Impact of Calcium Chloride Addition on the Microstructural and Physicochemical Properties of Pea Protein Isolate-Based Films Plasticized with Glycerol and Sorbitol

Abstract

Ca²⁺ can boost protein-protein interactions and, if present at an appropriate level, can potentially improve some physicochemical properties of protein-based gels and films. This study aimed to determine the effects of CaCl₂ (0%–0.05% w/w) on the microstructural, optical, water affinity, and mechanical characteristics of glycerol (Gly)- and sorbitol (Sor)-plasticized pea protein isolate (PPI)-based films. CaCl₂ caused darkening and a color shift of the films from yellow to yellow-green. Additionally, decreased light transmission, particularly in the UV range, acidification, and reduced moisture content were observed. CaCl₂ decreased the water vapor permeability of the Gly plasticized film by an average of 20% with no effect on the Sor-plasticized film. All films were completely soluble in water. CaCl₂ negatively impacted the mechanical integrity of the films, reducing the tensile strength of the Gly- and Sor-plasticized films by ~16% and 14%–37%, respectively. Further increases in CaCl₂ content (0.1% and 0.2% w/w) led to concentration-dependent microvoids resulting from protein over-crosslinking and/or coagulation. In summary, the incorporation of CaCl₂ into PPI-based films did not provide significant benefits and actually worsened key properties, such as transparency and mechanical strength. The type of plasticizer influenced how CaCl₂ affected some properties of the PPI-based film.

1. Introduction

Recently, the European Union (EU) has implemented the single-use plastics (SUP) directive [1] aimed at mitigating the environmental impact of SUP products (including certain packaging) and promoting a shift towards a circular economy. This includes replacing SUP with more sustainable options, such as bio-based and biodegradable materials derived from renewable resources. Using food-grade ingredients, such as proteins, polysaccharides, or lipids, and employing suitable manufacturing methods can result in packaging that is edible along with its contents. Edible packaging represents a niche alternative that is well suited for scenarios where plastic packaging is restricted. Notable examples include collagen casings for sausages, capsules made of gelatin, pullulan, starch, or hydroxypropyl methylcellulose, and certain food additives with coating-forming properties applied to fresh fruits [2].

Proteins offer a wider range of functions compared to polysaccharides, largely due to their structural complexity and dynamic nature. The properties of proteins can differ significantly depending on their origin and source. When selecting a protein for packaging production, factors such as barrier performance, mechanical strength, optical properties, processability, availability, and cost, as well as legal and environmental considerations, must be taken into account. Additionally, various structure-modifying methods and technologies can be considered to enhance or create novel attributes in protein-based materials.

The eco-design of packaging begins with the selection of raw materials. Approximately 80% of Earth’s biomass is comprised of plants [3]. Growing plants requires less energy than raising animals, making plant-based raw materials more cost-effective. Moreover, plants absorb CO₂ during growth, which helps offset the carbon footprint [4], and can be cultivated sustainably on a large scale, ensuring a continuous supply of raw materials. Legumes are considered an inexpensive source of high-quality proteins with the potential to produce edible packaging [5]. Among these, pea protein is gaining popularity in the global food industry, mainly as an alternative to soy protein. The reason is that pea plants can be grown in more temperate climates than soy [6,7], and unlike many protein-containing foods, such as cereals with gluten, eggs, fish, peanuts, soybeans, milk, nuts, and lupin, peas are less likely to cause allergies and are not listed as allergens that must be declared in the EU [8].

Previous studies have shown that factors such as plasticizer type and concentration, heating, pH, and the addition of lipids and polysaccharides significantly influence the barrier, optical, and mechanical properties of pea protein isolate (PPI)-based films [9,10,11,12]. For instance, films plasticized with sorbitol (Sor) were less permeable to water vapor, stronger, and less stretchable compared to glycerol (Gly)-plasticized films. Thermal treatment of film-forming solutions (FFSs) improved the mechanical strength and transparency of PPI-based films. Increasing the pH from 7 to 11 enhanced the mechanical strength of the Gly plasticized films, but not those plasticized with Sor. At the microscopic level, films obtained at neutral pH showed PPI particles conglomerating into a continuous layer [9,10]. As known, alkalization disrupts the protein’s native conformation, leading to unfolding or partial denaturation. Consequently, previously buried –SH groups and hydrophobic regions of the protein become exposed, facilitating the formation of a network among polypeptide chains and improving the cohesiveness of PPI-based films [10]. Nevertheless, considering that films produced under alkaline conditions may not be safe for consumption (as they could irritate mucous membranes and the digestive tract), producing films under neutral pH conditions appears to be the most reasonable option.

Some studies have shown that proteins can be customized using both chemical and enzymatic crosslinking agents to produce edible films with enhanced barrier and mechanical properties [13]. The use of calcium ions as counterions is a simple and cost-effective method to facilitate protein-protein interactions by altering the electrical charges on the protein molecules. It is believed that calcium-induced protein aggregation occurs via the following mechanisms:

• electrostatic shielding (ion binding neutralizes the repulsive charges, facilitating protein oligomerization),
• crosslinking of negatively charged carboxyl groups (in the C-terminus of each polypeptide chain and also the one in the aspartate and glutamate side chain) via protein-Ca²⁺-protein bridges,
• and ion-specific hydrophobic interactions [14,15].

Existing research on casein- [16,17,18], soy protein isolate (SPI)- [19], and whey protein isolate (WPI)-based films [20] indicates that the impact of calcium ions on the properties of protein-based films cannot be generalized, as it depends not only on the type of protein and calcium concentration but also on the type and amount of plasticizer used. It is worth mentioning that in addition to enhancing the functionality of protein films, the inclusion of calcium (a nutrient crucial for the mineralization of bones and teeth) can also improve their nutritional value. This is particularly relevant as calcium’s bioavailability increases when it is bound to proteins [21].

It has been demonstrated in some studies [14,22] that Ca²⁺, through a protein crosslinking mechanism, significantly increases the gelation ability, elastic modulus, and textural properties of PPI-based gels. It is worth noting that, similar to whey protein, pea protein is rich in acidic amino acids (i.e., aspartic and glutamic acids) [23], which suggests an abundance of potential binding sites for calcium.

Therefore, in the present study, we hypothesized that adding Ca²⁺ to a pea protein-based film recipe might enhance some of its functional properties. The objective was to compare the effects of increasing concentrations of CaCl₂ on the microstructural, optical, water affinity, and mechanical properties of Gly- and Sor-plasticized PPI-based films.

2. Materials and Methods

2.1. Materials

Propulse PPI (moisture content: 6% ± 1%; chemical composition on a dry basis: protein content: 82% ± 2%; total carbohydrates: 10.5% ± 1.5%; total fat: 2.5% ± 0.5%; ash: 3.5% ± 0.5%) was donated by Nutri-Pea (Portage la Prairie, Manitoba, Canada). Gly and D-Sor (min. 99.5%) were obtained from Sigma Chemical Co (St. Louis, MO, USA). Anhydrous CaCl₂ (analytical grade) was obtained from POCh (Gliwice, Poland).

2.2. Methods

2.2.1. Preparation and Conditioning of Films

The films were prepared by drying 10% (w/w) PPI solutions containing 5% (w/w) Gly or Sor and varying concentrations of CaCl₂ (0%, 0.01%, 0.025%, 0.05%, 0.1%, and 0.2% w/w). Firstly, the mixture (200 g) containing PPI, plasticizer (Gly or Sor), and distilled water was neutralized with concentrated NaOH solution to pH 7 and heated in a water bath at 90 °C for 20 min with constant stirring. Subsequently, CaCl₂ was added, and the FFSs were mixed using an H-500 homogenizer (Pol-Eko Aparatura, Wodzisław Śląski, Poland) at 20,000 rpm for 5 min. The FFSs were then cooled to 25 °C with continuous stirring, re-homogenized at 14,000 rpm for 1 min, degassed using a vacuum pump in a Büchner flask, and cast onto leveled polystyrene Petri dishes with an area of 145 cm² (Nunc, Roskilde, Denmark). To maintain a consistent film thickness of 100 ± 10 μm, 1.65 g of total solids (PPI + plasticizer + CaCl₂) was cast over the surface of a Petri dish. This corresponded to approximately 11 g of FFS. The FFSs were dried at ~25 °C and 50% ± 5% relative humidity (RH) for about 24 h. The resulting films were cut into samples, and their thickness was measured using a Mitutoyo No. 7327 micrometer (Mitutoyo, Tokyo, Japan). The samples were then conditioned at 25 °C and 50% RH for 48 h in a Sanyo Versatile Environmental Test Chamber MLR-350H (Sanyo Electric Biomedical Co. Ltd., Oizumi-Machi, Japan). Films without CaCl₂ were used as controls.

2.2.2. pH and Viscosity of FFSs

A glass electrode (Elmetron ERH-11S, Zabrze, Poland) connected to a pH meter (Elmetron CPC 401, Zabrze, Poland) was used to measure the pH of the FFSs at 25 ± 1 °C. The dynamic viscosity (η, mPa·s) of the FFSs was determined using a rotational viscometer ROTAVISC lo-vi (IKA, Staufen, Germany) under the following operating conditions: VOLS-1 adapter with spindle VOL-SP-6.7, 200 rpm, 25 °C and 6.7 mL of the sample. All analyses were performed in triplicate.

2.2.3. Microstructure

The microtopography of the films (and, for comparative purposes, also PPI powder) was analyzed using a scanning electron microscope (1430VP, LEO Electron Microscopy Ltd., Cambridge, UK). The film samples were re-dried under vacuum and coated with gold before observation. Additionally, the films were examined using a Leica 5500B microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a differential interference contrast optical system.

2.2.4. pH of the Films

A flat surface electrode (Elmetron EPX-3, Zabrze, Poland) connected to a pH meter (Elmetron CPC 401, Zabrze, Poland) was used to measure the pH of the films after slight surface hydration with 20.0 μL of deionized water.

2.2.5. Optical Properties

The color values (CIE L*a*b* and C*h) of the film samples (1 cm × 4 cm) were measured with a colorimeter X-RiteColor 8200 (X-Rite Inc., Grand Rapids, MI, USA) on a black background (L* = 25.63, a* = −0.12, b* = −0.47). The total color difference (ΔE*) was calculated using Equation (1).

$$\mathsf{\Delta }{E}^{*}=\sqrt{\mathsf{\Delta }{L}^{*2}+\mathsf{\Delta }{a}^{*2}+\mathsf{\Delta }{b}^{*2}}$$

(1)

where Δ is the difference between the color parameters of the films without CaCl₂ (control) and those with CaCl₂.

The light-barrier properties of the film samples (1 cm × 4 cm) were measured using a spectrophotometer (Lambda 40, Perkin–Elmer, Shelton, CT, USA) at selected wavelengths between 200 and 800 nm. The spectrophotometer was calibrated using air. The film sample was then placed in a special slit of the spectrophotometer, where the cuvette was typically positioned, and a UV/Vis spectrum scan was initiated. The opacity (Op) of the films was calculated using Equation (2):

$$\mathrm{Op}={\displaystyle \frac{{\mathrm{A}}_{600}}{\mathrm{t}}}$$

(2)

where A₆₀₀ is the absorbance of the film sample at 600 nm and t is the film sample thickness (mm).

Optical analyses were conducted five times.

2.2.6. Water Affinities

The specimens (2 cm × 2 cm) were dried in an oven at 105 °C for 24 h. The moisture content (MC) was determined by calculating the percentage of water removed from the samples. Solubility (So) was expressed as the percentage of the film solubilized in water containing 0.02% sodium azide. The specimens (2 cm × 2 cm) were shaken with 30 mL of distilled water in 50 mL Falcon test tubes in an ES-60 incubator (MIULAB, Hangzhou, China) at 25 ± 1 °C and 170 rpm for 24 h. Both the MC and So analyses were conducted in quadruplicate

Water vapor permeability (WVP, g·m·mm⁻²·day⁻¹·kPa⁻¹) was calculated as follows:

$$\mathrm{WVP}={\displaystyle \frac{\mathrm{WVTR}\times \mathrm{t}}{{\mathsf{\Delta }}_{\mathrm{p}}}}$$

(3)

where: WVTR is the water vapor transmission rate (g·m⁻²·day⁻¹) measured gravimetrically based on the ISO 2528 method [24], t is the mean film thickness (mm), and Δ_p is the difference in the water vapor pressure (kPa) between two sides of the film.

Briefly, poly(methyl methacrylate) permeation cell cups with an internal diameter of 7.98 cm (exposed film area = 50 cm²) and an internal depth of 2 cm were filled with 10 g of anhydrous calcium chloride (0% RH). The 10 cm diameter film samples were then placed over the circular openings and secured with O-ring rubber gaskets and screw tops. The cups were placed in a test chamber at 25 °C and 50% RH. Weight gain was monitored over 12 h, with weights recorded at 2-h intervals. The slopes of the steady-state (linear) portions of the weight gain versus time curves were used to calculate the WVTR.

The WVP analyses were performed in triplicate.

2.2.7. Mechanical Properties

Tensile strength (σ_max), elongation at break (ε_b), and elastic modulus (EM) were determined using a TA-XT2i texture analyzer equipped with a 50 kg load cell (Stable Micro Systems, Godalming, UK) following the procedure outlined in PN-EN ISO 527-1, 2, 3:1998, with some modifications [10]. The dumbbell-shaped film samples were mounted on the analyzer with an initial grip separation of 80 mm and stretched at a speed of 1 mm s⁻¹. The σ_max, ε_b, and EM were calculated using Equations (4)–(6), respectively:

$${\sigma }_{max}={\displaystyle \frac{{\mathrm{F}}_{\max }}{\mathrm{A}}}$$

(4)

where F_max is the maximum load for breaking the film (N), and A is the initial cross-sectional area (thickness × width, mm²) of the specimen,

$$\mathrm{E}={\displaystyle \frac{\mathsf{\Delta }\mathrm{L}}{{\mathrm{L}}_{\mathrm{i}}}}\times 100$$

(5)

where ΔL is the difference in the length at the moment of fracture, and L_i is the initial gage length (mm),

$$\mathrm{EM}={\displaystyle \frac{{\sigma }_2-{\sigma }_1}{{\epsilon }_2-{\epsilon }_1}}$$

(6)

where ε₁ is a strain of 0.0025 (0.25%), ε₂ is a strain of 0.01 (1%), σ₁ (MPa) is the stress at ε₁ and σ₂ (MPa) is the stress at ε₂.

2.2.8. Statistical Analysis

Differences among the mean values of the data were tested for statistical significance at the p < 0.05 level using analysis of variance (STATISTICA 13.3, StatSoft Inc., Tulsa, OK, USA) and Fisher’s test.

3. Results and Discussion

3.1. pH and Viscosity of the FFSs

Although the pH of the control FFSs was adjusted to 7.0, it dropped slightly after heating and cooling (

Table 1. Effect of CaCl₂ concentration on the pH and viscosity (η) of glycerol-(Gly) and sorbitol (Sor)-containing pea protein isolate-based film-forming solutions.

Plasticizer	CaCl2 (%)	pH	η (mPa·s)
Gly	0	6.90 ± 0.01 j	7.14 ± 0.07 g
	0.01	6.88 ± 0.01 i	6.32 ± 0.18 f
	0.025	6.81 ± 0.01 g	5.55 ± 0.18 e
	0.05	6.73 ± 0.01 e	5.06 ± 0.09 d
	0.1	6.59 ± 0.01 c	4.40 ± 0.07 c
	0.2	6.34 ± 0.01 a	3.39 ± 0.05 a
Sor	0	6.94 ± 0.01 k	7.71 ± 0.19 h
	0.01	6.89 ± 0.01 ij	6.30 ± 0.21 f
	0.025	6.86 ± 0.01 h	6.19 ± 0.21 f
	0.05	6.77 ± 0.01 f	5.76 ± 0.01 e
	0.1	6.64 ± 0.01 d	4.39 ± 0.15 c
	0.2	6.42 ± 0.01 b	3.77 ± 0.09 b

^a–k Values with the different superscript letters within one column are significantly different (p < 0.05).

). This likely resulted from the denaturation of proteins, which exposed hidden carboxyl and amine groups. The change in the availability of these groups in the solution could have affected the ionic balance and, consequently, the pH. Since CaCl₂ is an acidic salt [25] (because the anionic part of the salt, Cl⁻, is a stronger acid than the cationic part, Ca²⁺, which is a base), increasing the CaCl₂ content led to gradual acidification of the FFSs (Table 1). This result is consistent with the findings of Tarapata et al. (2020) [26], who showed that the addition of CaCl₂ results in a decrease in milk pH. FFSs containing glycerol generally had slightly lower pH than their sorbitol-containing counterparts (Table 1).

The increase in CaCl₂ concentration gradually decreased the viscosity of the FFSs (Table 1). Similarly, Peng et al. (2022) [27] observed that increasing calcium addition led to a significant decrease in the viscosity of soy protein dispersions, almost approaching the dynamic viscosity of water (0.89 mPa·s at 25 °C). It should be noted, however, that the cited study focused on Ca(OH)₂ added during the soy protein preparation, specifically during the neutralization step. According to the authors’ findings, this result was due to the cross-linking-mediated formation of larger aggregates that became denser, more insoluble, and less water-holding as calcium content increased. Consequently, these aggregates rarely interacted with water, and contributed less to the viscosity. Moreover, the decrease in viscosity can presumably be ascribed to the accompanying decrease in pH of the FFS (Table 1), which caused the protein to start precipitating out of the solution. As reported by Shin et al. (2021) [28], the viscosity of thermally-processed PPI solution exhibited a notable decrease in the pH range of 6.5 to 4.0, presumably driven by coagulum formation as the pH approached the isoelectric point (pI) of pea protein.

3.2. Microstructure

All the resultant films consisted of a conglomeration of PPI particles glued together by the dissolved portion of the protein, which is consistent with evidence from previous observations [9,12]. The particles in the films ranged from approximately one micrometer to 90 µm (Figure 1, Figure 2, Figure 3), and the larger maximum size compared to the PPI powder (Figure S1) likely indicates that they were in a swollen state. It was found that the Gly-containing FFSs tended to create more uniform microtopography with better compactness in comparison to their Sor-containing counterparts (Figure 1 and Figure 2). It is possible that in the Gly-plasticized films, the larger Gly-soaked PPI particles may have been positioned beneath smaller particles. The coherent PPI-based films were produced from FFSs containing up to 0.05% CaCl₂ (equivalent to a concentration of 4.5 mM) (Figure 1 and Figure 2). Within this range, no significant effect of CaCl₂ on the microstructure of the films was observed. Increasing the CaCl₂ concentration to 0.1% and 0.2% w/w led to the formation of crack-like holes in the films, with the severity depending on the concentration (Figure 2 and Figure 3). The detrimental impact of excessively high Ca²⁺ levels on the integrity of protein-containing gels and films is a well-documented phenomenon [20,29]. This issue likely arises from the over-crosslinking of the protein matrix, leading to coagulation. It should be noted that CaCl₂ also significantly reduced the pH of the FFSs (Table 1). Therefore, it is not surprising that the stability of the PPI suspension at a pH closer to its pI resulted in partial phase separation, as indicated by the gradual decrease in solution viscosity (Table 1) and the formation of a larger quantity of larger microvoids. This was likely due to the intensified coalescence of the supernatant, which hindered PPI particle adhesion. Scanning electron microscopy fractography evaluation did not reveal the so-called “rock candy fracture” in the microvoids (Figure 3), which was observed in a previous study when dehydrated PPI-based films were physically broken down [12]. Thus, the formed microvoids were not typical cracks. However, the presence of primarily slit-like microvoids (Figure 2 and Figure 3), i.e., no round dimples, suggests that internal shear stresses introduced during the drying process (evaporation causes volume reduction and consequently introduces stress within the material) played a significant role in their formation.

3.3. Optical Properties

Color and appearance are crucial factors that consumers consider when selecting a food product. The PPI-based films were transparent but had a yellow tint, which could limit their application possibilities. This color was due to the inherent creamy yellow hue of the PPI, indicating that some pea-origin color components, such as carotenoids and chlorophylls [30], were not removed during the isolation process. It’s important to note that the final color properties of PPI also strongly depend on the drying method used [31].

Regardless of CaCl₂ concentration, the Gly- and Sor-plasticized films did not show any significant differences in color parameters (p > 0.50) (

Table 2. Effect of CaCl₂ concentration on the color parameters of glycerol-(Gly) and sorbitol (Sor)-plasticized pea protein isolate-based films.

Plasticizer	CaCl2 (%)	L*	a*	b*	h (°)	C*	ΔE*
Gly	0	41.39 ± 0.01 cd	−0.21 ± 0.03 c	2.13 ± 0.07 cd	95.77 ± 0.39 a	2.14 ±0.07 bc	-
	0.01	37.60 ± 3.37 b	−0.69 ± 0.13 b	2.50 ± 0.16 d	105.45 ± 2.49 bc	2.60 ± 0.18 d	3.97 ± 3.15 a
	0.025	30.87 ± 1.24 a	−1.00 ± 0.09 a	1.79 ± 0.51 bc	120.09 ± 5.28 d	2.06 ± 0.49 bc	10.56 ± 1.23 b
	0.05	29.80 ± 0.40 a	−1.04 ± 0.02a	1.44 ± 0.20 ab	126.17 ± 4.42 d	1.78 ± 0.15 ab	11.64 ± 0.40 b
Sor	0	41.72 ± 0.04 d	-0.28 ± 0.02 c	2.30 ± 0.06 d	96.85 ± 0.32 ab	2.32 ± 0.06 cd	-
	0.01	38.46 ± 3.23 bc	−0.77 ± 0.24 b	2.27 ±0.48 d	109.61 ± 9.33 c	2.42 ± 0.39 cd	3.36 ± 3.19 a
	0.025	30.16 ± 0.62 a	−1.04 ± 0.08 a	1.80 ± 0.11 bc	126.69 ± 7.96 d	2.08 ± 0.05 bc	11.60 ± 0.61 b
	0.05	30.03 ± 0.44 a	−0.97 ± 0.08 a	1.25 ± 0.06 a	127.85 ± 2.80 d	1.58 ± 0.06 a	11.76 ± 0.45 b

^a–d Values with the different superscript letters within one column are significantly different (p < 0.05).

). Even at the lowest concentration, CaCl₂ caused a noticeable decrease in brightness (L* parameter) and red color contribution (a* parameter). A ΔE* value above 3 (Table 2) indicates that even an untrained observer could notice the difference [32] between films with and without CaCl₂. Increasing the concentration of CaCl₂ further reduced these parameters and additionally decreased the yellow color intensity (b* parameter). Consequently, the ΔE* increased above 11, meaning that an observer would notice two distinctly different colors (ΔE* > 5) [32]. The films containing intermediate and the highest amount of salt did not differ in color (Table 2). Compared to the control, these films exhibited an increased h (95.77°–96.85° vs. 120.09°–127.85°, Table 2), indicating a shift in color from yellow to yellow-green [33]. The darkening and reduced color saturation (C* parameter) of the CaCl₂-added films can likely be attributed to chemical reactions between the salt and the components of the material, including pigments. It is known that the dissolution of CaCl₂ involves the hydration of each ion: Cl⁻ combines with H⁺ in water to form HCl, a strong and corrosive acid, while Ca²⁺ combines with OH⁻ in water to form Ca(OH)₂, a weak but corrosive base.

The control Gly- and Sor- had similar UV/VIS light barrier properties (Figure 4). Consistent with previous works [9,11,12], the films exhibited excellent UV-B (280–315 nm) and UV-C (100–280 nm) blocking properties. Namely, the transmittance of control films noted at the UV-B spectrum was below ~5% (Figure 4). Barrier properties of protein films against UV light are mainly due to the tyrosine and tryptophan absorption (~280 nm) and peptide group absorption (190–220 nm spectral range) [34]. It is worth noting that the films also provided a certain barrier against long-wave UV (UV-A), which is important as this radiation is not absorbed by the ozone layer. The transmittance noted in this region was between ~5%–60% (Figure 4). In this respect, the PPI-based films were better UV blockers than films made from porcine gelatin or WPI [35,36]. The reason may be their yellow coloring (Table 2) and a nonhomogeneous microstructure that scattered light (Figure 1, Figure 2 and Figure 3).

The incorporation of CaCl₂ reduced light transmission, especially in the UV range (Figure 4). For example, at λ = 400 nm, the transmittance was reduced from approximately 60% to around 50% and 46% for Gly- and Sor-plasticized films, respectively. Presumably, the light was multiply scattered by the more precipitated proteins, as the pH of the CaCl₂-suplemented films was closer to the pI (

Table 3. Effect of CaCl₂ concentration on the pH, opacity (Op), moisture content (MC), water vapor permeability (WVP), and solubility (So) of glycerol-(Gly) and sorbitol (Sor)-plasticized pea protein isolate-based films.

Plasticizer	CaCl2 (%)	pH	Op (A600/mm)	MC (%)	WVP (*)	So (%)
Gly	0	6.50 ± 0.02 d	0.90 ± 0.02 a	29.46 ± 1.14 d	18.60 ± 0.88 c	100.00 ± 0.00 a
	0.01	6.49 ± 0.02 d	1.09 ± 0.05 bc	26.88 ± 1.42 c	14.91 ± 0.77 b	100.00 ± 0.00 a
	0.025	6.41 ± 0.01c	1.19 ± 0.06 d	27.63 ± 1.32 c	14.43 ± 0.98 b	100.00 ± 0.00 a
	0.05	6.35 ± 0.01b	1.19 ± 0.05 d	27.34 ± 1.50 c	15.51 ± 1.93 b	100.00 ± 0.00 a
Sor	0	6.54 ± 0.01 e	1.02 ± 0.08 b	11.50 ± 0.44 b	1.40 ± 0.04 a	100.00 ± 0.00 a
	0.01	6.48 ± 0.02 d	1.13 ± 0.06 cd	6.71 ± 0.12 a	0.81 ± 0.20 a	100.00 ± 0.00 a
	0.025	6.37 ± 0.01b	1.40 ± 0.08 e	6.62 ± 0.42 a	1.19 ± 0.16 a	100.00 ± 0.00 a
	0.05	6.23 ± 0.03a	1.43 ± 0.01 e	6.69 ± 0.57 a	1.43 ± 0.07 a	100.00 ± 0.00 a

^a–e Values with the different superscript letters within one column are significantly different (p < 0.05). * g·m·mm⁻²·day⁻¹·kPa⁻¹.

). A decrease in light permeability was also observed by Fang et al. (2002) [20] after incorporating CaCl₂ (5 and 10 mM) into WPI-based films.

Regardless of CaCl₂ concentration, the Gly-plasticized films were slightly more transparent compared to the Sor-plasticized counterparts (p < 0.05, Table 3). A possible explanation for this might be their slightly more homogenous microstructure, at least on the surface (Figure 1). It is known that as a material becomes less homogeneous, its opacity typically increases due to increased scattering and absorption of light caused by the inhomogeneities [37]. Consistent with the present results, previous studies have shown that a porcine gelatin-based film plasticized with Sor was more opaque compared to a film plasticized with Gly [35,38]. It is possible that Sor and Gly may show differences in compatibility with proteins, which could affect the optical properties of the resulting materials. Comparatively, the transparency of the PPI-based films is similar to the Sor-plasticized SPI-based film [38], but lower than that of porcine gelatin- and WPI-based films plasticized with Gly [35].

3.4. Water Affinities

The control film plasticized with Sor exhibited about 2.5 times lower MC and 16 times lower WVP than the film with Gly (p < 0.05, Table 3). These differences can be attributed to the distinct water-binding capacities of the plasticizers. Although Sor has six hydroxyl groups, they are less accessible for binding with water compared to the three hydroxyl groups of the more compact Gly. Specifically, at 25 °C and 50% RH, the hygroscopicity and water-holding capacity of Sor are 1 and 21 H₂O mg/100 mg, respectively, while for Gly these values are 25 and 40 H₂O mg/100 mg, respectively [39]. Since the Sor-plasticized films absorb less moisture [9], they consequently allow less water vapor to pass through (Table 3). It is important to note, however, that hygroscopicity is a key factor but not the sole determinant of a material’s WVP.

Regardless of the type of plasticizer used, the incorporation of CaCl₂ decreased the MC of the films in a concentration-independent manner (p < 0.05, Table 3). This reduction may be due to Ca²⁺-induced protein-protein linkages, which consequently inhibited the possibility of protein-water interactions. It has been shown that Ca²⁺ can alter the molecular conformation of proteins, contributing to the formation of hydrogen bonds, disulfide bonds, and hydrophobic interactions during gelation. Consequently, it can affect the water-binding capacity of the gel system, either increasing or decreasing it, depending on Ca²⁺ concentration [40,41]. To support this, Arabestani et al. (2013) [42] observed that the incorporation of CaCl₂ (0.1%–1%, w/w) significantly increased the surface hydrophobicity of vetch protein film. However, only the highest CaCl₂ concentration (1%, w/w) caused a significant reduction in the MC. The authors speculated that since the pH of the FFS was above the pI of the protein, the predominant negative charges on the protein molecules were bound by the calcium ions to form a compact network. These interactions might inhibit the charged sites from interacting with water, thereby enhancing the protein’s hydrophobicity.

The salt addition did not affect the barrier properties of the Sor-plasticized film but reduced the WVP of the Gly-plasticized PPI-based film by an average of 20% (Table 3). The differential impact of CaCl₂ on the WVP of films containing Gly and Sor suggests that the plasticizer type can influence the interactions between the salt ions and pea protein. Literature reports various effects of calcium ion addition on the WVP of protein films. Fang et al. (2002) [20] found that despite the crosslinking of the protein matrix, the addition of CaCl₂ (both 5 and 10 mM) to Gly-plasticized WPI-based films did not significantly influence WVP across the tested RH range (~30%–75%). Moreover, doubling the Gly concentration, combined with the protein aggregation effect at 10 mM CaCl₂, increased WVP due to pore formation. Park et al. (2001) [19] found that the addition of CaCl₂ did not affect the water barrier properties of Gly-plasticized SPI-based film, while a 30% reduction in WVP was observed with calcium sulfate incorporation. The authors attributed this to calcium bridges that maximized interactions between negatively charged soy protein molecules, enhancing network coherence. Also, the CaCl₂-induced tightening effect reduced the WVP by 7%–18% and 42% in vetch protein and sodium caseinate-based emulsion films, respectively [17,42]. It should also be mentioned that the calcium caseinate-based film exhibited lower WVP than the sodium caseinate-based film, which is also indirect evidence that the presence of calcium ions contributes to enhancing the water barrier properties [17,18].

All tested PPI-based films were completely soluble in water at 25 °C (Table 3). In fact, after 24 h of shaking, the films disintegrated into small, clammy pieces, making it very difficult to retrieve the remnants. Therefore, 100% So was assumed despite the presence of sediment. A similar finding was also reported for the Sor-plasticized SPI-based film [38]. A note of caution is due here since two methods for film So determination are used by researchers. Namely, the heat-dried (residue after oven drying for moisture content determination) or conditioned (25 °C, 50% RH) film samples are used. So (also known as total soluble matter) values obtained using these two methods are different. This is because the dehydro-thermo-treating (at 105 °C) of films develops tensional and compressional stresses in the material, which improves its integrity throughout the soaking procedure [36]. Therefore, only the second method, which was used in this study, shows an accurate behavior of films after coming into contact with water.

This study has been unable to demonstrate that CaCl₂ can reduce the So of the PPI-based films (Table 3). Such a possibility, however, was observed for whey protein concentrate- and vetch protein-based films, which, according to the authors, indicated some changes in the network cohesion due to the Ca²⁺-induced crosslinking of protein chains [42,43].

3.5. Mechanical Properties

The control film plasticized with Sor exhibited about four times higher σ_max and EM, but two times lower ε_b than the Gly-plasticized film (p < 0.05,

Table 4. Effect of CaCl₂ concentration on the tensile strength (σ_max), elastic modulus (EM) and elongation at break (ε_b) of glycerol-(Gly) and sorbitol (Sor)-plasticized pea protein isolate-based films.

Plasticizer	CaCl2 (%)	σmax (MPa)	EM (MPa)	εb (%)
Gly	0	1.70 ± 0.17 b	51.03 ± 3.35 b	73.79 ± 4.58 d
	0.01	1.43 ± 0.18 a	44.14 ± 8.00 ab	60.80 ± 4.89 c
	0.025	1.42 ± 0.16 a	42.52 ± 7.22 ab	57.52 ± 3.90 c
	0.05	1.41 ± 0.20 a	39.49 ± 4.00 a	59.49 ± 4.00 c
Sor	0	6.31 ± 0.24 e	219.15 ± 12.36 e	37.01 ± 3.70 a
	0.01	5.40 ± 0.27 d	193.52 ± 14.36 d	32.67 ± 2.72 a
	0.025	3.99 ± 0.26 c	129.23 ± 7.46 c	55.23 ± 3.06 c
	0.05	4.14 ± 0.25 c	133.39 ± 10.62 c	46.14 ± 8.88 b

^a–e Values with the different superscript letters within one column are significantly different (p < 0.05).

). Presumably less hydrated Sor-plasticized film (11.50% vs. 29.46%, Table 4) had a stronger and stiffer structure than the more moisturized Gly-plasticized film. Additionally, Gly, as a smaller compound, could more efficiently disrupt the intermolecular interaction among polypeptide chains than Sor, giving better plasticizing action and, thus, weaker but more stretchable films (Table 4). The different physical states of the plasticizers were also likely significant; liquid Gly could be a better lubricant for the film matrix than solid Sor [10].

CaCl₂ reduced the σ_max by ~16% and 14%–37% (Table 4) in the Gly- and Sor-plasticized films, respectively, and also caused a reduction of about 13%–23% and 12%–41% in EM, respectively (Table 4). This finding was unexpected, as it is believed that Ca²⁺ favors electrostatic interactions between two adjacent carboxylic groups of different polypeptide chains and/or shields electrostatic protein-protein repulsions, leading to a denser protein matrix structure, thus increasing the mechanical strength of gels and films [16,40,41,44,45,46]. The negative result indicates that CaCl₂ destroyed the integrity of the PPI-based films. It is difficult to explain this result, but it might be related to the fact that the films consisted of incompletely dissolved PPI particles (Figure 1, Figure 2 and Figure 3), which were not very susceptible to calcium-mediated crosslinking. On the contrary, the salt presumably interfered with the molecular association of the undissolved PPI particles. Pea proteins are similar in several ways to soy proteins [47]. Park et al. [19] found that CaCl₂ did not affect the σ_max of SPI film, while improvement was observed with the use of calcium sulfate. The inefficiency of CaCl₂ was explained by differences in the solubility of these salts. Specifically, CaCl₂, being more soluble, probably coagulated proteins faster than calcium sulfate, resulting in a more irregular microstructure in the CaCl₂-added film. The heterogeneous structure did not offer the same strength as the uniform structure of the film with sulfate. This clearly indicates the importance of microstructural features in shaping the mechanical properties of the films. Fang et al. (2002) [20] demonstrated that the effectiveness of CaCl₂ depends not only on its concentration but also on the plasticizer content. Although a 10 mM concentration of CaCl₂ increased the mechanical strength of WPI emulsion-based films, the same concentration with twice the amount of Gly resulted in films that were too fragile for tensile testing. Similarly, Mezgheni et al. [16] observed that the addition of CaCl₂ at a concentration of 0.25% weakened the strength of casein-based films, with the extent of weakening depending on the type and concentration of the plasticizer. In contrast, a lower concentration of CaCl₂ (0.125%) generally had no effect or improved the mechanical strength of these films. Despite the authors’ assumption of Ca²⁺-induced crosslinking through the formation of ionic bonds, the addition of CaCl₂ (0.05 g/100 g of dry matter) was insufficient to enhance the mechanical properties of the whey protein concentrate films [43]. Similarly, CaCl₂ (at concentrations of 0.1–1%, w/w) was unable to affect the mechanical properties (both σ_max and ε_b) of the Gly-plasticized vetch protein-based film [42]

It should be noted that CaCl₂ caused a greater deterioration in σ_max and EM in Sor-plasticized film compared to Gly-plasticized film (Table 4). This suggests that the more brittle and less elastic structure of the Sor film was more susceptible to CaCl₂-induced microdamage. It is difficult to explain this result, but it might be related to the more significantly lowered pH (Table 3), which could be less favorable for forming a cohesive film network.

Considering the above-discussed results, it is somewhat surprising that CaCl₂ at concentrations of 0.025–0.05% enhanced the ε_b of the Sor-plasticized film (p < 0.05, Table 4) while the opposite effect was observed for the Gly-plasticized film. This inconsistency may be due to the relaxation of the relatively strong and rigid structure of the Sor-plasticized film by the ions, which facilitated polypeptide chain slip movement. Most likely, the film plasticized with Gly did not exhibit this effect due to its already sufficiently high degree of plasticization (Table 4). In fact, CaCl₂ apparently exhibited an antiplasticization action in the Gly-containing PPI-based film. This outcome is contrary to that of Park et al. (2001) [19], who found that CaCl₂ significantly increased the ε_b of Gly-plasticized SPI-based film.

Comparing the mechanical properties of films produced by different researchers is challenging due to variations in several factors, including the concentrations of polymer in the FFSs, amounts and types of plasticizers used (i.e., polymer-to-plasticizer ratio), differences in the procedures for film preparation, and variations in testing methods. Nevertheless, considering the similar polymer:plasticizer ratio (i.e., ~1:0.5) and the method of preparation, the σ_max of the control Gly-plasticized PPI-based film (1.70 MPa, Table 4) is lower than that of the Gly-plasticized gelatin- (11.36 MPa), WPI- (6.10 MPa) [35], SPI- (5.5 MPa, pH of the FFS = 9) [19], or vetch protein-based films (≈ 5.0 MPa, pH of the FFS = 11) [42]. In turn, the σ_max of the PPI-based film plasticized with Sor (6.31 MPa, Table 4) is comparable to that of the Sor-plasticized SPI-based film (5.4 MPa) [38]. The relatively low strength of the PPI-based films, particularly the Gly-plasticized one (Table 4), is partially likely due to the low cohesion of the matrix formed by incompletely dissolved proteins at pH 7 (Figure 1, Figure 2 and Figure 3).

4. Conclusions

Taking into account that CaCl₂ at concentrations of 0.1% and 0.2% w/w induces serious protein over-crosslinking and/or coagulation, preventing the formation of a continuous PPI-based film, the amount of salt added should not exceed a critical concentration of 0.05% w/w in the FFS. CaCl₂ did not provide significant benefits to the PPI-based films and actually worsened their key properties, such as transparency and mechanical strength. Light transmission and some other film properties were dependent on the concentration of CaCl₂ but not to an impressive extent, suggesting a low sensitivity of the largely undissolved pea proteins to this salt. The results verified that the type of plasticizer differently affected how CaCl₂ influenced WVP and ε_b of the PPI-based film. Using pea proteins as an edible packaging ingredient requires methods beyond Ca²⁺-induced crosslinking to address existing challenges and fully exploit their potential. Alternatively, trials with FFS at a higher pH can be conducted to ensure better solubility of CaCl₂-added PPI and to minimize the coagulating effect of the salt. Although the films obtained in this study did not exhibit the expected improvements in key properties, they could potentially serve as a source of calcium in the diet.