Effect of Homogenization Rates on the Properties and Stability of Fish Gelatin Films with Cinnamon Essential Oil

Nomenclature

1  Introduction

Biodegradable polymers are increasingly studied as alternatives to conventional plastic due to their environmental benefits. Traditional petrochemical-based plastics are estimated to contribute approximately 15% of global carbon emissions, equivalent to around 1.7 gigatons of CO2 [1]. In contrast, biodegradable polymers can be derived from renewable sources such as agricultural feedstocks, marine industry by-products, animal, and microbial sources [2,3]. Common biopolymers used in packaging films include polysaccharides, lipids, and proteins, which may be combined to enhance their functional and mechanical properties [4].

Fish gelatin is a protein-based biopolymer and is extracted from the collagen in fish bones and skin. As food packaging, it is valued for its film-forming ability, biodegradability, biocompatibility, and excellent barrier properties against gases, volatile compounds, oils, and UV light [1,5–7]. Additionally, fish gelatin is a sustainable choice that aligns with religious and cultural dietary restrictions, avoiding health and ethical concerns linked to porcine and bovine sources [8]. Despite its benefits, fish gelatin has several limitations that must be addressed for broader application in food packaging. Pure gelatin films are typically brittle and are prone to cracking due to disulfide bonds, hydrogen bonds, and electrostatic interactions that decrease flexibility [9]. Secondly, its hydrophilic nature results in poor water vapor barrier and mechanical properties [10], particularly in high-humidity environments, limiting its application in food packaging [1,11]. To overcome this limitation, essential oils can be incorporated into gelatin to lower its hydrophilicity while improving its antimicrobial and antioxidant properties [12–14].

Essential oils are plant-based secondary metabolites which are approved by the Food and Drug Administration (FDA) as safe biological additives [8]. Due to the abundant bioactive compounds that are present in essential oils, especially those from aromatic spices such as cinnamon [15–17], clove [18,19], and oregano [20,21], they are known for their antimicrobial properties and are used to extend the shelf life of food products [22]. Cinnamon (Cinnamomum zeylanicum) essential oil (CEO), a volatile oil commonly obtained from the bark and branches of cinnamon, has been extensively studied for its broad-spectrum antimicrobial activity, including its inhibition of both Gram-positive and Gram-negative bacteria [23–26]. Furthermore, CEO exhibits antioxidant activity due to the bioactive compounds such as cinnamaldehyde, eugenol, and trans-cinnamaldehyde [27,28]. Additionally, CEO is categorized as Generally Recognized as Safe (GRAS) by USFDA and is commonly used as an additive in various food products [27]. However, the direct addition of essential oils into food can alter its organoleptic properties.

An alternative approach involves the creation of active packaging, where active compounds such as essential oils are incorporated into the packaging material itself, enhancing the polymer’s functional properties [29,30]. Furthermore, using the appropriate concentration of essential oil can mitigate undesirable odors in the films [14]. Previous studies have incorporated CEO into various biobased polymeric films, including polylactic acid [17], zein [31], and poly (butylene adipate-co-terephthalate)/thermoplastic starch composite [26]. In a recent study, Lim et al. [16] demonstrated the effectiveness of poly-ε-caprolactone/CEO films in inhibiting mold growth on bread for up to 21 days.

The process of incorporating essential oils into biobased polymer films or coatings typically involves the creation oil-in-water (O/W) emulsions, in which the oil is dispersed within an aqueous phase and stabilized by an emulsifier [32,33]. The emulsification process is critical to the quality of the resulting film, with droplet size and distribution being key factors [34]. Smaller droplet sizes have been shown to enhance emulsion stability and improve both barrier and mechanical properties. Alharbi et al. demonstrated that the homogenization rate and time significantly influenced droplet size in an emulsion, with smaller droplets (7 µm) exhibiting notably higher stability than larger droplets (27 µm) [35]. This effect is also evident in nanoemulsions. In a study by Chu et al. [36], pullulan films incorporating CEO nanoemulsions with smaller droplets (60 nm) displayed superior water vapor barrier and flexibility compared to those containing larger CEO droplets (>100 nm). This is because larger oil droplets could create micropores in the film matrix during drying, facilitating moisture transfer [18,21].

One common emulsification method is rotor-stator homogenization, which promotes uniform mixing of lipid and aqueous phases while reducing droplet size. This process relies on the shear forces generated between the rotating rotor and stationary stator to break down lipid droplets, ensuring homogeneous distribution within the film [37]. Previous research has shown that emulsion stability is influenced by factors such as homogenization rate, shearing intensity, pressure, temperature, and time [38,39], with higher homogenization rates producing emulsions with smaller droplets sizes and increased stability [35,39]. However, when the homogenization rate is too high, over-homogenization may occur which can lead to problems such as an increased interfacial tension, increased emulsion viscosity, and denaturation of proteins, and oxidation of thermolabile bioactive compounds [40]. Consequently, the emulsion stability may be affected and coalescence of oil droplets might occur during film drying.

Despite the existing research on emulsification processes, limited studies have explored the effect of homogenization rates on the properties of fish gelatin/CEO film. This study aims to address this gap by investigating how different homogenization rates influence the properties of fish gelatin/CEO films, particularly the barrier and mechanical properties.

2  Materials and Methods

2.1 Materials

Fish gelatin powder (240–260 bloom, 20–40 mesh, gelling temperature, Tgel = 25°C) was purchased from Custom Collagen (Addison, IL, USA). Food-grade pure cinnamon essential oil (CEO) from Ceylon cinnamon (Cinnamomum verum), steam-distilled with a cinnamaldehyde content of ≥40% and coumarin ≤0.01%, was obtained from Druera (Wilmington, UK). Glycerol was purchased from Sigma-Aldrich (St Louis, MO, USA). Nile Red, Tween 80, and polyethylene glycol were obtained from Acros Organics (Fairlawn, NJ, USA). Fast Green FCF was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

2.2 Preparation of Fish Gelatin/CEO Films

Fish gelatin powder was dissolved in distilled water to achieve a 3.5% (w/v) concentration. The solution was heated at 70°C for 30 min. Glycerol, at 30% (w/w) of the gelatin content, was incorporated as a plasticizer and stirred for another 30 min. These concentrations were selected based on preliminary studies, where a higher gelatin concentration produced overly rigid films, and increased glycerol concentration led to films that became excessively sticky after drying. To prepare the film-forming emulsion (FFE), 0.1% (v/v) CEO, relative to the gelatin solution, was mixed with Tween 80 as an emulsifier, set at 25% (w/w, based on CEO). The CEO concentration was determined based on preliminary studies, as higher concentrations resulted in brittle films. The FFE was homogenized using a rotor-stator homogenizer (Ultra-Turrax T25, IKA Work Inc., Wilmington, DE, USA) at different rates of 6500 rpm (B), 9500 rpm (C), 13,500 rpm (D), 17,500 rpm (E) and 21,500 rpm (F) for 3 min, as shown in Fig. 1. Control films (A) were prepared without homogenization. After homogenization, 20 mL of the FFE was cast onto a petri dish and air-dried at room temperature (25°C ± 2°C) for 48 h. The dried films were manually peeled off and stored at 25°C ± 2°C and 50% ± 5% RH prior to analysis.

Figure 1: Preparation of fish gelatin/cinnamon essential oil films

2.3 Characterization of Film-Forming Emulsion

The droplet size distribution of the FFE was measured using a laser diffractometer (MasterSizer 2000, Malvern Instruments, Worcestershire, UK). The samples were diluted in distilled water at 2000 rpm until an obstruction rate of 4% was obtained. The Mie theory was applied, considering the following optical property for CEO: a refractive index of 1.57 [41]. Each FFE was measured in triplicate. The surface area-weighted (Sauter) mean diameter (D3,2) and volume-weighted (De Brouckere) mean diameter (D4,3) were determined.

2.4 Film Thickness

The thickness of the film was expressed as the mean thickness at ten random positions on the film, measured using a hand-held digital micrometer (Mitutoyo 547–401 Thickness Gauge, Mitutoyo Co., Kawasaki, Japan).

2.5 Mechanical Properties

The tensile strength (TS) and elongation at break (EAB) of the films were measured using an INSTRON 4302 Series IX Machine (Singapore) equipped with a 10 N tensile load, following the ASTM-D882 standard method [42]. Test films were cut into strips (60 mm 20 mm) and mounted between two grips with an initial separation of 50 mm. The crosshead speed was set to 100 mm/min.

2.6 Water Vapor Permeability

Water vapor permeability (WVP) of the films was measured according to the ASTM E-96 standard method with slight modifications [43,44]. Distilled water (6 mL) was placed into each test cup, and the film sample was tightly secured over the cup opening. The cups were maintained under controlled temperature and RH (23°C ± 2°C, 50% ± 5% RH). Weight loss from the samples was monitored over a 6-h period, with weights recorded at 1-h intervals. The WVP of the film was then calculated as follows:

(1)

where m = weight loss of the cup (g); x = film thickness (mm); A = area of exposed film (m2); t = time (s); P = partial vapor pressure difference at 23°C, 2810.06 Pa [45].

2.7 Light Transmittance and Opacity

The percentage of light transmittance was measured at wavelengths of 200, 280, 400, 500, 600, and 800 nm using a UV–Vis spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA). The opacity of the film was calculated as follows:

(2)

where Abs600 = Absorbance value at 600 nm (AU); x = film thickness (mm).

2.8 Surface Microstructure

The surface microstructure of film samples was visualized using a scanning electron microscope (JSM 6400, JEOL, Akishima, Japan). The samples were mounted on a bronze stub and coated with gold using a sputter coater (SCD 005, BalTec, Canonsburg, PA, USA). All the samples were viewed at a voltage of 15 kV under the magnification of 1000 .

2.9 Confocal Laser Scanning Microscopy

A confocal microscope LSM 5 Pascal Exciter (Zeiss, White Plains, NY, USA) was used to examine the protein phase and oil distribution in the gelatin films, following the method by Auty et al. [46]. Film samples (10 mm 10 mm) were cut and placed on microscope glass covers, then stained with a 3:1 mixture of 0.02% (w/w) Nile Red in polyethylene glycol and 0.1% (w/w) aqueous Fast Green FCF. Samples were washed with water to remove excess stain before viewing. Fluorescence images were obtained using two separate channels: a Krypton/Argon laser (405 nm excitation) and a Helium/Neon laser (543 nm excitation). Micrographs of the XYZ layer projections were acquired using a 20 objective lens to represent a single image.

2.10 Atomic Force Microscopy

The surface morphology of the films was examined using atomic force microscopy (AFM) with a NanoScope scanning probe microscope (Digital Instruments CP-II, Inc., Santa Barbara, CA, USA). AFM scans were conducted at a scan size of 20 µm 20 µm, with a vertical range of 5 µm. A three-dimensional surface image (400 µm 400 µm) was obtained for each sample, with two images recorded per formulation. Two statistical parameters were used to quantify the surface roughness: average roughness (Ra), representing the mean of the absolute height deviations from the average surface level, and root-mean-square roughness (Rq), which calculates the root-mean-square of height deviations from the mean data plane.

2.11 Statistical Analysis

All experiments were conducted in triplicate, and the results are reported as mean ± standard deviation. Statistical analysis was performed using a one-way analysis of variance (ANOVA) with Minitab 16.1 software, at a significance level of 0.05. Post-hoc comparisons were made using the Tukey’s test.

3  Results and Discussions

3.1 Characterization of Film-Forming Emulsion

The D3,2 and D4,3 values of the FFE are shown in Table 1. The FFE for control (A) exhibited D3,2 and D4,3 values of 4.00 and 5.89 µm, respectively. The larger D4,3 value compared to D3,2 suggests a notable presence of larger droplets within the FFE, as D4,3 is more sensitive to larger droplet sizes, reflecting their significant contribution to the overall volume distribution [47].

Sample B exhibited the highest droplet size (p < 0.05), with droplet size decreasing as the homogenization rates increased. The low homogenization rate of 6500 rpm combined with the short homogenization time of three minutes likely provided insufficient shear force to effectively reduce droplet size. Longer residence times are often necessary when shear force is insufficient to achieve smaller droplet sizes. This finding is corroborated by Alharbi et al. [35], who reported that efficient droplet breakdown depends on both shear speed and residence time. An increase in homogenization rate beyond 9500 rpm resulted in a reduction in D3,2 and D4,3 values. At the highest homogenization rate (21,500 rpm), the difference between D3,2 and D4,3 was minimal, indicating a more uniform droplet size distribution compared to the other samples. This observation aligns with the findings by Vargas et al. [48], who observed that higher homogenization rates in a chitosan-oleic acid emulsion produced smaller droplets and more uniform size distributions.

Interestingly, the control samples exhibited D3,2 and D4,3 values similar (p ≥ 0.05) to those of the homogenized samples. This similarity may be attributed to the low concentration of CEO, resulting in fewer, sparsely distributed oil droplets within the emulsion. Consequently, the homogenization effect was minimal, as the limited amount of oil present was insufficient to significantly impact droplet size distribution, even with increasing homogenization rates.

3.2 Film Thickness

Table 1 shows the effect of varying homogenization rates on film thickness. The control film (A) exhibited the lowest (p < 0.05) thickness, with thickness increasing as homogenization rates increased. This may be attributed to changes in the arrangement of protein molecules in the film matrix. The higher shear forces during homogenization can disrupt the orderly alignment of protein molecules, as lipid droplets become dispersed and embedded within the protein network [49]. This disruption may result in a more protruded film structure, contributing to increased thickness. Additionally, excessive homogenization might have released the emulsifiers from the oil-water interface [39], leading to oil droplet aggregation during the drying process, which further increased the film thickness.

3.3 Mechanical Properties

Emulsion films often demonstrate unique mechanical properties resulting from the presence of dispersed lipid droplets. These properties significantly influence their strength and flexibility, which are essential for preserving product integrity during storage and transportation. The mechanical properties of the films are shown in Table 2. The control film exhibited TS and EAB values of 30 kPa and 144.95%, respectively. The TS and EAB demonstrated a complex, non-linear relationship with homogenization rates. While homogenization did not significantly impact TS (p ≥ 0.05), a slight increase was observed up to 9500 rpm. The highest (p < 0.05) EAB of 165.30% was also achieved at this rate, with EAB declining as homogenization rates further increased. While the highest EAB might suggest plasticizing effect of CEO [12,50,51], this same sample also showed the highest TS, which typically indicates a more rigid structure. This combination could be attributed to the roles of CEO as both a plasticizer and a filler. At this optimal rate (9500 rpm), CEO likely enhances both elasticity and tensile strength by filling spaces within the gelatin matrix, enhancing flexibility while strengthening interchain interactions. Beyond 9500 rpm, the negative impact on TS and EAB likely resulted from CEO aggregation due to the intensive shearing forces, which disrupts matrix continuity and weakens interchain cohesion.

3.4 Water Vapor Permeability

The WVP of biopolymer films is an important factor influencing food quality and shelf life. Low WVP is generally preferred for packaging dried products to prevent moisture absorption, while higher WVP is suitable for fresh produce packaging to prevent condensation. Table 2 shows that WVP in fish gelatin/CEO films slightly (p ≥ 0.05) increased with higher homogenization rates, increasing from 1.53 × 10−7 g m−1 Pa−1 s−1 in the control to 1.96 × 10−7 g m−1 Pa−1 s−1 at the highest rate (21,500 rpm). This increase in WVP may be due to the CEO droplets within the matrix, which can create free volume, facilitating water molecule movement across the film.

Though smaller lipid droplet sizes are generally expected to enhance water barrier properties by increasing the tortuosity of the matrix [52,53], interactions between the lipid droplets and protein network can also significantly affect WVP. At moderate homogenization rates, CEO droplets likely distribute more evenly, improving the barrier properties. However, the intense shear forces at the highest rate likely led to CEO droplet aggregation, disrupting the protein network and forming a more heterogeneous film structure. This disruption, as also reported by Masamba et al. [54], in a study where homogenization rates of 20,000 rpm resulted in an increase of 152% in WVP in zein films, compared to 10,000 rpm. The results have been attributed to the formation of heterogenous film microstructures which weakens the film matrix, allowing a greater transmission of water vapor molecules [54].

3.5 Light Transmittance and Opacity

Light transmittance and opacity are important optical properties in food packaging materials, as they influence the visual appeal and functional protection of the packaged contents. High transparency is often favored by consumers for clear visibility of the product, while increased opacity can serve to protect the food from potentially harmful UV radiation and photodegradation [55]. Table 3 shows the light transmittance and opacity values for the fish gelatin/CEO films. The control films exhibited light transmittance values of 3.32%, 20.30%, and 25.59% at 200, 280, and 400 nm, respectively, aligning well with the light transmittance values of gelatin film reported previously [7,56,57].

The influence of homogenization rate on light transmittance was minimal across all tested wavelengths (p ≥ 0.05), likely due to the low CEO concentrations in the films. The fish gelatin/CEO films demonstrated low UV transmittance, particularly at wavelengths of 200 and 280 nm. This reduced transmittance in the UV range can be attributed to the natural UV-absorbing properties of cinnamaldehyde, a major component of CEO, which absorbs at 286 nm [58,59]. Additionally, opacity values remained low and statistically similar (p ≥ 0.05) across all homogenization rates, as shown in Fig. 2. This consistent low opacity could be advantageous for packaging applications where moderate UV protection is desired without compromising product visibility.

Figure 2: Appearance of fish gelatin films incorporated with cinnamon essential oil produced using film-forming emulsion (FFE) with various homogenization rates (0, 6500, 9500, 13,500, 17,500, 21,500 rpm)

3.6 Surface Microstructure (SEM and CLSM)

Fig. 3 shows the scanning electron microscopy (SEM) images of fish gelatin/CEO films, prepared at different homogenization rates. The surface microstructures of these films were noticeably affected by the homogenization rates. The control film exhibited a dense and slightly rough structure, likely due to the presence of oil in the gelatin matrix. Similar findings have been reported previously, where the incorporation of palm wax into gelatin produced a rougher surface compared to pure gelatin films [60].

Figure 3: SEM surface microstructure of fish gelatin films incorporated with cinnamon essential oil produced using film-forming emulsion (FFE) with various homogenization rates (0, 6500, 9500, 13,500, 17,500, 21,500 rpm)

However, when the homogenization rate exceeded 13,500 rpm, the films developed irregular surface microstructures with visible clumps, likely caused by oil droplets disrupting the film surface. The high shear rates may have caused the aggregation of lipid droplets, reducing film stability [39], and forcing oil droplets out during the drying process. Since smaller droplet sizes were achieved at higher homogenization rates, as indicated in Table 1, it is likely that the aggregation of the CEO occurred during the film drying process. Similar results have been reported in a previous study [54], where increasing the homogenization rate from 10,000 to 20,000 rpm resulted in a coarse and heterogeneous microstructure in zein-oleic acid composite films.

At 21,500 rpm, microscopic pores were visible on the film surface, likely contributing to the significantly (p < 0.05) higher WVP compared to the other samples. Likewise, in a study by Almasi et al. [61], microemulsion films of calcium alginate and thyme essential oil exhibited high porosity, which resulted in elevated WVP.

Fig. 4 shows the CLSM images of the films. The red areas in the images represent the distribution of CEO within the gelatin matrix. In the control film (A), the essential oil was dispersed throughout the continuous gelatin phase, with lipid droplets exhibiting inconsistent sizes. At 9500 rpm, the oil droplets appeared to be homogeneously distributed, with more uniformly sized lipid droplets evenly dispersed within the gelatin matrix. This uniform distribution likely contributed to the enhanced mechanical properties observed at this homogenization rate.

Figure 4: Confocal laser scanning micrographs of fish gelatin films incorporated with cinnamon essential oil produced using film-forming emulsion (FFE) with various homogenization rates (0, 6500, 9500, 13,500, 17,500, 21,500 rpm)

3.7 Surface Topography

Fig. 5 shows the surface topographies of fish gelatin/CEO films at different homogenization rates. The corresponding roughness parameters, Ra and Rq, measured by AFM, are presented in Table 4. All the films share similar (p ≥ 0.05) Ra and Rq, regardless of the homogenization rate. Among the films, the control film (A) exhibited the highest Ra and Rq values, at 13.38 and 19.04 nm, respectively. The higher roughness may be attributed to the immiscibility between the hydrophilic gelatin and hydrophobic CEO, exacerbated by the inconsistent lipid droplet sizes, as observed in the CLSM images. Film C, produced at 9500 rpm, showed lower surface roughness, aligning with the SEM results, where the film displayed a smooth and homogeneous microstructure. This lower roughness likely reflects the uniform distribution of CEO droplets within the gelatin matrix.

Figure 5: AFM images of fish gelatin films incorporated with cinnamon essential oil produced using film-forming emulsion (FFE) with various homogenization rates (0, 6500, 9500, 13,500, 17,500, 21,500 rpm)

As the homogenization rate increased, the roughness values of the films C to F showed an increasing trend. The Ra values increased from 4.91 to 10.40 nm, while the Rq values increased from 6.46 to 13.11 nm. The Rq value, which gives more weight to larger peaks and valleys, is considered a better measure of surface irregularities [62]. The higher Rq values compared to Ra value supported the AFM observations, where occasional sharp peaks were visible on the film surfaces.

This increased roughness may be attributed to the entrapment of large CEO droplets in the film matrix or interactions between the oil molecules and gelatin chains that affect chain entanglements [63], suggesting that higher homogenization rates reduced emulsion stability during the drying process. Moreover, the increased surface tension between the oil and water phases, due to the presence of smaller oil droplets, may contribute to a rougher film morphology, as observed in a previous study [64]. The CEO droplets likely underwent agglomeration during drying, leading to surface roughness and irregularities, as also observed in the SEM images.

4  Conclusion

This study investigates the impact of homogenization rates on the physical and barrier properties of fish gelatin films containing cinnamon essential oil (CEO). The findings indicate that as the homogenization rate increased, droplet size and distribution in the film-forming emulsion (FFE) became significantly smaller and narrower. An optimal homogenization rate of 9500 rpm enhanced film flexibility and smoothness, likely due to a well-distributed CEO phase within the gelatin matrix. However, homogenization rates above 17,500 rpm led to CEO droplet aggregation, which disrupted the film matrix and increased water vapor permeability. These results contribute to the development of sustainable active packaging materials by demonstrating that adjusting homogenization rates can effectively tailor the mechanical and barrier properties of essential oil-enriched gelatin films. A limitation of this study is the focus on a single essential oil and specific homogenization parameters, which may not capture the broader behavior of other bioactive compounds or processing conditions. Future studies could explore a wider range of bioactive compounds and process variables for broader applicability. Additionally, the improvement of mechanical strength and water vapor barrier of the should also be studied. Such advancements can benefit the packaging industry by extending the shelf life of specific food products, particularly those requiring high moisture barriers.

Acknowledgement: The authors would like to thank the Faculty of Food Science and Technology, Universiti Putra Malaysia for the permission to use the laboratory and all of the science officers for their kind assistance.

Funding Statement: The authors received no specific funding for this study.

Author Contributions: The authors confirm contribution to the paper as follows: study conception and design: Nur Hanani Z. A.; data collection: See Cheng Lee; analysis and interpretation of results: See Cheng Lee, Han Lyn Foong; draft manuscript preparation: Han Lyn Foong. All authors reviewed the results and approved the final version of the manuscript.

Availability of Data and Materials: Data available on request from the authors.

Ethics Approval: Not applicable.

Conflicts of Interest: The authors declare no conflicts of interest to report regarding the present study.

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