Nozzle-less electrospun eugenol-loaded gelatin nanofibers: effect on fish preservation and quality enhancement

Abstract

This study evaluates the feasibility of producing nozzle-less eugenol-infused gelatin nanofibers as active coatings to improve fish quality and shelf life during cold storage. Nanofibers were fabricated using nozzle-less electrospinning with gelatin concentrations of 10–20% and gelatin:eugenol ratios of 100:0 to 50:50. The optimal formulation—20% gelatin with a 50:50 ratio—achieved >99.98% encapsulation efficiency and the smallest average fiber diameter (91.11 ± 18.53 nm). FTIR confirmed strong hydrogen bonding between gelatin and eugenol, while TGA indicated improved thermal stability. Higher eugenol loading enhanced antioxidant activity, limiting lipid oxidation to <0.15 mg MDA/kg compared with >1 mg MDA/kg in controls after 3 days. Microbial counts in coated fish at day 7 (2.80 log CFU/g) remained lower than uncoated samples at day 3 (3.88 log CFU/g). The coating also preserved texture and color throughout storage. Overall, these findings highlight the potential of bioactive nanofiber coatings for food preservation and waste reduction.

Introduction

Marine products are widely regarded as nutrient-dense foods because they provide high-quality proteins, essential minerals, vitamins, and polyunsaturated fatty acids¹. Among them, fish is especially important owing to its higher content of free amino acids, lower connective tissue, and greater enzymatic activity than other muscle-based foods. Paradoxically, these same characteristics accelerate post-harvest spoilage, particularly via protein denaturation and lipid oxidation, which compromise sensory attributes, reduce consumer acceptance, and shorten shelf life. Therefore, developing preservation strategies that extend storage while maintaining freshness remains a central objective in seafood processing².

Edible coatings derived from natural sources have been proposed as an efficient means of reducing post-harvest losses in aquatic products. These coatings form thin, uniform layers on food surfaces and are typically based on biopolymers such as carbohydrates, proteins, or lipids. Functioning as semi-permeable barriers, they limit oxygen and moisture transfer and thereby slow biochemical and oxidative reactions that drive quality deterioration³. The efficacy of edible coatings can be strengthened by integrating natural preservatives, particularly essential oils. Within this domain, eugenol has emerged as a promising compound for maintaining seafood freshness⁴.

Eugenol (C₁₀H₁₂O₂; 4-allyl-2-methoxyphenol) is a phenylpropene mainly extracted from clove oil⁵. It exhibits strong antioxidant capacity through scavenging free radicals and suppressing reactive oxygen species; the IC₅₀ of pure eugenol has been reported as 11.7 µg/mL, confirming its potent radical-quenching activity⁶. Mechanistically, its phenolic hydrogen and unsaturated double bonds allow trapping of chain-propagating peroxy radicals (ROO˙), interrupting lipid oxidation cascades⁷. In parallel, eugenol shows broad-spectrum antimicrobial effects. In Gram-negative bacteria it disrupts cytoplasmic membranes and causes leakage of intracellular components⁸. In Gram-positive bacteria, it weakens the peptidoglycan layer, increases membrane permeability, disturbs proton motive force, and induces oxidative stress, producing bactericidal outcomes even against resistant strains⁴. However, eugenol is chemically unstable and prone to oxidation, which limits its direct application; thus, encapsulation is considered necessary to improve stability, solubility, and controlled release⁹.

Encapsulation technologies at the nanoscale have advanced markedly, enabling more stable and efficient delivery of bioactive compounds. Nanofibers are advantageous carriers due to their high aspect ratio, large surface area, porosity, and mechanical strength¹⁰. They can be produced through several techniques, but electrospinning is particularly attractive because it is scalable and enables sustained release of entrapped antioxidants and antimicrobials¹¹. Gelatin is a suitable electrospinning polymer given its biocompatibility, biodegradability, availability, and excellent spinnability¹². A recent refinement, nozzle-less electrospinning, eliminates the conventional nozzle and generates multiple jets directly from the solution surface under an electric field, thereby increasing throughput, lowering costs, and preventing clogging while maintaining control over fiber morphology¹³. Its versatility has been demonstrated in prior encapsulation studies with essential oils and cinnamic aldehyde^14,15.

Accordingly, this study explores nozzle-less electrospinning as an advanced method for embedding eugenol into gelatin nanofibers. Gelatin concentration and eugenol-to-gelatin ratios will be optimized to evaluate encapsulation efficiency and physicochemical properties, and the preservative performance of the optimized nanofibers will be assessed in fish by monitoring microbial growth and oxidative degradation. This approach is expected to yield a practical antimicrobial and antioxidant coating strategy that enhances fish shelf life and supports sustainable active packaging for aquatic products.

Results and discussion

Viscosity, thermal conductivity and surface tension of the electrospinning solution

The key parameters of the spinning solutions—viscosity, electrical conductivity, and surface tension—were measured prior to electrospinning to understand their influence on nanofiber morphology and diameter¹⁶. The results are summarized in Table 1.

Table 1.

Viscosity, electrical conductivity and surface tension (mean ± SD) of solutions for different gelatin to eugenol with different volume ratios

Gelatin concentration (% w/v)	Gelatin/eugenol solution (v/v)	Viscosity (cp)	Electrical conductivity (μS/cm)	Surface tension (mN/m)
10	100:0	46.33 ± 0.57Ca	971.33 ± 8.08Ca	33.77 ± 0.28Aa
	90:10	36.66 ± 1.54Cb	903.33 ± 15.27Cb	32.54 ± 0.26Ab
	70:30	24.00 ± 1.00 Cc	801.66 ± 7.63 Cc	31.66 ± 0.25Ac
	50:50	19.33 ± 1.52Cd	501.66 ± 7.63 Cd	30.25 ± 0.28Ad
15	100:0	79.33 ± 1.52Ba	1179.00 ± 11.53Ba	32.99 ± 0.29Ba
	90:10	58.33 ± 0.57Bb	1155.00 ± 5.00Ba	32.00 ± 0.30Bb
	70:30	39.00 ± 1.00Bc	1050.33 ± 1.52Bb	30.50 ± 0.22Bc
	50:50	32./33 ± 0.52Bd	686.33 ± 5.50Bc	29.56 ± 0.27Bd
20	100:0	187.33 ± 0.57Aa	1257.33 ± 3.05Aa	31.30 ± 0.30Ca
	90:10	119.33 ± 0.55Ab	1223.33 ± 2.51Aa	30.69 ± 0.27Cb
	70:30	63.33 ± 0.57Ac	1157.33 ± 2.51Ab	29.38 ± 0.15 Cc
	50:50	47.36 ± 0.57Ad	763.66 ± 11.93Ac	28.66 ± 0.12 Cd

Different uppercase letters within the same column indicate significant differences between gelatin concentrations at a constant gelatin/eugenol ratio. Similarly, different lowercase letters within the same column denote significant differences between gelatin/eugenol ratios at a fixed gelatin concentration (P < 0.05).

As shown in Table 1, the viscosity of gelatin solutions increased with increasing gelatin concentration (10–20%). At varying gelatin-to-eugenol ratios, the viscosity decreased as the proportion of gelatin decreased from 90 to 50%. This reduction can be attributed to the lower viscosity of eugenol and the presence of ethanol in the mixture, which acts as a solvent. Similarly, Etxabide et al.¹⁷. reported that increasing the gelatin concentration (≥16% w/v) facilitates the formation of smooth, bead-free nanofibers, as higher viscosity dominates the electrospinning process compared to lower concentrations (≤14% w/v).

The electrical conductivity of gelatin solutions also increased with higher gelatin concentrations (10–20%) (Table 1), likely due to the greater concentration of suspended particles, charged ions, and hydrogen bonding interactions. However, in gelatin/eugenol solutions, a decrease in the gelatin ratio and the incorporation of eugenol led to a reduction in electrical conductivity (Table 1). This phenomenon can be explained by the low electrical conductivity of eugenol in ethanol, which does not ionize effectively, thereby resulting in fewer free ions being generated. In a related study, Mosayebi et al.¹⁸, demonstrated that increasing the gelatin concentration in electrospinning solutions enhances electrical conductivity. Conversely, increasing the Spirulina protein concentrate content from 20 to 80% caused a 40.8% reduction in conductivity (from 1271 to 752.5 μS/cm), which was attributed to the lower intrinsic conductivity of the Spirulina protein concentrate solution (400 ± 7 μS/cm) compared to the gelatin solution (1450 ± 12 μS/cm).

In contrast to the findings related to electrical conductivity and viscosity, the surface tension decreased as the gelatin content increased from 10 to 20% (Table 1). In polymer solutions such as gelatin, an increase in polymer concentration results in a higher number of polymer chains per unit volume. These polymer chains reduce the surface tension between water molecules and increase the solution’s density. Also, when the proportion of eugenol in the solution rose from 10 to 50%, the surface tension of gelatin/eugenol solutions with varying ratios declined (Table 1). This reduction can be attributed to the hydrophobic alkyl group present in eugenol, which repels water and causes its molecules to migrate toward the liquid’s surface. The accumulation of eugenol at the solution’s interface impedes the direct access of solvent molecules to the surface, thereby reducing surface tension—a phenomenon that has been corroborated in previous studies involving the incorporation of hydrophobic essential oils into gelatin solutions¹⁹.

Another contributing factor is the polarity of the eugenol molecule, which can form stronger hydrogen bonds with water molecules compared to acetic acid. This aligns with the findings of the FTIR analysis and partially disrupts the hydrogen bonding network between water molecules, diminishing their cohesive forces. According to the data, as the concentration of eugenol increased, the number of eugenol molecules at the interface also increased, leading to a corresponding reduction in surface tension²⁰. Similarly, prior investigations have demonstrated a decrease in surface tension with an increase in gelatin content¹⁸.

Rheological properties of the electrospinning solution

To investigate the rheological properties relevant to electrospinning, a 20% gelatin solution was selected. This concentration provides an optimal balance of viscosity, electrical conductivity, and surface tension, ensuring sufficient chain entanglement and viscoelasticity to maintain jet stability while allowing evaluation of the effects of eugenol incorporation on solution properties. The rheological properties of the 20% gelatin solution and gelatin/eugenol mixtures at varying ratios were subsequently analyzed by examining the influence of shear rate on apparent viscosity and the effect of frequency on storage modulus (G′) and loss modulus (G″). As shown in Fig. 1A, the apparent viscosity of all solutions decreased with increasing shear rate, indicating that the fluids exhibit non-Newtonian shear-thinning behavior. This behavior can be attributed to the breakdown of entangled polymer networks and macromolecular crosslinks under shear conditions. Specifically, the induced disruption interferes with the recovery of intermolecular entanglements, thereby reducing intermolecular resistance to flow²¹. Non-Newtonian fluids with shear-thinning properties are critical for applications involving injectable materials²².

Fig. 1. Rheological properties of gelatin and gelatin/eugenol solutions.

Changes in viscosity as a function of shear rate (A), and the effects of frequency on the storage modulus (G′) (B), loss modulus (G″) (C), and loss tangent (tan δ) (D) of gelatin and gelatin/eugenol solutions at different ratios.

As illustrated in Fig. 1A, as the proportion of eugenol increased, the extent of apparent viscosity reduction under shear stress diminished, and the fluid demonstrated reduced viscoelastic behavior, suggesting a decline in intermolecular interactions²³. Kutzli et al.²⁴, emphasized that weak shear-thinning behavior is essential for the successful electrospinning of polymer solutions. The viscoelastic properties of the electrospinning solutions are illustrated in Fig. 1B–D. As shown, the storage modulus (G’) of the pure gelatin solution increased with rising frequency, indicating the presence of robust network interactions within the gel capable of withstanding applied stresses at higher frequencies. This behavior highlights the elastic nature of the fluid. In contrast, for gelatin/eugenol solutions at varying ratios, the storage modulus (G’) decreased as the frequency increased. The incorporation of eugenol disrupts the bonds and attractive forces between polymer chains at higher frequencies, thereby weakening the gel structure. Consequently, the G’ values decline with increasing frequency, reflecting viscoelastic behavior and a shift toward liquid-like characteristics. The loss modulus (G”) increased with frequency across all solutions (Fig. 1C). At higher frequencies, the relative motion of polymer chains within the gel leads to energy dissipation, resulting in an increase in G”. The magnitudes of G’ and G”, along with their ratio, define the dominant elastic or viscous nature of a viscoelastic material. Specifically, when G’ exceeds G”, the material predominantly exhibits elastic solid-like behavior, whereas when G” surpasses G’, the material behaves more like a viscous liquid. According to Fig. 1D, Upon addition of eugenol, in the 10:90 and 30:70 gelatin/eugenol ratios, G′ remains higher than G″ but decreases with increasing frequency, indicating a weaker network and reduced viscoelasticity compared to pure gelatin (Zhang et al.). At the 50:50 ratio, G′ and G″ approach each other, reflecting a balance between elasticity and viscosity. This balance ensures sufficient viscosity to maintain fiber structure while allowing adequate fluidity for uniform jet stretching during electrospinning. Lower gelatin or higher eugenol ratios lead to unstable fibers or processing difficulties; hence, the 50:50 ratio was selected as optimal for electrospinning (Deng et al.; Zhang et al.).

Overall, the combined influence of viscosity, electrical conductivity, surface tension, and rheological behavior plays a crucial role in determining the electrospinnability and final morphology of the nanofibers. A balanced viscosity and electrical conductivity are essential to maintain a stable jet during electrospinning and to achieve continuous fiber formation, whereas excessively high viscosity may hinder jet stretching and lead to bead formation. The reduction in surface tension with increasing eugenol concentration lowers the cohesive forces at the liquid interface, facilitating smoother jet elongation and uniform fiber formation. Moreover, the observed shear-thinning behavior and viscoelastic characteristics, reflected by a higher storage modulus (G′) than loss modulus (G″), indicate sufficient chain entanglement and elasticity, which are critical for maintaining jet stability. The incorporation of eugenol slightly decreases G′ and increases G″, suggesting reduced intermolecular interactions that can influence fiber uniformity. Therefore, the optimized combination of increased viscosity and conductivity (due to higher gelatin content), decreased surface tension (from eugenol incorporation), and stable viscoelasticity collectively contributes to the formation of uniform, bead-free nanofibers. These results are consistent with previous studies emphasizing the synergistic influence of these parameters on fiber morphology and electrospinning performance^16,21

Morphological characteristics of fabricated nanofibers

Field emission scanning electron microscopy (FESEM) images of gelatin and gelatin/eugenol nanofibers are presented in Fig. 2. The fibers generated from a 10% gelatin solution exhibited irregular morphologies and were characterized by the presence of beads. This observation can be attributed to the low viscosity and electrical conductivity of the solution at lower gelatin concentrations, which likely resulted in insufficient stability of the gelatin polymer chains to form uniform fibers. Consequently, the 10% gelatin concentration was excluded from further investigation in this study. In contrast, the fibers produced from the 15% gelatin solution demonstrated greater uniformity, with an average diameter of 133.56 ± 26.25 nm. This improvement indicates that the 15% gelatin concentration outperformed the 10% concentration in terms of nanofiber production capabilities. Raising gelatin concentration from 10 to 15% increased solution viscosity, thereby stabilizing the electrospinning jet. Improved jet stability promotes more uniform stretching in the electric field, leading to thinner and more homogeneous fibers than those obtained from the 10% gelatin solution. Despite this improvement, fibers at 15% gelatin still exhibited a wider diameter distribution and less regular morphology, likely due to insufficient polymer chain entanglement at this concentration. SEM imaging of the 15% gelatin formulation revealed a clear reduction in fiber diameter as the eugenol loading increased. Fibers produced from neat 15% gelatin displayed the greatest mean diameter, whereas progressive eugenol addition yielded progressively finer fibers. This behavior is consistent with the plasticizing role of eugenol, which lowers the viscosity of the spinning dope and facilitates greater jet elongation during electrospinning⁹.

Fig. 2. Morphology and diameter distribution of gelatin and gelatin/eugenol nanofibers.

FESEM images and fiber diameter distributions of nanofibers obtained from gelatin solutions at 10% (w/v) (A), 15% (w/v) (B), and 20% (w/v) (C), as well as gelatin/eugenol solutions with 15% (w/v) gelatin at mixing ratios of 90:10 (D), 70:30 (E), and 50:50 (F), and gelatin/eugenol solutions with 20% (w/v) gelatin at mixing ratios of 90:10 (G), 70:30 (H), and 50:50 (I).

Finally, the fibers obtained from the 20% gelatin solution exhibited a reduced average diameter of 80.9 ± 53.18 nm compared to the other concentrations. This reduction suggests that the viscosity and electrical conductivity of the solution at this concentration were optimal, facilitating the formation of more uniform fibers with smaller diameters. According to Table 1, the 20% gelatin solution exhibited higher electrical conductivity than the other concentrations. The increased electrical conductivity of the polymer solution reduces surface tension, thereby promoting the necessary elongation of the jet toward the collector plate and resulting in a decrease in nanofiber diameter²⁵. Therefore, the 20% gelatin concentration provides optimal conditions for producing uniform and well-formed nanofibers. Similarly, Chi et al.²⁵, reported that increasing the concentration of polyvinyl alcohol (PVA) enhances fiber formation, with higher concentrations leading to thinner and less twisted fibers due to PVA’s lower viscosity, reduced electrical conductivity, and linear molecular structure.

At a constant gelatin concentration of 20%, increasing the eugenol ratio from 10 to 50% resulted in a reduction in the average diameter of the nanofibers. This phenomenon can be attributed to the plasticizing effect of eugenol, which decreases the solution’s viscosity, thereby facilitating the formation of thinner jets and producing fibers with smaller diameters. Additionally, the hydroxyl functional groups in eugenol may form hydrogen bonds with the functional groups in gelatin, leading to a more compact polymer network and, consequently, smaller fiber diameters. These findings are consistent with the results obtained from FTIR analysis²⁰.

Enhanced electrical conductivity, driven by increased electrostatic forces and jet elongation, generally contributes to the formation of fibers with reduced diameters. However, in this study, the observed decrease in nanofiber diameter was more closely aligned with the trend in solution viscosity than with electrical conductivity. This suggests that solution viscosity played a dominant role in determining the nanofiber diameter. Shi et al.²⁶, reported similar findings in their investigation, where polymer solutions with 20% gelatin produced fibers with lower average diameters and more uniform morphologies compared to those at 15%. Based on these results, a constant gelatin concentration of 20% was selected as the optimal condition for further experiments. Similarly, Gupta et al.²⁷, demonstrated that acacia gum nanofibers containing eugenol exhibited smaller diameters than those without eugenol. This effect was likely due to the plasticizing properties of eugenol, which reduce solution viscosity and enhance fiber formation.

Encapsulation efficiency and loading capacity

As illustrated in Fig. 3A, the encapsulation efficiency of gelatin/eugenol nanofibers at a 15% gelatin concentration ranged from 95.97 to 96.98%, while at a 20% gelatin concentration, it ranged from 98.99 to 99.32%. Increasing the eugenol ratio from 10 to 50% at both 15% and 20% gelatin concentrations significantly enhanced the encapsulation efficiency (P < 0.05). Furthermore, at equivalent gelatin-to-eugenol ratios, the encapsulation efficiency was significantly higher at a 20% gelatin concentration compared to 15%.

Fig. 3. Encapsulation performance of eugenol in gelatin nanofibers.

Encapsulation efficiency (A) and loading capacity (B) of eugenol in gelatin nanofibers at different eugenol/gelatin ratios and various gelatin concentrations. Different uppercase letters indicate significant differences among gelatin concentrations, while different lowercase letters indicate significant differences among eugenol/gelatin ratios at the 95% confidence level (p < 0.05).

According to Li et al.²⁸, gelatin/poly(lactic acid) nanofibers demonstrated an encapsulation efficiency of approximately 90%, with loading efficiency increasing from 48.87 to 64.07% as the eugenol concentration rose from 2 to 4 mg/g. They concluded that the high encapsulation efficiency and loading capacity were likely attributable to the nanofibers’ ability to inhibit eugenol crystallization, thereby enhancing its retention and stability. Figure 3B also depicts the encapsulation loading of gelatin/eugenol nanofibers at various ratios and gelatin concentrations (15 and 20%). As the eugenol ratio increased from 10 to 50%, the loading capacity significantly improved (P < 0.05). Additionally, at equivalent gelatin-to-eugenol ratios, the encapsulation loading was significantly higher at a 20% gelatin concentration compared to 15%.

In conclusion, the 50:50 gelatin-to-eugenol ratio at a 20% gelatin concentration exhibited the highest encapsulation efficiency and loading capacity (P < 0.05). Similarly, Aydogdu et al.²⁹, reported that increasing the concentration of gallic acid from 2 to 10 g per 100 g resulted in a rise in the loading efficiency of electrospun hydroxypropyl methylcellulose nanofibers containing gallic acid, from 61 to 69%. The improved encapsulation efficiency and loading capacity observed at higher eugenol ratios and gelatin concentrations are consistent with previous reports, which attribute this effect to strong hydrophobic interactions and hydrogen bonding between eugenol and gelatin³⁰. These molecular interactions stabilize the polymer network and minimize surface migration of eugenol, resulting in enhanced encapsulation and retention within the nanofiber matrix. Furthermore, the denser gelatin network at higher concentrations provides additional binding sites for eugenol molecules, thereby improving the overall structural integrity and encapsulation performance³⁰.

Fourier transform infrared spectroscopy (FTIR)

Fourier transform infrared spectroscopy (FTIR) was employed to analyze the chemical structure and functional groups present in the samples. Figure 4 illustrates the FTIR spectra of gelatin powder, eugenol, and eugenol microencapsulated within gelatin nanofibers, recorded in the wavenumber range of 4000–350 cm⁻¹. The spectrum obtained from gelatin powder reveals a broad absorption band in the range of 3700–3100 cm⁻¹, which is attributed to the stretching vibrations of O–H bonds from free water molecules and N–H stretching vibrations associated with amide A. The absorption band at 2929 cm⁻¹ corresponds to the stretching vibrations of the methyl (CH₃) group. Additionally, the absorption band observed around 1638 cm⁻¹ is associated with the C = O stretching vibrations of amide I, indicating the presence of peptide bonds within the gelatin structure. The absorption band at approximately 1525 cm⁻¹ is assigned to the N–H bending vibrations and C–N stretching vibrations characteristic of amide II. Furthermore, the absorption bands in the range of 1400–1450 cm⁻¹ are ascribed to symmetric and asymmetric bending vibrations of methyl groups along the main gelatin chain. Finally, the absorption band around 1239 cm⁻¹ is attributed to the N–H stretching vibrations of amide III³¹.

Fig. 4. FTIR spectra.

Fourier Transform Infrared (FTIR) spectroscopy of gelatin powder, eugenol, and gelatin/eugenol nanofibers at a 50:50 ratio.

In the FTIR spectrum obtained from eugenol essential oil, a distinct peak corresponding to O–H stretching vibrations is observed at 3517 cm⁻¹, which is indicative of the presence of hydroxyl (OH) groups in the essential oil. The peaks at 2970 and 2841 cm⁻¹ are attributed to the stretching vibrations of the C–H group and the methyl (CH₃) group, respectively. Additionally, the wavenumber at 1643 cm⁻¹ is assigned to the stretching vibrations of aromatic C=C bonds. The absorption band at 1514 cm⁻¹ corresponds to C–H bending vibrations, while the band at 1269 cm⁻¹ is associated with C–O stretching vibrations³². In the FTIR spectrum of gelatin/eugenol nanofibers, the broad band observed at 3306 cm⁻¹ corresponds to O–H stretching vibrations, overlapping with N–H vibrations. The peak at 1653 cm⁻¹ is attributed to amide I, which arises from C=O stretching vibrations in the gelatin structure. The band at 1523 cm⁻¹ corresponds to the stretching vibrations of aromatic C=C bonds, confirming the presence of eugenol within the nanofiber matrix. Furthermore, the transmission peak at 1451 cm⁻¹ is associated with the stretching and bending vibrations of C–N and N–H bonds. The peaks in the range of 1380–1270 cm⁻¹ are attributed to CH₃ stretching vibrations, O–H bending vibrations, and C–O stretching vibrations in the phenolic group of the eugenol molecule, further validating the successful incorporation of eugenol into the gelatin nanofibers.

These spectral changes suggest that gelatin likely interacts with the hydroxyl groups of eugenol through hydrogen bonding, potentially forming amide bonds. FTIR analysis confirms that hydrogen bonding serves as the primary interaction between eugenol and gelatin protein, thereby demonstrating the successful microencapsulation of eugenol within the gelatin nanofiber structure.

Thermal properties of nanofibers

(TGA) was employed to evaluate the thermal stability and heat resistance of the nanofibers. The first derivative (DTG) mass loss curves, obtained by differentiating the TGA curve, reveal the temperature at which maximum weight loss occurs, indicating the material’s highest thermal stability³³. The results are presented in Fig. 5. A significant weight loss (approximately 92%) was observed in the temperature range of 70–160 °C, corresponding to the degradation of eugenol. The maximal rate of eugenol degradation was indicated by a distinct peak in the DTG curve at 140 °C, with a weight loss of approximately 99.23% at this temperature. Since eugenol is an organic compound, no residual mass was detected after the test, consistent with the findings reported by Matykiewicz and Skórczewska³⁴.

Fig. 5. Thermal degradation behavior of gelatin and gelatin/eugenol nanofibers.

The TGA (A) and DTG (B) curves correspond to (1) eugenol, (2) gelatin powder, (3) gelatin nanofibers with a 20% concentration (G100E0), and (4) gelatin/eugenol nanofibers at a 50:50 ratio with a 20% gelatin concentration (G50E50).

The TGA curve of pure gelatin exhibits four distinct stages of weight reduction. In the first stage (80–170 °C), an 8.3% weight loss was observed, primarily due to moisture evaporation. The second stage (172–257 °C) resulted in a 3.65% weight loss, likely attributed to melting. The third stage (253–433 °C) demonstrated a significant 52.68% weight loss, potentially due to the degradation of gelatin into smaller fragments. The final stage (430–570 °C) showed an 8.95% weight loss, likely associated with continued degradation. The maximum thermal stability of gelatin was observed around 325 °C, where the highest rate of weight loss occurred.

The TGA curve of gelatin nanofibers without eugenol (Ge/Eu 100:0) also exhibited four distinct stages of weight reduction. In the first stage (70–150 °C), a 15.9% weight loss was observed, attributed to the evaporation of water and acetic acid. The second stage (150–237 °C) resulted in a 2% weight loss, likely due to melting. The third stage (237–475 °C) demonstrated a significant 72.57% weight loss, ascribed to the degradation of the nanofiber structure. The final stage (475–598 °C) showed a 63.3% weight loss, likely due to continued decomposition. The maximum thermal stability occurs around 325 °C, where the highest weight loss is observed. The TGA curve of 20% (w/v) gelatin nanofibers containing 50% eugenol (Ge/Eu 50:50) exhibits four distinct stages of weight loss. In the first stage, a 5.84% weight loss occurs between 30 and 100 °C due to the evaporation of water and solvents. The second stage, characterized by a 9.17% weight loss, occurs between 115 and 255 °C and is attributed to the decomposition of eugenol. Notably, while pure eugenol decomposes within the temperature range of 70–160 °C, encapsulation within gelatin nanofibers shifts this range to 115–255 °C, indicating enhanced thermal stability.

The DTG curve of the gelatin/eugenol 50:50 nanofibers reveals that the initial peak corresponds to the disintegration of eugenol at approximately 187 °C (Fig. 5B). This represents an approximate 60 °C improvement in thermal stability compared to pure eugenol, which can be attributed to its encapsulation within the gelatin nanofiber matrix. Celebioglu et al.³⁵, similarly observed that the evaporation and weight loss of pure eugenol occurred between approximately 50 and 190 °C. However, encapsulating eugenol in pullulan/eugenol-cyclodextrin nanofibers significantly enhanced its thermal stability, shifting the evaporation range from 50–190 to 125–300 °C.

The lower weight loss rate of gelatin nanofibers (approximately 20% less than pure gelatin) indicates their superior thermal resistance. Moreover, the shift of the eugenol decomposition peak to higher temperatures within the gelatin nanofibers confirms the efficiency of encapsulation and the resulting improvement in thermal stability. Liu et al.³⁶, that pure eugenol showed significantly greater weight loss during degradation compared to gelatin/zein nanofibers, demonstrating its lower thermal stability.

Antioxidant activity of nanofibers

The antioxidant capacity of eugenol and gelatin nanofibers containing eugenol at varying ratios and concentrations was evaluated using the DPPH assay. The antioxidant activity of pure eugenol and nanofibers without eugenol was measured at 96.86 ± 0.41% and 25.00 ± 0.54%, respectively. The results of the antioxidant activity for different gelatin concentrations and gelatin-to-eugenol ratios are presented in Table 2. As shown, the antioxidant activity of the nanofibers increased with the rise in eugenol content from 10% to 50%. These findings align with those reported by Bonilla et al.⁷, who demonstrated that the antioxidant activity of gelatin/eugenol nanofibers significantly improves with higher eugenol content. This enhancement can be attributed to the inherent antioxidant properties of eugenol, as well as the contribution of specific amino acids such as glycine and proline present in the gelatin matrix.

Table 2.

The antioxidant activity (%) of the fish nanofibers with different concentrations of gelatin and various eugenol /gelatin ratios

Gelatin/eugenol ratio	Gelatin concentration (w/v)
	15	20
50:50	60.21 ± 0.10Ba	75.99 ± 0.43Aa
70:30	44.25 ± 0.28Bb	66.23 ± 1.23Ab
90:10	36.39 ± 2.52Bc	55.50 ± 2.25Ac

Different uppercase letters indicate significant differences between gelatin concentrations, while different lowercase letters denote significant differences across different eugenol/gelatin ratios (P < 0.05).

Furthermore, the antioxidant activity of gelatin/eugenol nanofibers derived from a 20% gelatin concentration was significantly higher than those produced from a 15% gelatin concentration (P < 0.05). This difference is attributed to the smaller diameter of nanofibers formed at the higher gelatin concentration, as confirmed by FESEM analysis. The reduced diameter increases the surface-to-volume ratio, which facilitates faster and more efficient release of eugenol, thereby enhancing the antioxidant activity. In addition, higher eugenol concentrations in the nanofibers lead to neutralization of more free radicals, further increasing total antioxidant activity. These findings are consistent with Celebioglu and Uyar³⁵, who demonstrated that reducing nanofiber diameter significantly improved the antioxidant activity of encapsulated eugenol in pullulan/eugenol-γCD nanofibers. Similarly, in this study, the smaller diameter of gelatin/eugenol nanofibers at 20% gelatin contributed to higher antioxidant activity. This improvement is mainly due to the higher surface-to-volume ratio, which promotes greater interaction between the nanofibers and eugenol, enhancing antioxidant efficiency. Overall, the results indicate that the 50:50 gelatin-to-eugenol ratio at 20% gelatin concentration exhibited the highest antioxidant activity.

The sterility of nanofibers containing eugenol

All eugenol-loaded nanofiber samples, including those with different gelatin concentrations and eugenol ratios, were aseptically cultured under both aerobic and anaerobic conditions. For each sample, six serial 10-fold dilutions (10⁻¹ to 10⁻⁶) were prepared and plated on appropriate media. After 48 h of incubation at 37 °C, no microbial growth was observed in any of the dilutions, confirming that all eugenol-containing nanofibers were sterile.

Chemical composition of fish

The moisture, dry matter, protein, fat, and ash content of the fish were 77.50 ± 0.13%, 22.41 ± 0.13%, 6.43 ± 0.13%, and 1.34 ± 0.00%, respectively.

Fish pH

pH is a critical determinant of fish quality and chemical deterioration, often associated with a decline in freshness. Figure 6A illustrates the changes in pH levels of uncoated fish and fish coated with gelatin/eugenol nanofibers during 7 days of storage at 6 °C. In both samples, the pH increased over time; however, the changes were not statistically significant in the uncoated sample until day 1 and in the coated sample until day 3. Beyond this point, significant increases in pH were observed. On days 0 and 1, no significant difference was noted between the two samples. However, from day 3 to day 7, the uncoated sample exhibited a significantly higher increase in pH compared to the coated sample at a 95% confidence level (P < 0.05). This disparity can be attributed to the antimicrobial properties of eugenol, which, due to its hydroxyl (OH) groups and hydrophobic nature, inhibits bacterial growth and prevents proteolytic degradation of fish tissue, thereby mitigating pH increases during storage⁶.

Fig. 6. Physicochemical quality changes of fish samples during refrigerated storage.

pH (A), drip loss (B), and TBARS (C) of uncoated and gelatin/eugenol nanofiber-coated fish samples during 7 days of storage at 6 °C. Superscripts with different uppercase letters indicate significant differences between uncoated and coated samples, while superscripts with different lowercase letters denote significant differences among storage times (p < 0.05).

Xia et al.³⁷, investigated the use of chitosan-based nanofibers loaded with tea tree leaf oil to extend the shelf life of fresh rainbow trout fillets. They observed a significant increase in pH across all groups over time, driven by post-mortem microbial activity that degrades fish proteins and produces alkaline compounds such as ammonia and amines. Their findings further demonstrated that chitosan-based nanofibers effectively delayed the pH increase in rainbow trout fillets, underscoring their potential for preserving fish quality. Similarly, Ruelas et al.³⁸, reported that guar gum incorporated with thyme essential oil significantly delayed pH increases in tilapia fillets during storage compared to untreated fillets or those treated with agar gum. This delay was attributed to the reduction in bacterial and enzymatic activity, which typically contributes to pH elevation.

Drip loss of fish

Drip loss serves as a significant indicator of fish meat quality, as it reflects the extent of water loss from the meat during storage. Excessive drip loss can lead to undesirable alterations in the appearance and flavor of fish meat³⁹.

Figure 6B illustrates the changes in drip loss observed in uncoated fish and fish coated with gelatin-eugenol nanofibers during 7 days of storage at 6 °C. As shown, a significant difference between the two samples was evident from day 1 of storage. The uncoated sample exhibited a higher drip loss compared to the coated sample. In the uncoated sample, drip loss increased significantly with prolonged storage time. Notably, a sudden and substantial increase in drip loss was observed on day 3, which may be attributed to microbial contamination. This finding aligns with the microbial deterioration reported in Fig. 7, where a sharp rise in microbial contamination on day 3 likely contributed to the heightened drip loss due to proteolytic tissue degradation caused by microbial activity⁴⁰. In contrast, the drip loss in the nanofiber-coated sample remained relatively stable throughout the storage period, with no discernible variations observed (p > 0.05), except for a minor increase on day 3. This slight rise may also be linked to microbial contamination that occurred on the same day. These results are consistent with the findings of Roshnak, Yarabbi, Movaffagh, and Shahidi⁴¹, who demonstrated that coating fresh beef with CS-PEO nanofibers containing Buforin I significantly reduced weight loss during storage. After 9 days of storage, weight loss in the coated samples was less than 8%, compared to 16.43% in the uncoated samples. The coating acted as an effective barrier against moisture evaporation and capillary water flow, thereby preserving the meat’s freshness and texture. Additionally, the coating reduced enzymatic activity by limiting exposure to oxygen and CO₂.

Fig. 7. Microbial spoilage of fish samples during refrigerated storage.

Bacterial (A) and yeast and mold (B) spoilage variations in uncoated and gelatin/eugenol nanofiber-coated fish samples during 7 days of storage at 6 °C. Superscripts with different uppercase letters indicate significant differences between uncoated and coated samples, while superscripts with different lowercase letters denote significant differences among storage times (p < 0.05).

Oxidative stability of fish

Lipid oxidation is initiated by free radicals degrading polyunsaturated fatty acids, a process that can occur either enzymatically or non-enzymatically through a chain reaction. This phenomenon adversely affects fish quality by producing undesirable odors and flavors⁴¹.

As illustrated in Fig. 6C, lipid oxidation in the uncoated fish sample increased over time, peaking on day 3, followed by a gradual decline until day 7. This trend may be attributed to the generation of malondialdehyde (MDA), an intermediate by-product of lipid oxidation. MDA can further oxidize into other compounds, such as organic acids and alcohols, which do not react with thiobarbituric acid (TBA). Consequently, the reduction in TBARs index values observed over the storage period may be explained by the inability of this method to detect these newly formed compounds⁴². The increase in lipid oxidation on day 3 could be linked to several factors, including heightened drip loss (Fig. 6B), proteolytic tissue degradation, and microbiological contamination (Fig. 7). These factors likely contribute synergistically to the accelerated oxidation observed during this period. The oxidation levels in fish samples coated with gelatin/eugenol nanofibers did not exhibit significant changes (P < 0.05) between days 0 and 7 of storage. In contrast, the oxidation levels in uncoated fish samples were significantly higher (P < 0.05) from day 1 to day 7 compared to the coated samples. These findings indicate that gelatin/eugenol nanofiber coatings were considerably more effective in controlling lipid oxidation. The results of the antioxidant activity assessment of gelatin nanofibers containing eugenol (Table 2) demonstrated the antioxidant properties of this compound, which corresponded to its observed protective effects on fish fillets. These findings are consistent with those reported by Zhou et al.⁴³, who investigated the impact of eugenol-enriched gelatin coatings on Chinese sea bass during superchilled storage (−0.9 °C). They observed an initial increase in malondialdehyde (MDA) levels, followed by a decline after 15 days. Eugenol reduced MDA levels, delayed lipid oxidation, and the subsequent decline was attributed to MDA reacting with aldehydes and ketones, forming by-products that may influence TBARS measurements. Similarly, Najafi et al.⁴², utilized saffron extract encapsulated in electrospun zein nanofibers to extend the shelf life of European seabass (Dicentrarchus labrax) fillets. TBARS analysis revealed that while the control sample exhibited high oxidation levels initially, coating with saffron-infused nanofibers significantly reduced lipid oxidation and MDA levels. The MDA levels peaked by day 4 before gradually declining.

Microbial spoilage of fish

The number of bacterial and fungal colonies that appeared in the culture media on days 0, 1, 3, 5, and 7 was meticulously counted and expressed as log CFU/g. The results, presented in Fig. 7, reveal significant differences (P < 0.05) in microbial deterioration between coated and uncoated samples. Additionally, microbial spoilage in both sample types increased significantly over time from day 0 to day 7 of storage (P < 0.05). As illustrated in Fig. 7, the initial microbial load on day 0 was 0.04 ± 0.03 log CFU/g for the uncoated samples and coated samples. Over time, the microbial load in the uncoated samples increased significantly, peaking on day 3. A slight decline was observed on day 5, followed by another increase on day 7. The reduction in microbial spoilage on day 5, compared to day 3, may be attributed to the production of oxidation by-products, such as alcohols and organic acids, which acted as inhibitory agents and temporarily suppressed microbial growth. This observation aligns with the lipid oxidation findings presented in Fig. 6C⁴². It may also be associated with contamination on day 3, which resulted in heightened spoilage compared to day 5. In the coated sample containing gelatin nanofibers infused with eugenol, fungal and bacterial spoilage progressed at a slower rate than in the uncoated control sample. A significant disparity in microbial deterioration was observed between the two samples at a confidence level of P < 0.05 on each sampling day. Furthermore, no fungal colonies were detected on the culture medium in the coated sample on days 0 and 1, with the microbial load remaining undetectable during this period. The acceptable microbial limits were considered according to the International Commission on Microbiological Specifications for Foods⁴⁴ as reported by Al-Saadi et al.⁴⁵, with ≤7 log₁₀ CFU/g for Total Viable Count (TVC) and ≤5 log₁₀ CFU/g for yeast and mold counts (TFC). Throughout the 7-day storage period, the microbial counts of both uncoated and coated samples remained below these limits; still, the TVC of coated fish was 1.7 log₁₀ CFU/g lower than uncoated sample, confirming that the coating effectively controlled microbial growth. Zhou et al.⁴³, demonstrated that the incorporation of eugenol as an antimicrobial agent effectively extended the shelf life of sea bass samples during superchilled storage. Eugenol suppressed the growth of total viable bacteria, H₂S-producing bacteria, Pseudomonas species, and psychrophilic bacteria, thereby preventing the production of undesirable metabolic compounds that compromise product quality.

Similarly, Ceylan et al.⁴⁶, investigated the effects of grape seed oil nanofibers on limiting microbial growth and lipid oxidation in Kashar cheese and fish meat. Fish fillets coated with nanofibers exhibited significantly lower bacterial counts over a 9-day storage period (P < 0.05) and effectively inhibited mold and yeast growth after day 1 of cold storage. These findings are consistent with the results of the present study, underscoring the efficacy of nanofiber coatings in controlling microbial spoilage and preserving product quality.

Fish color

Changes in the surface color of fish serve as a direct indicator of its freshness. The color parameters of the fish surface are presented in Table 3, where lightness, redness, and yellowness are denoted by L*, a*, and b*, respectively.

Table 3.

Color parameters of uncoated and coated fish fillet samples during 7 days storage

Color parameters	Storage time (Days)	Uncoated	Coated
L*	0	54.66 ± 2.17Aa	54.66 ± 2.17Aa
	1	53.32 ± 0.80Aa	55.02 ± 1.44Aa
	3	52.63 ± 0.56Aa	54.37 ± 1.64Aa
	5	50.40 ± 0.61Bb	53.17 ± 0.48Aa
	7	49.56 ± 0.17Bb	52.89 ± 0.21Aa
a*	0	3.48 ± 1.50Ab	3.48 ± 1.50Aa
	1	7.74 ± 2.54Aa	2.30 ± 3.29Ba
	3	6.91 ± 0.64Aab	2.97 ± 2.01Ba
	5	3.69 ± 1.10Ab	2.93 ± 2.73Aa
	7	4.26 ± 2.97Aab	4.43 ± 0.97Aa
b*	0	11.31 ± 1.20Aa	11.31 ± 1.20Aa
	1	10.20 ± 1.66Aa	9.02 ± 0.51Aa
	3	8.83 ± 3.69Aab	9.58 ± 1.50Aa
	5	6.57 ± 2.30Ab	9.02 ± 1.97Aa
	7	8.49 ± 1.91Aab	9.09 ± 0.56Aa
ΔE	1	5.00 ± 2.03Aa	3.93 ± 10.29Ba
	3	5.43 ± 1.77Aa	2.75 ± 1.63Ba
	5	6.64 ± 1.28Aa	3.67 ± 1.70Ba
	7	6.59 ± 0.89Aa	3.23 ± 0.67Ba

All data are shown as mean ± standard deviation (SD). Different capital letters indicate significant differences between uncoated and coated samples, while different lowercase letters denote significant differences across different storage times (P < 0.05).

Charette et al.⁴⁷, reported that the color of fish fillets is influenced by the physical structure of muscle tissue, heme-based pigments, and the amount of free water, all of which affect light distribution. Color changes in fish during storage can result from enzymatic reactions that degrade myofibrillar proteins, as well as non-enzymatic processes such as oxidation and microbial activity. These processes can lead to visible changes in appearance, including the development of brown and green discoloration. The L* and a* values of the uncoated and coated samples exhibited significant differences, whereas no significant difference was observed in the b* values between the two samples (P > 0.05). According to the Table 3, the L* values of the uncoated samples gradually decreased during storage, indicating fish darkening caused by myoglobin oxidation. In the coated samples, the initial L* values were not significantly different from those of the uncoated samples. However, over time, on days 3 and 5, the L* values of the coated samples were significantly higher than those of the uncoated samples (P < 0.05). Furthermore, the L* values of the coated samples remained relatively stable throughout the storage period, with no significant changes observed. Over the storage period, the a* values in the uncoated samples exhibited a substantial increase on day 1 and day 3, likely due to microbial proliferation, protein degradation, and lipid oxidation³⁸. In contrast, the changes in a* values in the coated samples were not statistically significant over time (P > 0.05). Similarly, the b* values in both coated and uncoated samples showed no significant differences (P > 0.05). However, in the uncoated samples, the b* value decreased on day 5, potentially attributable to lipid oxidation.

Alongside the individual color coordinates, the total color difference (ΔE) provides an integrated measure of the coating’s impact on visual quality. ΔE values were significantly higher in uncoated than in coated samples across the storage period (P < 0.05), reflecting greater discoloration in the absence of coating. However, within each treatment group, ΔE did not change significantly over time (P > 0.05), suggesting that the observed differences were established early and did not intensify during storage. Consistently, the coated fish exhibited highly stable L*, a*, and b* values over 7 days, with no significant upward or downward trends (P > 0.05). Collectively, these results indicate that coating fish with eugenol-loaded gelatin nanofibers effectively preserves visual quality and retards color deterioration during refrigerated storage.

The findings of this study are consistent with those reported by Duan et al.⁴⁸, who developed electrospun pullulan–carboxymethyl chitosan/PEO core–shell nanofibers loaded with nanogels. They observed that the L* values of all samples decreased during storage; however, the L* value in the treatment group coated with core-shell nanofibers containing 8 mg/mL of nisin nanoparticles was significantly higher than that of the control group. Additionally, they noted that the a* and b* values in both treated and control samples increased noticeably over the storage period. Nevertheless, the treated samples exhibited a markedly smaller rise in both a* and b* values compared to the control groups.

Texture of fish

Hardness, springiness, cohesiveness, gumminess, and chewiness are the five fundamental texture-determining parameters used to evaluate the texture of both coated and uncoated fish samples. The changes in hardness over time are presented in Table 4. Variations in fish muscle hardness are generally influenced by several factors, including fish size and biological parameters⁴⁹.

Table 4.

Textural parameters of uncoated and coated fish fillet samples during 7 days storage

Textural parameters	Storage time (Days)	Uncoated	Coated
Hardness (N)	0	54.66 ± 2.17Aa	54.66 ± 2.17Aa
	1	53.32 ± 0.80Aa	55.02 ± 1.44Aa
	3	52.63 ± 0.56Aa	54.37 ± 1.64Aa
	5	50.40 ± 0.61Bb	53.17 ± 0.48Aa
	7	49.56 ± 0.17Bb	52.89 ± 0.21Aa
Springiness (mm)	0	5.28 ± 0.10Aa	5.28 ± 0.10Aa
	1	4.76 ± 0.28Aa	5.08 ± 0.38Aa
	3	3.65 ± 0.35Bb	4.86 ± 0.48Aa
	5	3.88 ± 0.74Bb	4.88 ± 0.46Aa
	7	3.72 ± 0.12Bb	5.25 ± 0.14Aa
Cohesiveness	0	0.43 ± 0.09Ab	0.43 ± 0.09Aa
	1	0.50 ± 0.02Ab	0.40 ± 0.01Aa
	3	0.51 ± 0.02Ab	0.44 ± 0.02Aa
	5	0.57 ± 0.01Ab	0.43 ± 0.08Aa
	7	0.99 ± 0.05Aa	0.50 ± 0.01Aa
Gumminess (N)	0	21.96 ± 2.06Aa	21.96 ± 2.06Aa
	1	20.70 ± 1.96Aa	19.85 ± 6.90Aa
	3	12.73 ± 5.85Bb	18.86 ± 2.14Aa
	5	14.41 ± 3.89Ab	18.20 ± 1.87Aa
	7	4.67 ± 0.96Bc	16.26 ± 1.90Aa
Chewiness (N/mm2)	0	97.85 ± 10.30Aa	97.85 ± 10.30Aa
	1	95.55 ± 8.10Aa	96.95 ± 9.89Aa
	3	90.95 ± 12.72Aa	92.12 ± 10.44Aa
	5	51.10 ± 10.00Bb	102.60 ± 6.30Aa
	7	34.03 ± 2.83Bc	95.58 ± 9.97Aa

All data are shown as mean ± standard deviation (SD). Different capital letters indicate significant difference between uncoated and coated samples, while different lowercase letters denote significant differences across different times (P < 0.05).

As shown in Table 4, the hardness values of the uncoated and coated samples ranged from 38.9 to 53.8 and 48.33 7 to 53.38, respectively. A significant decline in hardness was observed in the uncoated samples over the storage period. In contrast, the hardness of the coated samples remained relatively constant from the 3 to day 7 of storage. Additionally, no significant difference in hardness was detected between the coated and uncoated samples until day 3; however, the coated samples exhibited significantly higher hardness values thereafter. Consistent with our results, carvacrol-loaded gelatin nanofiber coatings on trout fillets were reported to delay hardness loss during refrigeration, indicating that gelatin-phenolic nanofibers help preserve fish textural integrity⁹.

This disparity, particularly after day 3, may be attributed to increased microbial spoilage in the uncoated samples, where microbial growth exceeded 3.8 log CFU/g. In contrast, the microbial load in the coated samples remained below 3 log CFU/g until day 7 (Fig. 7). These findings suggest that the eugenol-containing nanofiber coating effectively preserves the hardness of fish tissue and delays microbial spoilage. This preservation may result from the interaction of phenolic compounds, such as eugenol, with proteins, leading to enhanced tissue rigidity. The results of this study are consistent with those reported by Ceylan et al.⁴⁹, who investigated the impact of tissue degradation in fish flesh coated with electrospun nanofibers. They observed that the hardness of the control samples decreased significantly by 68% during cold storage, compared to the treated samples.

Springiness refers to the extent of height recovery of a food substance between two compression cycles during TPA and reflects the degree of internal bond formation within protein gel networks⁵⁰. The springiness of the uncoated and coated samples differed significantly (P < 0.001). As shown in Table 4, the springiness of the uncoated samples decreased markedly over the storage period, whereas the coated samples remained stable, with no significant changes observed. During the first three days, no significant difference in springiness was noted between the two groups. However, from day 3 onward, the coated samples exhibited significantly higher springiness values, underscoring the protective effect of the coating on texture preservation. After the fish’s death, autolysis and microbial growth lead to tissue softening, which ultimately reduces springiness⁵¹. Ceylan et al.⁴⁹, reported that the springiness of control samples decreased by 24% over a 12-day period, ranging from 0.755 to 0.935. In contrast, fish samples coated with nanofibers demonstrated greater stability in springiness, with minimal changes observed. Similarly, Xia et al.³⁷, utilized electrospun chitosan-based nanofibers loaded with tea tree oil to extend the shelf life of rainbow trout fillets. While hardness and springiness declined in the control groups, the treated fish maintained significantly higher values. They attributed the decline in hardness and springiness in the control groups to protein denaturation caused by microbial growth and water loss. In contrast, the tea tree oil nanofibers preserved the texture by inhibiting microbial activity.

Variations in tissue cohesiveness in fish flesh are influenced by several factors, including protein content, collagen levels, tissue characteristics, fish species, protein solubility, storage conditions, and processing methods⁵². No significant difference was observed between the cohesiveness of uncoated and coated samples up to day 3 of storage (P > 0.05). However, the cohesiveness of the uncoated sample increased notably on day 7, whereas the coated sample remained stable throughout the storage period. Similarly, Ceylan et al.⁴⁹, observed in their study that the texture cohesiveness of fish meat slightly increased during cold storage in the control group compared to the treated samples.

The gumminess of the uncoated and coated samples differed significantly (P < 0.05). According to Table 4, gumminess in the control samples decreased markedly over the storage period, while the coated samples maintained stable gumminess values. On day 1, no significant difference in gumminess was observed between the two groups; however, from day 3 onward, the coated samples exhibited significantly higher gumminess. These findings align with those reported by Yilmaz et al.³⁰, who observed a significant decline in the gumminess of beef samples in the control group during storage (ranging from 0.73 to 3.14). In contrast, the gumminess of samples coated with eugenol-loaded nanofibers remained stable throughout the storage period (ranging from 1.21 to 1.66).

Similarly, the chewiness of the coated and uncoated samples differed significantly (P < 0.05). As shown in Table 4, the chewiness of the uncoated samples decreased significantly over time, whereas the coated samples retained stable chewiness without any notable changes. Initially (days 1–3), no significant difference was observed between the two groups; however, after this period, the coated samples demonstrated significantly higher chewiness. Chewiness reflects the energy required to break down semi-solid food into a state suitable for swallowing. Over time, the energy needed to break down fish muscle in the control samples decreased, likely due to reductions in gumminess and muscle firmness⁵³. Ceylan et al.⁴⁹, demonstrated that the application of nanofiber coatings can effectively preserve the quality and mechanical properties of fish muscle during storage. In the control samples, firmness and chewiness declined significantly over time, whereas these properties remained stable in the nanofiber-coated samples. The results of this study are also consistent with the findings of Yilmaz et al.³⁰, who reported that the chewiness of beef samples in the control group decreased significantly during storage (from 7.60 to 3.22). In contrast, the chewiness of nanofiber-coated samples containing eugenol remained stable throughout the storage period. Li et al.⁵⁴, investigated the preparation of linalool/polycaprolactone coaxial electrospun films and their application in preserving salmon slices. Their results showed that the use of nanofibers containing linalool effectively preserved the chewiness of salmon fillets during storage. In contrast, chewiness declined significantly in the control samples due to microbial growth and the activity of autolytic enzymes. This preservation effect was attributed to linalool’s ability to slow down protein denaturation and hydrolysis by inhibiting microbial activity, thereby delaying texture degradation and spoilage during cold storage.

In conclusion, this work demonstrates that electrospun gelatin nanofiber coatings loaded with eugenol can effectively preserve fish quality during refrigerated storage. Incorporation of eugenol into the gelatin nanofibers—especially at higher gelatin concentration and eugenol loading—produced stable coatings with high loading capacity and practical applicability for food systems. Compared with uncoated samples, coated fish showed a clear delay in spoilage progression, evidenced by slower microbial growth and reduced physicochemical deterioration during storage, resulting in a shelf-life extension. In parallel, the coating helped limit lipid oxidation and pH increase and improved the retention of color and texture-related attributes.

Overall, gelatin/eugenol nanofiber coatings represent a promising natural preservation strategy to enhance refrigerated fish stability and reduce post-harvest losses. Future studies should address scale-up of electrospinning, cost-effectiveness, and validation under industrial handling and longer storage scenarios, as well as testing with other seafood products and real packaging conditions.

Methods

Material

Bovine gelatin powder (Type A; Bloom number of 160) was obtained from the MTRoyal company (Istanbul, Türkiye). Eugenol (≥99%, C₁₀H₁₂O, CAS No: 97-53-0, Merck, Darmstadt, Germany), glacial acetic acid, and absolute ethanol (purity > 99%, Merck, Darmstadt, Germany) was employed as the solvent. DPPH was provided from Sigma-Aldrich (St. Louis, MO, USA). Tryptic soy broth (TSB), Sulfite Polymyxin Sulfadiazine (SPS) agar, Potato Dextrose agar (PDA), and Plate Count agar (PCA) were purchased from Merck Company (Darmstadt, Germany). All reagents were of analytical grade.

Preparation of solutions

A eugenol solution (20% v/v) was prepared by dissolving eugenol in absolute ethanol at room temperature (25 ± 1 °C) due to its poor water solubility, ensuring a homogeneous solution for electrospinning. Gelatin solutions with concentrations of 10, 15, and 20% (w/v) were separately prepared by dissolving gelatin in an acetic acid/distilled water mixture (8:2 v/v) at 40 °C for 2 h. The solutions were then stored overnight in a refrigerator to ensure complete hydration. Subsequently, the gelatin and eugenol solutions were mixed at different gelatin-to-eugenol ratios (100:0, 90:10, 70:30, and 50:50), stirred at room temperature (25 ± 1 °C) for 30 min, and utilized for the electrospinning process. In these mixtures, the final concentrations of eugenol in the spinning solutions were 0%, 2%, 6%, and 10% (v/v), corresponding to the 100:0, 90:10, 70:30,

Electrical conductivity

The electrical conductivity (µS/cm) of the solutions was measured at room temperature (25 ± 1 °C) using a digital conductometer (Jenway, model 3540, Staffordshire, UK).

Surface tensions

The surface tension (mN/m) of the solutions was determined at room temperature using a tensiometer (Dataphysics DCAT 11EC, Filderstadt, Germany) employing the Wilhelmy plate method⁵⁵.

Viscosity

The steady viscosity (cP) of the solutions was assessed using a rotational viscometer (Brookfield, model R VDV-II, Middleboro, MA, USA) equipped with spindle 21 at a rotational speed of 50 rpm.

Rheological properties

Frequency sweep tests were conducted to investigate the viscoelastic properties of the solutions at room temperature using a rheometer (Anton Paar, Physica MRC 301, Graz, Austria). The tests were performed over a frequency range of 0.1–100 rad/s, with a fixed strain amplitude of 1%. The storage modulus (G′), loss modulus (G″), and loss tangent (tan δ) were recorded and calculated according to the Eq. 1¹².

$$\tan \mathrm{\delta }={\mathrm{G}}^{\prime \prime }/{\mathrm{G}}^{\prime }$$ 1

Nanofiber fabrication

A custom-designed needleless electrospinning setup equipped with a rotating spiked cylindrical spinneret (developed in the Department of Food Science & Technology, Isfahan University of Technology, Iran) was employed for the electrospinning process. The solutions were poured into the pan of the device, which operated at an applied voltage of 13 kV, a tip-to-collector distance of 10 cm, and a rotational speed of 30 rpm. The electrospinning process was conducted at room temperature (25 ± 1 °C) for 30 min and the resulting nanofiber mats were collected on drum collectors covered with aluminum foil for further analysis.

Characterization of nanofibers

The following experiments were performed to evaluate the physicochemical characteristics of the gelatin nanofibers containing eugenol.

Sterility of nanofibers

To confirm the sterility of the fabricated nanofibers, they were cultured aerobically in TSB medium and anaerobically in SPS medium. The cultures were incubated at 37 °C for 48 h. A total plate count was subsequently performed using the pour plate method.

Morphological evaluation of nanofibers

The morphological properties of the fabricated gelatin and eugenol-loaded gelatin nanofibers were analyzed using (FESEM; Quanta FEG-450 model, FEI, Hillsboro, OR, USA) at an accelerating voltage of 5–25 kV. Prior to imaging, 1 × 1 mm samples of the nanofibers were deposited onto stubs and coated with gold (10 nm thickness) for 200 s using a high-resolution sputter coater (Agar, model AGB7234-DRY, UK). The fiber diameters were measured at a minimum of 50 random locations in the FESEM images using ImageJ software (National Institutes of Health, Maryland, USA).

Determination of encapsulation efficiency and loading capacity

The encapsulation efficiency (EE%) and loading capacity (LC%) were evaluated by quantifying the surface concentration of eugenol. Initially, a nanofiber sample was mixed with 10 mL of ethanol for 1 min, and the resulting suspension was filtered through filter paper to achieve a final volume of 50 mL. The solution was then filtered using a nylon syringe filter with a pore size of 0.45 µm, and the absorbance was measured at 281 nm using a UV–Vis spectrophotometer (T60 UV, England). The concentration of eugenol was determined using a standard calibration curve of eugenol. The EE% and LC% were calculated using the following equations¹⁵:

$$\mathrm{EE}(\%)=\frac{\mathrm{Total\; eugenol\; amount}-\mathrm{Free\; surface\; eugenol}}{\mathrm{Total\; eugenol\; amount}}\times 100$$ 2

$$\mathrm{LC}\left(\%\right)=\frac{\mathrm{Total\; eugenol\; amount}-\mathrm{Free\; surface\; eugenol}}{\mathrm{Total\; nanofiber\; amount}}\times 100$$ 3

Fourier-transform infrared (FTIR) spectroscopy

Nanofibers with the smallest average diameter and highest encapsulation efficiency were selected and mixed with potassium bromide (KBr) at a 1:100 ratio to prepare transparent pellets. The pellets were used for FTIR spectroscopy (Bruker, Tensor 27, Germany) to analyze the functional groups and potential chemical interactions between eugenol and gelatin. Spectra were recorded over the wavenumber range of 400–4000 cm⁻¹ with a resolution of 4 cm⁻¹⁵⁶.

Thermal gravimetric analysis (TGA) of nanofibers

The thermal stability and degradation profiles of eugenol, gelatin powder, and eugenol-loaded gelatin nanofibers were investigated using thermogravimetric analysis (NETZSCH STA 449F3, Selb, Germany). Approximately 5 mg of each sample was analyzed over a temperature range of 25–600 °C at a heating rate of 10 °C/min under a nitrogen atmosphere with a flow rate of 30 mL/min. The weight loss of the samples was monitored as a function of temperature to evaluate their thermal behavior⁵⁷.

Antioxidant activity of nanofibers

The antioxidant potential of eugenol and eugenol-loaded nanofibers was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay²⁸. Initially, 2 mL of a DPPH-free radical solution (prepared by dissolving 2.5 mg of DPPH in 100 mL of ethanol) was mixed with 0.2 mL of ethanol. The absorbance of this mixture (A1) was measured at 517 nm using a UV-Vis spectrophotometer (T60 UV, Leicester, UK). Subsequently, 5 mg of each sample (eugenol, gelatin nanofibers, and eugenol-loaded gelatin nanofibers) was dissolved in 5 mL of an ethanol solution (7:3, v/v) and incubated in a shaking incubator at 45 °C and 280 rpm for 1 h. Following incubation, 0.2 mL of the clear supernatant was collected and mixed with 2 mL of the DPPH-free radical solution. The mixture was then incubated in the dark for 30 min to allow complete interaction between the free radicals and antioxidants. After centrifugation at 8000 g for 5 min, the absorbance of the resulting solution was measured at 517 nm to determine the remaining concentration of DPPH free radicals, and the absorbance value (A2) was recorded. The corresponding concentrations of A1 and A2 were determined using the standard calibration curve of the DPPH solution. The antioxidant capacity was then calculated using the Eq. 4.

$$\mathrm{Radical\; scavenging\; activity}(\%)=\frac{\mathrm{A}1-\mathrm{A}2}{\mathrm{A}1}\times 100$$ 4

Fish coating

Raw fish (Scomberomorus commerson) was obtained from a local market (Isfahan, Iran). The chemical composition of the fish samples, including moisture, fat, protein, and ash content, was determined using the official methods outlined by the Association of Official Analytical Chemists⁵⁸. Fish fillets (2 × 10 cm²) were prepared and wrapped in aluminum foil (18 × 12 cm²) containing gelatin nanofibers loaded with eugenol (50:50 ratio, 20% w/v gelatin). The 50:50 eugenol-to-gelatin ratio with 20% w/v gelatin was selected based on its optimal fiber formation and highest encapsulation efficiency. Control samples were wrapped in plain aluminum foil without nanofibers. Subsequently, the wrapped fish samples were placed in polyethylene bags and stored at 6 °C for 7 days. All subsequent analyses were performed at intervals of 1, 3, 5, and 7 days during storage.

Determination of fish pH

The pH of the fish samples was determined by homogenizing a 10% (w/v) suspension of fish in distilled water. The pH was measured using a laboratory pH meter (Jenway 3330, Staffordshire, UK).

Drip loss of fish

Drip loss was evaluated by weighing the fish fillet samples at different time points during storage and comparing their weights with the initial weight recorded on day 0. The drip loss percentage was calculated using the Eq. 5.

$$\mathrm{Drip\; Loss}(\%)=\frac{\mathrm{initial\; weight\; of\; fish\; fillet\; sample}-\mathrm{Final\; weight\; of\; fish\; fillet\; sample}}{\mathrm{initial\; weight\; of\; fish\; fillet\; sample}}\times 100$$ 5

Thiobarbituric acid reactive substances (TBARS)

The thiobarbituric acid reactive substances (TBARS) were determined according to the method described by Li et al.⁵⁹, with slight modifications. Briefly, approximately 4 g of fish sample was homogenized with 10 mL of a stock solution containing 20% trichloroacetic acid (TCA) in 2 M phosphoric acid. Subsequently, 10 mL of distilled water was added to the homogenate. The mixture was filtered through filter paper, and 3 mL of the filtrate was collected and mixed with 3 mL of a 0.01 M thiobarbituric acid (TBA) solution prepared in 90% acetic acid.

The resulting mixture was heated in a boiling water bath for 30 min to develop a pink color. After cooling to room temperature, the absorbance of the solution was measured at 532 nm using a UV-Vis spectrophotometer. The TBARS values, expressed as mg malondialdehyde (MDA) per kg of sample, were calculated by applying a conversion factor of 5.4 to the absorbance readings.

Microbial spoilage of fish

One gram of fish samples was aseptically transferred into 9 mL of sterile sodium phosphate buffer (0.1 M, pH 7.0) and homogenized using a stomacher (MRC, Harlow, England) at 250 rpm for 2 min. From the homogenate, six 10-fold serial dilutions were prepared using the same sterile buffer. For microbiological enumeration, 1 mL aliquots from appropriate dilutions were aseptically spread in triplicate onto Petri dishes containing 15 mL of Plate Count Agar (PCA; Merck, Darmstadt, Germany) for Total Viable Count (TVC) and 15 mL of Potato Dextrose Agar (PDA; Merck, Darmstadt, Germany) for yeast and mold counts. The inoculated samples were evenly spread using a flame-sterilized glass spreader, starting from the highest dilution to the lowest, to minimize cross-contamination. All PCA plates were incubated aerobically at 37 ± 1 °C for 48 h, while PDA plates were incubated aerobically at 25 ± 1 °C for 3–5 days. After incubation, visible colonies were counted using a digital colony counter, and the results were expressed as colony-forming units per gram (CFU/g) of fish sample. For statistical analysis, microbial counts were converted to log₁₀ CFU/g values

Fish color

The color changes of the fish fillets during refrigerated storage were evaluated using a Hunter-Lab Colorflex colorimeter (Nippon Denshoku, model ZE6000, Tokyo, Japan). The following parameters were recorded: lightness (L*), which ranges from black to white on a 0–100 scale; redness (a*), where positive values indicate red hues and negative values indicate green hues; and yellowness (b*), where positive values correspond to yellow and negative values to blue. The total color difference (ΔE*) of the samples was calculated using the Eq. 6.

$$\mathrm{\Delta }\mathrm{E}=\sqrt{{(\mathrm{\Delta }\mathrm{L}*)}^2+(\mathrm{\Delta }{\mathrm{a}*)}^2{+(\mathrm{\Delta }\mathrm{b}*)}^2}$$ 6

where ΔL*, Δa* and Δb* represent the color differences between the coated fish fillets and the fresh fish, used as the reference (L* = 54.66, a* = 11.31, b* = 3.48).

Texture profile analysis

Texture profile analysis (TPA) was performed instrumentally using a texture analyzer (SANTAM, STM-20, Tehran, Iran) to evaluate the textural properties of fish fillet. Fish fillets, cut into uniform dimensions of 2 × 2 × 2 cm, were placed on the instrument’s platform and subjected to two consecutive cycles of compression and decompression. A cylindrical probe with a diameter of 150 mm was used to achieve 30% deformation of the samples at a constant speed of 60 mm/min. The force-time curves were recorded during the process, and key textural parameters—hardness (N), cohesiveness, gumminess (N), and chewiness (N/mm²)—were determined. Hardness was defined as the maximum force required to compress the samples. Cohesiveness was calculated as the ratio of the area under the force-time curve during the second compression cycle to that of the first cycle. Gumminess was computed by multiplying hardness by cohesiveness, while chewiness was determined by multiplying gumminess by springiness (mm). Springiness was quantified as the ratio of the time required to reach the maximum force during the second compression cycle to that of the first cycle¹⁴.

Statistical analysis

Two separate factorial designs were employed in this study. For the electrospinning process, a 3 × 4 full factorial design was implemented to evaluate the effects of gelatin concentration (10, 15, and 20% w/v) and gelatin-to-eugenol ratio (100:0, 90:10, 70:30, and 50:50). For the fish preservation experiment, a 2 × 4 factorial design was utilized to assess the effects of nanofiber coating (coated vs. uncoated) and storage duration (1, 3, 5, and 7 days). The effects of the variables were analyzed using two-way ANOVA at a significant level of P < 0.05. Statistical analyses were conducted using SAS software (Version 9.0), and all treatments were performed independently in triplicate.

Author contributions

Parya Shirmohammadi: Formal analysis, Investigation, Writing—original draftNafiseh Soltanizadeh: Funding acquisition, Project administration, Supervision, Validation, Writing—review and editingMilad Fathi: Data curation, Methodology, Project administration, Writing—review and editingAlireza Allafchian: Data curation, Formal analysis, Writing—review and editing.

Data availability

Data are available upon reasonable request.

Competing interests

The authors declare no competing interests.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used Qwen2.5-plus in order to improve the language and readability of the manuscript. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.