Rosehip Seed Oil-Incorporated Chitosan Films for Potential Fruit Packaging Applications

Featured Application

The developed chitosan films can be used as edible coatings to prolong the shelf-life of food products. Our results show that the rosehip oil-loaded chitosan films with an optimized composition possess good color, mechanical, barrier, antioxidant, and antimicrobial characteristics, which assist the preservation of minimal processed fruits.

Abstract

The concept of food packaging plays a crucial role in ensuring consumer satisfaction and extending the shelf life of food products. The rising trend of introducing innovative materials for food packaging has become prominent in recent years. The present study aims to investigate the impact of rosehip seed oil (RSO) on the physical, physicochemical, antioxidant, and antimicrobial properties of edible films based on chitosan for potential fruit packaging applications. Scanning electron microscopy revealed a uniform distribution of the incorporated emulsion throughout the edible film. The addition of RSO increased the deformation at break in both tensile and puncture test, thereby improving the elastic properties of the films. The resulting films exhibited a light-yellow color with high opacity. The immobilization of RSO led to a decrease in water content by almost two times and an increase in water vapor permeability of the films. The films showed enhanced antioxidant activity and retained good protective properties against the yeast S. cerevisiae. Consequently, these newly formulated multicomponent films are found to be suitable for applications in the development of active food packaging because of their physical, antioxidant, and antimicrobial properties.

1. Introduction

In recent years, a tendency to replace plastic packaging with environmentally friendly, edible, and biodegradable films using natural resources has been observed [1]. This shift is primarily driven by concerns about environmental pollution caused by plastic packaging and the desire to minimize food waste. Plastic pollution has become a pressing global issue, prompting governments, businesses, and consumers to seek alternative packaging solutions that are more sustainable [2].

Edible films are thin layers of edible material derived from sources such as polysaccharides, proteins, or lipids [3,4]. These films can be formulated to have properties similar to traditional plastic packaging, such as barrier properties and mechanical strength, while being edible and biodegradable. In addition, they have the advantage of containing various biologically active substances, such as coloring agents, antimicrobial agents, and antioxidants [5].

Chitosan stands out as a highly promising biomaterial for substituting synthetic counterparts, especially in the realm of food and packaging applications [6,7]. It is characterized as a linear polysaccharide composed of (1,4)-linked 2-amino-deoxy-β-D-glucan. Commercially, chitosan is obtained through the chemical alkali deacetylation process of chitin, which ranks as the second most abundant polysaccharide in nature, following cellulose [8]. Chitosan has some advantages over other biomaterials because of its antimicrobial activity against a wide variety of microorganisms including fungi, algae, and some bacteria [7]. One common theory about the way in which chitosan exhibits antibacterial activity suggests that the positively charged amine group in chitosan binds to the negatively charged bacterial cell wall, affecting membrane permeability and disrupting normal cell function. Chitosan may also act as a chelating agent, binding to metal elements and inhibiting toxin production and microbial growth. Another proposed mechanism involves lower molecular weight chitosan entering the bacterial cell and binding to DNA, preventing DNA replication and causing cell death [8].

Another well-known function of chitosan is its antioxidant activity. Chitosan can scavenge the excessive free radicals due to the presence of functional amino and hydroxyl groups on its backbone [9]. Chitosan is reported to present radical scavenging activity, including OH•, O2•−, DPPH•, and ABTS•+. The scavenging of these radicals is negatively correlated with the degree of acetylation (DA) of chitosan due to the stabilization of oxidized structures provided by the amino group. Based on Schreiber et al., native chitosan (MW = 307 kDa, DDA = 80%) had a scavenging ability against the DPPH radical (9.4%) [10].

Similar to many polysaccharide-based films, chitosan films possess a strong hydrophilic nature, granting them favorable barrier properties against gases and lipids but presenting a challenge in terms of water vapor permeability [11]. This limitation restricts the potential applications of chitosan films, as effective moisture control is often essential in food packaging scenarios [12].

The addition of essential oils (EOs) to chitosan films can lead to an improvement in both their antibacterial activity and water-resistant properties [13]. They are a promising alternative to conventional antimicrobials in food packaging applications due to their ability to sterilize headspace and food surfaces [14]. They are approved by the FDA and are generally recognized as safe [15]. Moreover, incorporating EOs can influence the mechanical, barrier, and thermal properties of the final composite, potentially improving their antimicrobial properties [16]. For instance, EOs can reduce the water vapor permeability (WVP) of hydrophilic materials, decrease tensile strength (TS), and increase elongation at break. Different cellulose esters, such as lemongrass, basil, and rosemary pepper, behave like plasticizers in the matrix, affecting Young’s modulus, tensile strength, and elongation at break of the films [17].

Among the considered potential sources, rosehip seed oil (RSO) emerges as a promising alternative due to its richness in unsaturated fatty acids, particularly oleic, linoleic, and linolenic acids [18]. Additionally, RSO boasts a high content of vitamins, minerals, carotenoids, tocopherols, phytosterols, flavonoids, tannins, pectin, sugars, organic acids, and amino acids [19]. Thanks to the high content of biologically active substances, RSO demonstrates antibacterial properties against a range of bacteria and fungi, such as Aspergillus niger, Candida albicans, Escherichia coli, and Staphylococcus aureus [20]. Different approaches regarding the mechanism of antibacterial and antifungal action have been proposed in the literature—bacterial inhibition due to the deterioration of membrane integrity, loss of cell content (molecules and ions) due to damage of the selective permeable structure of membranes, secondary metabolites (phenolic compounds) in volatile oil composition causing damage to cell membranes, and cell vital activities (energy production and protein synthesis) [21]. Because rosehip oil has a significant amount of vitamins, especially C, E, and A, it is well known for having antioxidant qualities [22,23]. Considering the mentioned benefits and wide availability, RSO has garnered significant attention across cosmetic [24], pharmaceutical [25,26], and food industries [24,25,26,27].

Previously Butnaru et al. have reported that the presence of rosehip seed oil in chitosan films led to the formation of flexible films with improved mechanical, gas, and water vapor barrier properties, antioxidant activity, and antimicrobial properties against Escherichia coli, Salmonella enterica subsp. enterica ser. Typhimurium, and Bacillus cereus [28]. Conducted by Butnaru, FTIR studies indicate interaction by hydrogen bonds between chitosan and rosehip seed oil. Additionally, when shifting to higher wavenumbers, the vibration bounds of chitosan were caused by inhibited inter/intra molecular hydrogen bonding, which existed in the neat chitosan film.

One of the main disadvantages of chitosan in its application in the food industry is its dissolution in an acidic environment, mainly in acetic acid. In this case, some precautions are necessary due to the potential migration of the acid into the food matrix. Although volatile organic acids are usually removed during solvent evaporation, residual amounts might remain. Acetic acid is known for its strong odor and taste, which can affect the sensory qualities and decrease the consumer acceptance of the final product [29]. To overcome these limitations, an alternative approach to use water-soluble chitosan could be adopted [30].

To the best of our knowledge, there are no studies on the formulation of edible films on the basis of water-soluble chitosan and rosehip seed oil emulsion. The present article aims to investigate the effect of rosehip seed oil concentration on the morphology, rheological properties, hydrophilic–hydrophobic balance, and antioxidant and antibacterial activities of the multicomponent films based on water-soluble chitosan.

2. Materials and Methods

2.1. Materials

Water-soluble chitosan hydrochloride (viscosity of 1% in water, 20 °C is 10–120 cps, and degree of deacetylation is >85%) from fungal origin with viscosity ranging from 10 cps to 120 cps was purchased from Glentham life sciences Ltd. (Corsham, UK) and was used without further purification and characterization. Tween 20, DPPH (2,2-diphenyl-1-picrylhydrazyl), and Trolox [(±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylicacid] were bought from Sigma-Aldrich (Taufkirchen, Germany). Folin–Ciocalteau (FC) reagent was delivered from Merck (Darmstadt, Germany); Gallic acid monohydrate was acquired from Fluka (Buchs, Switzerland). Rosa Canina fruits were collected from gardens near the town of Kazanlak, Bulgaria. Rosehip seed oil was produced by an international producer (Green Gold International Ltd., Plovdiv, Bulgaria) for the needs of the Institute of Food Preservation and Quality—Plovdiv, Agricultural Academy of Bulgaria. For this purpose, the following production protocol was used. The oilseed cake (waste product of cold pressed rosehip oil production) was ground to a particle size of less than 40 μm and sent to a subcontractor for extraction with supercritical Freon R134. The extraction pressure was set to 140 bar and the received yield was 9.1%. The described method provides an extract devoid of tocopherols but rich in carotenes (45.32 mg/kg). Some of the physicochemical characteristics of the oil are presented by Iliev et al. [31]. These parameters are also confirmed by other authors [32,33]. All other chemicals were of analytical grade.

2.2. Preparation of Rosehip Seed Oil Emulsion

The rosehip seed oil (RSO) emulsion was prepared using 10% w/w RSO, 10% w/w Tween 20, used as an emulsifier, and 80% w/w deionized water. The compounds were stirred for 3 min at 3000 rpm and a temperature of 25 °C with the use of a PV-1 Vortex Mixer (Grant Instruments, Chelmsford, UK). The resulting mixture was ultrasonicated for 3 min using UP100H—Compact Ultrasonic Laboratory Device (Hielscher Ultrasonics GmbH, Teltow Germany.

2.3. Preparation of Chitosan Films Loaded with Rosehip Seed Oil Emulsion

Different amounts of the already prepared emulsion, with corresponding concentrations of 1%, 2%, 3%, 4%, and 5%, were added to a 1% water solution of chitosan containing 0.15% (v/v) glycerol and sonicated for 5 min. The final emulsion was degassed for 30 min in an ultrasound bath (BANDELIN electronic GmbH & Co. KG, Berlin, Germany), cast in glass Petri dishes (d = 100 mm), and dried in an oven (Faithful Technology Park, Cangzhou, China) at 30 °C for 12 h. The resulting dry films (Figure 1) were stored in a desiccator at a relative humidity of 55% for further use.

2.4. Film Morphology

The morphology of the films was studied using scanning electron microscopy (SEM) (Prisma E SEM, Thermo Scientific, Waltham, MA, USA). The film was attached onto an aluminum holder and subsequently coated with carbon and gold using a vacuum evaporator Quorum Q150T Plus (Quorum Technologies, West Sussex, UK). All collected images were captured using a back-scattered electron detector (Prisma E SEM, Thermo Scientific, Waltham, MA, USA) at an accelerating voltage of 15 kV at different levels of magnification. SEM microphotographs were used to evaluate the oil droplet size using the free software ImageJ.

2.5. Mechanical Testing

The tensile properties of the films were examined using an LS1 universal testing machine (Lloyd Instruments Ltd., Bognor Regis, UK) according to standard ASTM D882-91 [34]. Film strips (width: 10 mm, length: 100 mm, instrument gap: 50 mm) were fastened with rubber sealed pneumatic clumps and subjected to a load with a constant deformation rate of 0.1 mm/s up to breaking. The tensile modulus, the force and deformation at the break, and the work to break were investigated.

The mechanical properties of the films were also analyzed with a puncture test using a Stable Micro Systems texture analyzer equipped with a film support rig (HDP/FSR) (Stable Micro Systems Ltd., Godalming, UK). The cut film pieces, with a diameter of 50 mm, were fixed above a rounded hole and punctured with a ball probe (5 mm in diameter, P/5S) with a loading speed of 1 mm/s. The puncture force and deformation, puncture energy, and puncture modulus were analyzed [35].

Both mechanical tests were repeated seven times for each type of film in order to obtain a statistical evaluation of the results.

2.6. Color Parameters

The color parameters of the films were measured with a PCE-CSM 5 portable colorimeter (PCE Deutschland GmbH, Meschede, Germany). The CIELAB color parameters L, a, and b were determined at a measuring geometry of 8°/d, Ø 8 mm, light source D65. A white control plate (L* = 94.3; a* = −0.92; b* = −0.67) was used as a calibration plate [36]. The color coordinates CIE L*, a*, b*, and Y were detected in reflection mode, with samples placed on the surface of a standard white and black plate (L* = 21.04; a* = 0.10; b* = −0.24), allowing for the calculation of both opacity differences [37] and the total color difference between the chitosan and the rosehip oil-loaded films.

Chroma (C*) was evaluated based on the formula:

$$C^{*}=\sqrt{a^{*2}+b^{*2}},$$

(1)

The total color difference ΔE was calculated based on the equation:

$${∆E}_{ab}^{*}=\sqrt{{\left(L_0^{*}-L_x^{*}\right)}^2+{\left(a_0^{*}-a_x^{*}\right)}^2+{\left(b_0^{*}-b_x^{*}\right)}^2},$$

(2)

where $L_0^{*}$, $a_0^{*}$, and $b_0^{*}$ are the color parameters of the chitosan film without added rosehip oil, and $L_x^{*}$, $a_x^{*}$, and $b_x^{*}$ are the color parameters of the chitosan film with rosehip oil (1–5%).

The whiteness index (WI) [38] was also calculated as follows:

$$WI=100-\sqrt{{\left(100-L^{*}\right)}^2+a^{\mathrm{*}2}+b^{\mathrm{*}2}},$$

(3)

The tolerance of color differences is given based on the recommendations of ASTM D2224-02 (2016 standard) [39,40] (

Table 1. Tolerance of color differences [39,40].

${∆\mathit{E}}_{\mathit{a}\mathit{b}}^{\mathit{*}}$	Perceived Difference
0–0.5	Trace level
0.5–1.5	Slight
1.5–3.0	Noticeable
3.0–6.0	Appreciable
6.0–12.0	Large
>12	Very obvious

).

The opacity of the films was calculated based on the Hunterlab method 2008 [41]:

$$opacity={\displaystyle \frac{Y_b}{Y_w}}·100$$

(4)

where Y_b is measured on the film with a standard black background and Y_w is measured on the film with a standard white background.

2.7. Water Sorption Isotherms

Water sorption isotherms were measured at 25 °C through a gravimetrical method [41]. Film samples (20 × 10 mm²) were placed in desiccators with different relative humidities, achieved with saturated salt solutions. The samples were equilibrated in the desiccators for 72 h. After that, they were weighed using an analytical balance and dried at 105 °C for 4 h. The moisture content of the samples was calculated as the relative change in their mass before and after drying. All tests were repeated 5 times.

2.8. Water Vapor Permeability and Transmission Rate

The vapor permeability and transmission rate of the films were measured gravimetrically at 38 °C using the instrument W3/031 (Labthink, Jinan, China). A constant humidity difference of 80% was generated between the two sides of the tested film. Before the test, the samples were stored for 72 h in a desiccator at a relative humidity of 55% and a temperature of 25 °C.

2.9. Water Contact Angle Measurement

Water contact angle measurements were carried out with the use of the static droplet method under standard atmospheric conditions. Contact angle measurement equipment was used to measure the water contact angle on the surface of chitosan films. For all measurements, droplets of distilled water and diiodomethane were deposited onto the surface of the chitosan films. Five measurements were performed on different parts of the surface for each type of film. In total, 2 μL droplets were used in order to decrease the impact of the surface roughness on the water contact angle measurements.

The drops were carefully placed on the surface with the use of a 10 μL glass microsyringe (Innovative Labor System GmbH, Ilmenau, Franz-Ferdinand-Greiner Str. 37, Germany). The contact angles were determined by measuring the tangent of the drop profile from images, captured with a high-resolution camera. The image processing was performed with the use of public domain ImageJ software (ImageJ v1.51k software, developed at the National Institutes of Health, Bethesda, MD, USA).

2.10. Film Phase State and Thermal Stability

Thermal properties and the stability of the chitosan-based films were studied with the use of the differential scanning calorimetry method. DSC 204F1 Phoenix produced by Netzsch Gerätebau GmbH, Selb, Germany was used in these measurements. The instrument was calibrated using indium standard (T_m = 156.6 °C, ΔH_m = 28.5 J/g) for both heat flow and temperature. The samples were placed in aluminum pans and hermetically sealed. An identical empty pan was used as a reference. The following temperature protocol was followed:

• Cooling down from 25 °C to −70 °C with a cooling rate of 2 K/min;
• Isothermal step at −70 °C for 15 min;
• Heating from −70 °C up to 100 °C with a heating rate of 10 K/min.

The experimental data was evaluated using Netzsch Proteus—Thermal Analysis software (Version 6.1.0B, Selb, Germany).

2.11. Total Phenolics Content

The content of total polyphenols (TPP) was determined with Folin–Ciocalteu reagent according to Moradi et al. [42], using gallic acid as a standard. Results were calculated as mg Gallic acid equivalents (GAE) per gram of dried film, according to the following formula:

$$TPP={\displaystyle \frac{C·V}{M}}$$

(5)

where TPP is measured in mg/g dried film, in GAE; C is the concentration of Gallic acid established from the calibration curve, mg/mL; V is the volume of film extract, mL; and M is the mass of dried film, g.

2.12. Film Antioxidant Activity

The efficacy of the films to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was determined spectrophotometrically on the basis of the bleaching of the bluish-red or purple colour of the DPPH solution as a reagent, according to Moradi et al. [42]. Every sample (25 mg) from each film was dissolved in 3 mL of distilled water, and then 2.8 mL of film extract solution was mixed with 0.2 mL of 1 mM methanolic solutions of DPPH. The absorbance at 517 nm was measured after the solution had been allowed to stand in the dark at ambient temperature for 30 min. The percentage of DPPH radical-scavenging activity was calculated using the equation:

$${DPPH}_{Scavengingeffect}(\%)={\displaystyle \frac{{Abs}_{DPPH}-{Abs}_{filmextract}}{{Abs}_{DPPH}}}·100$$

(6)

2.13. Testing the Surface Resistance of Film Samples to Fungi

Two types of solid-phase nutrient medium, specific for the cultivation of yeasts and molds, were used. The first culture medium was malt extract agar base (HIMEDIA, Thane, India). The second one was sabouraud medium with 2% agar (Merck, Boston, MA, USA). The mediums were poured into Petri dishes. Film discs with a diameter of 15 mm were placed on them. The film discs represented 6 variants based on the amount of rosehip oil. Four discs were placed in each Petri dish. The Petri dishes holding the discs were incubated at 30 °C for 72 h. Monitoring was carried out at 24 h, 48 h, and 72 h.

The design of the second type of experiment repeats the first variant in which a yeast culture of Saccharomyces cerevisiae 0527 (collection of the Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria), the most common agent of fruit fermentation in nature, was inoculated on the discs in the Petri dishes. Monitoring was carried out in the manner mentioned above. The experiments were conducted in triplicate.

2.14. Statistical Analysis

The statistical analysis was performed using the statistical software Statistica (TIBCO Software Inc. ver. 14, Palo Alto, CA, USA). ANOVA analysis was used to find significant differences (p = 0.05) between investigated film parameters at different RSO emulsion concentrations.

3. Results and Discussion

3.1. Chitosan/RSO Film Morphology

The morphology of the samples was investigated using scanning electron microscopy (SEM). SEM examination reveals the morphology of the film’s surface and bulk (via cross-section) obtained by the incorporation of rosehip oil into chitosan films (Figure 2). The obtained microstructures were qualitatively analyzed, aiming to describe the role of rosehip oil and the homogenization procedure in the structure of the chitosan matrix.

As illustrated in Figure 2, the pure chitosan film (Figure 2a) presented a compact and homogeneous structure. However, when RSO is introduced to the chitosan film, particularly at higher concentrations, the microstructure becomes heterogeneous. The presence of RSO caused a heterogeneous structure in which oil droplets were entrapped in the continuous carbohydrate network. The number and size of the lipid droplets were increased with the RSO concentration.

Spherical pores with variable dimensions, attributed to rosehip oil droplets distributed randomly within the matrix, can be observed, which could reduce hydrophobic aspects and increase flexibility, providing the film with a more amorphous structure [28,43]. The presence of these pores is likely due to the flocculation and coalescence of small drops of emulsified essential oil during the drying of the film. The observed structural differences could be explained by the differences in essential oil concentration in the films during drying and caused by the intricate interactions between lipids, polysaccharides, and the solvent [44]. Such behavior likely arises from the deformation forces exerted during the polymer chain aggregation, induced by solvent evaporation [45].

The droplet sizes were calculated, and the results are listed in

Table 2. Droplet sizes based on measurement of randomly selected droplets.

RSO Emulsion Concentration, %	Pore Size Diameter, µm
1	5.60 ± 0.17 a
2	6.30 ± 0.20 b
3	6.70 ± 0.21 c
4	7.20 ± 0.22 d
5	8.10 ± 0.24 e
R	0.9905

^a–e: different letters in the same column indicate significant differences (p < 0.05).

. The table presents averaged values with standard deviations for each value.

As it can be seen, the droplet size increases at bigger concentrations of the RSO. Similar surface microstructures, namely discontinuities associated with the formation of two phases, was found for a quinoa protein–chitosan–sunflower oil film by Valenzuela et al. [46].

3.2. Physico-Mechanical Properties of Chitosan/RSO Edible Films

The mechanical properties of edible films are of great importance, as they determine the capability of the material to be used as packaging. The mechanical properties of chitosan/RSO in this research were investigated using two different methods: a tensile test for the elongation properties and a puncture test for the perpendicular deformation tolerance—see

Table 3. Mechanical properties of chitosan/RSO films.

RSO cc, %	Tensile Strength, MPa	Strain at Break, %	Tensile Energy, N·mm	Tensile Modulus, GPa	Puncture Force, N	Deformati-on at Break, mm	Puncture Energy, N·mm	Puncture Modulus, N/mm
0	62.27 ± 6.75 c	2.87 ± 0.33 a	4.22 ± 0.62 a	1.016 ± 0.547 c	20.53 ± 1.17 d	0.70 ± 0.09 a	14.46 ± 1.76 a	5.46 ± 0.78 c
1	19.27 ± 1.90 b	6.89 ± 0.21 b	12.85 ± 2.68 b	0.588 ± 0.432 b	16.99 ± 0.61 c	1.09 ± 0.17 b	18.53 ± 2.36 b	4.77 ± 0.72 c
2	19.08 ± 3.36 b	9.15 ± 1.54 c	16.00 ± 2.57 bc	0.556 ± 0.913 ab	15.89 ± 1.06 b	1.24 ± 0.15 c	19.38 ± 3.82 b	4.03 ± 0.42 b
3	19.74 ± 2.21 b	11.58 ± 1.89 d	18.82 ± 3.19 c	0.544 ± 0.725 ab	15.59 ± 0.82 b	1.47 ± 0.22 d	23.30 ± 3.00 c	3.84 ± 0.54 ab
4	17.23 ± 1.88 b	11.07 ± 1.24 cd	23.56 ± 4.33 d	0.488 ± 0.552 a	15.32 ± 0.82 b	1.78 ± 0.17 e	27.29 ± 2.32 d	3.91 ± 0.33 ab
5	11.93 ± 1.33 a	12.52 ± 2.34 d	27.43 ± 0.30 e	0.470 ± 0.852 a	13.02 ± 1.67 a	2.21 ± 0.27 f	28.78 ± 3.82 d	3.30 ± 0.37 a
R	−0.7420	0.9344	0.9838	−0.7987	−0.9245	0.9892	0.9889	−0.9423

^a–f: different letters in the same column indicate significant differences (p < 0.05).

.

The tensile strength of pure chitosan film was (62.27 ± 6.75) MPa. The addition of the RSO emulsion up to 3% decreased the tensile stress by about three times—(19–20) MPa. The further increase in oil concentration up to 4–5% caused its bigger decrease down to (11.93 ± 1.33) MPa. A similar trend can be observed in the values of the tensile modulus, which reduced by almost half—from (1.016 ± 0.547) GPa to (0.544 ± 0.725) GPa when 3% oil emulsion was added. Above this emulsion concentration, the tensile modulus dropped to (0.470 ± 0.852) GPa.

The presence of oil in the chitosan films causes a change in their elasticity (deformability), which is expressed as an increase in the strain at break. The pure chitosan film is brittle, with strain at break less than 3%. It rises up to 11% when RSO emulsion with a concentration in the range of 1–3% is added and remains constant (11–12%) when increasing the emulsion concentration up to 5%. These values are comparable with the result of Butnaru et al. [19], who studied chitosan-based bionanocomposite films with rosehip oil and montmorillonite nanoclay. The tensile energy of chitosan film was (4.22 ± 0.62) N·mm. The inclusion of RSO emulsion up to 3% resulted in a three to four times increase up to (12.85 ± 2.68) N·mm, (16.00 ± 2.57) N·mm, and (18.82 ± 3.19) N·mm, respectively. At higher emulsion concentrations, it rose 6–8 times, up to (27.43 ± 0.30) N·mm.

Similar trends can be observed in the puncture properties of the films. The puncture force decreased from (20.53 ± 1.17) N for the pure chitosan film to (13.02 ± 1.67) N for the film containing 5% RSO emulsion. The puncture modulus was also affected by the oil addition and dropped from (5.46 ± 0.78) N/mm down to (3.30 ± 0.37) N/mm.

A measure of the elasticity in the puncture test is the deformation at break. It shows a linear increase in the full concentration interval from (0.70 ± 0.09) mm to up to three times higher at (2.21 ± 0.27 mm).

Similar to the tensile energy, the puncture energy increased with increasing oil content from (14.46 ± 1.76) N·mm to (28.78 ± 3.82) N·mm.

The decrease in strength and the increase in deformability and fracture energy are associated with a change in film properties from stiffer to more elastic. One possible explanation for the observed dependence is the plasticizing effect of the oil [47]. The hydrogen bonds within and between the chitosan chains undergo partial disruption, leading to a decrease in film strength. Similar results were reported by Kanani et al., who investigated a chitosan–PLA film containing cinnamon and ginger essential oil [48]. Based on their investigation, oil incorporation into the film can induce the replacement of stronger polymer–polymer interaction by a weaker polymer–oil interaction. This interaction may have caused embrittlement of the network structure, hence decreasing film tensile strength. The other possible reason is the increase in the polyphenol content, which also results in the formation of more hydrogen bonds between the polyphenol and polysaccharide molecules [49]. Consequently, there is an increase in molecular mobility and polymer flexibility.

The observed differences are significant between the 0 and 1% RSO emulsion and the above 4–5% RSO emulsions, but all of the investigated physico-mechanical parameters are stabile between the 1 to 3% concentration. That phenomenon may be attributed to the oil drop size and distribution in the chitosan matrix [50]. The addition of essential oils into films may reinforce the development of the heterogeneous film matrix, which may in turn lead to discontinuity in the film network. The faster change in the elastic properties of the films, when the oil emulsion concentration increases from 3 to 5%, is related to the rapid growth of the oil droplet sizes (see Table 2). A larger oil droplet size destroys internal structure integrity and causes an inhomogeneous structure that leads to the deterioration of the physico-mechanical properties of the films. Similar results were reported by Zhao et al., who studied anthocyanidin/chitosan nanocomposite-based edible films containing cinnamon–perilla essential oil Pickering nanoemulsions [51]. They found an optimal concentration of the loaded essential oil, at which the smallest drop size corresponds to the best mechanical properties. Castro and co-authors also reached an optimal concentration of tea tree oil in a chitosan/polyvinyl alcohol film, after which the mechanical properties deteriorate sharply [52].

Based on the obtained physico-mechanical indicators of the here investigated films, an emulsion concentration of 3% can be deduced as optimal.

3.3. Color Parameters of Chitosan/RSO Edible Films

The values of the lightness (L*), chroma (C*), the whiteness index, and the total color differences (${∆E}_{ab}^{*}$) between chitosan and rosehip oil-loaded films were measured on the surface of a standard black plate. For the evaluation of the opacity values, the color parameters were detected on the surfaces of both standard black and white plates. The opacity was calculated using the software algorithm of the PCE-CSM 5 portable colorimeter. The opacity of the white and black plates without films is (5.8 ± 0.1). The opacity of the pure chitosan film (15.2 ± 0.4), the lightness (42.49 ± 0.66), and the whiteness index (14.20 ± 0.74) are represented in

Table 4. Color parameters of chitosan films with added RSO with different concentrations.

cc, %	L*	C*	WI	Opacity	${∆\mathit{E}}_{\mathit{a}\mathit{b}}^{\mathit{*}}$	Difference
0	42.49 ± 0.66 a	1.26 ± 0.16 d	14.20 ± 0.74 b	15.2 ± 0.00 b
1	41.96 ± 0.63 a	1.27 ± 0.15 d	14.00 ± 0.50 b	15.7 ± 0.3 b	0.66 ± 0.16 a	slight
2	41.60 ± 0.77 a	0.76 ± 0.12 ab	13.49± 0.50 b	15.1 ± 0.4 b	0.97 ± 0.14 b	slight
3	41.37 ± 0.22 a	0.67 ± 0.07 a	13.30 ± 0.30 b	14.8 ± 0.1 b	1.24 ±0.17 c	slight
4	40.65 ± 1.07 a	0.85 ± 0.12 b	13.22 ± 0.33 b	13.8 ± 1.4 ab	1.87 ± 0.20 d	noticeable
5	35.93 ± 6.60 b	1.04 ± 0.13 c	12.04 ± 1.59 a	11.5 ± 1.8 a	6.56 ± 0.82 e	very obvious

^a–e: different letters in the same column indicate significant differences (p < 0.05).

.

The addition of rosehip oil causes significant differences in the lightness, the whiteness, and the opacity of the films only at higher concentrations. The opacity values of the films loaded with up to 3% RSO emulsion are not significantly different from those of the pure chitosan film. At a 5% RSO emulsion, the opacity significantly decreased to 11.5 ± 1.8.

Opacity is an important property of edible packaging since it controls the light that enters the food and has a direct effect on its appearance. Its decrease with increasing RSO content in the films is at first glance surprising, since the presence of lipid droplets formed during the coating formulation that are dispersed in the polymer matrix is usually related to the difficult penetration of light [53]. On the other hand, the rise of plasticizer concentration leads to an increase in the mobility of the polymer chains and a decrease in the opacity by permitting a better penetration of light [54]. The plasticizing effect of RSO has already been established in the study of the mechanical properties of the films and is confirmed by the change in the optical characteristics.

The values of the chrome show a minimum (colorless) for the films with 0.3% oil concentration, which can be explained with the subtraction of the color-opponents and results in a film with an achromatic color. The decrease in chrome value for films with up to 0.3% RSO loading means discoloration of the film. Above this concentration, the film becomes darker (L*) and the chrome value increases again with significant differences. The values of the total color differences were slightly noticeable under a 0.3% rosehip oil concentration, but for higher concentrations, the differences were increased to a noticeable or very obvious level. These results confirm the results from Bonilla and Sobral [55]. These authors worked with different concentrations of ethanolic extract (from cinnamon, guarana, rosemary, and boldo-do-chile)-loaded gelatin-chitosan films and noticed no significant differences between pure chitosan and gelatin, as well as blended and loaded films in low concentrations.

3.4. Chitosan/RSO Edible Films’ Water Affinity

Water affinity is a crucial characteristic for edible films due to its direct impact on the film’s functionality and food preservation. Understanding and controlling the water content, water vapor barrier properties, and hydrophobicity of edible films are essential for designing effective packaging materials that can extend the shelf life, maintain quality, and ensure the safety of packaged products [56].

Understanding the moisture sorption properties of packaging films is essential for predicting their performance under various humidity conditions [57]. Moisture sorption isotherms illustrate the correlation between relative humidity (RH) and the equilibrium moisture content.

The water adsorption isotherms of the investigated samples are presented in Figure 3. The curves are considered type III isotherms [57]. The moisture adsorbed by chitosan films increased when subjected to high levels of RH and decreased with the increase in RSO concentration at a given RH value. At low relative humidity levels, water strongly adheres to the binding sites on the surface of the film. As relative humidity increases, the film swells, creating additional binding sites and resulting in an increase in equilibrium moisture content. The incorporation of different concentrations of RSO into the polymer matrix leads to a decrease in equilibrium moisture content, indicating improved water resistance. This phenomenon can be attributed to the reduced availability of amino groups in chitosan, likely due to electrostatic neutralization with the carboxylate groups present in RSO. Similar observations were made by Butnaru for chitosan–rosehip oil films [28] and Pereda for chitosan–olive oil films [58]. In summary, the addition of RSO emulsion diminishes water-binding sites and enhances the hydrophobic properties of the films.

A crucial role of edible coatings is to minimize the transfer of water between food and its surroundings or between different food components [59]. This entails ensuring that the coatings exhibit minimal water vapor permeability.

The dependence of the water vapor transmission rate (WVTR) and water vapor permeability (WVP) on the RSO concentration are presented in Figure 4. The pure chitosan film is characterized with the lowest WVTR—(107 ± 4) g/m²·24 h, and WVP—(7.57 ± 0.26) ·10⁻¹⁴ g·mm/m²·day·kPa. The increase in RSO concentration leads to an increase in both investigated parameters.

According to the literature data, the embedment of essential oils in edible hydrocolloid films can lead to both an increase and a decrease in the vapor permeability of the films [60,61]. It is widely recognized that lipids have the ability to improve the water resistance and reduce the water absorption capacity of polymer-based films. The presence of an oil phase leads to an increase in the tortuosity factor for water movement within the film matrix, effectively increasing the distance that water molecules must travel through the films [44]. Some exceptions to this have also been observed. For example, in a study by Zhang et al. [62], a gellan gum–chitosan multilayer film was created with thyme essential oil (TEO) nanoemulsion. The findings indicated that the addition of TEO increased water vapor permeability (WVP) due to the formation of a porous structure. Furthermore, the incorporation of turmeric essential oil (TurEO) notably raised the WVP of the films [63]. TurEO, being a hydrophobic and complex mixture, enhanced the hydrophobic nature of the packaging films. Nevertheless, the presence of a substantial number of micropores on the film’s surface, formed during the evaporation of TurEO, facilitated the penetration of water vapor through the hydrophilic region of the films. In a similar way, the rise in the water vapor permeability (WVP) of our films was impacted by the formation of pores, which induced structural alterations in the polymer network and facilitated the transfer of water vapor. In the present study, the occurrence of aggregates from the incorporated oil droplets, which give rise to an inhomogeneous structure of the films, is demonstrated by the visualization of the morphology of the films.

The wettability of the investigated samples was measured using the static water contact angle method. The water contact angle is a good indicator of the hydrophilic or hydrophobic nature of films and gives information about the wetting of the film by water. The contact angle is defined as the angle between a film surface and the tangent leading from the contact position of a liquid drop on the surface [64]. To analyze the effect of rosehip oil content in chitosan-based films, contact angle values were measured. The final value of the contact angle was the mean value of six measurements. The estimated error was less than 5%.

The contact angles of chitosan films with different concentrations of RSO emulsion are presented in Figure 5.

The results show that the pure chitosan film revealed a relatively high contact angle of about 75°, being classified as hydrophilic. The hydrophilicity of chitosan is attributed to the hydroxyl and amino groups present in its structure [65]. The positive charges that arise when the amino groups are protonated decrease the free energy of the surface, improving the wettability of the films [66]. According to the literature, the contact angle values of chitosan with water are quite different. Some authors [67,68] found values close to 80° and others [69] reported values close to 70°. This discrepancy in the results should be associated with the difficulty of measuring the contact angle at the exact moment when the liquid comes into contact with the surface of the film and by the same token, the rapid absorption of the water. Factors such as the degree of deacetylation of chitosan and the pH of water may also affect this measurement. From the results presented in Figure 5, it can clearly be seen that a drop in the contact angle occurred. While at a 1% RSO emulsion concentration, the differences in contact angle values were statistically indistinguishable, at higher concentrations, the contact angle rapidly decreased. With the addition of rosehip oil at different ratios, the contact angle decreased from 75° (0 wt.%) to 44° (5 wt.%). The incorporation of the rosehip oil results in more hydrophilic films. These results are in agreement with the surface wettability studies of chitosan–soy protein [70] and chitosan–grape seed extract films [41]. The reason may be due to the composition of the used rosehip oil. For example, a higher amount of polyphenols with their hydrophilic groups can lead to increased interactions of the film surface with water, followed by a decrease in contact angle. This phenomenon was also discussed by other authors [71]. It is important to point out that the measured values are strongly dependent on the location of a droplet’s placement on the film surface. In the case where the sample is not ideally homogeneous, the results may be substantially affected [71].

Following the theory of Owens and Wendt [72], the total surface free energy of all investigated samples was calculated. The results obtained are presented in Figure 5. It was established that the surface free energy of chitosan films with different concentrations of rosehip oil showed an increase when compared to the pure chitosan film. The value of the surface free energy significantly increased with an increase of rosehip oil emulsion concentration above 3%. In other words, the films with different concentrations of rosehip oil were more hydrophilic than the original chitosan film [43].

3.5. Thermal Properties and Stability of Chitosan/RSO Edible Films

The DSC analysis in Figure 6 illustrates the thermal properties and stability of the chitosan/RSO edible films.

The endothermic peak at −24.2 °C is associated with the melting phenomena of the RSO. Since such a peak is not observed in the edible films, it can be assumed that in the immobilized state, the oil does not crystallize. The broad endothermic peak at approximately 100 °C was associated with the moisture composition and water evaporation from the films [72]. The increase in RSO emulsion concentration up to 3% let to an appearance of an exothermic peak above 120 °C, which may be associated with some thermal instability of the multicomponent films. However, all of the studied films are characterized with thermal stability up to 100 °C, which makes them suitable for packaging foods that are not subject to heat treatment, such as minimally processed fruits and vegetables.

3.6. Total Phenolics Content and Antioxidant Properties of Chitosan/RSO Edible Films

Food oxidation is a deteriorating process that leads to changes in its chemical, sensory, and nutritional attributes over time. The antioxidant potential of edible films is typically determined by their composition. By incorporating natural antioxidants, such as essential oils (EOs), the oxidation of sensitive components like proteins and lipids can be slowed down, thereby significantly improving the quality, stability, and shelf life of food products. Additionally, the inclusion of natural antioxidants can help preserve or enhance the sensory characteristics of the food. Utilizing essential oils, particularly when loaded into edible films, has shown promising results in extending shelf life, improving stability, and enhancing sensory properties of packaged foods [73]. Essential oil components act on the food’s surface, contributing to its shelf life and imparting a characteristic odor and flavor. Furthermore, the incorporation of essential oils can confer antioxidant and antimicrobial effects to the films.

Phenolic compounds containing polyhydroxyl groups are often responsible for the overall antioxidant activity. These components possess significant abilities to scavenge and inhibit lipid peroxidation.

Table 5. Antioxidant potential and total phenolic content in chitosan/RSO films.

RSO Emulsion Concentration, %	Antioxidant Activity DPPH, μmolTE/100 g	Total Polyphenol Content mgGAE/100 g
0	0.717 ± 0.012 a	10.300 ± 0.100 a
1	106.453 ± 0.595 b	65.480 ± 0.660 b
2	221.577 ± 0.611 c	107.833 ± 1.216 c
3	325.663 ± 0.883 d	181.867 ± 0.952 d
4	428.693 ± 0.594 e	238.447 ± 6.456 e
5	543.720 ± 1.357 f	271.423 ± 0.810 f
R	0.9999	0.9959

^a–f: different letters in the same column indicate significant differences (p < 0.05).

shows that films containing only chitosan exhibit minimal antioxidant effects due to synergetic action between the amino group and hydroxyl groups, which produce stable macromolecules. Similar results were reported by Al-Harrasi et al. [47] and Liu et al. [74]. The addition of RSO improved the antioxidant capacity of the films significantly like the effect of other essential oils [13]. For example, the antioxidant activity obtained in the present study of chitosan with added 0.5% rosehip oil (5% emulsion) was comparable to that of a chitosan film containing the same amount of cinnamon oil [75]. Moreover, a linear dependence is observed between the antioxidant potential and the total polyphenol content as the concentration of RSO emulsion in the films increases.

It is clear that the presence of polyphenolic compounds in the films is due to the added RSO, which predominantly contains catechin, epicatechin and rutin [76]. They far exceed the polyphenol values found in chitosan films, which contain the same amount of canola, clove, basil, and lemongrass oils [77]. The obtained values for the polyphenol content correspond to the content of polyphenols in pure oil [78]. Therefore, it can be concluded that polyphenols do not interact with the chitosan matrix and retain their activity.

The antioxidant activity of the films is again directly related to the antioxidant activity of the RSO. Considering the amount of oil incorporated in the films and the values quoted in the literature for non-incorporated oil, it can be concluded that the incorporation of oil into the chitosan matrix does not alter its antioxidant activity [79].

3.7. Testing the Surface Resistance of Chitosan/RSO Edible Film Samples to Fungi

All variants were analyzed sequentially after 24, 48 and 72 h of incubation. Due to contact with the surface of the culture medium in the Petri dishes, some of the film discs changed their shape. In the variant with 4% added rosehip oil in one of the replicates, mold growth was observed on the sabouraud medium. No such growth was observed in the rest of the replicates. In general, yeast and mold growth were not observed for all other variants, regardless of the percentage of oil phase content. No halos were formed around the films in the Petri dishes (Figure 7a,b).

In the second test investigating film surface resistance, zones formed by the films that had changed their shape were observed. Dense growth from the added yeast culture was observed around them. Yeast did not develop on the film surface (Figure 7c). Halos were formed around the films.

Since no molds and yeasts were observed after 72 h of incubation of the samples (with one exception), it was indicative that the film surface did not retain such microorganisms, despite their non-sterile preparation. This is the reason for the absence of introduced and germinated microorganisms in the Petri dishes. The film surface does not retain fungal agents responsible for fruit fermentation or decay.

The described results were also confirmed in the second design of the film resistance test. The films did not allow the development of the yeast culture on their surface. Clear boundaries of growth were outlined around the films regardless of the presence or absence of rosehip oil. For this reason, it can be concluded that the newly obtained films are resistant to the growth and development of fungal agents responsible for fruit fermentation. This property of maintaining a sterile surface is extremely important when used in the industrial and food and nutrition sectors to increase the shelf life of food, etc. The evaluation of the chitosan film showed significant antimicrobial activity [80]. Some chitosan-based films inhibited the growth of various types of bacteria [81,82]. Data have been reported for over 99% antifungal activity of a film containing chitosan and cinnamon oil against several fungal representatives [83].

4. Conclusions

The physicochemical, antioxidant, and antifungal properties of casted chitosan films, containing different amount of rosehip seed oil emulsion (1%, 2%, 3%, 4%, and 5%) were investigated in the present research. The loaded RSO decreased the films strength and improved their mechanical properties by increasing their elasticity. The total color difference was slight for a RSO emulsion concentration under 3%, and sharply increased at higher concentrations. The addition of oil led to a reduction in water content. However, water vapor permeability and surface free energy increased due to the presence of inhomogeneity and micro-sized oil droplets. With respect to improving the hydrophobicity of the films, an optimization of the composition and method of formulation are necessary. Based on the thermal analysis, the incorporation of RSO does not affect the film stability up to 100 °C and therefore, the films are suitable for the food packaging of fresh or minimally processed food. At the same time, RSO significantly improves antioxidant activity. Based on the obtained results, it can be concluded that all of the measured parameters of the films remain stable up to a 3% concentration of the oil emulsion, after which they change significantly. For these reasons, this concentration can be used as optimal for the creation of edible coatings to prolong fruit shelf life. In addition, the evaluation of the antifungal activity of the new films showed that their surface did not retain fungal cells or their spores and provided an opportunity to maintain a sterile area of the coatings. Our future plans include the application of the chitosan/RSO coatings on concrete foodstuff and the investigation of the effect of the packaging on food safety and quality parameters (physical, physicochemical, and sensory). In this way, the best optimization of the composition and formulation method could be achieved.