Encapsulation of Pink Pepper Essential Oil (Schinus terebinthifolius Raddi) in Albumin and Low-Methoxyl Amidated Pectin Cryogels

Abstract

This study evaluated cryogels from albumin (ALB) and albumin–pectin (ALB:PEC) as carriers for pink pepper (Schinus terebinthifolius Raddi) essential oil. Cryogels were evaluated through infrared spectrophotometry, X-ray diffraction, scanning electron microscopy, thermogravimetric analysis, and differential scanning calorimetry. The bioactivity of the cryogels was analyzed by measuring their encapsulation efficiency (EE%), the antimicrobial activity of the encapsulated oil against S. aureus, E. coli, and B. cereus using the agar diffusion method; total phenolic content and antioxidant activity were analyzed by UV-vis spectrophotometry. The EE% varied between 59.61% and 77.41%. The cryogel with only ALB had the highest total phenolic content with 2.802 mg GAE/g, while the cryogel with the 30:70 ratio (ALB:PEC) presented a value of 0.822 mg GAE/g. A higher proportion of PEC resulted in a more significant inhibitory activity against S. aureus, reaching an inhibition zone of 18.67 mm. The cryogels with ALB and 70:30 ratio (ALB:PEC) presented fusion endotherms at 137.16 °C and 134.15 °C, respectively, and semicrystalline structures. The interaction between ALB and PEC increased with their concentration, as evidenced by the decreased intensity of the O-H stretching peak, leading to lower encapsulation efficiency. The cryogels obtained can be considered a suitable matrix for encapsulating pink pepper oil.

1. Introduction

The interest in technological applications of essential oils (EOs) has been increasing due to their antioxidant properties and antimicrobial action against foodborne pathogens [1]. Among the EOs, pink pepper essential oil (Schinus terebinthifolius Raddi) stands out for its antioxidant, antimicrobial, and anti-inflammatory properties, and anticancer capacity [2], which are attributed to its major constituents that include α-pinene, β-pinene, β-myrcene, β-cubebene, and limonene, in addition to monoterpenes and sesquiterpenes [3,4]. Pink pepper essential oil can be affected by external factors such as oxygen, light, humidity, and pH, which reduce biological activity [5], so encapsulation processes have become a tool to increase its stability and even improve its release profile [6]. Different technologies, including spray drying [7,8,9,10,11,12,13,14], complex coacervation [15,16,17,18,19,20,21], emulsion extrusion technique [15], and emulsification-ionic gelation [22,23,24] may be used for encapsulating pink pepper essential oil. However, recently, researchers have become interested in protecting essential oils using hydrogels and cryogels because they are produced from biopolymers without using crosslinker agents, while at the same time allowing the design of a controlled release system. Cryogels are obtained from hydrogels prepared from proteins or polysaccharides dried by lyophilization [25]. Cinnamon essential oil encapsulated in gelatin hydrogels offers a constant release over a long period while it maintains a high antimicrobial capacity against S. aureus and E. coli [26]. Also, the microgels obtained from chitosan maintain the biological activity of the essential oil of Gaultheria procumbens against the secretion of aflatoxins from A. flavus [27]. The cryogels offer advantages due to high load capacity and encapsulation efficiency. Volić et al. [28] reported that calcium alginate used as wall material reached an encapsulation efficiency of about 85% for thyme essential oil, and the combination of calcium alginate and soy protein resulted in an efficiency of 80%.

Egg albumin, a globular protein, possesses excellent gel-forming properties. Heat treatment above 82 °C induces protein denaturation, disrupting its structural integrity and facilitating the formation of high molecular weight aggregates. These aggregates, stabilized by disulfide, hydrogen, hydrophobic, and electrostatic interactions, contribute to increased solution viscosity and subsequent gelation [29,30,31]. Low-methoxyl amidated pectin, a polysaccharide containing amino groups, exhibits superior gelling characteristics compared to standard low-methoxyl pectin. It forms strong gels at low calcium concentrations and can gel under acidic conditions (pH < 3) [32]. There are few studies on the encapsulation of essential oils in cryogels of egg albumin and low-methoxyl amidated pectin; these biopolymers stand out for their ability to form gels and protect bioactive compounds [33]. From these considerations, this study aimed to encapsulate pink pepper essential oil in cryogels prepared from egg albumin and low-methoxyl amidated pectin, as well as to evaluate the inhibitory properties of this encapsulation system on S. aureus, E. coli, and B. cereus. In addition, to better understand the performance of the supramolecular assembly, we analyzed the microstructure and bioactivity of the produced cryogels.

2. Materials and Methods

Pink pepper (Schinus terebinthifolius Raddi) essential oil extract from fruits was obtained from Ferquima (Vargem Grande Paulista, São Paulo, Brazil). Powdered egg albumin (ALB) was purchased from Neovita Foods Eirelli (São Paulo, São Paulo, Brazil; 83.3% protein, 5.0% carbohydrates, and 0.0% fat). Amidated low-methoxyl pectin (PEC) was purchased from Danisco (GRINDSTED LA 210; 34% esterification degree and 17% amidation, Barueri, São Paulo, Brazil). Sodium hydroxide, calcium chloride, and hydrochloric acid were obtained from Panreac (Química, S.A, Castellar de Vallès, Barcelona, Spain). Mueller–Hinton agar (HiMedia, Sumaré, São Paulo, Brazil). Ethanol (95%, vol/vol) was supplied by Synth (Diadema, São Paulo, Brazil). All reagents were analytical grade.

2.1. Encapsulation of Pink Pepper Essential Oil in Cryogels

The encapsulation of pink pepper essential oil was carried out according to the methodology proposed by Chaux-Gutiérrez et al. [33] and Volić et al. [28] with some modifications. Individual dispersions of ALB (5% w/w, based on the total mass of the dispersion) and PEC (5% w/w, based on the total mass of the dispersion) were prepared at room temperature, maintaining constant stirring for 3 h until complete dispersion using a magnetic stirrer. For preparing ALB hydrogels, the ALB dispersion was adjusted to pH 8 using HCl (0.1 M) and NaOH (0.1 M) solutions, heated to 85 °C, kept under constant stirring for 15 min, and cooled to 40 °C. Then, 3% of pink pepper essential oil (% w/w, based on the total mass of the dispersion) was dispersed into the ALB hydrogel at 14000 rpm for 5 min (Ultra-Turrax^® IKA T25, IKA-Werke GmbH, Staufen, Baden-Württemberg, Germany). Finally, the mixture was stored in a Petri dish at 4 °C until gel formation. For preparing ALB-PEC gels, ALB and PEC dispersions were first prepared as described above, and then mixed at different ALB:PEC ratios—70:30, 50:50, and 30:70—maintaining constant stirring for 5 min. After that, 3% of the pink pepper essential oil (% w/w, based on the total mass of the dispersion) was added, and the mixture was homogenized at 14,000 rpm for 5 min (Ultra-Turrax^® IKA T25, IKA-Werke GmbH, Staufen, Baden-Württemberg Bodense, Germany). Then, 2% (w/w) calcium chloride solution (prepared previously at 2% w/v) was incorporated, maintaining constant stirring for 10 min. The mixture was stored in a Petri dish at 4 °C until gel formation. Finally, the gels were frozen at −18 °C for 24 h and then freeze-dried (model L-101, Liotop, São Carlos, São Paulo, Brazil) at 40 μmHg for 48 h. The lyophilized samples were stored in metalized bags within desiccators at 25 °C. (Figure 1).

2.2. Encapsulation Efficiency (EE%)

The encapsulation efficiency of the cryogels was determined according to the methodology of Abreu et al. [34] with some modifications. One hundred milligrams of cryogels was dispersed in 5 mL of ethanol. The mixture was centrifuged at 9000 rpm for 5 min at 25 °C using a centrifugal separation (Z 326 K, HERMLE Labortechnik GmbH, WehinGen, Baden-Würtemberg, Germany). The pink pepper essential oil content was measured using a UV-vis spectrophotometer (SP-200, Biospectro, Curitiba, Paraná, Brazil) at a wavelength of 291 nm, using a calibration curve (y = 0.7269x + 0.223, R² = 0.9949) of essential oil in ethanol solution (0.2–1.2 mg/mL). The encapsulation efficiency (EE%) was calculated according to Equation (1):

$$EE\left(\%\right)={\displaystyle \frac{M}{M_0}}\times 100$$

(1)

where M is the amount (mg) of oil in loaded cryogels and M_o is the initial oil amount (mg) added to cryogel preparation.

2.3. Total Phenolic Content

The phenolic content of the cryogels was determined using the Folin–Ciocalteu method [35]. Extracts were prepared using methanol, and the absorbance was read at 725 nm in a UV/VIS spectrophotometer (SP-200, Biospectro, Curitiba, Paraná, Brazil). Quantification was performed using a calibration curve of aqueous solutions of gallic acid (0–500 ppm). The results were expressed in mg of gallic acid equivalent per gram (mg GAE/g).

2.4. Antioxidant Activity

The antioxidant activity was determined by radical ABTS*⁺ capture methodology, according to a method proposed by Re et al. [36]. Extracts were prepared using methanol and the absorbance was measured at 730 nm (SP-200, Biospectro, Brazil). The antioxidant activity was determined using a calibration curve prepared with an aqueous solution of Trolox (0–200 μmol/L) and expressed as micromoles of Trolox equivalents (TEs) per gram (μmol TEs/g).

2.5. In Vitro Antimicrobial Activity

Evaluation of the inhibitory effects of essential oil encapsulated in cryogels on selected bacteria was performed in vitro using the agar diffusion method according to the Clinical and Laboratory Standards Institute [37], with some adaptations. Three bacteria were selected: two Gram-positive (Staphylococcus aureus ATCC6538 and Bacillus cereus ATCC11778), and one Gram-negative (Escherichia coli ATCC8739). Suspensions were prepared for each of the bacteria at a concentration of 10⁸ CFU/mL. The culture medium used in the Petri dishes for microbial strains was MMA Growth Agar. In the inoculated agar, wells of 7 mm diameter were made using a sterile metal cylinder; subsequently, these were filled with different amounts of the cryogels (1.25, 2.5, 5, 10, 20, and 30 mg). Cryogel without essential oil was used as blank. The inoculated material was incubated at 37 °C for 24 h. After incubation, inhibition zone diameters were measured in millimeters.

2.6. Fourier Transform Infrared Spectrometry (FT-IR)

FT-IR spectra of cryogels were recorded using a Spectrum One (Perkin-Elmer Corp., Shelton, CT, USA) device with an attenuated total reflectance accessory with a ZnSe crystal. Cryogels were ground into a fine powder using mortar and pestle. Then, samples were analyzed directly after pressing them on the crystal (80 psi). The scanning was conducted from 4000 to 400 cm⁻¹ using 20 scans, with a resolution of 4 cm⁻¹ [33].

2.7. X-ray Diffraction

The X-ray diffraction (XRD) patterns of cryogels were determined using a RINT 2000 diffractometer (Rigaku, Tokio, Japan) equipped with a Cu Kα radiation source (λ = 1.542 Å). Operating conditions were 45 kV and 30 mA. Data were collected from 5° and 50° (2θ), with step sizes of 0.02° and a scan rate of 1 s per step. Before analysis, cryogel samples were ground into a fine powder using a mortar and pestle.

2.8. Thermal Analysis

Thermogravimetric analysis (TGA) (SDT Q600 Thermogravimetric Analyzers, TA Instrument, New Castle, DE, USA) was used to estimate the thermal stability of cryogels. Each 5 mg sample was placed in the platinum pan and heated up by a TGA furnace at 10 °C/min under a nitrogen atmosphere from 25 to 200 °C. Differential scanning calorimetry was carried out using a calorimeter (DSC-25, TA Instrument, New Castle, DE, USA) previously calibrated with indium. An empty aluminum pan was used as a reference. Each 3 mg sample was weighed, cooled from 25 to −50 °C at 35 °C/min, maintained at this temperature for 1 min, and subsequently heated at 35 °C/min from −50 to 200 °C.

2.9. Scanning Electron Microscopy (SEM)

The morphology of the cryogels was examined using scanning electron microscopy (SEM, EVO LS15, Zeiss, Carl Zeiss, Ostalbkreis, Baden-Württerberg, Germany) at 20 kV. Samples were prepared by mounting cryogels on double-sided carbon tape and coating them with a thin layer of gold using a sputter coater (Quorum, model Q150 T, Lewes, UK) for 1.5 min. Micrographs were acquired from both the surface and the cross-section of cryogels.

2.10. Statistical Analysis

All measurements were performed in triplicate and results were expressed as mean ± standard deviation. Statistical analyses were performed with one-way analysis of variance (ANOVA) using the Minitab 21^® program, considering a significance level of 5%. Tukey’s test was used to compare differences among the mean values of samples.

3. Results and Discussion

3.1. Encapsulation Efficiency (EE%)

Encapsulation efficiency is crucial for assessing the potential of ALB and PEC as suitable materials for encapsulating pink pepper essential oil. Higher efficiency indicates a better encapsulation process. It is also considered a measure of the essential oil that could reach its target site. However, this depends on wall structural characteristics and its affinity for the essential oil. The encapsulation efficiency of the cryogels with varying ALB:PEC ratios ranged between 59.6 and 77.4% (

Table 1. Encapsulation efficiency, total phenolic content, and antioxidant activity of pink pepper essential oil encapsulated in ALB and ALB: PEC cryogels.

Sample	EE (%)	Total Phenolic Content (mg GAE/g)	ABTS (μmol TEs/g)
ALB	75.0 ± 5.5 a	2.80 ± 0.23 a	6.28 ± 0.49 a
70:30 ALB:PEC	75.2 ± 4.9 a	2.31 ± 0.18 b	1.16 ± 0.16 b
50:50 ALB:PEC	77.4 ± 1.9 a	1.41 ± 0.11 c	0.84 ± 0.05 b
30:70 ALB:PEC	59.6 ± 2.3 b	0.82 ± 0.17 d	0.99 ± 0.24 b

Results expressed as mean (n = 3) ± standard deviation. Different lowercase letters in the column indicate significant differences between treatments. GAE: gallic acid equivalent. TEs: Trolox equivalents.

), being almost constant for the cryogels with higher contents of ALB (pure ALB, 70:30 and 50:50 ALB:PEC). However, an increase in the PEC concentration, as in the 30:70 (ALB:PEC) ratio, led to a decrease in efficiency. This suggests a limited concentration for pectin, beyond which the EE% decreases. These results are attributed to the superior ability of proteins to form films and trap oil compared to polysaccharides [38]. Studies on oregano essential oil (Origanum vulgare Linneus) encapsulation exemplify this effect: a 79% encapsulation efficiency was achieved using zein at 0.2% (w/v) in the wall material [21]. In contrast, chitosan at 1% (w/v) resulted in a lower efficiency of 24.72% [39]. Similarly, increasing the protein concentration in systems composed of whey protein isolate (WPI) and carboxymethylcellulose (CMC) for encapsulation of orange essential oil leads to higher efficiency: the encapsulation efficiency increased from 3% in the 1:1 (WPI:CMC) ratio to 86% in the 3:1 (WPI:CMC) [40]. Bastos et al. [41] reported a similar trend when encapsulating black pepper essential oil (Piper nigrum L) using a wall material composed of gelatin and alginate: a ratio of 0.3:0.05 (gelatin: alginate) resulted in an efficiency of 52.26%, while increasing the protein content to a ratio of 0.9:0.15 (gelatin:alginate) increased efficiency to 82.20%. The encapsulation efficiency obtained in the present study is within a similar range to those reported by Chen and Zhong [42] and Rajkumar et al. [43] for encapsulation of mint essential oil in chitosan nanoparticles, for which they achieved an efficiency of 64%.

3.2. Total Phenolic Compound Content and Antioxidant Activity

Phenolic compounds, abundant secondary metabolites in plants and recognized for their antioxidant properties, include flavonoids, phenolic acids, and terpenes such as carvacrol, eugenol, and p-cinene [44,45]. This study examined the influence of wall material composition on total phenolic content and their antioxidant activity in cryogels prepared with either ALB or ALB:PEC. Phenolic content ranged from 2.80 to 0.82 mg GAE/g (Table 1), with higher values observed in the cryogels with greater ALB content. This result could be related to protein denaturation during the production of the cryogels. Thermal treatment enhances protein–polyphenol interactions via hydrogen bonding [46]. Unfolded polypeptide chains in the heat-treated protein expose hydrophobic amino acid residues, creating a hydrophobic site for entrapped phenolic compounds within the three-dimensional network [47]. Conversely, the negatively charged PEC electrostatically interacts with positively charged sites on ALB [29], altering protein–phenolic compound interaction and reducing encapsulation efficiency; this explains the decreasing total phenolic content observed in cryogels with increasing PEC ratios (70:30, 50:50, 30:70 ALB:PEC). Similarly, Volić et al. [28] reported greater encapsulation efficiency (80% of the total phenolics) for thyme essential oil in a 1:1.5 (% w/w) alginate:soy protein blend compared to alginate alone (72% of the total phenolics). On the other hand, the antioxidant activity did not show significant differences in cryogels prepared with PEC (p < 0.05). It was observed that the presence of PEC in the wall material, even at the lower level, decreased the encapsulation of phenolic compounds contained in the essential oil, which then contributed to a decrease in the antioxidant activity, ranging from 6.28 to 0.99 μmol TEs/g (Table 1). Authors such as Arslan and Çelik [48] report that the antioxidant activity of Salvia cidronella Boiss essential oil is associated with its phenolic compound content.

3.3. Antimicrobial Activity

Pink pepper oil has known antimicrobial properties due to the presence of monoterpenes such as α-pinene, D-Limonene, α-phellandrene, γ-terpene, and p-cymene, and sesquiterpenes such as caryophyllene, germacrene-D, and α-Muurolene that can damage microbial cell walls [49,50]. In the present study, the antimicrobial activity of the proposed encapsulation system was evaluated by measuring inhibition zones formed around cryogels in contact with gram-positive and gram-negative bacteria cultures [37]. The inhibition zone size reflects the amount of essential oil diffusing through the cryogel and inhibiting microbial growth. The results showed that none of the cryogels were able to inhibit B. cereus or E. coli growth even at the higher amounts applied. The resistance of E. coli (gram-negative) cells is due to the envelope; the complex structure, including the lipopolysaccharide membrane, hinders the penetration of monoterpenes like D-limonene and γ-terpinene found in pink pepper oil [50,51]. The B. cereus, an endospore-forming bacterium, offers increased resistance to monoterpenes found in pink pepper essential oil [52]. However, Dannenberg et al. [53] reported pink pepper essential oil inhibiting B. cereus, suggesting variations in essential oil composition. Encapsulated pink pepper oil inhibited the growth of S. aureus, but this effect depended on the mass of cryogels used. The sensitivity of S. aureus is likely due to its cell wall, which is around 90% peptidoglycan. This composition makes it less resistant to hydrophobic molecules, allowing them to destabilize the cell wall and cytoplasm [51]. Phenolic compounds in the essential oil can further alter the bacteria cell membrane by binding to proteins and disrupting their function [52]. The study also found a relationship between the concentration of ALB and PEC in the cryogel wall material and the size of the inhibition zone (Figure 2 and

Table 2. Inhibitory activity (agar disk diffusion) against S. aureus (ATCC 6538) of pink pepper essential oil encapsulated in ALB and ALB:PEC cryogels.

	Inhibition Zone (mm)
Mass Cryogel (mg)	ALB	70:30 ALB:PEC	50:50 ALB:PEC	30:70 ALB:PEC
1.25	n.d	n.d	n.d	n.d
2.5	n.d	n.d	n.d	n.d
5	n.d	n.d	n.d	9.33 ± 1.12 d
10	0.50 ± 0.43 ef	n.d	9.44 ± 0.88 d	12.44 ± 3.54 c
20	1.33 ± 0.50 ef	1.89 ± 0.60 ef	15.56 ± 1.94 b	17.22 ± 2.59 ab
30	1.17 ± 0.41 ef	2.44 ± 0.73 e	18.22 ± 1.39 a	18.67 ± 2.83 a

Results expressed as mean (n = 6) ± standard deviation. Different lowercase letters in the row indicate significant differences between treatments. n.d = not detected.

). Cryogels with a 30:70 (ALB:PEC) ratio required the least mass (only 5 mg) to show inhibition. Conversely, cryogels with 50:50 (ALB:PEC) ratio, ALB alone, and 70:30 (ALB:PEC) required a minimum of 10 and 20 mg, respectively. Interestingly, although the cryogels prepared with ALB alone and with 70:30 and 50:50 (ALB:PEC) ratios had a higher encapsulation efficiency (Table 1), their required mass for the same inhibitory effect was higher, suggesting that some of the essential oil was unavailable as an antimicrobial agent, possibly due to interaction with the unfolded protein in the cryogel wall. Evans et al. [38] suggested that these interactions correspond to hydrophobic bonds. A higher pectin ratio in the cryogel wall material led to larger pores, as observed in the SEM analysis (as discussed in Section 3.7), which facilitated the release and diffusion of the encapsulated oil. The cryogel with a 30:70 (ALB:PEC) ratio showed a significant increase in the inhibition zone (from 9.33 mm to 18.67 mm) when the mass increased from 5 mg to 30 mg, compared to cryogels with only ALB or a 70:30 (ALB:PEC) ratio. These results align with previous findings of S. aureus inhibition by pink pepper oil encapsulated in soy protein isolate/high methoxyl pectin [4]. These results suggest that amidated low methoxyl pectin is crucial in designing a controlled release system for pink pepper oil by acting as the release trigger.

3.4. FT-IR Spectroscopy

The Fourier-transform infrared (FTIR) spectra of the cryogels loaded with essential oil revealed characteristic peaks from the stretching of the C–H bond at 2934 and 2872 cm⁻¹, typically associated with alkenes; peaks between 1440 and 1447 cm⁻¹, indicative of C–H and C–O stretching; as well as a peak at 884 cm⁻¹ associated with the C–H out-of-plane bending vibration due to the presence of monoterpenes present in essential oil (Figure 3) [54,55]. The intensity of the peak at 3275 cm⁻¹, assigned to O-H stretching corresponding to the stretching of the O–H hydroxyl group [56], decreased with increasing PEC concentration in the 50:50 and 30:70 (ALB:PEC) cryogels, suggesting a more significant interaction between the ALB and PEC components in the wall material; this could explain the lower encapsulation efficiency observed for these formulations. Conversely, in the ALB and 70:30 (ALB:PEC) cryogels, the absorption bands at 1600 cm⁻¹ corresponding to the amide I group, which represents the secondary structure of the protein, were shifted to lower wavenumber. This shift, associated with the stretching vibration of C-N bonds [57] in the cryogels, suggests an interaction between polyphenols and protein, as evidenced by the higher phenolic compounds content in these cryogels.

3.5. X-ray Diffraction

Figure 4 shows the X-ray diffraction (XRD) of the cryogels. All cryogels showed characteristic diffraction patterns of semicrystalline structures. Compared to pure albumin, the disappearance of the broad peak at 9° in the cryogels indicates alterations to the secondary structure of the protein [58]. The intensity of the peaks increased with the presence of PEC, due to the more crystalline nature of this polysaccharide (Figure 4f). This behavior resembles that observed in studies of edible films produced with egg albumin protein (5% w/v) and pectin (5% w/v) [59]. However, the characteristic diffraction pattern of pure PEC (Figure 4f) practically disappears in ALB:PEC cryogels due to interactions between the unfolded protein chains and the polysaccharide, which alters the crystalline regions of the PEC and decreases the crystallinity of the sample, as was also observed by Mebarki et al. [60], who found that the casein–pectin interaction leads to a decrease in crystallinity. This reduction in crystallinity is generally advantageous for encapsulation systems, as it can improve bioactive compound release and bioavailability, according to Chen et al. [58] and Ghobadi et al. [61].

3.6. Thermal Analysis

Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were employed to investigate the thermal behavior of the cryogels. These techniques provide valuable insights into the material thermal stability and potential first-order or second-order transitions [62]. TGA analysis showed distinct mass loss patterns between cryogels (Figure 5a). The cryogel containing only albumin (ALB) exhibited the lowest overall mass loss (7.66%) and the most excellent thermal stability above 80 °C. Conversely, cryogels formulated with ALB and PEC displayed higher mass losses of 11.5% (70:30), 12.4% (50:50), and 12.5% (30:70). The derivative TGA curves indicated a single significant mass loss event for the ALB-only cryogel. In contrast, PEC-containing cryogels showed multiple events, suggesting a more complex degradation process (Figure 5b). The analysis of DTG curves (the first derivative of TGA) revealed distinct mass loss patterns for the cryogels (Figure 5b). The cryogel with only ALB exhibited the highest mass loss rate within the 40–70 °C temperature range, followed by cryogels with ALB:PEC ratios of 50:50, 70:30, and 30:70, respectively. Notably, the 70:30 ALB:PEC cryogel displayed two distinct peaks at 59.13 °C and 154.87 °C, while the others showed three events at 54.59 °C, 101.82 °C, and 151.48 °C for the ratio 50:50, and 53.84 °C, 122.39 °C, and 148.80 °C for the 30:70 ratio (ALB:PEC). The presence of PEC influenced the degradation profile. Cryogels with PEC exhibited lower mass loss rates between 20 and 80 °C than the ALB-only cryogel, indicating enhanced stability in this range. However, ALB provided superior thermal stability at higher temperatures (>80 °C). Finally, all PEC-containing cryogels underwent degradation between 170 and 190 °C, with higher PEC content correlating to a lower degradation temperature (176.6 °C); this suggests the depolymerization and decomposition of PEC units within the cryogel matrix, as previously reported by Liu et al. [63].

Table 3. Thermal behavior of the ALB and ALB: PEC cryogels loaded with pink pepper essential oil.

Sample	Tpeak onset (°C)	Tpeak (°C)	Tpeak endset (°C)	ΔH (W/g)
ALB	79.58	137.16	193.05	62.83
70:30	51.18	134.15	169.81	70.96
50:50	110.7	159.54	193.05	29.52
30:70	114.89	168.69	193.05	56.43

shows the T_{peak onset}, T_{peak endset} values, and enthalpy of cryogels. The thermograms (Figure 5c,d) for cryogels prepared only with ALB and with a 70:30 ALB:PEC ratio revealed a single endothermic event at 137.16 °C and 134.15 °C, respectively, attributed to the formation of agglomerates during cryogel preparation via intermolecular hydrophobic and hydrogen-bonding interactions between unfolded ALB chains (ALB-ALB and ALB-PEC). As Jacob et al. [64] suggested, these intermolecular interactions are essential in stabilizing the denatured protein. These structures exhibit high fusion temperatures and fusion enthalpies (Table 3) and enhanced thermal stability. In cryogels prepared with ALB:PEC ratios of 50:50 and 30:70, peaks at 159.54 °C and 168.69 °C, respectively, were observed. This peak corresponds to the thermal degradation of the wall material rather than protein unfolding, suggesting the disruptive effect of PEC on protein assembly within the cryogel matrix.

3.7. Scanning Electron Microscopy

Figure 6 shows the cross-sectional micrographs of the cryogels containing pink pepper essential oil. The microparticles exhibit irregular morphology and varied sizes. The cryogel only with ALB displays a dense, heterogeneous structure (Figure 6(a1,a2)) with essential oil droplets on its surface (Figure 6(a4)). As the proportion of PEC increases, the cryogels develop a more porous structure, entrapping essential oil particles within their cavities (Figure 6(c3,d3)). These structural changes correspond to the essential oil release profiles, particularly in the 50:50 and 30:70 ALB:PEC cryogels, which exhibited enhanced antimicrobial activity against S. aureus. The soy protein isolate–pectin encapsulation system demonstrates increased porosity due to pectin presence, enhancing the release of encapsulated bioactive compounds [65,66].

4. Conclusions

ALB and ALB:PEC cryogels are suitable matrices for encapsulating the essential oil of pink pepper (Schinus terebinthifolius Raddi) and demonstrated antimicrobial activity primarily against S. aureus. The inhibition zone size was influenced by both cryogel composition and mass, with higher pectin content correlating to larger inhibition zones. The pectin plays a crucial role in oil release and diffusion. PEC acts as a trigger in the proposed encapsulation system. Its concentration in the wall material significantly influences the encapsulation efficiency and total phenolic compound protection. More importantly, it can be harnessed to control the release of the essential oil, thereby enhancing its inhibitory effects against S. aureus. The interaction between ALB, PEC, and essential oil alters the wall material, thermal properties, and morphology. The PEC introduced led to multiple degradation steps and a more complex degradation process, reducing the thermal stability and increasing its porosity. It is still necessary to establish the optimal concentrations of PEC in the proposed encapsulation system to determine the kinetics and release profile and to establish its use in food matrices as an alternative for food preservation.