted basil oil (BO) is used in tapioca starch films as additive to enhance the properties.

Tapioca-based edible films were standardized using a patented film-making machine (patent no. 480705, Indian Patent, dated 12th December 2023), designed by the Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore, Tamil Nadu, India. Initially, 10 g of tapioca starchand 2 ml of glycerol, serving as the matrix and plasticizer, respectively, were dissolved in 290 ml of water and heated to 100°C for 10–12 min to form a clear, transparent casting solution. To enhance and optimize the active film composition, various concentrations of citric acid (0.1–1 g), carboxymethyl cellulose (0.1–1 g), and extracted basil oil (1–5 ml) were tried and the best proportion was selected (based on visual appearance, stretchability, thickness, seal-ability and surface smoothness) and evaluated for its sensory attributes, structural integrity, and barrier properties. The standardized formulaton was: 0.1 g of food-grade citric acid as a cross-linker, carboxymethyl cellulose (0.5 g) as a filler, and 1 ml of extracted oil from shade-dried basil leaves as an additive. The film casting solution was then kept undisturbed for 15 min to remove air bubbles, and the solution was casted onto the conveyor belt of the film-making machine, where an adjustable thickness scale ensured the uniform spreading of the solution. The film was passed through three heating coils set at controlled temperatures of 70, 65, and 55°C, moving at a speed of 0.5–1.5 rpm in a closed chamber for 4 h. Then, the dried film was kept undisturbed for 7 min in the end of conveyor belt (out of heating coils), to reach ambient room temperature, allowing for easy peeling (from the conveyor belt) without cracking. Attemptingto peel the film while it was still at 55°C, leads to cracking of film.

The tapioca starch film (TSF) refers to filmsstandardized withtapioca starch and glycerol. Subsequently, to improve the film properties, carboxymethyl cellulose (CMC) was added to TSF resultng in TSF-CMC formulation. Further, the incorporation of citric acid (CA) to TSF-CMC to standardizethe TSF-CMC-CA film. To further enhance the propeerties, basil oil extract (BO) to TSF-CMC-CA was added to form TSF-CMC-CA-BO film.

Smooth, dry starch films were analyzed using a 3D Optical Profilometer at a Z range of 404 μm, 200 steps, and a step size of 2.08 μm. The analysis, conducted at a resolution of 95 μm × 71 μm, revealed the surface patterns and trends critical for film characterization.

Non-perishable food products were packed in edible films, and weight changes were monitored for 120 days at 5-day intervals and stored at room temperature (28°C). The ingredients selected for the studyincludes non-perishable food items from each category, such as cereals, pulses, spices, condiments, and extruded products. Weight change refers to the moisture and oxygen absorption by the standardized filmsfrom the environment. For the study, 10 g of rice, ragi, bengal gram dhal, salt, sugar, spiral pasta were taken in 10 g and 5 g of ragi flour, chili powder, cumin seeds, tea powder, marshmellows, and candies (sweetened and flavored candies, cumin candies) wereweighed. Then the weighed food items were sealed (a single press) at 80–90°C using a sealing machine (Ekavir home scale sealing machine).

The toxicitywas determined using brine shrimp lethality assay (BSLA) for all the four standardized films (TSF, TSF-CMC, TSF-CMC-CA, and TSF-CMC-CA-BO). For each film, a stock solution was prepared by weighing 1 mg of the film that was dissolved in 1 ml of distilled water. Stock solutions were prepared at a concentrations of 100, 250, 500, 1,000, and 1,500 μl by adding saline solution. Thirty shrimps were introduced in 25 ml of saline solution and the mortality rate was observed at 2, 4, 6, and 24 h. Potassium dichromate (K₂Cr₂O₇) (1 mg/ml) was used as a positive control, and a brine solution without sample was used as the blank. The mortality rate of brine shrimp was estimated using the number of dead shrimp during a set period.

The colony-forming units for standardized films were measured using IS 5402 procedures on days 1, 30, 60, 90, and 120 as shelf-life indicators for standardized edible tapioca films (IS 5402, 2012; Makawi et al., 2019) by dissolving one gram of the sample in 9 ml of sterile peptone water (0.1%). Serial dilutions from 10¹ to 10⁹ in duplicate were made using homogenate water with 0.1% peptone and poured into plate count agar. The dilutions were incubated at 35°C for 48 h, and the colonies were counted.

A soil burial test was employed to assess the biodegradability of the films (Ferreira-Filipe et al., 2022). Square-shaped standardized tapioca films, 5 cm in size, weighing 2 g (approx.) were buried in a beaker which contains 100 g of soil which was wetted with 20 ml of waterand stored at room temperature (28°C with 55% humidity) for 24 days to check their biodegradability. Weight changes were recorded every 3 days until the samples were completely degraded. The weight loss percentage was computed by dividing the change in the initial and final weights (g) by the initial weight, multiplied by 100.

All experiments were performed in triplicate, and the results were expressed as the mean value (standard deviation). One-way Analysis of Variance (ANOVA) and Duncan's Multiple-Range Test (p ≤ 0.05) were performed using the Statistical Package for Social Sciences (SPSS) software version 25. Graph was plotted using Microsoft Office Excel, version 2021.

One hundred gram of fresh tapioca resulted in the extraction of 62 g of starch through a decanting technique, leading to a total starch yield of 62%. The physical and chemical properties of tapioca starch andactive films, highlighting asignificant difference (p < 0.05) in moisture, ash content, and pH values are shown in Table 1. Tapioca starch exhibited the highest moisture content (8.12%), the lowest ash content (1.99%), and a mildly acidic pH of 5.45. The edible tapioca film, measuring, 30 cm × 21 cm × 0.25 mm (length × breadth × thickness) was standardized and optimized from the standardized proportions. The tapioca starch film (TSF) showed a reduced moisture content of 5.51%, an increased ash content of 2.34%, and a slight increase in pH to 5.79 compared to the raw starch. Incorporating carboxymethyl cellulose (CMC) further decreased the moisture content to 4.32%, increased the ash content to 2.7%, and brought the pH closer to neutrality at 6.21. The addition of citric acid (TSF-CMC-CA) lowered moisture to 3.50%, raised ash to 2.86%, and resulted in a pH of 5.74. The combination of basil oil, citric acid, and CMC in the starch film (TSF-CMC-CA-BO) achieved the lowest moisture content (3.47%), the highest ash content (2.98%), and the most neutral pH of 6.32. Increasing the concentration of cross-linker, filler and additive resulted in a poor physical properties and deformed films. Supporting these findings, Ratnaduhita et al. (2021) reported that tapioca starch contains 9.32% moisture and 0.09% ash content. In contrast, Kaur et al. (2023) found that starch extracted from Curcuma angustifolia rhizomes had moisture and ash contents of 5.9 and 0.5%, respectively. Furthermore, Pérez-Vergara et al. (2020) indicated that the moisture content of native cassava starch used in beeswax and plasticizer-based films ranged between 8.60 and 20%, with corresponding ash percentages.

Figure 1 shows the surface morphology of tapioca starch film through a 3D optical profilometer. The grayscale variations indicate differences in height or depth across the surface. Lighter areas represent higher elevations, and darker areas indicate lower elevations. It shows an uneven and textured surface with peaks and valleys, highlighting the topographical features of the starch film. The microscopic view illustrates the composition and structure of the film, allowing for an analysis of its properties and characteristics. The surface roughness of the tapioca film is observable through the differences in color intensity. The imaging was conducted at a field of view measuring 95 μm × 71 μm with a step size of 1.37 μm. The 3D imaging reveals that the film has a slight roughness, and the bonding of all the compounds is visible. The average roughness and Z-range value for the tapioca film was 404 μm. The present study aligns with the results of Biswal et al. (2024) reported that the film formulated with starch, carboxymethylcellulose, and glycerol exhibited a uniform microstructure, showing no signs of phase separation among its components. Similarly, Kaur et al. (2023) found that the starch film displayed an ordered, compact, and homogeneous structure.

The exposure test of the tapioca film for packaging various dry food ingredients was assessed over a 120-day storage period, and the results are tabulated in Table 4 and Figure 2. Across all samples, the films retained slightly higher weights than their initial values, suggesting minimal moisture absorption. Films containing basil oil (TSF-CMC-CA-BO) exhibited slightly lower weights, likely due to the volatile nature of essential oils. Among the packed ingredients, rice and pasta showed high weight retention, while whole and ground ragi exhibited moderate interaction with the films. Salt and sugar resulted in notable variations, with salt contributing to higher weight retention, possibly due to moisture absorption. Spices such as chili powder, cumin, and tea powder exhibited the lowest weight retention, indicating stronger interactions with the films.

Tapioca starch films, particularly those enhanced with fillers, crosslinkers, and additives such as carboxymethylcellulose, citric acid, and basil oil, demonstrated good stability and strong potential for eco-friendly, biodegradable, and edible packaging. The films proved suitable for packing a variety of dry food products, including cereals, pulses, spices, sugar, and salt in both whole and powdered forms. Additionally, candies, marshmallows, and sugar confectioneries wrapped in these edible films showed minimal weight gain, remaining within a 0.3 g increase over 120 days. The 120-day exposure test confirmed that tapioca starch films are effective for packaging non-perishable foods, including cereals, pulses, spices, condiments, extruded products, and sugar-based confectioneries, making them a promising eco-friendly alternative to conventional packaging materials. The findings of the present study were supported by the studies, Otero-Herrera et al. (2025) found that, sweet potato starch films were suitable for packing candies. Roidoung et al. (2025) stated that, tapioca starch was heat sealable and suitable for sachet packaging.

The Brine Shrimp Lethality Assay was conducted to assess the toxicity of standardized active tapioca films with fillers, additives, and crosslinkers (Table 5). The findings showed that all tested film formulations displayed low toxicity when compared to potassium dichromate (K₂Cr₂O₇) and the blank (brine solution) has not exhibited any mortality till 24 h. At a concentration of 1,000 μl, the mortality rate of brine shrimp remained low across all samples, with a maximum of two deaths recorded in the formulation containing starch, glycerol, carboxymethyl cellulose, citric acid, and basil oil (TSF-CMC-CA-BO). After 24 h, the highest recorded mortality rate was only 7%, indicating that the films are largely non-toxic. The low lethality rate of the developed tapioca films indicates their non-toxic nature, reinforcing their suitability as biodegradable and eco-friendly alternatives to conventional plastic-based food packaging. This aligns with the findings of Lazim et al. (2021), which state that D. pentaphylla starch is non-toxic and biocompatible, as its LC50 value is below the toxicity threshold of 29.27 mg/ml in a zebrafish embryo study.

The microbial growth assessment of tapioca starch-based films over a period of 120 days revealed that the incorporation of crosslinkers (such as citric acid) and additives (like basil oil) effectively reduced microbial proliferation. On the first day, no colony-forming units (CFUs) were detected in any of the four standardized films. By day 120, the CFU counts varied among the formulations: the tapioca starch film (TSF) exhibited 5.7 × 10¹ CFU/g, TSF with carboxymethyl cellulose (TSF–CMC) had 4.1 × 10¹ CFU/g, TSF combined with citric acid (TSF-CMC-CA) recorded 3.7 × 10¹ CFU/g, and the formula with both citric acid and basil oil (TSF-CMC-CA-BO) showed the lowest count, 3.1 × 10¹ CFU/g. The findings of the present study are consistent with previous research, Pérez-Vergara et al. (2020), which showed that tapioca starch films containing beeswax and ethanolic propolis extract had strong antifungal activity against Aspergillus niger, creating an inhibition zone of about 25 mm. It confirms that adding bioactive compounds to starch films can improve their antimicrobial properties. Kaur et al. (2023) found that crosslinking with organic acids led to stronger antimicrobial activity than films without these acids. On the other hand, Lazim et al. (2021) reported that Dioscorea pentaphylla starch did not show significant antibacterial activity against E. coli, S. aureus, S. cerevisiae, and S. typhi within 24 h. And also suggests that the antimicrobial effectiveness of starch films depends on the specific additives used. Combining citric acid and Basil leaf oil is important in reducing microbial count in tapioca films.

The biodegradability study, using a soil burial test, shows that all the tapioca films break down effectively over time. Their weight decreases gradually, with complete degradation occurring by Day 24 as shown in Table 6. The tapioca starch film (TSF) degraded the fastest, leaving no residue by Day 20. The TSF with carboxymethylcellulose (TSF-CMC) followed a similar pattern but took slightly longer to degrade. Films containing citric acid (TSF-CMC-CA) and basil oil (TSF-CMC-CA-BO) were more stable at first, leaving small residues (0.02 g) by Day 20, but they fully degraded by Day 24. The enhanced structural stability of the films with citric acid and basil oil can be attributed to crosslinking, which delays the degradation process. The biodegradability of tapioca films fortified with fillers, crosslinkers, and additives shows complete degradation within 24 days, confirming their status as environmentally friendly and suitable for sustainable packaging applications. An earlier study by Amaraweera et al. (2021) noted that 58% of the total weight loss occurred by the 15 day in native tapioca starch thin films. Meanwhile, Biswal et al. (2024) reported that a film standardized with starch, carboxymethylcellulose, and glycerol degraded by 32% within seven days. Additionally, Kaur et al. (2023) indicated that weight loss during film degradation is due to soil bacteria breaking down starch, resulting in the production of ethanol and methane through fermentation. They also noted that water sorption and crosslinking modify the water absorption properties, which delays degradation.