Ultrasound-Regulated Molecular Reorganization and Property Enhancement in Gelatin–Glycerol Films

TL;DR

This study shows that low-power ultrasound can improve the structure and performance of gelatin-glycerol films, making them suitable for sustainable packaging and biomedical uses.

Contribution

Introduces low-power direct-probe ultrasonication as a green method to enhance gelatin-glycerol films without additives.

Findings

Low-power ultrasound promotes helix-like molecular packing and smooth film surfaces.

Treated films show improved hydrophilicity, reduced defects, and better thermal stability.

High-power ultrasound leads to porous structures and reduced performance.

Abstract

The replacement of petroleum-based plastics with sustainable and biodegradable materials remains a critical challenge for food packaging and biomedical applications. Gelatin is an attractive natural biopolymer for film fabrication; however, its inherent brittleness, moisture sensitivity, and limited structural stability restrict practical use. In this work, for the first time, low-power direct-probe ultrasonication is introduced as a green and additive-free strategy to regulate molecular organization and enhance the performance of gelatin–glycerol composite films. Systematic variation in ultrasonic power and treatment duration revealed a strong dependence of film structure and properties on processing conditions. Low-power ultrasonication (20 W) promoted gelatin–glycerol interactions, induced a transition from loosely organized molecular arrangements to helix-like molecular packing at the nanometer scale, and produced smooth, compact microscale surface morphologies. As a result, these films exhibited enhanced hydrophilicity, reduced surface defects, and improved thermal stability. In contrast, high-power ultrasonication generated excessive cavitation, leading to large-scale porous structures and diminished thermal and surface performance. Therefore, this work identifies a distinct low-power ultrasonic window that enables controlled molecular reorganization and hierarchical structure formation in gelatin–glycerol systems. Structural and physicochemical analyses using SEM, FTIR, XRD, water contact angle measurements, and thermogravimetric analysis collectively elucidate the ultrasound-driven structure–property relationships within the gelatin–glycerol matrix. Overall, this study demonstrates that controlled ultrasonication enables precise tuning of gelatin-based film architecture and properties, offering a scalable and environmentally friendly route to high-performance biodegradable materials for sustainable packaging and biomedical applications.

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Full text

1. Introduction

The widespread use of petroleum-based plastics has raised significant environmental and health concerns due to their poor degradability and the release of persistent microplastics or toxic additives [1]. These issues are particularly critical in biomedical, pharmaceutical, and food-packaging applications, driving the development of biodegradable, non-toxic, and environmentally compatible polymeric alternatives. Among the many candidates, natural polymers such as starch, chitosan, cellulose, and gelatin have received considerable attention. They are renewable, biodegradable, and often inexpensive byproducts of existing industries. However, replacing conventional plastics remains challenging, as packaging and biomedical applications require a combination of strength, flexibility, transparency, and moisture resistance—properties that most single biopolymers do not fully provide [2]. Gelatin, produced by the partial hydrolysis of collagen, is one of the most promising protein-based materials for edible and biodegradable films. It is widely available from animal byproducts, inexpensive, and already used in food and pharmaceutical applications. Films made from gelatin are transparent, easy to form, and exhibit excellent oxygen-barrier properties under dry conditions [3]. These characteristics make gelatin attractive for both food-packaging and biomedical uses.

Despite these advantages, gelatin also has notable drawbacks. Gelatin films are brittle and prone to cracking unless modified, and their strong affinity for water leads to poor performance in humid environments. These limitations restrict the direct use of gelatin films in practical applications, particularly for food and biomedical systems involving moisture exposure [4,5,6]. Various strategies have been explored to overcome these challenges. Chemical crosslinkers can enhance mechanical strength and reduce water sensitivity, but their potential toxicity raises concerns for food- and biomedical-related applications [7]. Blending gelatin with natural polymers such as chitosan, starch, or alginate can improve barrier properties but often compromises film transparency and biodegradability. The incorporation of lipids or essential oils can enhance water resistance, yet may result in heterogeneous textures or reduced mechanical strength. Reinforcement with nanofillers such as nanocellulose or clays has also shown promise, although issues related to large-scale production and safety remain under evaluation [8].

One effective approach to improving the flexibility of gelatin films is the incorporation of plasticizers such as glycerol. Glycerol molecules interpose between gelatin chains, weakening intermolecular hydrogen bonding and increasing molecular mobility within the polymer matrix. This plasticizing effect produces softer and more flexible films that are less prone to fracture [5]. However, glycerol is highly hydrophilic, and its incorporation increases water uptake while often promoting macroscopic phase separation between gelatin and glycerol. This phase separation, combined with excessive plasticization, can induce microcracks and significantly reduce the mechanical integrity of the films [6]. Studies have shown that incorporating a moderate amount of glycerol, such as ~7 wt%, can achieve a balance between flexibility and microstructural homogeneity, which remains a central strategy in the design of gelatin–glycerol composite films.

Ultrasonication has emerged as a promising physical technique to address these challenges. When high-frequency sound waves propagate through a liquid medium, they generate cavitation bubbles that collapse violently, producing localized regions of extreme pressure, temperature, and shear. These effects can disrupt molecular aggregates, enhance mixing, and alter polymer conformations [9]. In food and biopolymer research, ultrasound has been widely used to homogenize emulsions, improve extraction efficiency, and modify protein structures [10]. For protein systems such as gelatin, ultrasonication can induce chain unfolding, expose functional groups, and promote the rearrangement of secondary structures, including α-helices [11]. Such structural modifications can enhance solubility, stability, and network-forming capability.

Recent studies have demonstrated that ultrasonication can improve the properties of gelatin-based films [12,13,14,15,16,17,18]. High-intensity ultrasound has been shown to promote hydrogel network formation in gelatin, while mild ultrasound treatment can enhance material properties without causing significant morphological damage [12,19]. However, most prior studies have focused on high-intensity ultrasound or have not systematically distinguished the fundamentally different structural responses between low- and high-power regimes in gelatin–glycerol systems. As a result, the unique structure–property behavior that occurs under mild ultrasonic conditions remains poorly understood.

In this study, we systematically investigate, for the first time, the contrasting effects of low- and high-power ultrasonication on the molecular organization, morphology, and functional properties of gelatin–glycerol films. Direct-probe ultrasonication was applied during film preparation, and samples fabricated under different ultrasonic conditions were characterized using Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), water contact angle measurements, and thermogravimetric analysis (TGA). These complementary techniques provide a comprehensive assessment of molecular interactions, microstructure, surface wettability, and thermal behavior. The ultimate objective is to evaluate whether ultrasonication can serve as a clean, efficient, and scalable method for producing biodegradable gelatin-based films with balanced morphology, flexibility, thermal stability, and hydrophilicity, which are key attributes required for both food-packaging and biomedical applications.

2. Results and Discussion

2.1. Morphological Analysis

SEM micrographs were used to evaluate changes in the surface morphology of gelatin–glycerol films induced by ultrasonic treatment. Previous studies have reported that high-intensity ultrasound can promote hydrogel formation in gelatin-based biomaterials. Therefore, low-power ultrasound (20 W) was first employed to investigate the initial effects of ultrasonication on gelatin–glycerol films. Figure 1 presents SEM images of films treated at 20 W for different durations (3, 5, 10, and 15 min), with untreated films shown as the control.

The control films exhibited relatively rough but flat surfaces without observable pores. As the ultrasound treatment time increased from 3 to 5 min, the films developed submicron- to micron-scale surface features accompanied by increased surface roughness. This observation suggests that a phase or structural transition occurs with increasing ultrasonication time. Further insight into this transition is provided by the structural analyses discussed later. With continued ultrasonication from 5 to 15 min, the surface morphology became more stable and uniform. The films treated for 10–15 min exhibited smoother and more continuous surfaces, with the most ordered submicro-scale surface features observed at higher magnification (10 μm scale). These features are morphological consequences of ultrasound-induced molecular reorganization rather than direct visualization of molecular nanostructures. This morphology reflects improved phase dispersion and reduced surface defects [12]. Notably, no porous structures were observed in films treated at 20 W, even at extended treatment times.

To further evaluate the influence of ultrasonic power on film morphology, Figure 2 shows SEM images of gelatin–glycerol films treated at higher power levels (40, 65, 90, and 120 W) for a fixed duration of 15 min. At 40 W, the film surfaces remained generally smooth but lacked the ordered submicro-scale patterns observed in the 20 W-treated samples. Upon increasing the ultrasonic power to 65 W, significant morphological changes were observed. The films transitioned from transparent to opaque white, and SEM images at the 200 μm scale revealed the formation of large pores with diameters of approximately 10–40 μm. This indicates that the gelatin–glycerol solution began to form gel-like or foam-like structures as ultrasonic power increased. Further increases in ultrasonic power to 90 and 120 W intensified this effect, with pore sizes expanding to approximately 20–60 μm for the 90 W-treated films and 30–120 μm for the 120 W-treated films. These morphological changes were primarily evident at larger length scales (200 μm). At smaller length scales (10 and 30 μm), the film surfaces remained relatively smooth despite increasing ultrasonic power. However, no ordered submicro-scale surface features, similar to those observed in the 20 W-treated samples, were detected at these higher power levels.

In summary, SEM analysis indicates that low-power ultrasonication promotes the formation of ordered micro-/submicro-scale surface morphologies in gelatin–glycerol films. These patterns arise from the aggregation of nanoscale molecular rearrangements into larger surface motifs, as supported by the structural analyses discussed later. In contrast, high-power ultrasound generates intense shear forces and turbulence through cavitation, which can enhance phase separation at larger length scales and promote the formation of dense porous structures [14].

2.2. Hydrophilicity Measurement

Surface hydrophilicity is a critical parameter for biomaterials, as it strongly influences interactions with biological components such as cells, biomarkers, and tissues. To correlate the morphology of ultrasound-treated gelatin–glycerol films with their surface wettability, water contact angle measurements were performed on films treated at different durations (3–15 min) at 20 W (black line) and at varying ultrasonic power levels (40–120 W) for 15 min (red line), as shown in Figure 3a,b, respectively. Representative contact angle images corresponding to each treatment condition are also presented alongside the data points.

Under low-power ultrasonication (20 W, Figure 3a), ultrasound-treated films exhibited significantly lower water contact angles compared to the untreated samples, indicating enhanced surface hydrophilicity. As the treatment time increased, the contact angle decreased continuously, from approximately 95° for the untreated film to about 60° for the film treated for 15 min. This improvement in wettability is attributed to the more uniform surface structure and redistribution of polar functional groups induced by ultrasonic cavitation. Specifically, this redistribution may involve increased surface exposure of hydroxyl groups from glycerol and amide groups from gelatin, which enhances hydrogen-bonding interactions with water molecules. Among all ultrasound-treated samples shown in Figure 3, films treated at 20 W for 15 min exhibited the highest hydrophilicity. This behavior is consistent with the SEM observations, where uniform molecular dispersion and the formation of ordered nanostructures increased the surface exposure of hydrophilic functional groups, thereby strengthening interactions with water molecules and resulting in reduced contact angles [6].

In contrast, increasing the ultrasonic power disrupted this trend. At 40 W for 15 min, the contact angle decreased to approximately 85° compared to the untreated film (~95°), but remained higher than that of the 20 W-treated samples (~65°). This result indicates that low-power ultrasonication is more effective for tuning the surface wettability of gelatin–glycerol films. Further increases in ultrasonic power from 40 to 120 W reversed the wettability trend, with contact angles increasing to approximately 104°, 110°, and 112° for films treated at 65, 90, and 120 W, respectively. As revealed by SEM analysis, these films developed large-scale porous surface structures, which significantly alter surface topology and reduce effective wettability, making the surfaces more water-repellent compared to smooth films.

Overall, the contact angle results demonstrate that low-power ultrasound treatment produces gelatin–glycerol films with superior surface hydrophilicity. Such surfaces are particularly advantageous for biomedical applications, where enhanced wettability can promote cell attachment, proliferation, and interactions with biological environments relevant to tissue regeneration and drug delivery.

2.3. Structural Analysis

Figure 4 presents the FTIR spectra of gelatin–glycerol films treated with ultrasound at different treatment durations (3–15 min) at 20 W and at varying power levels (40–120 W) for 15 min, with the untreated film shown as the control. Characteristic absorption bands of gelatin were observed in all spectra, including the amide I (~1650 cm^−1^), amide II (~1550 cm^−1^), and amide III (~1240 cm^−1^) regions, confirming the preservation of the protein backbone [20]. FTIR analysis revealed notable changes in the secondary structure of gelatin within the gelatin–glycerol films as a result of ultrasonic treatment. In particular, variations were observed in the amide I region (1600–1700 cm^−1^), which is highly sensitive to protein conformational changes. Both the untreated gelatin–glycerol film (Untreated) and the pure gelatin film (Gelatin) exhibited a weak band centered around 1634 cm^−1^ with a small shoulder near 1650 cm^−1^, characteristic of loosely organized α-helical structures commonly associated with partially denatured collagen [11]. However, clear differences were observed in the amide II region between the gelatin–glycerol film and the pure gelatin film. In the gelatin–glycerol sample, the peak at 1532 cm^−1^ decreased significantly, and the band originally located at 1545 cm^−1^ shifted to 1553 cm^−1^. The amide II region is highly sensitive to side-chain interactions and the local chemical environment of proteins [11]. These changes therefore may indicate that the incorporation of glycerol alters the tertiary structure and local bonding environment of gelatin through hydrogen-bond interactions. Importantly, the absence of major changes in the amide I region before ultrasonication suggests that glycerol primarily affects higher-order molecular organization rather than the protein secondary structure at this stage.

Upon low-power ultrasonication at 20 W (Figure 4a), a clear shift toward higher wavenumbers and an increase in peak intensity around ~1650 cm^−1^ were observed with increasing treatment time, indicating enhanced α-helical content [11]. This transition became pronounced after 5 min of treatment, with only minor changes in peak intensity observed between 10 and 15 min. This trend is consistent with the SEM observations, which shows that the microscale morphological changes associated with underlying molecular reorganization occurred within the first 5 min of ultrasonication. Prolonged treatment further stabilized the formed nanostructure but did not induce significant additional changes in the secondary structure of the gelatin–glycerol films. The transition from loosely organized to more ordered α-helical structures can be attributed to the localized shear forces and cavitation effects generated during low-power ultrasonication. These effects disrupt weak intramolecular hydrogen bonds associated with random coils or disordered helices and facilitate chain alignment into more thermodynamically stable, longer-range α-helix-like domains.

In contrast, increasing the ultrasonic power from 40 to 120 W did not promote further α-helical formation. As shown in Figure 4b, higher power treatments did not enhance the intensity of the ~1650 cm^−1^ band. Instead, a slight increase in the peak near 1634 cm^−1^ was observed with increasing power, suggesting stabilization of loosely organized α-helical structures or the possible formation of short-range β-sheet-like domains. However, these structural changes did not indicate long-range molecular reorganization comparable to that induced by low-power (20 W) ultrasonication.

In addition, ultrasound-treated films exhibited increased intensity in the O–H stretching region (~3300 cm^−1^), indicative of enhanced hydrogen bonding interactions resulting from improved gelatin–glycerol molecular interactions. This observation suggests that ultrasonication promotes exposure of functional groups and strengthens intermolecular interactions within the gelatin–glycerol matrix [11,12]. Overall, the FTIR results confirm that low-power ultrasonication is an effective physical strategy for modulating the molecular architecture and performance of gelatin-based biopolymer films.

2.4. Crystalline Analysis

To further understand the molecular reorganization of gelatin–glycerol films, X-ray diffraction (XRD) was employed to examine structural features and transitions induced by ultrasonic treatment. Wide-angle powder XRD was performed on free-standing dried films in reflection mode. Figure 5 shows the XRD patterns of gelatin–glycerol films treated with ultrasound at different treatment durations (3–15 min) at 20 W and at varying power levels (40–120 W) for 15 min with the untreated gelatin–glycerol film and pure gelatin film used as controls.

The untreated gelatin–glycerol film exhibited a broad diffraction halo centered at approximately 19–20° (2θ), which is characteristic of predominantly amorphous regions or loosely ordered polypeptide segments in gelatin-based materials [17,18]. A weak diffraction peak was observed in the range of 6–6.5° (corresponding to d ≈ 13–14 Å), which is partially masked by background scattering from the primary beam. In addition, a small diffraction peak was also observed in the range of 12–13° (d ≈ 7.0–7.4 Å). Previous XRD studies have reported that a low-angle diffraction peak or shoulder at approximately 2θ ≈ 7–8° (d-spacing ≈ 11 Å) is associated with residual triple-helical structures in gelatin [17,18]. It was also found that adding glycerol into the gelatin matrix reduces the intensity of this peak, as it breaks the hydrogen bonds holding the triple-helix together, resulting in a more disorganized matrix [18]. Increasing glycerol concentrations also cause this diffraction peak to shift to a lower angle value, indicating increased intermolecular spacing [18]. In the present study, the diffraction peak position shifted from 2θ ≈ 7.8° (d ≈ 11.3 Å) for pure gelatin to 2θ ≈ 6.3° (d ≈ 14.0 Å) for the untreated gelatin–glycerol sample, and its intensity relative to the baseline was also reduced with the addition of glycerol, confirming the findings of previous studies [18]. However, the third small peak observed around 2θ ≈ 12.6° (d ≈ 7.0 Å) lies outside the canonical 7° peak region, suggesting the formation of a distinct intermediate ordered phase involving gelatin–glycerol interactions. This interplanar spacing is smaller than the typical α-helix diameter (10~15 Å) and larger than the helix pitch (~5 Å), indicating that the feature is more likely related to interchain or intermolecular packing distances rather than the dimensions of individual helices. This feature may reflect modified lamellar spacing or interchain distances resulting from glycerol incorporation, rather than the presence of intact gelatin triple helices. Scherrer analysis of the broad ~12.6° peak (FWHM ≈ 4.5°) also suggests that the corresponding coherent scattering domains are on the order of ~1–2 nm, indicating that this ordering is limited to short-range molecular packing rather than long-range crystallinity. Overall, the absence of sharp diffraction peaks in the untreated films confirms the lack of long-range crystalline order, which is consistent with the partially denatured nature of gelatin and the plasticizing effect of glycerol. The broad halo pattern indicates predominantly disordered molecular packing with only limited short-range associations [3].

The influence of ultrasound was first examined for the low-power (20 W) treated samples (Figure 5a). Short ultrasonication times (3–5 min) resulted in only slight reductions in halo intensity near 20° (d ≈ 4.4 Å), indicating that limited cavitation enhanced chain mobility and molecular mixing without inducing major structural rearrangement. In contrast, extended ultrasonication (10–15 min) produced broader and weaker halos at 20°, accompanied by a noticeable increase in the diffraction intensity near 12° and a gradual decrease of the triple-helix peak around 2θ ≈ 6.3°. These changes suggest that prolonged low-power ultrasound promotes structural reorganization by disrupting initial gelatin molecular interactions and facilitating the formation of a more ordered helix-like packing incorporating both gelatin and glycerol molecules. Similar ultrasound-induced structural transitions have been reported in other protein–plasticizer systems, such as soy protein isolate and whey protein films, where cavitation-driven forces enhanced molecular ordering [19,21,22].

A comparable but distinct trend was observed for power-dependent treatments (40–120 W for 15 min), as shown in Figure 5b. At lower power levels (40 and 65 W), the amorphous halo intensity increased slightly compared to the untreated film, indicating partial disruption of residual gelatin associations and improved compatibility of glycerol within the gelatin network. However, the strong contrast between the 20 W and 40 W samples may reflect the non-linear nature of acoustic cavitation: 20 W enables gradual molecular reorganization between gelatin and glycerol molecules, whereas ≥40 W produces violent cavitation that rapidly locks the initial gelatin structure and prevents long-range reassembly. At higher ultrasonic powers (90 and 120 W), the halo became more intense and slightly sharper, suggesting enhanced intramolecular hydrogen bonding within loosely ordered helical domains. The increased intensity near 20° may indicate enhanced local packing or component association, although this does not necessarily imply long-range crystallization. Although the 120 W-treated sample also exhibited an increase in the diffraction feature near 12°, its intensity remained significantly lower than that observed in the 20 W-treated films. This observation indicates that while high-power ultrasonication increases molecular ordering, it does not promote the same degree of ordered packing formed under mild ultrasound conditions. Similar behavior has been reported in ultrasound-treated gelatin systems, where excessive cavitation disrupts initial hydrogen bonding while limiting organized structural reassembly [19].

Overall, XRD analysis confirms that gelatin–glycerol films are predominantly amorphous with loosely ordered crystalline domains and that ultrasonic treatment can modulate their crystalline organization in a controlled manner. Mild ultrasonication enhances structural homogeneity and gelatin–glycerol compatibility, promoting the formation of a more ordered molecular packing. In contrast, higher ultrasonic amplitudes increase molecular ordering without significantly altering crystalline packing or spacing. These structural modifications are important because enhanced molecular order and compatibility are often correlated with improved flexibility, plasticizer distribution, and functional performance in gelatin–glycerol films for food packaging, pharmaceutical, and biomedical applications [19,21,22].

2.5. Thermal Analysis

Figure 6 shows the thermogravimetric (TG) curves of gelatin–glycerol films treated with ultrasound for different durations (3–15 min) at 20 W and at varying power levels (40–120 W) for 15 min, recorded during heating from room temperature to 800 °C. The TGA results reveal three main stages of weight loss: moisture evaporation (25–120 °C), glycerol volatilization (180–300 °C), and gelatin decomposition (300–500 °C).

For films treated with low-power ultrasound (20 W), the ultrasound-treated samples exhibited a delayed onset of thermal degradation and a higher residual mass compared to the untreated film. The remaining mass at 800 °C increased progressively with increasing ultrasound treatment time, from 22.9% for the untreated film to 25.4%, 28.9%, 30.6%, and 32.9% for films treated for 3, 5, 10, and 15 min, respectively. This gradual increase in residual mass suggests enhanced thermal stability, which can be attributed to tighter molecular packing and stronger intermolecular interactions, as supported by the FTIR and XRD results. These findings indicate that low-power ultrasonication promotes the formation of a more cohesive polymer network, improving resistance to thermal decomposition [12]. As a result, ultrasound-treated films exhibit improved uniformity, thermal resistance, and overall material performance [12].

In contrast, high-power ultrasound treatments showed a different trend. While treatment at 40 W moderately improved thermal stability, increasing the residual mass at 800 °C to 27.2%, further increases in ultrasonic power led to a pronounced reduction in thermal stability. The residual mass decreased to 22.1%, 18.5%, and 16.5% for films treated at 65, 90, and 120 W, respectively. As observed in the SEM analysis, these samples developed large-scale porous structures, with pore size increasing as ultrasonic power increased. The formation of such porous morphologies significantly increases the effective surface area of the films, facilitating heat penetration through the pores and accelerating thermal degradation across the film thickness. Therefore, despite the presence of enhanced molecular packing and intermolecular interactions indicated by FTIR and XRD for high-power ultrasound-treated samples, their three-dimensional porous morphology renders them more susceptible to thermal decomposition. These results suggest that high-power ultrasonication is not favorable for enhancing the thermal stability of gelatin–glycerol films, whereas low-power ultrasound treatment offers a more effective and controlled approach for improving thermal performance.

2.6. Mechanism of Ultrasound on Gelatin–Glycerol Material

Gelatin is composed of polypeptide chains stabilized by extensive inter- and intramolecular hydrogen bonding between amide groups and side-chain functionalities. These interactions provide structural rigidity but inherently limit molecular flexibility. When glycerol is introduced as a plasticizer, its multiple hydroxyl (–OH) groups form hydrogen bonds with gelatin’s amide, carbonyl, and amino residues, partially disrupting the native hydrogen-bonding network and increasing the free volume within the polymer matrix. This molecular insertion enhances chain mobility, reduces intermolecular cohesion, lowers the glass transition temperature, and ultimately results in more flexible and mechanically durable films [20,23,24,25,26,27,28].

In the present study, films containing a low glycerol content (~7 wt%) remained predominantly amorphous or loosely ordered, as indicated by the broad XRD halo, exhibiting smooth and homogeneous microstructures without macroscopic phase separation (as observed in SEM, Figure 7) [15,16,25,26]. Under these conditions, low-power ultrasonication (20 W) exerted a pronounced influence on the gelatin–glycerol matrix. SEM observations revealed the formation of ordered microscale surface features associated with underlying nanoscale molecular reorganization, while water contact angle measurements confirmed enhanced surface hydrophilicity. Complementary FTIR (amide I shift) and XRD (new diffraction feature at ~12°) further demonstrated that prolonged low-power ultrasonication promotes deeper incorporation of glycerol into the gelatin network. This process facilitates molecular reorganization into a helix-like ordered structure that is distinct from the canonical triple-helix structure of native gelatin. The formation of this ultrasound-induced nanostructure is consistent with the improved thermal stability observed in TGA, contributing directly to the enhanced thermal stability of the gelatin–glycerol films compared to untreated samples.

In contrast, high-power ultrasonication (40–120 W) produced markedly different effects. At elevated ultrasonic intensities, strong cavitation and shear forces rapidly promote hydrogen bond formation within the gelatin–glycerol matrix, reinforcing the initially loosely ordered structure through strong intermolecular interactions, as suggested by FTIR and XRD trends. However, this rapid stabilization prevents the matrix from undergoing the initial disruption and subsequent reorganization necessary to form a well-defined nanoscale, helix-like structure with effective glycerol integration [6,22,27,28]. Furthermore, ultrasonic powers above 40 W induce the formation of large-scale porous structures (10–120 μm), as directly observed in SEM images. These pores are likely generated by the collapse and entrapment of cavitation-induced air bubbles within the polymer matrix during high-intensity ultrasonication [12,24]. The emergence of such porous morphologies significantly increases surface area and disrupts matrix continuity, which correlates with the reduced hydrophilicity (contact angle) and lower thermal stability (TGA). These effects counteract the benefits of enhanced molecular interactions and render high-power ultrasound treatments less favorable for applications requiring controlled wettability and thermal resistance.

Collectively, these results demonstrate that glycerol plasticizes gelatin films by disrupting native hydrogen bonding, increasing chain mobility, and modulating molecular organization. Ultrasonication further governs these effects in a power- and time-dependent manner: low-power ultrasound enables controlled molecular rearrangement and the formation of stable, ordered nanostructures, as supported by SEM, FTIR, XRD, contact angle, and TGA results, while excessive ultrasonic energy promotes porous morphology and compromises functional performance. This mechanistic understanding underscores the importance of optimized ultrasonic conditions for designing gelatin–glycerol biomaterials suitable for green packaging and biomedical applications.

3. Materials and Methods

3.1. Preparation of Materials

Gelatin powder (Type A, laboratory grade) was purchased from Acros Organics (Fair Lawn, NJ, USA) and used as received. A total of 1.0 g of gelatin was dissolved in distilled water to prepare a 10% (w/v) gelatin solution. Specifically, gelatin powder was added to distilled water in a glass beaker and heated to 60 °C for 30 min under continuous magnetic stirring to ensure complete dissolution. After gelatin dissolution, glycerol (≥99.5%, EMD Millipore Corporation, Burlington, MA, USA) was added as a plasticizer at a gelatin/glycerol mass ratio of 93/7. The mixture was stirred until a homogeneous solution was obtained. This relatively low glycerol content was selected based on our preliminary optimization studies, in which glycerol concentrations ranging from 3 wt% to 15 wt% in gelatin matrix were evaluated, and is consistent with values reported in the literature for gelatin–glycerol films that balance flexibility and mechanical integrity [13,14,15,16]. Previous studies have demonstrated that glycerol contents of approximately 7 wt% provide an effective compromise between enhanced elongation and controlled water uptake, while maintaining tensile strength and film transparency [13,14,15,16]. Together, these considerations support the selection of 7 wt% glycerol as a practical and scalable formulation for gelatin-based biopolymer films.

Following complete mixing, the gelatin–glycerol solution was subjected to direct-probe ultrasonication (Figure 8). Ultrasonic treatment was carried out using a probe sonicator operating at a frequency of 20 kHz, with the titanium probe immersed directly into the solution to ensure efficient energy transfer. Ultrasonication was performed in pulsed mode (1 s on/1 s off) to minimize excessive heating and prevent thermal degradation. To further control temperature rise during sonication, the beaker containing the solution was placed in a water chilling bath, maintaining the solution temperature close to ambient conditions throughout treatment. Therefore, the observed structural changes can be attributed primarily to acoustic cavitation and mechanical effects rather than to thermal heating.

To systematically investigate the effects of ultrasonic energy input, two sets of parameters were explored: (i) varying ultrasonic power levels (0, 40, 65, 90, and 120 W) at a fixed treatment duration of 15 min, and (ii) varying treatment durations (0, 3, 5, 10, and 15 min) at a fixed ultrasonic power of 20 W. These conditions were selected to span mild to relatively high ultrasonic intensities, enabling identification of optimal processing parameters for enhanced polymer interactions and film performance. A detailed discussion of the influence of ultrasonic amplitude and duration on film morphology and structure is provided in Section 2.

After ultrasonication, the treated solutions were cast into polydimethylsiloxane (PDMS) molds to obtain films with uniform thickness and geometry. PDMS molds were chosen due to their flexibility, chemical inertness, and non-stick properties, which facilitate easy demolding. The cast solutions were allowed to dry at room temperature (22–25 °C) under ambient conditions for 48 h. Once fully dried, the films were carefully peeled from the molds and conditioned under standard laboratory atmosphere (approximately 50% relative humidity) for at least 24 h prior to characterization.

3.2. Fourier-Transform Infrared Spectroscopy (FTIR)

Fourier-transform infrared (FTIR) spectra were collected using a Bruker Tensor 27 FTIR spectrometer (Bruker Optics, Billerica, MA, USA) equipped with a deuterated triglycine sulfate (DTGS) detector and a multiple-reflection horizontal MIRacle attenuated total reflectance (ATR) accessory fitted with a germanium (Ge) crystal (Pike Technologies, Madison, WI, USA). Spectra were recorded over the wavenumber range of 4000–400 cm^−1^ with a spectral resolution of 4 cm^−1^, averaging 64 scans per measurement. To account for potential heterogeneity, each film sample was measured at a minimum of four different locations (n ≥ 4). Representative spectra were obtained by averaging these measurements. The ATR crystal was thoroughly cleaned with ethanol between measurements to prevent cross-contamination.

3.3. Water Contact Angle Measurements

Surface wettability of the gelatin–glycerol films treated under various ultrasonic conditions was evaluated using a contact angle goniometer (DSA30S, KRÜSS GmbH, Hamburg, Germany) equipped with a high-resolution U5-series camera. A droplet of distilled water (2 µL) was gently deposited onto a randomly selected area of the film surface using a microsyringe. Measurements were performed at room temperature, and multiple measurements (n ≥ 3) were conducted for each sample to ensure reproducibility.

3.4. Thermogravimetric Analysis (TGA)

Thermal stability of the gelatin–glycerol films was analyzed using a Discovery SDT 650 thermogravimetric analyzer (TA Instruments, New Castle, DE, USA). Film samples weighing approximately 4–6 mg were placed in alumina crucibles and heated from 25 °C to 800 °C at a constant heating rate of 10 °C min^−1^ under a nitrogen atmosphere. The nitrogen flow rate was maintained at 100 mL min^−1^ to prevent oxidative degradation. Weight loss as a function of temperature was continuously recorded throughout the heating process.

3.5. Scanning Electron Microscopy (SEM)

The microstructural features of the gelatin–glycerol films were examined using a field-emission scanning electron microscope (Leo 1530 VP, Carl Zeiss SMT, Oberkochen, Germany). Prior to imaging, film samples were mounted on aluminum stubs and sputter-coated with a thin gold layer for 60 s to enhance surface conductivity. SEM images were acquired at magnifications of 1000×, 5000×, and 10,000×, using an accelerating voltage of 5.0 kV.

3.6. X-Ray Diffraction (XRD)

The crystalline structure of the gelatin–glycerol films was analyzed by X-ray diffraction (XRD) using a PANalytical Empyrean diffractometer (Malvern Panalytical, Almelo, The Netherlands). Measurements were conducted at an operating voltage of 30 kV and a current of 10 mA. Diffraction patterns were collected across the relevant 2θ range characteristic of gelatin–glycerol materials.

4. Conclusions

This work establishes direct-probe ultrasonication as an effective physical approach for enhancing the performance of gelatin–glycerol films. Under controlled ultrasonic processing [29,30,31,32], the gelatin–glycerol matrix becomes more structurally organized, as supported by combined FTIR, XRD, and SEM observations. Notably, films treated with low-power ultrasound exhibited reduced water contact angles, smoother surfaces, and superior thermal stability relative to untreated controls, with optimal properties achieved at 20 W for 15 min. Conversely, excessive ultrasonic energy introduced unfavorable effects, including the formation of large-scale porous structures (10–100 μm) and limited molecular reorganization, ultimately compromising wettability and thermal resistance. Taken together, these findings demonstrate that moderated ultrasonication effectively improves morphology uniformity, hydrophilicity, and thermal stability without chemical additives [24,25,32]. Consequently, gelatin–glycerol films processed under optimized ultrasonic conditions show strong potential for sustainable food-packaging applications and, importantly, may also be promising for biomedical and pharmaceutical uses requiring controlled degradation, biocompatibility, and reliable functional performance.