Bambara Groundnut Pericarp–Derived Carbon Dots: Reinforcement of Gelatin/PLA Bilayer Films and Their Function as Preservative in Packed Asian Seabass Slices

ABSTRACT

Conversion of agro‐waste into carbon dots (CDs) transforms biomass into sustainable nanomaterials, enabling advanced applications in packaging and ensuring food safety. Bambara groundnut pericarp powder (BGPP) was hydrothermally processed at 200°C for varying durations (3–12 h) to synthesize BGP‐CDs. BGP‐CDs exhibited differences in size (1.34–15.02 nm), shape, and chemical composition, as characterized by transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) analyses. All samples exhibited excellent optical properties, including photoluminescence and UV barrier capability. Notably, BGP‐CDs/3 (synthesized for 3 h) at 100 µg/mL demonstrated the highest UV‐blocking efficiency against UV‐A (35.62%) and UV‐B (92.64%) lights. BGP‐CDs synthesized for 12 h exhibited remarkable antimicrobial activities against selected species (Escherichia coli, Listeria monocytogenes, Aspergillus flavus, and Aspergillus parasiticus) due to their nanoscale size; however, their reduced size negatively impacted human cell viability. Antioxidant activity was highest in BGP‐CDs synthesized for 6 h (BGP‐CDs/6), as determined by different assays. Incorporating BGP‐CDs/6 (4%, w/w) into gelatin/polylactic acid (PLA) bilayer film enhanced UV and water barrier properties, antioxidant capacity, and antibacterial efficacy compared to the control film. Additionally, Asian seabass slices (AS‐S) packaged in pouches made from active bilayer film had the maintained color with negligible pH change. Microbial growth and lipid oxidation in AS‐S were retarded as indicated by the lower rise in total viable count (TVC) and psychrophilic bacterial count (PBC) as well as thiobarbituric acid reactive substances (TBARS) values over 12 days of storage at 4°C. Thus, the developed multifunctional packaging film offers an innovative strategy that could impede the quality loss of perishable fish slices.

1. Introduction

Circular bioeconomy is increasingly recognized for its role in transforming waste into value‐added products for sustainability of natural resources (Ajayi and Lateef 2023). Among the numerous resources, agri‐food waste and by‐products are commonly found during crop processing. These accessible materials can be converted into valuable products (Baraketi and Khwaldia 2024). Various marketable products from agricultural wastes, including bioenergy, organic acids, biopolymers, single‐cell proteins, nutraceuticals, and bioactive substances, are launched annually (Capanoglu et al. 2022). Among these, carbon dots (CDs) derived from agro‐waste can be of value‐added products via potential valorization of leftovers (Murugan et al. 2024a). These nanoscale CDs (<10 nm) with unique photoluminescence (PL), high water solubility, biocompatibility, and low toxicity make them superior to traditional quantum dots (Kousheh et al. 2020). Their strong surface interactions enable wider applications in food packaging as active fillers with antioxidant, antimicrobial, and UV‐blocking properties. CDs can be employed in biomedical fields like bioimaging, biosensing, and drug delivery (Ghosal and Ghosh 2019).

Processsing of bambara groundnut (Voandzeia subterranea), cultivated in sub‐Saharan Africa and Southern Thailand (Agboeze et al. 2022; Palamae et al. 2024), generates significant agro‐waste, including pericarp and seed coats, which are often discarded, burned, or buried, contributing to environmental pollution (Ajayi and Lateef 2023). With the rising demand for nuts as functional foods, valorizing these by‐products for sustainable applications is crucial (Klompong and Benjakul 2015). Groundnut pericarp, rich in carbonaceous substances, can be potentially utilized for synthesizing CDs, providing an eco‐friendly approach to waste management. These CDs can serve as the active components in food packaging, combating the drawback of biopolymers and enabling their use as active packaging materials.

Gelatin has been used in food and packaging due to its high glycine, proline, and hydroxyproline contents, yielding flexible biodegradable film (Wang et al. 2021). It serves as an excellent oxygen barrier property but has poor water resistance and mechanical properties, likely owing to its hydrophilicity. These limitations can be resolved by incorporating polymers, plant extracts, or additives (Murugan et al. 2023). Poly(lactic acid) (PLA) is a biodegradable, biocompatible thermoplastic polyester with superior mechanical strength, hydrophobicity, and thermal plasticity (Murugan et al. 2023) commonly used in packaging, biomedical and agricultural fields. However, its brittleness and low toughness limit its performance (Yeh et al. 2009). Blending PLA with flexible gelatin offers a cost‐effective solution to enhance flexibility and sealability, thus widening its applicability in various fields. Lamination/coating enhances the property of single‐polymer films by integrating multiple layers. As a consequence, their unique properties can be combined for improved protection and durability (Alias et al. 2022).

Conventional films with excellent barrier properties are insufficient for optimal product preservation. Enhancement of film functionality and bioactivity is crucial and can be achieved through the incorporation of nano‐fillers or natural additives (Mir et al. 2018). Among these, CDs have emerged as beneficial fillers, offering multifunctional benefits to improve the performance of packaging films (Riahi et al. 2025). Incorporation of naturally derived CDs enhanced both the barrier properties and bioactivities of films, which can be used as the potential substitute for commercial plastic packaging (Khan et al. 2023a; Murugan et al. 2024a, 2024b). Although numerous CDs have been produced from natural sources by hydrothermal processes, mainly under specific conditions (200°C for 6 h), those CDs, as active fillers, were mostly incorporated in the blend films and applied for wrapping perishable foods (Khan et al. 2023a, 2023b; Riahi et al. 2024). However, the attention on the use of CDs into bilayer gelatin/PLA films for real food preservation, particularly in the form of bags or pouches, is limited. Moreover, the dehulling of bambara groundnut generates a significant amount of waste (pericarp), which serves as an excellent source for CDs synthesis. Nonetheless, this pericarp has not been exploited. To address this gap, CDs from bambara groundnut pericarp powder (BGPP) were synthesized at 200°C for varying durations (3, 6, 9, and 12 h), and their properties and characteristics were investigated. This is the first report on the incorporation of CDs into gelatin/PLA bilayer films and their usage as pouches for packing Asian seabass slices (AS‐S). The novelty of this work lies in both the waste valorization and the practical application of CDs to enhance film properties and preserve real food products, addressing a significant advancement in sustainable packaging through material innovation and effective food preservation.

2. Materials and Methods

2.1. Fish Sample, Chemicals, and Tested Microorganisms

Fresh AS‐S (Lates calcarifer) were gotten from a local supermarket in Hat Yai, Songkhla province, Thailand. AS‐S were packed in a polyethylene bag, placed on three volumes of crushed ice, and transported to the laboratory. Fish skin gelatin (∼250 bloom) and PLA pellets were procured from Vihn Hoan Corp. (Dong Thap Province, Vietnam) and Nature Work Co. Ltd. (Blair, NE, USA), respectively. All the chemicals for antioxidant assays and thiobarbituric acid reactive substances (TBARS) analysis were supplied by Sigma‐Aldrich Inc. (St. Louis, MO, USA). Chemicals were of analytical grade. All the microbial media were purchased from HiMedia (Mumbai, India).

Escherichia coli (EC) (American Type Culture Collection [ATCC] 25922) and Listeria monocytogenes (LM) (Food Safety Lab [FSL] J1 208) were gifted by the ATCC (Manassas, VA, USA) and FSL, Cornell University (Ithaca, NY, USA), respectively. Fungal strains, namely, Aspergillus flavus (AF) (PSRDC‐4) and Aspergillus parasiticus (AP) (TISTR3276), were obtained from the Thailand Institute of Scientific and Technological Research (Khlong Luang, Pathum Thani, Thailand). Human BJ fibroblasts (ATCC.CRL‐2522), fibroblast basal media (FBM), and fibroblast supplemental growth factors were acquired from the ATCC (Manassas, VA, USA).

2.2. Preparation of BGPP and Its CDs

Bambara groundnuts (V. subterranea) were bought from a local plantation in Narathiwat province, Thailand. Pericarps were separated manually and used for CDs preparation. Pericarps were cleaned using tap water to remove the foreign matter, drained, and dried to possess 3–4% moisture content using a tray dryer (60°C). Dried pericarps were ground and finally sieved (80 mesh). The obtained powder was named “BGPP,” packed in a zip‐lock bag, and kept in a desiccator.

Bambara groundnut pericarp carbon dots (BGP‐CDs) were synthesized from BGPP following a hydrothermal procedure (Murugan et al. 2024a). A suspension of BGPP (2%) in distilled water was stirred intermittently at 25 ± 3°C for 10 min, placed in a Teflon‐lined cylinder, sealed in a stainless‐steel reactor, and subsequently heated in a muffle furnace (200°C) for varying times (3, 6, 9, and 12 h). After cooling to room temperature, the yellow‐brownish mixture was then filtered using Whatman filter paper (100 µm). The filtrate was centrifuged (5000 × g, 15 min). Thereafter, a membrane filter (pore size: 0.22 µm) was used to filter the nano‐sized CDs. The filtrate was kept at 4°C until analyzed. The resulting CDs prepared for 3, 6, 9, and 12 h were named BGP‐CDs/3, BGP‐CDs/6, BGP‐CDs/9, and BGP‐CDs/12, respectively. All CDs were analyzed.

2.3. Analyses

2.3.1. Appearance and Transmission Electron Microscopic (TEM) Images of BGP‐CDs

The visual appearance of BGP‐CDs under normal and UV light exposure was captured by a smartphone camera. The morphology of BGP‐CDs was visualized by field emission transmission electron microscope (Thermo Scientific Co. Ltd., Model: Talos F200i, Waltham, MA, USA) (Khan et al. 2023a).

2.3.2. UV–Vis Spectrophotometric and Spectrofluorometric Spectra

The absorption spectrum was examined by UV–vis spectrophotometer (200–800 nm) (Shimadzu, Model UV‐1800, Kyoto, Japan). Fluorescence spectral measurement of BGP‐CDs solution was done using a spectrofluorometer (Hitachi, Model F‐7100 FL, Tokyo, Japan) at different excitation wavelengths (25°C) (Khan et al. 2024).

2.3.3. Fourier Transform Infrared (FTIR) Spectra

FTIR spectra of BGP‐CDs (freeze‐dried powder) and BGPP were obtained with the aid of a Bruker FTIR spectrometer (Model Equinox 55, Bruker Co., Ettlingen, Germany) (Gulzar et al. 2022). Functional groups and secondary structure were determined in the range of 4000–400 cm⁻¹, in which 32 scans and 4 cm⁻¹ resolution were used. Samples were mixed with KBr at a 1:10 ratio, blended using a mortar and pestle, and compressed into pellets for analysis. OPUS 3.0 data collecting software was utilized to normalize the spectra prior to interpretation.

2.3.4. UV‐Blocking Property

The UV barrier property was examined (Koutchma et al. 2016), and the blocking ability of UV‐A and UV‐B was computed using the following equations (1 and 2):

$$\mathrm{UV}\mathrm{-}\mathrm{A}\mathrm{blocking}\left(\%\right)=100-\frac{{\int }_{320}^{400}T\left(\lambda \right)d\lambda }{{\int }_{320}^{400}d\lambda }$$ (1)

$$\mathrm{UV}\mathrm{-}\mathrm{B}\mathrm{blocking}\left(\%\right)=100-\frac{{\int }_{280}^{320}T\left(\lambda \right)d\lambda }{{\int }_{280}^{320}d\lambda }$$ (2)

where T(λ) is the average transmittance of BGP‐CDs at the wavelength λ and dλ is the bandwidth interval.

2.3.5. Antioxidant Activities

BGP‐CDs at varying concentrations (25–100 µg/mL) were determined for antioxidant activities. ABTS radical scavenging activity (ABTS‐RS‐A), DPPH radical scavenging activity (DPPH‐RS‐A), and ferric reducing antioxidant power (FRA‐P) were examined and reported as µmol Trolox equivalent (TE)/mL sample (Benjakul et al. 2014). Metal chelating activity (MC‐A) was tested (Benjakul et al. 2014) and expressed as µmol EDTA equivalent (EE)/mL sample.

2.3.6. Antimicrobial Activity

Antifungal activities of all CDs at various levels (1–3 mg/mL) against AF and AP were tested (Buatong et al. 2023) in comparison with the control (distilled water). The growth of fungi was expressed in diameter (mm).

The antibacterial efficacy of BGP‐CDs against LM and EC was tested using the total viable colony count (TVCC) method (Ezati et al. 2022; Roy et al. 2019). Bacteria were cultured in tryptic soy broth (TSB) at 37°C for 18–24 h and diluted properly. Subsequently, the inoculum (200 µL) was added to 20 mL of TSB containing 1 mL of BGP‐CDs (3 mg/mL). Samples were incubated (37°C, 12 h) with mild shaking. Viable colonies (Log CFU/mL) were quantified by plating 100 µL of aliquots on tryptic soy agar (TSA) plates at 3 h intervals. A control without CDs was also included.

2.3.7. Cytotoxicity

The cytotoxicity of BGP‐CDs was determined using the MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyl tetrazolium bromide) assay toward BJ dermal fibroblast cells. Cells were cultured in EMEM (Eagle Minimum Essential Medium) containing 10% FBS and 1% antibiotic–antimycotic at 37°C in a 5% CO₂ incubator. After seeding (10⁴ cells/well) in 96‐well plates and incubating for 24 and 48 h, the cells were treated with BGP‐CDs (0.125–1 µg/mL). Following the incubation, MTT solution (10 µL) was added and further incubated for another 4 h. Thereafter, MTT solution was drained and solubilization buffer was introduced. Absorbance at 570 nm of the obtained solution was read using a FLUOstar Omega microplate reader, and cell viability was computed to evaluate cytotoxicity (Saetang et al. 2023).

BGP‐CDs/6 was selected for further study based on the optimal yield, strong bioactivity (antioxidant, antibacterial, and antifungal activities), non‐toxicity to human cells, and uniform nanoscale size. It was incorporated into gelatin/PLA bilayer films for food packaging applications.

2.4. Preparation of Gelatin/PLA Bilayer Films Containing BGP‐CDs

Bilayer films were prepared using PLA as the bottom and gelatin as the top layer at the PLA/gelatin ratio of 40/60 (1.6/2.4 g of PLA and gelatin [4%, w/w of total solid content]) by using the solvent casting method (Murugan et al. 2023). Prior to use, PLA pellets were dried (60°C, 24 h). Then, pellets (1.6%, w/v) were mixed with chloroform, stirred until complete solubilization was achieved, and the solution was named PLA film‐forming solution (P‐FFS). P‐FFS (100 mL) were poured into an aluminum tray (20 × 20 cm²) and dried. Gelatin film‐forming solution (G‐FFS) was attained by dispersing gelatin (2.4%, w/v) in distilled water and incubating at 60°C for 30 min. To plasticize the film, glycerol (25%, w/w of gelatin) was employed. Furthermore, BGP‐CDs/6 (4%, w/w, based on gelatin) was introduced as the active filler into the G‐FFS. G‐FFS (100 mL) without and with BGP‐CDs/6 was cast over the dried PLA film and kept dried for 24 h. Dried films were manually peeled off and kept in an environmental chamber (E‐C) (50 ± 5% RH at 25 ± 0.5°C) for 48 h. The resulting films were referred to as ‘“GP” and “GP‐CD” (containing BGP‐CDs/6 at the level of 0 and 4%, respectively). On the basis of the different exposure sides, PLA sides (PG and PG‐CD) and gelatin sides (GP and GP‐CD) of the films were named differently. Films were further analyzed for barrier properties and bioactivities.

2.4.1. Appearance and Water Vapor Permeability (WVP) of Films

Photographs of all films (both sides) were captured with the aid of a smartphone camera. WVP of films was evaluated by placing film samples over the aluminum permeation cup filled with dried silica before sealing, in which both sides were faced to the cup (Murugan et al. 2023). After keeping it in the E‐C (25 ± 0.5°C; 50% RH), the weight of the cup was measured (Equation 3), and the calculation of WVP was done every 1 h up to 10 h:

$$\mathrm{WVP}\left(\mathrm{g}\mathrm{m}{\mathrm{m}}^{-2}{\mathrm{s}}^{-1}\mathrm{P}{\mathrm{a}}^{-1}\right)=wl{A}^{-1}{t}^{-1}{(P_2-P_1)}^{-1}$$ (3)

where w stands for the weight gain of the cup (g); ldenotes the film thickness (m); A represents the exposed film area (m²); t is the time of gain (s); (P ₂ − P ₁) stands for vapor pressure difference across the film (Pa).

2.4.2. Color and Optical Properties

Color of films was measured with the aid of a colorimeter (Hunterlab, Reston, VA, USA) (Murugan et al. 2024a). L* (lightness), a* (redness or greenness), and b* (yellowness or blueness) values were determined. ΔE* (total color difference) was computed (Equation 4):

$$\mathrm{\Delta }{E}^{\ast }=\sqrt{{(\mathrm{\Delta }{L}^{\ast })}^2+{(\mathrm{\Delta }{a}^{\ast })}^2+{(\mathrm{\Delta }{b}^{\ast })}^2}$$ (4)

where ΔL* (lightness), Δa*, and Δb* are the differences of each parameter in comparison with the corresponding values of a white standard plate.

The opaqueness value was computed (Murugan et al. 2024a) using Equation 5 and expressed as mm^−1.

$$\mathrm{Opaqueness}\mathrm{value}=\frac{\log T_{600}}{X}$$ (5)

where T ₆₀₀ and X represent fractional transmittance at 600 nm and film thickness (mm), respectively.

The UV barrier property was also examined (Section 2.3.4).

2.4.3. Thickness and Mechanical Properties

Film thickness was measured using a digital micrometer (Mitutoyo Corp., Kawasaki‐shi, Japan). Films were subjected to tensile testing using the Universal Testing Machine (Lloyd Instruments, Hampshire, UK). Young's modulus (YM), tensile strength (TS), and elongation at break (EAB) of the films were tested and recorded (Khan et al. 2023a).

2.4.4. ATR–FTIR Spectra

FTIR spectra of all films were analyzed in the range of 500–4000 cm⁻¹ using 32 scans and 4 cm⁻¹ resolution (Murugan et al. 2024a). Spectra were normalized and recorded.

2.4.5. Antioxidant and Antibacterial Activities

To 80% methanol (20 mL), films cut into small pieces (0.2 g) were mixed and stirred continuously for 16 h at room temperature (28–30°C) using a magnetic stirrer. The mixture was centrifuged (8500 × g, 15 min), and the resulting supernatants were determined for DPPH‐RS‐A, ABTS‐RS‐A, and FRA‐P as previously described. Activities were expressed as µmol TE/10 g sample. MC‐A was also determined and recorded as µmol EE/10 g sample (Murugan et al. 2024a).

The antibacterial efficacies of all films against LM and EC were analyzed using TVCC protocol (Roy et al. 2021). All film samples were irradiated using UV light for 15 min before the analyses. TVCC in the absence and presence of different films was monitored every 3 h for up to 12 h.

2.5. Effects of Gelatin/PLA Bilayer Film Pouch Containing BGP‐CDs on Shelf‐Life Extension of AS‐S

AS‐S (5 g) were packaged in pouches (50 × 50 mm²) made of GP‐based bilayer films, both GP and GP‐CD, where gelatin film was located inside the pouch (contact with the AS‐S). After the air in the pouches was manually removed, the pouches containing AS‐S were heat‐sealed. All samples were stored at 4°C for 12 days. The control samples were kept on the polystyrene tray and covered with shrink film. During storage, all parameters were determined at 3‐day intervals, for up to 12 days, except color, which was recorded on days 0, 6, and 12.

The pH of AS‐S homogenate (2:10, w/v) was determined using a pH meter. The TBARS value of the AS‐S samples was also determined. A standard curve of malonaldehyde (MDA) prepared from 1,1,3,3‐tetramethoxypropane (0–10 mg/L) was used. The values were expressed as mg MDA equivalent per kg of sample (Ahmad et al. 2024).

Total viable count (TVC) and psychrophilic bacterial count (PBC) were determined. AS‐S (5 g) was homogenized with 45 mL of peptone water (0.85%), serially diluted, and plated on sterile petri dishes. TVC was incubated at 37°C for 24–48 h, whereas PBC was incubated at 4°C for 7 days (Arfat et al. 2015). Colonies were counted and expressed as Log CFU/g.

2.6. Statistical Analyses

Experiments and analyses were done in triplicate. Three different lots of pericarp powder were used for CDs preparation. Completely randomized design (CRD) was applied for all studies. Data were analyzed using the SPSS package (SPSS 27.0 for Windows, SPSS Inc., Chicago, IL, USA) and reported as mean ± standard deviation (SD). One‐way analysis of variance (ANOVA) was done, and Duncan's multiple range test (DMRT) was employed to compare the means.

3. Results and Discussion

3.1. Characteristics and Bioactivities of BGP‐CDs

3.1.1. Appearance

BGP‐CDs were synthesized successfully by the hydrothermal method. The resultant CDs were yellowish brown in color (Figure 1a), but the intensity varied with the reaction time used. The yield of CDs (3.8–4.3 mg/mL) was slightly varied. The yellowish‐brown color of CDs arose from hydrothermal effect, which enhanced conjugation and light absorption. In addition, surface functional groups, especially oxygenated groups, affected light scattering property, whereas larger CDs particles absorbed lower‐energy light. Furthermore, broad UV–vis absorption influenced color perception. It was noted that all BGP‐CDs samples exhibited fluorescence under UV light exposure because of quantum confinement (Figure 1b), surface functional groups, emissive traps, and π–π*/n–π* transitions, thereby enabling excitation‐dependent emission (Miao et al. 2018). Unlike non‐fluorescent distilled water, CDs’ structural defects and conjugated aromatic systems enhanced light absorption and visible emission, thus creating their distinctive glow. Both UV and sunlight, therefore, influenced the color and fluorescent behavior of the resulting BGP‐CDs.

FIGURE 1.

Photographs under normal light (a) and UV light exposure of distilled water and BGP‐CDs. Numbers following BGP‐CDs/ (3, 6, 9, and 12) represent the various synthesis time of BGP‐CDs (3, 6, 9, and 12 h). BGP‐CDs, bambara groundnut pericarp carbon dots.

3.1.2. TEM Images

TEM images revealed that the obtained BGP‐CDs fell within the quantum range (< 10 nm), except for BGP‐CDs/3. BGP‐CDs/3 had the sizes of 15.02 ± 7.31 nm. All BGP‐CDs were spherical and monodispersed. However, BGP‐CDs/3 had a non‐linear structure and differences in size (Figure 2a). It was postulated that the synthesis time of 3 h was not sufficient to obtain homogeneity of CDs. BGP‐CDs/6, BGP‐CDs/9, and BGP‐CDs/12 h had the sizes of 2.28 ± 0.26, 1.46 ± 0.20, and 1.34 ± 0.05 nm, respectively. BGP‐CDs/6, BGP‐CD/9, and BGP‐CD/12 samples did not have any obvious agglomerations. This was different from BGP‐CDs/3. The size of the CDs was reduced with increasing reaction time. Insufficient pyrolysis temperature and time might hinder the full modification of the carbon source into CDs (BGP‐CDs/3). Conversely, excessively high temperatures could lead to over‐oxidation, thereby damaging the surface structure of CDs. This might affect the optical properties (Kang et al. 2020). The result indicated that the temperature had profound influences on the size and uniformity of the CDs. The higher temperatures could promote more nucleation sites, break larger structures, and prevent excessive aggregation, thus resulting in uniform CDs with smaller sizes.

FIGURE 2.

TEM images (a), UV–vis absorption spectrum (b), fluorescence excitation and emission spectra (c), 3D‐fluorescence responses at different excitation wavelengths (200–400 nm) and emission wavelengths (300–700 nm) (d), and FTIR (e) of BGP‐CDs. Key: see Figure 1 caption. BGPP, Bambara groundnut pericarp powder.

3.1.3. UV–Vis Spectrophotometric and Spectrofluorometric Spectra

The optical properties of BGP‐CDs in aqueous dispersions were extensively analyzed through UV–vis absorption, excitation, and emission spectroscopy (Figure 2b–d). Different BGP‐CDs showed UV absorption peaks (Figure 2b) in various regions, which appeared at, 279 nm (BGP‐CDs/3, BGP‐CDs/6, and BGP‐CDs/9) and 277 nm (BGP‐CDs/12). Generally, the peaks detected between 200 and 300 nm denoted the π–π* (sp² aromatic/alkenyl C═C bond). The peak at 270–280 nm indicated the effective hydrothermal modification of BGPP and conversion to BGP‐CDs. The observed peaks between 277 and 279 nm had varying intensity and broadness, which was plausibly caused by the hydrothermal method. The similar variation was observed for 4‐aminoantipyrine CDs synthesized for different reaction times at 180°C (Mohammed et al. 2023). This phenomenon was plausibly caused by the diverse functional groups on the CDs surface, highlighting their crucial influence on optical properties (Sasikumar et al. 2024).

In spectrofluorometric spectra, BGP‐CDs/3 showed the maximum excitation (EX) and emission (EM) at 312 and 384 nm, respectively (Figure 2c). By increasing the time of synthesis, CDs fluorometric spectra exhibited the variations in wavelength. Maximum EX was found at 320 nm for the remaining BGP‐CDs, whereas maximum EM was recorded at 402 (BGP‐CDs/6) and 406 nm (BGP‐CDs/9 and BGP‐CDs/12), as depicted in Figure 2d (3D spectra). On the basis of the maximum EM and EX of all samples, the redness in the spectra denoted the maximum wavelength. The slight differences in EM and EX were related with the hydrothermal synthesis of CDs for various times. Extended reaction time led to over‐hydrothermal process, damaging the CDs surface, whereas short reaction time produced CDs with weak fluorescence owing to incomplete hydrothermal modification of the carbon source (Kang et al. 2020). The PL of CDs is influenced by multiple factors, including surface functional groups, carbon excitations, emissive traps, size effects, aromatic structures, edge defects, and free zigzag sites (Miao et al. 2018). With increasing temperature, their fluorescence intensity began to decrease as reported by previous documentation (Liu et al. 2025). This was mostly due to the formation of a carbon nucleus at a specific temperature. Surface groups, such as hydroxyl, carboxyl, and amine, influence the electronic transitions, whereas carbon excitations contribute to light emission. Emissive traps caused by structural defects affect energy levels, and the size effect determines quantum confinement, altering emission wavelengths (Kang and Lee 2019). Aromatic structures enhance conjugation and have an impact on fluorescence intensity, whereas edge defects and zigzag sites create localized energy states. CDs’ properties were tunable, depending on the precursor, synthesis time, method, and temperature (Liu 2020). Despite these insights, the exact mechanisms governing CDs’ PL behavior remain uncertain and continue to be an active area of investigation (Shabbir et al. 2023). Thus, the presence of fluorescence properties made them a promising alternative for future applications, particularly in bioimaging and sensing areas.

FIGURE 3.

UV–vis light transmittance spectra (a) and % UV‐A (320–400 nm) and % UV‐B (280–320 nm) blocking properties (b) of BGP‐CDs at various concentrations (25–100 µg/mL). Vertical bars represent the standard deviation (n = 3). For each UV light source, different lowercase letters on the bars within the same concentration of different CDs indicate significant difference (p < 0.05). Key: see Figure 1 caption.

3.1.4. FTIR Spectra

FTIR spectra of BGP‐CDs synthesized for various times and BGPP are shown in Figure 2e. Broad peaks observed for all samples at the wavenumbers of 3414 (BGPP), 3390 (BGP‐CDs/3), 3394 (BGP‐CDs/6), 3314 (BGP‐CDs/9), and 3316 cm⁻¹ (BGP‐CDs/12) represent the stretching vibration of –OH group (Santos et al. 2020). The CH asymmetric stretching vibrations of aliphatic groups were associated with the wavenumbers of 2924 cm⁻¹ for BGPP and 2934 cm⁻¹ for all BGP‐CDs (Amaral et al. 2020). Peak detected at 2373–2375 cm⁻¹ was caused by gaseous or physically adsorbed CO₂ (Tuerhong et al. 2023). C–H bending at 1737 and 1768 cm⁻¹ for BGPP and BGP‐CDs confirmed the occurrence of aromatic compounds (Pandhi et al. 2022). An absorption band (C═O) was recorded at 1628 (BGPP) and 1600–1602 cm⁻¹ (BGP‐CDs), which was associated with vibration in the stretch characteristic of aromatics (Santos et al. 2020). The absorption peaks around ∼1740–1630 cm⁻¹ recorded for green tea CDs (GT‐CDs) were mostly ascribed to the C═O/C═C bending vibrations (Khan et al. 2023a). Moreover, due to the hydrothermal effect, the presence of C═O stretching vibrations was found for BGP‐CDs/3 and BGP‐CDs/6 at 1664 cm⁻¹, which was merged together with peak 1600–1601 cm⁻¹ for BGP‐CDs/9 and BGP‐CDs/12. Distinctive peaks recorded at 1512–1520 cm⁻¹ for all the samples denoted the stretching bands of C═C (Amaral et al. 2020). Peaks recorded at 1441 and 1400–1403 cm⁻¹ for BGPP and BGP‐CDs were owing to the antisymmetric in‐plane bending of CH₃ group (Eevera et al. 2023). Bending symmetric vibration of C–H bonds of CH₃ groups was recorded at 1377 cm⁻¹ for BGPP (Eevera et al. 2023), which disappeared in BGP‐CDs samples, plausibly due to the hydrothermal synthesization. Peaks at 1251 and 1203–1213 cm⁻¹ for BGPP and BGP‐CDs corresponded to the CO stretching vibration (Santos et al. 2020). Stretching vibration of the C–O–C bond was indicated by the adsorption peaks positioned around ∼1281–1283 and ∼1059–1060 cm⁻¹ for BGP‐CDs (Khan et al. 2023a). Peaks ranging from 920 to 1190 cm⁻¹ corresponded to the C–O and C–C bonds and the structural deformations of the CH₂OH ring (Santos et al. 2020). Peak shifts in CDs arose from surface functionalization, altering bond strength, quantum confinement as affected by vibrational energy, and structural rearrangement of aromatic domains, which could modify bonds. Hydrogen bonding variations influenced intermolecular interactions and absorption frequencies. These factors collectively changed the CDs’ spectral properties compared to BGPP.

3.1.5. UV‐Blocking Property

Both UV‐A and UV‐B lights were blocked by the BGP‐CDs. Spectra showed low transmittance in UV region (200–400 nm) (Figure 3a). CDs might exhibit strong scattering and absorption properties, contributing to the conversion of UV photons into heat (Deepika et al. 2023). At lower concentrations (25 µg/mL), the highest UV‐A and ‐B blocking properties were found for BGP‐CDs/3, whereas the lowest blocking ability was found for BGP‐CDs/9 (Figure 3b). The similar result was found at higher concentrations (100 µg/mL), in which highest property was observed for BGP‐CDs/3. UV blocking properties of BGP‐CDs were enhanced as the concentration upsurged (p < 0.05). The small‐sized CDs with high abundance effectively blocked short‐wavelength UV light while permitting the transmission of long‐wavelength visible light (Murugan et al. 2025). The UV blocking properties of CDs synthesized at different reaction times exhibited irregular trends. The highest UV blocking was observed for BGP‐CDs/3, followed by the remaining BGP‐CDs samples. This variation was plausibly caused by the differences in particle size, surface functionalization, and temperature, which had an influence on UV absorption efficiency. Notably, the blocking efficiency of BGP‐CDs (100 µg/mL) was 28.35–35.63% of UV‐A and 87.41–92.63% of UV‐B. The result revealed that the blocking toward UV‐B was higher than UV‐A (p < 0.05), which was caused by the differences in unique absorption peaks (π–π* transition) in conjugated domains, as well as surface functional groups in absorbing UV light and quantum confinement effects.

3.1.6. Antioxidant Activities

Antioxidant activity of synthesized BGP‐CDs was tested for the scavenging capability of free radicals. BGP‐CDs (25–100 µg/mL) showed excellent antioxidant capacity as assayed by RS‐As (DPPH and ABTS), FRA‐P, and MC‐A (Table 1). At 100 µg/mL, all BGP‐CDs showed the highest scavenging effects toward both radicals tested. This might be due to the optimal functionalization and electron transfer capability of CDs. Higher antioxidant activities (DPPH‐RS‐A, FRA‐P, and MC‐A) at various levels were achieved by the BGP‐CDs/6, compared to others. This was probably owing to the optimal surface functionalization and balanced hydrothermal destruction/modification of BGP‐CDs/6. BGP‐CDs/3 lacked uniformity in structure and size. BGP‐CDs/9 and BGP‐CDs/12 underwent over‐hydrothermal process, thus reducing their active functional groups (–OH, –COOH) essential for antioxidant activity. Conversely, BGP‐CDs/9 showed higher ABTS‐RS‐A than other CDs. Overall, ABTS‐RS‐A of BGP‐CDs was higher than DPPH‐RS‐A, probably caused by the hydrophilicity and better dispersibility of CDs in aqueous medium (Murugan et al. 2025). As a result, the synthesized CDs acted as an excellent antioxidant. Its scavenging activities were in a dose‐dependent manner.

TABLE 1.

Antioxidant activities of BGP‐CDs at different concentrations (25–100 µg/mL).

Samples	DPPH‐RS‐A (µmol TE/mL sample)Concentration (µg/mL)				ABTS‐RS‐A (µmol TE/mL sample)Concentration (µg/mL)
	25	50	75	100	25	50	75	100
BGP‐CDs/3	1.81 ± 0.08d	4.34 ± 0.11d	6.11 ± 0.04c	7.30 ± 0.01d	18.92 ± 0.26c	25.67 ± 0.07c	31.61 ± 0.34c	40.80 ± 0.21c
BGP‐CDs/6	3.87 ± 0.15a	6.37 ± 0.12a	8.68 ± 0.06a	9.47 ± 0.02a	20.11 ± 0.04b	29.34 ± 1.90b	36.46 ± 0.16b	44.41 ± 0.29b
BGP‐CDs/9	2.71 ± 0.13b	5.88 ± 0.30b	7.84 ± 0.13b	9.18 ± 0.04b	23.28 ± 0.65a	32.36 ± 0.14a	38.42 ± 0.33a	51.45 ± 0.48a
BGP‐CDs/12	2.32 ± 0.03c	4.82 ± 0.01c	6.16 ± 0.05c	7.47 ± 0.01c	12.65 ± 0.84d	19.91 ± 0.21d	28.31 ± 0.15d	36.00 ± 0.32d

Samples	FRA‐P (µmol TE/mL sample)Concentration (µg/mL)				MC‐A (µmol EE/mL sample)Concentration (µg/mL)
	25	50	75	100	25	50	75	100
BGP‐CDs/3	4.96 ± 0.21d	10.45 ± 0.07c	15.61 ± 0.34c	20.26 ± 1.09b	0.83 ± 0.04a	1.16 ± 0.03c	1.53 ± 0.12c	2.10 ± 0.07d
BGP‐CDs/6	10.70 ± 0.10a	20.12 ± 0.02a	25.45 ± 0.37a	31.78 ± 0.76a	0.85 ± 0.09a	1.81 ± 0.01a	2.41 ± 0.06a	3.59 ± 0.01a
BGP‐CDs/9	7.84 ± 0.71b	15.50 ± 0.35b	22.46 ± 0.59b	29.38 ± 2.10a	0.89 ± 0.02a	1.42 ± 0.09b	2.16 ± 0.01b	3.28 ± 0.05b
BGP‐CDs/12	6.54 ± 0.25c	10.78 ± 0.81c	16.98 ± 2.10c	22.15 ± 1.95b	0.18 ± 0.06b	0.72 ± 0.02d	1.29 ± 0.02d	2.19 ± 0.01c

Note: Mean ± SD (n = 3). Different lowercase superscripts in the same column indicate significant differences (p < 0.05). Numbers following BGP‐CDs/(3, 6, 9, and 12) represent the various synthesis times of BGP‐CDs (3, 6, 9, and 12 h).

Abbreviations: BGP‐CDs, bambara groundnut pericarp carbon dots; ABTS‐RS‐A, ABTS radical scavenging activity; DPPH‐RS‐A, DPPH radical scavenging activity; FRA‐P, ferric reducing antioxidant power; MC‐A, metal chelating activity; TE, Trolox equivalent; EE, EDTA equivalent.

3.1.7. Antifungal Activity

Antifungal properties are crucial for preventing mold growth and maintaining food quality by inhibiting spoilage fungi. Antifungal activities of BGP‐CDs at various levels (1–3 mg/mL) against selected species (AF and AP) are depicted in Figure 4. Antifungal activities of all BGP‐CDs were in a concentration‐dependent manner. Higher concentration (3 mg/mL) exhibited potent activity, whereas lower activity was found at lower concentration (1 mg/mL). Control (distilled water) had no activity due to the lack of antifungal agent. Among all BGP‐CDs, higher activity was found for BGP‐CDs/12, followed by BGP‐CDs/9, BGP‐CDs/6, and BGP‐CDs/3 against both selected species, respectively. The small diameter was related to the water solubility, effective dispersion, and high diffusion capacity, which are crucial for their antimicrobial efficacy (Ezati et al. 2022). Generally, CDs exert antifungal activity by generating ROS, disrupting membranes, interfering with intracellular processes, and inhibiting biofilm formation, eventually leading to cell death (Koul et al. 2024). Smaller CDs enhanced these effects through improved cellular penetration. This was matched well with potent activity observed for small‐sized CDs (BGP‐CDs/12), when compared to the larger one (BGP‐CDs/3). CDs with nanoscale size and functionalized surfaces enabled penetration into fungal cell walls by promoting electrostatic or hydrophobic interactions with the fungal membrane (Li et al. 2024). The lower growth was achieved by BGP‐CDs/12 at 3 mg/mL against both AP (21.20 mm) and AF (16.50 mm). Moreover, all BGP‐CDs samples exhibited stronger antifungal activity against A. flavus than A. parasiticus. This was plausibly due to the differences in cell wall permeability, membrane structure, and metabolic pathways between both fungi. A. flavus might be more susceptible to ROS‐induced oxidative stress and membrane disruption. As a result, BGP‐CDs could act as antifungal agents in food or as the active filler in food packaging.

FIGURE 4.

Growth of Aspergillus parasiticus (a) and Aspergillus flavus (b) as affected by treatment with different BGP‐CDs at various concentrations (1–3 mg/mL) and their growth reported in terms of diameter (mm) (c). Vertical bars represent the standard deviation (n = 3). Different lowercase letters on the bar within the same BGP‐CDs sample indicate significant difference (p < 0.05). Key: see Figure 1 caption.

3.1.8. Antibacterial Activity

Antimicrobial activity is a key feature of active fillers, which can extend shelf life and provide protective functionality. Control (distilled water) showed no activity against either strain. For both gram strains, BGP‐CDs/6, BGP‐CDs/9, and BGP‐CDs/12 showed excellent bactericidal activity, in which BGP‐CDs/12 completely terminated the growth of microbes within 9 h of incubation (Figure 5a). Higher activity was observed with the extended synthesis time of CDs. BGP‐CDs/3 showed the lower inhibitory effect, more likely due to insufficient heat and limited ROS generation. In contrast, BGP‐CDs/6, BGP‐CDs/9, and BGP‐CDs/12 exhibited a bactericidal effect since CDs with extended synthesis time could enhance ROS production, oxidative stress, and membrane disruption, leading to complete bacterial cell death. Notably, the size of CDs was also essential for antibacterial activity. Lower size resulted in higher antibacterial activity, which allowed them easily penetrate into bacterial membranes. This led to intracellular protein leakage, oxidative stress, and ROS generation, thereby ultimately causing cell disruption and death (Travlou et al. 2018). In other words, smaller CDs also exhibited stronger electrostatic interactions with negatively charged bacterial surfaces, further improving their antibacterial effectiveness. Thus, the synthesis time and size of CDs greatly influenced the antibacterial activity.

FIGURE 5.

Antimicrobial activity of BGP‐CDs at different concentrations (1–3 mg/mL) against Escherichia coli and Listeria monocytogenes (a), viability of BJ cells after treatment with different BGP‐CDs at different concentrations (0.125–1 µg/mL) for 24 and 48 h (b). Vertical bars represent the standard deviation (n = 3). Different lowercase letters on the bar within the same concentration of different CDs (b) indicate significant difference (p < 0.05) (b). Key: see Figure 1 caption.

3.1.9. Cytotoxicity

Cytotoxicity testing is crucial for assessment of biocompatibility of CDs for food packaging and biomedical applications, ensuring their safe use and identifying potential toxic effects of CDs‐based nanocomposites (Ezati et al. 2022). Synthesized CDs at various levels (0.125–1 µg/mL) were tested for cytotoxicity using BJ cells (Figure 5b), whereas the control (without CDs) was used as a reference. For both 24 and 48 h of incubation, the CDs synthesized for 3 h (BGP‐CDs/3) showed higher cell viability when the same level was used, compared to other CDs samples. Higher cell viability decreased with extended synthesis time. This indicated that prolonged synthesis enhanced surface oxidation and ROS generation, thus increasing cytotoxicity. Less oxidized and large‐sized CDs (BGP‐CDs/3) exhibited better biocompatibility, whereas the extended synthesis resulted in structural changes, reducing cell viability. Regardless of incubation time, all CDs with lower concentrations (0.125–0.5 µg/mL) showed higher cell viability or no cytotoxic effects. The result indicated that the CDs below the critical level were safe to use due to the non‐toxicity. Conversely, CDs at high concentrations resulted in lower cell viability (Ezati et al. 2022; Liu et al. 2021). The primary cytotoxic mechanism involves the induction of oxidative stress, which triggers lipid peroxidation and increases membrane permeability (Kuznietsova et al. 2023). Notably, higher synthesis time increased those ROS levels and surface oxidation, leading to oxidative stress, cell leakage, ATP depletion, and apoptosis, ultimately enhancing cytotoxic effects. Other factors such as surface modification, functional groups, and nano‐size also influenced the CDs toxicity (Kuznietsova et al. 2023). CDs with a larger size (BGP‐CDs/3) had higher viability, whereas CDs having the lesser size (BGP‐CDs/12) exhibited lower viability (p < 0.05). Both BGP‐CDs/3 and BGP‐CDs/6 at various levels and different incubation times resulted in >90% cell viability, which represented its excellent biocompatibility and minimal cytotoxic effects, whereas BGP‐CDs/9 and BGP‐CDs/12 exhibited toxic effects after 48 h of incubation at the level of 1 mg/mL. Therefore, cytotoxicity of CDs was primarily dependent on the synthesis time, size, and concentration.

Among all samples, BGP‐CDs/3 exhibited non‐homogeneity, larger particle size, and lower bioactivities (antioxidant, antibacterial, and antifungal), making it less effective for potential applications. In contrast, BGP‐CDs/9 and BGP‐CDs/12 demonstrated nanoscale dimensions with superior bioactivities but showed lower cell viability on tested human cells when compared to BGP‐CDs/6, limiting their practical use. Notably, BGP‐CDs/6 emerged as the optimal candidate, offering a balanced combination of high bioactivity, uniform morphology, and higher cell viability. It exhibited excellent antioxidant, antibacterial, and antifungal properties while maintaining a safer profile for biological applications. Furthermore, its uniform size distribution and stability made it a promising active filler for food packaging applications, contributing to the extended shelf life and improved safety.

3.2. Properties and Bioactivities of Gelatin/PLA‐Based Bilayer Films Containing BGP‐CDs/6

The prepared bilayer films incorporated without (GP film) and with 4% of BGP‐CDs/6 (GP‐CD film) were analyzed. Selected parameters such as WVP, optical properties and ATR‐FTIR were analyzed on both exposure sides.

3.2.1. Appearance and WVP of Films

Pure biopolymer‐based films have high transparency due to the homogeneous polymer matrix (Abdullah et al. 2022). GP films were transparent and clear due to the homogeneity of each polymer that was achieved by the solvent casting. Polymer interaction between layers could be connected properly (Figure 6a). The chosen solid content for FFS provided the sufficient transparency and fine dispersibility in the solvents (water/chloroform for G/P). Inclusion of BGP‐CDs altered the transparency and color to some extent. Yellowness of the films was attained after the addition of CDs to the gelatin side of the film. Notably, the addition of CDs to the GP‐CD (gelatin) side alters the transparency of the PG‐CD (PLA) side due to refractive index mismatch, increased light scattering, structural changes in the bilayer, interfacial interactions, and energy transfer effects, which influenced the optical properties across the film. Moreover, the incorporation had no negative effect on the binding between gelatin and PLA films.

FIGURE 6.

Appearance (a), UV–vis light transmittance spectra and UV blocking properties (b), thickness and mechanical property (tensile strength [TS] and elongation at break [EAB]) (c), FTIR (d), antioxidant activity (radical scavenging activities [DPPH‐RS‐A and ABTS‐RS‐A], FRA‐P and MC‐A) (e), and antibacterial activity against Escherichia coli and Listeria monocytogenes (f) of gelatin/PLA bilayer film containing without and with 4% of BGP‐CDs. Vertical bars represent the standard deviation (n = 3). G: Gelatin, P: PLA, GP and PG: Gelatin and PLA side of the bilayer film, respectively. GP‐CD and PG‐CD: Gelatin and PLA sides of the bilayer film containing BGP‐CDs, respectively. BGP‐CDs, bambara groundnut pericarp carbon dots; CD, carbon dots; ABTS‐RS‐A, ABTS radical scavenging activity; DPPH‐RS‐A, DPPH radical scavenging activity; FRA‐P, ferric reducing antioxidant power; MC‐A, metal chelating activity. TE, Trolox equivalent; EE, EDTA equivalent;

Water resistance is essential for preventing food deterioration and is a key property of films. However, gelatin is highly hydrophilic, limiting its use in packaging foods with high water activity (Abdullah et al. 2022). WVP was analyzed for both exposure sides (gelatin/PLA) (Table 2). In GP film, PLA (PG) exposure side had lower WVP (1.26 × 10⁻¹² g m m⁻² s⁻¹ Pa⁻¹) than gelatin (GP) exposure side (1.48 × 10⁻¹² g m m⁻² s⁻¹ Pa⁻¹) (p < 0.05). Lower value of WVP indicated the higher water vapor barrier property. The similar reduction of WVP was noticed in GP‐CD film, in which higher WVP (1.36 × 10⁻¹² g m m⁻² s⁻¹ Pa⁻¹) was observed for gelatin (GP‐CD) exposure side than PLA (PG‐CD) exposure side (1.15 × 10⁻¹² g m m⁻² s⁻¹ Pa⁻¹) (p < 0.05). Notably, gelatin films had low water barrier properties due to polar peptides and plasticizers added, whereas the addition of bioplastic (PLA) layers on the gelatin film significantly reduced WVP of bilayer film (Murugan et al. 2023). The enhanced water vapor barrier property was greatly achieved by the addition of natural filler (CDs). This was plausibly due to the BGP‐CDs enhancing the water resistance by creating a tortuous pathway in the GP film, limiting water penetration. Their interaction with gelatin reduced the hydrophilicity, as indicated by the lowered WVP value. In addition, the presence of BGP‐CDs in one exposure (gelatin) side effectively improved the water vapor barrier property of the other exposure (PLA) side. Overall, the inclusion of nano‐filler (CDs) into gelatin film improved the hydrophobicity of film and also enhanced polymer interactions.

TABLE 2.

WVP, color and opaqueness value of gelatin/polylactic acid (PLA) bilayer films without and with 4% of BGP‐CDs.

Film samples	Properties
	WVP*	L*	a*	b*	ΔE*	Opaqueness value (mm−1)
GP	1.48 ± 0.03 a	91.65 ± 0.15b	−1.40 ± 0.01c	2.08 ± 0.14c	1.94 ± 0.09c	0.56 ± 0.02b
PG	1.26 ± 0.02c	92.15 ± 0.14 a	−1.44 ± 0.04c	1.25 ± 0.19c	1.01 ± 0.02d	0.54 ± 0.04b
GP‐CD	1.36 ± 0.02b	74.35 ± 0.01d	1.87 ± 0.36 a	39.41 ± 0.84 a	43.16 ± 0.72 a	0.85 ± 0.02 a
PG‐CD	1.15 ± 0.03d	79.98 ± 0.16c	−0.45 ± 0.03b	29.02 ± 0.27b	31.26 ± 0.31b	0.82 ± 0.03 a

Note: Values are presented as mean ± SD (n = 3). Different lowercase superscripts in the same column indicate significant differences (p < 0.05). G: Gelatin, P: PLA, GP and PG: Gelatin and PLA side of the bilayer film. GP‐CD and PG‐CD: Gelatin and PLA side of the bilayer film containing BGP‐CDs.

Abbreviation: BGP‐CDs, Bambara groundnut pericarp carbon dots; CD, Carbon dots; WVP, water vapor permeability.

^*WVP was examined at 50% RH and 25°C and expressed as ×10⁻¹² g m m⁻² s⁻¹ Pa⁻¹.

3.2.2. Color and Optical Properties

The color of food packaging films enhances the visibility and consumer preference. Among both exposure sides, PLA side of films (PG and PG‐CD) had highest L* (92.15 and 79.98) values than gelatin side (GP [91.65] and GP‐CD [74.35]). Different amounts of the polymers used (PLA: 1.6% and gelatin: 2.4%) might contribute to the varying color of the resulting film (Table 2). In GP‐CD film, the addition of yellowish‐brown nano‐filler (CDs) effectively altered the appearance and transparency of the resulting film. Moreover, the presence of CDs reduced L* value and increased b* (yellowness) value to some extent (p < 0.05). The increased yellowness (b* value) in CD‐added films was owing to the optical properties of CDs, which absorb and scatter the light, introducing chromophores that enhance yellow coloration. Interactions with film components also affected pigment dispersion, intensifying the yellow hue. Natural extracts interact with biopolymers in various ways, producing diverse colors (Murugan et al. 2025). Changes in L* and b* values were also related with the alteration of a* and ΔE* values. Yellowness of the films was increased by 1794.7% and 2221.6% for gelatin and PLA side in GP‐CD film, respectively. As a result, the addition of CDs to the gelatin‐exposed side also impacted the transparency of the PLA side.

Transparency is a key optical property of biopolymer‐based films, influencing product appearance and consumer acceptance (Abdullah et al. 2022). Lower opaqueness value was recorded for PG film (Table 2), which represented the most transparent film among the rest of films. Transparency was slightly affected by the introduction of fillers (CDs). Addition of CDs increased the yellowness (Table 2) of films, which could further impact the transparency. The enhanced yellow hue resulted from the optical properties of CDs, which absorb and scatter the light, thus reducing film transparency by altering light transmission and increasing the color intensity of films. Addition of functional nanomaterials into the biopolymer matrix modifies film transparency and influences consumer satisfaction (Murugan et al. 2025). Opaqueness value was increased from 0.56 to 0.85 mm⁻¹ for GP film and 0.54 to 0.82 mm⁻¹ for PG film after addition of 4% BGP‐CDs (p < 0.05). The GP‐CD bilayer film became opaquer with the addition of BGP‐CDs, aligning with alterations in L* and b* values. Overall, incorporation of yellowish CDs slightly affected the opaqueness of GP bilayer film.

The UV protection of packaging films is a crucial property that safeguards the quality of packaged foods from photochemical reactions. UV light exposure deteriorates food quality and shortens its shelf life, mainly via induction of oxidation (Mathew et al. 2019). From light transmission spectra (Figure 6b), gelatin exposure side showed lower transmittance range when compared to PLA exposure side (p < 0.05). Gelatin film containing a high content of aromatic amino acids could absorb UV light, thus showing high UV barrier properties (Ahmed and Ikram 2016). The similar result was observed for monolayer film, whereas a better UV barrier property was observed for gelatin film than PLA (Murugan et al. 2023). At the visible light region, highest transmission spectra were recorded for PLA exposure side due to the transparent nature. The similar effect was observed for CDs added to GP‐CD film. However, the transmission in UV and visible regions was decreased by the presence of yellowish color and UV‐blocking nature of CDs in GP‐CD film.

The UV protection of composite films depends on the type and amount of UV‐absorbing nanomaterials used. UV suppression is a key feature of packaging films, shielding food from harmful UV radiation and external environment (Ramadas et al. 2024). Due to the UV barrier nature of gelatin, GP film had UV blocking properties, in which highest UV‐A (11.17%) and UV‐B (19.71%) light blockage was achieved for the gelatin exposure side (p < 0.05). Generally, natural sunlight, especially in the UV‐A range (320–400 nm), has strong penetration ability, thereby accelerating lipid oxidation in food and causing the formation of undesirable flavors (Riahi et al. 2025). The addition of CDs actively improved the UV blocking properties of GP film. GP‐CD film increased the blocking efficiency of both UV‐A and UV‐B by 647.8% and 402.6% (Figure 6b), respectively, compared to GP film without CDs incorporation. CDs could block UV‐A (320–400 nm) and UV‐B (280–320 nm) through strong optical absorption, surface functional groups, and quantum confinement effects. π–π* and n–π* transitions absorb UV‐A, whereas CDs’ high bandgap captures UV‐B. Thus, the CDs’ UV‐blocking property could enhance their potential in protecting products from UV‐induced degradation, thus minimizing oxidative and photochemical damages.

3.2.3. Thickness and Mechanical Properties

Thickness of the film was generally influenced by the addition of fillers (Figure 6c). GP film had the thickness of 94.3 µm (p < 0.05). The thickness of layered films depends on polymer binding ability and network compactness in each film matrix (Murugan et al. 2023). Enhanced thickness (104.3 µm) was observed for CD‐added film (GP‐CD), which was plausibly caused by the introduction of foreign particles/fillers into the film matrix. This enhancement in thickness affected the mechanical, barrier, and optical properties of film matrix. Increased thickness likely resulted from cross‐linking and the broader distribution of hydrophilic CDs, which expanded the spatial distance within the film matrix. Enhanced thickness was also noticed for CD‐added films, in which the thickness was raised, depending on the concentration of CDs (Khan et al. 2023a; Murugan et al. 2024a). Thus, the inclusion of CDs directly influenced the thickness of the film matrix.

Mechanical properties like TS and EAB are crucial for food packaging films, ensuring durability and protection during handling, storage, and distribution (Bangar et al. 2021). Highest TS of 15.9 MPa (Figure 6c) was recorded for GP film (p < 0.05), whereas the least value was found for GP‐CD film (13.37 MPa). The reduction in TS of CD‐incorporated films corresponded with an increase in EAB value. This result was in line with the previous documentation (Murugan et al. 2024a). Addition of guava leaf CDs decreased the TS in a dose‐dependent manner. This reduction in TS was plausibly caused by the rearrangement in the film matrix via the introduction of fillers. The lower TS in gelatin film added with BGP‐CDs resulted from a loosened structure through reducing polymer interactions. However, the PLA side remained unaffected due to its rigid and hydrophobic nature, thus maintaining its mechanical strength. The mechanical properties of the film are generally influenced by the distribution and density of inter‐ and intramolecular interactions between polymer chains (Mir et al. 2018).

For EAB, the highest value was found for GP‐CD film (48.25%), and the lowest value was observed for GP film (39.25%). Satisfactory EAB of GP film (39.25%) was achieved by PLA layer and glycerol (plasticizer) added in gelatin film. Enhanced EAB was documented previously for gelatin layered with bioplastics (PLA and PBAT) (Murugan et al. 2023). Films from natural hydrocolloids are generally brittle. Therefore, plasticizers are commonly used to enhance flexibility by increasing the EAB value (Abdullah et al. 2022). Notably, the inclusion of CDs in the gelatin film increased the flexibility of GP film to 48.25% (p < 0.05). Enhancement of EAB was mainly caused by the hydrophilic CDs with small sizes, which might interfere with the arrangement of film matrix (Murugan et al. 2025). The addition of additives like plasticizers and antimicrobial agents can alter the mechanical properties of biopolymer‐based films by modifying the intermolecular arrangement or crosslinking within the biopolymer in film structure (Abdullah et al. 2022). The increased EAB was also observed for chitosan/PVA films added with tangerine peel CDs (Murugan et al. 2024a). The flexibility and mechanical strength of films are largely influenced by the casting technique, polymer type, structure, concentration, and the inclusion of plasticizers and other additives (Murugan et al. 2023).

3.2.4. ATR‐FTIR Spectra

FTIR spectra of gelatin sides of both films (without and with CDs) are depicted in Figure 6d. Amide‐A band, which corresponds to the combination of N–H stretching and H‐bond, appeared at the wavenumbers of 3284 and 3283 cm⁻¹ for GP and GP‐CD films, respectively (Murugan et al. 2023). Amide‐B was detected at 3081 (GP) and 3083 cm⁻¹ (GP‐CD), owing to the asymmetric stretching vibration of ═C–H as well as –NH₃ ⁺ (Nagarajan et al. 2017). Peaks detected at 2934 and 2931 cm⁻¹ (asymmetrical) for GP and GP‐CD films and 2877 cm⁻¹ (symmetrical) for both films demonstrated C–H stretching vibrations of the –CH₂ groups (Nilsuwan et al. 2018). Amide‐I (C═O stretch), ‐II (N–H and C–N vibrations), and ‐III (CN and NH in‐plane bending of amide bonds) appeared at various wavenumbers for GP (1631, 1541, and 1238 cm⁻¹) and GP‐CD films (1632, 1542, and 1238 cm⁻¹), respectively (Murugan et al. 2023; Nilsuwan et al. 2018). The characteristic peaks observed at 1449 and 1450 cm⁻¹ primarily represent the C–N and N–H stretching peaks (Murugan et al. 2025). Peaks at 1036 and 1037 cm⁻¹ for GP and GP‐CD films corresponded to –OH groups mainly from plasticizers such as glycerol (Murugan et al. 2023).

In both PG and PG‐CD films, a broad absorption band was noticeable over the wavenumber ranging from 3200 to 3700 cm⁻¹, mostly due to the highly interacted O–H group of carboxylic acids (Nagarajan et al. 2017). Peaks detected at 2945 and 2995 cm⁻¹ for both PLA exposure sides of films (without and with CDs) were attributed to the C–H stretching of CH₃ group in the saturated hydrocarbon (Murugan et al. 2023). Peaks were detected at 1452 and 1360 cm⁻¹ for both PG and PG‐CD films, denoting the asymmetric and symmetric –CH₃ deformation vibrational peaks of PLA (Nilsuwan et al. 2018). In both PLA exposure sides of films, the characteristic peaks at 1748 and 1381 cm⁻¹ were observed, owing to the C═O stretching vibration of the ester group and deformation of the CH group peak (Murugan et al. 2023). In addition, PLA ester group's symmetric C–O–C stretching vibrational peaks were detected at 1181, 1081, and 1044 cm⁻¹ for both PG and PG‐CD films (Murugan et al. 2023). Distinctive peak at 1127 cm⁻¹ demonstrates the stretching vibration of –C–O or –C–OH deformation vibration (Nilsuwan et al. 2018). For both PLA exposure sides of films, absorption of the (O–CH–CH₃) ester group was noticeable at 867 cm⁻¹ and the peak at 757 cm⁻¹ was recorded due to the rocking vibration absorption of α‐methyl group (Weng et al. 2013). Notably, shifting of the peaks appeared only in GP‐CD films when compared with the gelatin exposure side of GP film, which was plausibly caused by the intermolecular interaction between CDs and gelatin molecules in the film matrix. Conversely, no alteration was recorded in both PLA exposure sides of the films (PG and PG‐CD). The result indicated that the presence of CDs in the gelatin matrix did not penetrate into the other side (PLA side). Additionally, the bilayer film was formed homogeneously by the solvent casting method, thus preventing IR rays from passing through from one side to the other side.

3.2.5. Antioxidant and Antibacterial Activities

Antioxidant properties of GP and GP‐CD films as assayed by RS‐As toward ABTS and DPPH, FRA‐P, and MC‐A are illustrated in Figure 6e. RS‐As against ABTS and DPPH were detected for both films, in which highest RS‐As were found for GP‐CD film (p < 0.05). Functional groups presented in gelatin were responsible for the radical neutralization of GP film to some extent. However, the addition of BGP‐CDs upsurged the antioxidant activity of GP film. Antioxidant activity of CDs was plausibly due to the existence of oxygenated and amide functional groups on their surface (Murugan et al. 2025). Negligible FRA‐P and MC‐A activities were detected for GP film, which was effectively increased by the addition of BGP‐CDs containing active functional groups (like hydroxyl and carboxyl groups). These groups exhibited antioxidant activity by scavenging ROS and donating electrons. Their quantum confinement effect enhanced the electron transfer and prevented lipid peroxidation (Priyadarshi et al. 2024). As a consequence, the release of CDs from the film matrix directly affected their capability as radical scavengers or antioxidants.

Antibacterial activity of selected microbes is shown in Figure 6f. Control (without films) and GP film had no antibacterial actions against tested microbes, due to the lack of active agents present in film. Moreover, GP‐CD film had excellent activity against both gram strains. The inhibitory activity might be linked to CDs’ water solubility, high dispersion, and strong diffusion, which are essential for their antibacterial effectiveness (Travlou et al. 2018). Stronger activity was found against gram‐positive bacteria when compared to gram‐negative bacteria. The presence of lipopolysaccharide in the latter more likely acted as a barrier against CDs. Moreover, various factors, including electrostatic interactions, nanoscale size, concentration, surface area, functional groups, and ROS generation, influence the antibacterial properties of CDs (Travlou et al. 2018). The ultrasmall size ( <3 nm) of CDs played a key role in bacterial penetration, enabling entry and inducing internal protein leakage. This led to cell membrane disruption, oxidative stress, and ultimately bacterial cell death (Roy et al. 2022). Thus, the incorporated active filler effectively improved the antimicrobial activity of GP‐CD film.

3.3. Packaging Application

Fish and fish products are highly susceptible to spoilage due to rapid microbial growth, enzymatic autolysis, and oxidative degradation of lipids and proteins. These processes are accelerated by the high nutrient and moisture content of fish (Rathod et al. 2023). AS‐S samples were unpacked in pouch (covered with shrink film) and packed in pouches made from gelatin/PLA bilayer film without and with BGP‐CDs incorporated (GP and GP‐CD pouches) and analyzed for chemical and microbial changes during 12 days (4°C) of storage period. Photographs of AS‐S packaged with the pouch made from gelatin/PLA bilayer (GP) films for 12 days at 4°C are depicted in Figure 7a. Control samples turned brownish and yellowish over the storage time. Notably, AS‐S samples packed in GP pouches remained fresh until the sixth day of storage, whereas AS‐S samples packed with GP‐CD pouches preserved AS‐S in good condition until the final day of storage (Figure 7a).

FIGURE 7.

Image of Asian seabass slices packaged with the gelatin/PLA bilayer pouches for 12 days at 4°C (a), changes in color (a* and b*) values (b), pH (c), TBARS value (d), total viable count (TVC), and psychrophilic bacteria count (PBC) (e) of Asian seabass slices packaged without and with pouches made from the gelatin/PLA bilayer films in the absence and presence of 4% BGP‐CDs/6 during 12 days of storage at 4°C. Vertical bars represent the standard deviation (n = 3). Control: seabass meat covered with shrink film; GP and GP‐CD: Asian seabass slices packed in pouches made from GP and GP‐CD films, respectively. Key: see Figure 6 caption.

3.3.1. pH and Color

At the initial stage, the pH of AS‐S samples was 5.99 (p < 0.05). By extending the storage time, the pH of all AS‐S samples increased gradually, which indicated spoilage. Elevated pH levels were associated with the degradation of muscle proteins and connective tissues, causing a loss of texture (Figure 7c). Among three AS‐S samples, the control showed higher pH during the entire storage period. The pH increase was closely linked to bacterial growth in AS‐S sample. As the bacterial population upsurged, the production of basic products was intensified, resulting in a further rise in pH (Riahi et al. 2025). Alteration in pH was lower for the AS‐S samples packed in both pouches (GP and GP‐CD pouches). Changes in pH also matched well with the color changes (Figure 7b). At day 0, an a* value of −2.93 and b* value of −5.10 were found, respectively. At the sixth day, the a* and b* values of the control AS‐S samples decreased and increased, respectively (p < 0.05). Increased b* value represented the appearance of yellow coloration in AS‐S sample, which indirectly indicated the deterioration. Moreover, the b* value remained constant for AS‐S samples packed in GP and GP‐CD pouches. Throughout the storage period, the a* value was decreased at the 6th day, followed by an increase at the 12th day, which indicated the appearance of brown coloration of AS‐S samples. The b* value increased for AS‐S samples packed in GP‐CD pouches at the final day of storage, which was plausibly due to the release of yellowish CDs from the film into AS‐S samples (Figure 7a).

3.3.2. Thiobarbituric Acid–Reactive Substances

TBARS value has been employed to assess the effectiveness of antioxidants in inhibiting lipid oxidative degradation (Wei et al. 2019). Among all AS‐S samples, the control had the highest TBARS throughout the storage period (Figure 7d), which was mostly due to the absence of potential packaging material/antioxidant agents. On Day 3, there was no significant difference between both AS‐S samples packed in GP and GP‐CD pouches. From 6th to 12th day of storage period, the TBARS value increased rapidly (p < 0.05). The rise in TBARS value was likely due to the liberation of lipid caused by microbial growth and the enhanced lipid oxidation (Wei et al. 2019). On Day 12, AS‐S samples packed in GP‐CD pouches attained lower TBARS values when compared to others. The lower TBARS value in AS‐S samples packed in GP‐CD pouches was due to CDs’ antioxidant properties, which scavenged free radicals and inhibited lipid oxidation. Additionally, their UV‐blocking ability reduced photo‐oxidation, whereas their barrier properties limited oxygen and moisture migration, collectively enhancing lipid stability. Moreover, the acceptable limit of TBARS value was recorded on Day 12 for AS‐S samples packed in GP and GP‐CD pouches, which was plausibly due to the barrier properties of pouches. Thus, the presence of active filler in GP‐CD pouch effectively prevented lipid oxidation.

3.3.3. TVC and PBC

According to the International Commission on Microbiological Specifications for Food (ICMSF), a total microbial count of 7 Log CFU/g was considered a threshold for fish spoilage (Gui et al. 2024). In this study, microbial levels of TVC and PBC for all AS‐S samples exhibited a rising trend throughout the storage period (Figure 7e). At Day 0, TVC and PBC were found to be 2.84 and 2.45 Log CFU/g, respectively, which indicated that AS‐S samples were still in a fresh condition. TVC reached the limit on the 9th day for the control and on the 12th day for AS‐S samples packed in GP pouches, whereas AS‐S samples packed in GP‐CD pouches maintained acceptability throughout the storage period. This was likely due to the antimicrobial action of the GP‐CD pouch, thus effectively slowing bacterial growth. During the storage, CDs from the inner film were released into the surface of AS‐S and acted as an antimicrobial agent. This effectively prevented the microbial load from exceeding the limit, unlike the control and AS‐S samples packed in GP pouches, which lacked strong antimicrobial protection. At the final day of storage, the lowest TVC (5.57 Log CFU/g) and PBC (5.25 Log CFU/g) were detected for AS‐S samples packed in GP‐CD pouches. This result was in line with the previous reports (Riahi et al. 2025), which found that shrimp samples packed in sodium alginate/cellulose nanofibril pouches containing CD‐functionalized polyaniline complex had an extended shelf life of up to 15 days under refrigerated conditions, which was plausibly caused by the synergetic effect of the antibacterial and UV barrier properties of CDs. In addition, lower TVC was observed in pork samples packaged with chitosan/gelatin pouches containing GT‐CDs (Khan et al. 2023a) and for shrimp samples packaged with carrageenan pouches containing kohlrabi peel–derived CDs (Khan et al. 2024) when compared to the corresponding control counterparts. Although the release of CDs from the pouch into the product slightly altered the color of the AS‐S samples (Figure 7a), those released CDs enhanced preservation by providing a preventive effect against microbial growth and lipid oxidation. As a result, the pouch made from bilayer films containing CDs acted as a promising means for quality maintenance of the refrigerated AS‐S samples.

4. Conclusions

CDs (BGP‐CDs) from BGPP were successfully synthesized through the hydrothermal method for varying durations. All CDs were proven as nanomaterials ( <15 nm) with remarkable optical and high antioxidant properties as assayed by ABTS and DPPH RS‐As, FRA‐P, and MC‐A. It also showed antifungal activity against AF and AP and antibacterial properties against LM and EC. Among those, BGP‐CDs/6 prepared via the hydrothermal process for 6 h having the satisfactory properties was utilized as the active nano‐filler, which effectively improved the elasticity, water vapor barrier, UV‐blocking ability, and antioxidant and antibacterial activities of gelatin/PLA bilayer films. Moreover, the pouches made from functionalized (GP‐CD) film effectively preserved AS‐S by lowering the increase in pH, reducing the microbial growth (TVC and PBC), and inhibiting lipid oxidation (TBARS value) during 12 days of refrigerated storage. Overall, bambara groundnut pericarp–derived CDs could be used as the active filler in a gelatin/PLA layered biopolymer system, highlighting the novelty in terms of agro‐waste utilization for sustainable and functional food packaging. The finding contributes to the development of bio‐based nanomaterials, in which a promising alternative to synthetic preservatives can be applied for the preparation of active food packaging.

Author Contributions

Gokulprasanth Murugan: investigation, methodology, data curation, writing – original draft. Krisana Nilsuwan: writing – review and editing, data curation, formal analysis. Jun Tae Kim: writing – review and editing, formal analysis, data curation. Jong‐Whan Rhim: data curation, formal analysis, writing – review and editing. Tao Yin: data curation, formal analysis, writing – review and editing. Bin Zhang: data curation, formal analysis, writing – review and editing. Soottawat Benjakul: conceptualization, funding acquisition, resources, writing – review and editing, supervision.

Ethics Statement

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Murugan, G. , Nilsuwan K., Kim J. T., et al. 2025. “Bambara Groundnut Pericarp–Derived Carbon Dots: Reinforcement of Gelatin/PLA Bilayer Films and Their Function as Preservative in Packed Asian Seabass Slices.” Journal of Food Science 90, no. 8: 90, e70469. 10.1111/1750-3841.70469

Data Availability Statement

The authors have nothing to report.