Enzyme-Modified Soy Protein/Maltodextrin Microcapsules Embedding Cinnamaldehyde: Structure, Release Characteristics, and Antibacterial Activity

Abstract

Cinnamaldehyde (CIN) is a natural organic compound known for its antimicrobial and antioxidant properties. However, its susceptibility to environmental degradation has restricted its practical application. This study aimed to microencapsulate CIN using soy protein isolate hydrolysates and maltodextrin as wall materials through emulsion preparation and spray drying, and to characterize the microstructure, controlled-release properties, antibacterial efficacy, and preservation performance of the resulting microcapsules. Under optimized condition, the encapsulation efficiency reached 70.72%. The microcapsules displayed smooth spherical structures, improved thermal stability, and an average particle size of 291.01 ± 33.64 nm. They demonstrated enhanced storage stability and sustained-release characteristics. Furthermore, the microcapsules exhibited significant antibacterial and antioxidant activity, which effectively delayed lipid and protein oxidation in pork loin for up to 6 days. Collectively, the results confirm the successful encapsulation of CIN and indicate the strong potential of these microcapsules for food industry applications requiring preservative and controlled-release functions.

1. Introduction

Cinnamon is derived from the bark, roots, and leaves of Cinnamomum species and is valued for its flavor, aroma, and potential health benefits. Its bioactive ingredients include cinnamic acid, cinnamaldehyde (CIN), and various polyphenols. Among these, CIN is a chemically reactive active ingredient extracted from cinnamon essential oil, which is famous for its strong antimicrobial properties and edibility [1]. However, such bioactive compounds are inherently susceptible to environmental factors, such as light, heat, oxygen, and humidity. Encapsulation has been widely adopted as an effective technique to overcome these difficulties, protecting and delivering cinnamon bioactives while enhancing the efficient delivery of active ingredients in food applications.

Various encapsulation delivery carriers have been used to overcome the instability of sensitive substances, including liposomes, micelles, emulsions, nanoparticles, microcapsules, and others. Among these, microencapsulation helps retain the original color, flavor, and biological activity of the core material, while preventing nutrient degradation or loss [2]. In particular, proteins from diverse sources are widely used for microcapsule preparation due to their high biocompatibility, amphiphilic properties, and non-toxicity. Soybean isolate protein (SPI), a by-product of the soybean oil industry, exhibits excellent emulsifying and gelling properties. As a plant-based protein, it is promising due to biodegradability, high availability, low cost, and long-term stability, making it a potential wall material for microencapsulation [3].

However, SPI exhibits low antioxidant capacity in emulsion systems. Numerous studies have reported that enzymatic hydrolysis can improve its functional properties by reducing molecular weight, unfolding the protein structure, enhancing flexibility, exposing hydrophobic groups, and releasing antioxidant peptides, thereby boosting both antioxidant performance and adsorption capacity at the oil-water interface [4]. Meanwhile, moderate enzymatic hydrolysis of soy protein not only regulates the encapsulation efficiency of CIN within the wall material but also enables sustained release of CIN through diverse structural configurations. Although enzymatically hydrolyzed SPI addresses certain limitations of native proteins as wall materials, single-protein-based wall materials still suffer from drawbacks such as poor solubility, high viscosity, and compromised processability and emulsifying activity.

Microcapsules with enhanced stability and improved structural properties can be prepared by complexing proteins with other natural molecules, such polysaccharides and polyphenols. Maltodextrin (MD) is a starch hydrolysate commonly used for microencapsulation of food ingredients. The incorporation of polysaccharides facilitates the formation of a secondary protective layer deposited over the primary protein layer, thus enhancing the spatial component of inter-droplet repulsive forces [5]. MD offers advantages including favorable solubility, neutral aroma and flavor, low viscosity at high concentrations, and ideal antioxidant stability for the core material. However, it exhibits poor emulsifying capacity and low lipid retention. Therefore, MD is typically used in combination with additional wall materials, such as proteins, to improve the encapsulation performance of microcapsule.

In summary, CIN is prone to oxidation, insoluble in water, and highly volatile. These limitations significantly hinder its broad application in the food industry. Consequently, there is an imperative need to develop effective methods to inhibit its oxidation, enhance its controlled-release preservation efficacy, and improve its processability. This study designed CIN-loaded microcapsules using spray drying, an efficient encapsulation technique employing enzymatically hydrolyzed SPI and MD as wall materials. The microstructure, antibacterial activity, and fresh-keeping performance of the microcapsules were evaluated, along with key physical properties of the microcapsule powder. This study innovatively employs enzymatically modified protein-polysaccharide wall materials and establishes an experimental foundation for the sustained application of CIN functionality, thereby supporting future development of novel bioactive compound controlled-release systems.

2. Materials and Methods

2.1. Materials and Regents

Food-grade SPI was obtained from Linyi Shansong Biological Products Co., Ltd. (Linyi, China). Papain (800 U/mg) was purchased from Yuanye Co., Ltd. (Shanghai, China). MD was acquired from Shandong Xiwang Sugar Industry Co., Ltd. (Binzhou, China). Disodium tetraborate decahydrate was kindly provided by Sigma Chemical Co. (St. Louis, MO, USA). DPPH (2,2-diphenyl-1-picrylhydrazyl) was sourced from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). CIN purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). All other materials and regents used were listed in the Supplementary Materials.

2.2. Preparation of Enzyme-Modified Soy Protein

The preparation of soy protein isolate hydrolysates (SPIHs) was adapted from a previous method [6] with modifications. SPI (5% w/v) was dispersed in deionized water and stirred at room temperature for 2 h to ensure complete hydration. Under optimal enzymatic hydrolysis conditions (50 °C, pH 7.5), papain (800 U/mg) was added at an enzyme-to-substrate (E/S) ratio of 0.2% to initiate hydrolysis. Aliquots were collected at 0, 20, 40, 60, 90, 120, and 180 min, then the reaction was immediately terminated by heating at 100 °C for 5 min to inactivate the enzyme. After cooling to room temperature (25 °C), the hydrolysate was centrifuged at 5000 rpm for 20 min. The supernatant was collected and freeze-dried for subsequent use.

2.2.1. Degree of Hydrolysis

The degree of hydrolysis (DH) of SPI was measured using the o-Phthalaldehyde (OPA) method [7]. The OPA reagent was prepared by dissolving 3.81 g of sodium tetraborate and 0.1 g sodium dodecyl sulfate in 80 mL deionized water, followed by the addition of OPA (160 mg dissolved in 4 mL ethanol) and dithiothreitol (176 mg dissolved in 1 mL deionized water). The final volume was adjusted to 100 mL with deionized water. The hydrolysate samples were diluted to 2 g/L with deionized water. 200 μL of the hydrolysate was mixed with 1.5 mL of OPA reagent and allowed to react for 2 min. The absorbance at 340 nm was recorded as OD_sample using a UV spectrophotometer (UV-2100, Purkinje General Instrument Co., Ltd., Beijing, China). A standard reaction was performed using 200 μL of serine standard solution (0.1 mg/mL) mixed with 1.5 mL OPA reagent for 5 s, and the absorbance was recorded as OD_standard.

$$DH \left(\%\right)={\displaystyle \frac{h}{h_{\mathrm{tot}}}}$$

(1)

$$h={\displaystyle \frac{\mathit{Serine} N{H}_2-\beta }{\alpha }}$$

(2)

$$\mathit{Serine} N{H}_2={\displaystyle \frac{{\mathit{OD}}_{\mathit{sample}}-{\mathit{OD}}_{\mathit{blank}}}{{\mathit{OD}}_{\mathit{standard}} -{ \mathit{OD}}_{\mathit{blank}}}} \times 0.9515 \times 0.1 \times {\displaystyle \frac{1}{X}} \times P$$

(3)

where, h_tot = 7.8 (mmol/g protein) for SPI; α = 0.970; β = 0.342; X is the sample (g); P is the protein content in sample (%).

2.2.2. DPPH Radical Scavenging Activity

The antioxidant activity of SPIHs prepared at different hydrolysis times was evaluated using a modified DPPH radical scavenging assay [8].

A mixture of 4 mL of DPPH reagent (0.1 mmol/L in 95% methanol) and 1 mL of protein sample solution (1 mg/mL) was incubated in the dark for 30 min. The absorbance was measured at 517 nm using a microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Glutathione was used as the positive control.

The DPPH radical scavenging activity was calculated using the following formula:

$$\mathit{DPPH} \mathit{radical} \mathit{scavenging} \mathit{capacity} \left(\%\right) = {\displaystyle \frac{A_{\mathit{blank}} - \left(A_{\mathit{sample}} - A_{\mathit{control}}\right)}{A_{\mathit{blank}}}} \times 100$$

(4)

where, A_blank is the absorbance of solution without a tested sample; A_sample is the absorbance od the tested sample; A_control is the absorbance of the sample solution itself.

2.3. Preparation of Microcapsules

SPIHs that exhibited the strongest antioxidant activity (obtained on the optimal enzymatic hydrolysis time) were selected as one of the wall materials for microencapsulation. The wall solution was prepared by mixing varying ratios of SPIHs and MD in deionized water with constant stirring for 2 h, followed by overnight hydration at 4 °C to ensure complete dissolution. Under vigorous stirring at room temperature, CIN was added dropwise into the wall solution, and the mixture was further stirred for 1 h. A primary emulsion was obtained by homogenizing the mixture at 10,000 rpm for 5 min. Subsequently, a secondary emulsification was performed using a high-pressure homogenizer (KS-1800L, KBS Nanotechnology Co., Ltd., Shanghai, China). Finally, the microcapsules were prepared via spray drying (BUCHI Labortechnik AG, Flawil, Switzerland).

2.4. Characterization of Microcapsules

The encapsulation efficiency, particle size, thermal gravimetric characteristic, surface morphology, crystalline structures, Fourier transform infrared (FTIR) spectra, thermogravimetric analysis (TGA), and storage stability of the microcapsules were measured. The specific methods were provided in the Supplementary Materials.

2.5. Antibacterial Properties

2.5.1. Determination of the Inhibition Zone

The antibacterial activity of microcapsules was evaluated against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) following established methodology [9]. Bacterial strains were activated and subcultured on Luria Bertani (LB) agar plates and inoculated into liquid medium. Single colonies were inoculated into liquid medium and incubated overnight at 37 °C with shaking to reach mid-logarithmic phase. A 100 μL aliquot of bacterial suspension (10⁸ CFU/mL) was evenly spread onto agar plates. After absorption, wells (6 mm diameter) were punched into the agar and filled with microcapsule powder. Plates were inverted and incubated at 37 °C for 24 h. Antibacterial activity was quantified by measuring inhibition zone diameters (mm).

2.5.2. Determination of Sustained Release of Antibacterial Activity from Microcapsules

Due to the strong volatility of CIN, it can exhibit spatial inhibitory effects on bacteria through the vapor phase. Using vapor fumigation, the persistence of initial antibacterial effects of CIN and SPIHs/MD/CIN against E. coli and S. aureus was evaluated over a 14-day period. Bacterial suspensions of both strains were adjusted to a concentration of 1 × 10⁵ CFU/mL and spread on nutrient agar plates, followed by uniform spreading using a spreader. A 0.2 g sample of microcapsules and an equivalent amount of free CIN (based on encapsulated amount) were placed on the inner lid of petri dishes [10]. Plates were sealed with sterile parafilm, inverted, and incubated at 37 °C. Observations were recorded photographically at specific time intervals.

2.6. Evaluation of Preservative Effect of Microcapsules on Fresh Pork

Fresh pork loin was used in this study, chilled to 0–4 °C within 12 h post-slaughter and maintained at this temperature range during transportation. Samples were prepared following the Chinese National Standard GB/T 9695.19-2008 [11]. Under laminar flow conditions, the loin was aseptically cut into appropriately sized pieces and placed in plastic petri dishes. Microcapsules were loaded into non-woven gauze bags and placed adjacent to pork samples. All samples were stored at 4 °C and sealed with plastic wrap. The entire process was conducted under sterile conditions within a laminar flow hood. All utensils were sterilized with 75% ethanol and ultraviolet-irradiated for 20 min prior to use.

Pork loin samples were recorded and analyzed for appearance, pH, texture profile analysis, drip loss, peroxide value, and the degree of secondary oxidation of lipids and fatty acids on days 0, 2, 4, 6, 8, 10, 12, and 14 days of storage. The detailed methods were introduced in the Supplementary Materials.

2.7. Statistical Analysis

All treatments were prepared in triplicate to ensure statistical reliability and reproducibility. The results were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was performed using SPSS 24.0 statistical software to determine significant differences among groups. A p < 0.05 was considered statistically significant.

3. Results and Discussion

3.1. Characterization of SPIHs

3.1.1. The Hydrolysis Degree of SPIHs

Representing the proportion of cleaved peptide bonds in protein hydrolysates, DH is extensively employed as a pivotal indicator to assess hydrolysate characteristics. As illustrated in Figure 1A, the DH of SPIHs demonstrated in a time-dependent manner during enzymatic hydrolysis. This trend can be attributed to the enzymatic properties of papain. As a cysteine endopeptidase, papain exhibits broad substrate specificity with preferential cleavage at lysine (Lys), arginine (Arg), and phenylalanine (Phe) residues, rendering it particularly efficient in enhancing the DH of protein [12]. Previous studies [13] have indicated that papain-treated proteins exhibit increased α-helix content, decreased β-sheet content, and elevated random coil content. These structural modifications contribute to enhanced molecular disorder and improved molecular flexibility. Consequently, the exposure of internal hydrophobic amino acids is facilitated, thereby increasing protein hydrophobicity. The gradual plateauing of the DH curve suggests a deceleration in the hydrolysis rate, possibly resulting from either progressive exhaustion of hydrolysable substrates or gradual diminution of enzymatic activity over time.

Figure 1. Changes in the DH of SPIHs (A) and DPPH radical scavenging activity of SPIHs (B) at different enzymatic hydrolysis times; FTIR spectra (C) and XRD patterns (D) of (a) SPIHs/MD/CIN microcapsules, (b) SPIHs/MD wall, (c) MD, (d) SPIHs, (e) CIN; TGA (E) and DTG (F) curves of (a) SPIHs/MD wall, (b) SPIHs/MD/CIN microcapsules, (c) CIN; DSC curves (G) of (a) SPIHs/MD/CIN microcapsules, (b) SPIHs/MD wall, (c) MD, (d) SPIHs.

3.1.2. Antioxidant Activity of SPIHs

DPPH is a stable free radical that can accept a hydrogen atom from antioxidant compounds to form a stable diamagnetic molecule. As illustrated in Figure 1B, the DPPH radical scavenging activity of SPIHs underwent time-dependent fluctuations throughout enzymatic hydrolysis. The maximum antioxidant activity (23.76 ± 1.11%) was observed at 120 min, substantially exceeding values obtained at other intervals and is consistent with the findings of previous studies [14]. The native (unhydrolyzed) protein sample exhibited relatively low antioxidant activity, while limited proteolysis markedly improved DPPH radical scavenging capacity, presumably through the generation of soluble bioactive peptides. Notably, the DPPH scavenging activity fluctuated during the 60–90 min hydrolysis period, potentially attributable to differential sensitivity of DPPH radicals to specific water-soluble peptide fractions generated during this phase [15]. Consequently, the SPIHs with the highest antioxidant activity (obtained at 120 min of enzymatic hydrolysis) were selected as one of the wall materials for subsequent microencapsulation.

3.2. Characterization of Microcapsules

3.2.1. Fourier Transform Infrared Spectroscopy

Figure 1C showed the peaks appearing at 2927–3493 cm⁻¹, which were primarily attributed to O-H bond stretching vibrations, C-H bond stretching vibrations, and residual water. A distinct absorption band observed at 1067.65 cm⁻¹ was identified as a characteristic of MD, corresponding to C-O-C stretching vibrations within the polysaccharide fingerprint region, which exhibited multiple peak features [16]. For SPIHs, absorption peaks at 1393.53 cm⁻¹, 1512.94 cm⁻¹, and 1647.06 cm⁻¹ were assigned to amide III, amide II, and amide I bands, respectively [17]. Broadening of the amide I band suggested partial enzymatic hydrolysis leading to shorter peptide chains and disrupted secondary protein structures. Comparative analysis confirmed that Maillard-induced covalent bonding between MD and SPIHs during microencapsulation altered the vibrational characteristics of the amide groups. Notably, CIN exhibited a distinct FTIR profile, with characteristic peaks at 1670.59 cm⁻¹ and 1621.18 cm⁻¹, assigned to C=O and C=C stretching vibrations, respectively. Additionally, weak doublet peaks at 2737.06 cm⁻¹ and 2824.71 cm⁻¹ were assigned to aldehyde C-H stretching vibrations. It could be observed through comparison that the peak intensities of the C=O and C-H bonds in the CIN aldehyde group weakened or disappeared after microencapsulation. This indirectly indicated that SPIHs undergo a Schiff base reaction with CIN, which differs from displacement phenomena involving non-covalent interactions. It enhanced the stability of CIN. Meanwhile, attenuated peak intensities suggested new hydrogen-bonding interactions between wall materials and CIN, while enhanced C-H stretching vibrations confirmed successful encapsulation of the hydrophobic core material. Collectively, the FTIR results demonstrate effective encapsulation of CIN within the SPIHs/MD wall matrix, supported by specific molecular interactions and spectral modifications.

3.2.2. X-Ray Diffraction

As shown in Figure 1D, the XRD pattern of MD exhibited a strong diffraction peak at 18.61°, while SPIHs displayed two broad and weak diffraction peaks at 19.94° and 9.38°. In comparison, the SPIHs/MD composite wall material showed a more intense diffraction peak at 19.34°. After encapsulation of CIN, the dominant diffraction peak shifted to 19.15° with a notable increase in intensity. The microcapsules also exhibited sharper peaks, narrower full-width at half-maximum, and shifts toward lower angles, indicating improved ordering and structural compactness, consistent with the previous report [9]. Amorphous microcapsules, which lack long-range molecular order, display inferior stability, with CIN oil showing pronounced volatility throughout storage. Such amorphous systems are also prone to moisture absorption, which leads to capsule swelling, sugar crystallization, and eventual agglomeration into larger aggregates [18]. Conversely, no significant phase transformation or new crystalline arrangements were observed after microencapsulation, suggesting that interaction between wall materials and CIN did not alter the fundamental structure. The microcapsules retained largely amorphous with minor crystalline inclusions, which confirmed structural preservation of the original components. Ultimately, these observations verify that CIN is effectively encapsulated within a tightly bound SPIHs/MD wall matrix, enhancing core stability and functional performance.

3.2.3. Thermogravimetric Analysis

Figure 1E,F present the TGA and derivative thermogravimetric (DTG) curves of the core material, wall material, and microcapsules over a temperature range of 30 °C to 550 °C. The DTG profiles illustrate the correlation between the rate of weight loss and temperature, where each discernible peak represented the onset and conclusion of a specific thermal degradation event. As indicated in Figure 1F, the temperatures corresponding to the maximum thermal degradation rates were 173.40 °C for the core material, 300.15 °C for the wall material, and 302.71 °C for the microcapsules.

The microcapsules underwent three distinct thermal decomposition stages. An initial mass loss of 18% occurred between 105 °C and 166 °C, during which the weight decreased sharply with increasing temperature. This stage was ascribed to the evaporation of residual surface moisture, the release of structurally bound water, and the volatilization of unencapsulated CIN. The second stage, from 166 °C to 243 °C, was characterized by a conspicuous and rapid decrease in mass, due to the initial decomposition of the wall material and the accelerated release of CIN. These processes collectively resulted in the structural degradation and eventual disruption of the microcapsules architecture. The third stage (243–359 °C) corresponded to the advanced decomposition and carbonization of residual components. CIN was entirely volatilized at approximately 200 °C. As reflected in the DTG curves, the latter two decomposition stages of both the wall material and microcapsules displayed notable concordance. Moreover, the mass loss rate of the microcapsules was significantly subdued compared to that of CIN, underscoring the role of microencapsulation in enhancing thermal stability. TGA further revealed that microencapsulation shifted the endothermic peak to higher temperatures, attributable to the protective effect of the wall material against thermal exposure. This phenomenon is similar to a previous finding [19]. In conclusion, the microencapsulated system exhibited superior thermal stability, as evidenced by the shift of mass loss to elevated temperatures. This improvement effectively delays the thermal degradation process and significantly enhances the stability of encapsulated CIN.

3.2.4. Particle Size, Polydispersity Index, and ζ-Potential of Microcapsules

As illustrated in Table 1, SPIHs, SPIHs/MD wall, and the resulting microcapsules exhibited distinct particle sizes, polydispersity index (PDI), and ζ-potential values. The formation of wall materials led to reduced particle size and increased ζ-potential. A similar trend was observed upon the incorporation of core material, i.e., during microencapsulation. The relatively large size of SPIHs facilitated extensive cross-linking and aggregation [20]. However, the introduction of core substance reduced the microcapsule size from 420.53 nm to 291.01 nm. This diminished dimension suggested significant advantages in terms of enhancing absorption and bioavailability of encapsulated compounds, especially for systems requiring either rapid or controlled-release. Research indicates that morphological changes in spray-dried microcapsules are associated with rapid water droplet evaporation and solute diffusion during atomization. These particles are prone to shrinkage during high-temperature drying or low-temperature cooling, forming ultimately contracted structural granules. Moreover, the microcapsules exhibited a normal particle size distribution with uniform and concentrated dispersion. PDI defines the breadth of microcapsule size distribution, with values between 0.01 and 0.5 indicating a narrow distribution and values exceeding 0.7 suggesting a wide range. Microcapsules with a PDI consistently above 0.7 demonstrate poor homogeneity, increasing the risk of aggregation and sedimentation, which adversely impacts product performance, stability during storage, and application [21]. A higher ζ-potential generally indicates stronger electrostatic repulsion between droplets, effectively preventing flocculation and coalescence. Although polysaccharides and proteins were combined, their like charges resulted in mutual electrostatic repulsion. In contrast, SPIHs/MD/CIN microcapsules possessed a moderate ζ-potential along with improved size uniformity, making them promising candidates for applications requiring consistent encapsulation and quantifiable controlled-release behavior.

Table 1. Physical properties of SPIHs/MD/CIN microcapsules.

Samples	Particle Size (nm)	Polydispersity	ζ-Potential (mV)
SPIHs	769.00 ± 18.99 a	0.35 ± 0.02 b	−44.41 ± 1.11 c
SPIHs/MD	420.53 ± 57.61 b	0.38 ± 0.06 b	−33.39 ± 1.18 b
SPIHs/MD/CIN	291.01 ± 33.64 c	0.59 ± 0.14 a	−5.90 ± 0.66 a

Each value represents the mean ± SD of three independent measurements. Lowercase letters indicate differences in the same indicator between different groups (p < 0.05).

3.2.5. Differential Scanning Calorimetry

The temperature measured during the glass-to-rubber transition (typically determined by the midpoint of the change in specific heat capacity) is called the glass transition temperature (T_g). Meanwhile, the peak degradation temperature (T_d) represents the endothermic degradation peak of the sample. As shown in Figure 1G, SPIHs sample exhibited a pronounced endothermic peak at approximately 139.65 °C, corresponding to its thermal denaturation. The MD sample displayed an endothermic transition around 130 °C, likely associated with its T_g. T_d and enthalpy of degradation (ΔH) for each wall material component and corresponding microcapsules were calculated from the thermograms. The SPIHs/MD wall material exhibited reduced T_d and ΔH (T_d = 145.82 °C, ΔH = −140.713 J/g) relative to the SPIHs/MD/CIN microcapsules (T_d = 114.35 °C, ΔH = 3.649 J/g). Furthermore, the interaction between MD and SPIHs shifted the endothermic peak to a higher temperature. This shift indicated that the protein-polysaccharide complexation enhanced thermal stability. This phenomenon can be primarily attributed to structural reorganization following SPIHs and MD complexation, along with synergistic attractions and hydrogen bonding between the two components. This finding aligns with previous reports that polysaccharide complexation improves protein thermal stability [22].

T_g represents the critical temperature at which the transition from a glassy to rubbery state occurs. The endothermic peak observed at 114.35 °C for the SPIHs/MD/CIN microcapsules is primarily ascribed to the volatilization of CIN triggered by wall matrix rupture, which also indicates the successful encapsulation of CIN. Furthermore, CIN demonstrated sustained release from the microcapsules, and its thermal stability was improved through encapsulation. These findings are consistent with previous studies [23]. The elevated thermal denaturation temperature of SPIHs/MD/CIN microcapsules clearly indicates that the microcapsules prepared by protein/polysaccharide complexation exhibited improved thermal stability. This enhancement is primarily attributed to strong interactions between the wall material and CIN, which enhanced thermal resistance. This observation aligns well with earlier research [22]. Importantly, these observations constitute compelling evidence of successful encapsulation. These findings provide critical insights for further optimization of microcapsule formulations and preparation processes.

3.2.6. Sustained Release Analysis

Volatilization generally refers to the physical phenomenon where a substance spontaneously changes from a liquid (or solid) state to a gaseous state at room temperature or under heating conditions, escaping from the sample system. The release of CIN is an example of volatilization. As shown in Figure 2, the release kinetics of CIN from SPIHs/MD/CIN microcapsules were monitored over a 10-day period at two storage temperatures (25 °C and 4 °C), with temperature identified as a critical factor influencing microcapsule stability. Following 10 days of storage at both 25 °C and 4 °C, cumulative CIN release increasing markedly. These findings align with previously reported behavior of CIN encapsulated within Pickering emulsions [24]. Further analysis of Figure 2 revealed a rapid decrease in CIN retention during the initial two days post-encapsulation. The early-stage loss resulted from the relatively facile release of CIN molecules located near the inner surface of the microcapsule wall, combined with potential oxidation of any unencapsulated core material. The subsequent deceleration in release kinetics may be explained by a diminished concentration gradient between the microcapsule interior and the external environment. CIN is susceptible to autoxidation, a two-stage process involving peroxide formation via oxygen attack on the unsaturated aldehyde group, followed by thermal decomposition of these peroxides [25]. The enhancement of CIN stability through microencapsulation primarily stems from the physical sequestration of CIN molecules from pro-oxidative factors in the environment.

Figure 2. Cumulative release kinetics of CIN at 4 °C and 25 °C over ten days: (A) Percentage cumulative release over time; (B) Zero-order model fit of cumulative release; (C) First-order kinetic model fit of cumulative release; (D) Ritger-Peppas model fit of cumulative release.

To understand the release mechanism of CIN, three kinetic models were employed to analyze the release curve. As shown in Figure 2, the release behavior of SPIHs/MD/CIN microcapsules followed the Ritger-Peppas kinetic model, with a release exponent of n ≤ 0.45 and R² values of 0.9880 and 0.9973, confirming Fickian diffusion as the dominant release mechanism. This approach encapsulates active CIN within a protective matrix, effectively shielding it from external factors to achieve controlled-release and maximizing its application potential across diverse food systems. However, significant limitations remain to be overcome. Specifically, the high raw material cost of SPIHs and the absence of industry standards for cinnamaldehyde content testing pose substantial challenges for industrial-scale conversion. Future efforts will focus on process optimization and scaled-up validation to achieve synergistic improvements in controlled-release performance and industrial applicability.

3.2.7. Scanning Electron Microscopy

As shown in Figure 3A, SPIHs exhibited an irregular, flake-like morphology, likely resulting from the disruption of rigid structures during hydrolysis. The porous architecture observed in SPIHs may originate from cavities formed by ice crystals or trapped air bubbles during freeze-drying. MD displayed irregular bulk structures (Figure 3B), while blank microcapsules based on SPIHs/MD blends showed larger aggregates (Figure 3C). CIN-loaded microcapsules presented a regular spherical morphology with superficially smooth surfaces with an overall rough texture (Figure 3D). Simultaneously, microcapsules exhibited the higher shrinkage and dense surface characteristics, resulting from droplet contraction caused by rapid moisture loss during the early stages of spray drying. According to numerous studies, the characteristic surface indentations of microcapsules primarily attributed to shrinkage induced by dehydration of the interfacial film assembled from protein and polysaccharide components during drying and cooling processes [26]. Furthermore, these surface indentations were caused by particle shrinkage during spray drying, which occurred at both high and low inlet temperatures. At low inlet temperatures slower water diffusion allowed more time for undergo shrinkage. Conversely, at high inlet temperatures, rapid internal moisture evaporation and associated high pressure within the particles also induced significant contraction. Surface defects such as cracks or fractures observed on the microcapsules could potentially facilitate oxidation or degradation of encapsulated core material. Most studies have demonstrated that microcapsules with different wall material compositions exhibit irregular spherical shapes of varying sizes, which is characteristic of spray-dried powders [27]. Finally, the adhesion of small particles on the microcapsule surface suggested incomplete encapsulation when SPIHs/MD were used as the wall material. The incorporation of protein hydrolysates improved morphological characteristics, yielding more spherical microcapsules distinct from those prepared with SPIHs or MD alone. Collectively, these structural observations confirm the successful preparation of microcapsules via the spray drying process.

Figure 3. Scanning electron micrographs of (A) SPIHs, (B) MD, (C) SPIHs/MD wall, (D) SPIHs/MD/CIN microcapsule.

3.3. Antibacterial Properties

3.3.1. Inhibition Zone Assay

The results are shown in Figure 4A and Table 2. The inhibition zones of CIN-loaded microcapsules against E. coli and S. aureus were 26.13 ± 5.18 mm and 31.51 ± 2.40 mm, respectively. As both diameters exceeded 25 mm, the microcapsules can be considered to possess significant antimicrobial activity [28]. This activity is mainly ascribed to the ability of CIN to interfere with enzymatic activity, particularly that of key enzymes involved in energy metabolism, leading to protein denaturation and altered permeability of microbial cell walls. Furthermore, CIN generates reactive oxygen species through redox reactions, disrupting intracellular homeostasis and ultimately causing content leakage and cell death.

Figure 4. Inhibitory zones (A) and sustained antimicrobial efficacy (B) of blank and CIN-loaded microcapsules against E. coli and S. aureus.

Table 2. Zone-of-inhibition assay demonstrating the antimicrobial efficacy of both loaded and blank microcapsules.

Microbial Strain	SPIHs/MD	SPIHs/MD/CIN
E. coli	8.63 ± 0.12 mm	26.13 ± 5.18 mm
S. aureus	7.42 ± 0.09 mm	31.51 ± 2.40 mm

In rows, data are presented as the mean ± SD (n = 3).

It is noteworthy that the antimicrobial activity against S. aureus is stronger than that against E. coli. This difference arises from the absence of an outer membrane in Gram-positive bacteria, whereas Gram-negative bacteria possess an outer membrane composed of polar terminals and lipopolysaccharides. These structural characteristic renders Gram-negative bacteria less susceptible to CIN cell wall-targeting action. As a result, the diffusion of CIN into Gram-negative bacterial cells is significantly restricted [29].

3.3.2. Sustained-Release Antibacterial Activity of Microcapsules

The study assessed whether encapsulating antimicrobial CIN enables SPIHs/MD/CIN microcapsules to maintain the persistence of initial microbial inhibition and thereby effectively prevent spoilage in prepared meat products. As depicted in Figure 4B, both free CIN and SPIHs/MD/CIN microcapsules exhibited the highest inhibitory activity against S. aureus and E. coli on the first day of storage, demonstrating the rapid initial antibacterial action of CIN. However, the antimicrobial effect of free CIN progressively declined over time, whereas the microencapsulated form effectively reduced CIN volatility and extended the persistence of this initial inhibitory effect. From day 10 onward, a pronounced reduction in the inhibitory efficacy of free CIN against E. coli was observed, a phenomenon attributable to the gradual evaporation of volatile CIN constituents. Comparative analysis further confirmed that free CIN exhibited superior activity against S. aureus to that against E. coli. In contrast, SPIHs/MD/CIN microcapsules maintained consistently high inhibition rates against both bacterial strains throughout the storage period. This stable maintenance of initial antimicrobial activity is attributed to the microencapsulation matrix and associated intermolecular interactions, which limited the diffusion of CIN, preserved its bioactive volatile components. These results are consistent with the findings of Yuan [10] and Yang [30], indicating that CIN suppresses amino acid decarboxylase activity via interactions with protein carbonyl groups. The sustained retention of initial antimicrobial activity of SPIHs/MD/CIN microcapsules highlights their potential application in food preservation systems, where controlled CIN release contributes to maintaining long-term freshness.

3.4. Evaluation of the Preservative Effect of Microcapsules on Frozen Pork

3.4.1. Appearance

As visually documented in Figure 5A, all experimental groups maintained relatively stable color during the first 4 days of refrigerated storage. Beyond this period, the blank control group demonstrated progressive quality deterioration characterized by substantial moisture loss, a color transition from bright red to deep reddish brown, and diminished surface lightness. These degenerative changes became particularly evident after 6 days, accompanied by noticeable darkening and surface wrinkling. Comparative analysis revealed that SPIHs/MD wall material delayed visual darkening by approximately 2 days compared to the blank control, suggesting a modest protective function. In contrast, the free CIN group showed limited practical efficacy, as the characteristic CIN aroma became undetectable after 4 days due to masking by spoilage-derived odors. Additionally, progressive CIN volatilization during storage resulted in simultaneous surface darkening and yellowish pigmentation. Both CIN formulations successfully extended the shelf life of pork, with the microencapsulated treatment demonstrating superior performance by delaying color deterioration by 6 days relative to the control. This result confirms that the controlled-release mechanism effectively preserved the desirable red coloration of pork throughout refrigerated storage.

Figure 5. Effects of different treatments on the appearance (A), pH (B), drip loss (C), PV (D), and TBARS (E) of pork loins. Lowercase letters indicate differences in the same indicator between different groups (p < 0.05).

3.4.2. pH Measurement

As shown in Figure 5B, the blank control group reached a pH of 6.76 within 6–8 days, exceeding the established spoilage threshold. Both microencapsulated SPIHs/MD/CIN and free CIN treatments-maintained pH below the spoilage threshold throughout the 14 days experimental period, demonstrating effective shelf-life extension. This result is consistent with previous study [31]. The gradual pH increases in chilled pork during storage primarily results from microbial and enzymatic degradation of muscle proteins, which leads to the accumulation of alkaline compounds including ammonia and amines. This biochemical process was consistent with existing literature on post-mortem muscle deterioration [32]. Notably, all experimental groups exhibited an initial pH decline during the first 2 days of storage, followed by a progressive alkaline shift. This biphasic pattern corresponds to characteristic biochemical transitions in post-mortem muscle: initial acidification from glycogenolysis and ATP degradation producing lactic and phosphoric acids, followed by alkalization from microbial production of nitrogenous compounds. Although the free CIN treatment showed superior pH stabilization during the initial 4 days compared to the microencapsulated group, its preservative efficacy diminished thereafter due to volatile losses of CIN. The subsequent pH elevation in the free CIN treatment group relative to the microencapsulated formulation demonstrates that the controlled-release mechanism effectively retards protein decomposition and microbial proliferation. These findings substantiate that microencapsulation significantly enhances the preservative efficacy of CIN in chilled pork loin preservation systems.

3.4.3. Texture Profile Analysis

As summarized in Table S3 in the Supplementary Materials, the hardness, cohesiveness, springiness, and resilience of pork loins progressively deteriorated across all treatments during storage. This decline is principally ascribed to structural weakening resulting from muscle protein hydrolysis, with reduced hardness further correlated with protein denaturation processes [33]. Microencapsulated CIN consistently preserved the hardness, cohesiveness, springiness, and resilience of chilled pork throughout the experimental period, as evidenced in Table S3 of the Supplementary Materials. These findings are consistent with previous reports by other researchers [34]. The alignment was attributed to the well-established mechanism by which plant essential oils modulate meat texture through the suppression of lipid and protein oxidation. The microencapsulated CIN consequently inhibited microbial proliferation in chilled pork, retarding spoilage progression and mitigating the rate of textural deterioration. The controlled-release kinetics of CIN from SPIHs/MD/CIN microcapsules mitigated oxidative damage to both lipids and proteins, thereby enhancing textural preservation and extending the shelf life of pork loins. This conclusion is supported by parallel investigations of pH evolution, drip loss, and peroxide value trends. Overall, the SPIHs/MD/CIN system effectively suppressed proteolytic activity and delayed textural degradation in refrigerated pork, demonstrating comprehensive preservation efficacy.

3.4.4. Drip Loss Determination

Analysis of the data presented in Figure 5C established that extended storage duration resulted in progressive microbial colonization within the pork surface microenvironment. This microbial proliferation consumed essential nutrients and induced gradual exudate release across all pork loin samples. The nutrient-rich exudate, containing guanidine compounds, nucleotides, minerals, and vitamins, contributed significantly to both mass loss and quality degradation. Substantial drip loss is observed to induce multiple detrimental effects: visual lightening of pork coloration, deterioration of sensory characteristics, development of off-odors, and diminished consumer acceptability. Both the SPIHs/MD formulation and blank control displayed progressively increasing drip loss rates throughout storage. This phenomenon can be partially explained by structural alterations in muscle tissue, including protein denaturation and myofibrillar network contraction, which collectively compromised water-holding capacity. In distinct contrast, the SPIHs/MD/CIN treatment maintained substantially lower drip loss rates compared to other experimental groups. This preservation effect is attributed to the controlled-release of CIN from microcapsules, which effectively retards protein and lipid degradation while enhancing moisture retention capabilities [33]. Notably, the free CIN treatment exhibited minimal drip loss during the initial 4 days period before experiencing rapid efficacy decline due to compound volatility. The subsequent acceleration in drip loss correlated with increased membrane permeability induced by lipid oxidation and subsequent intracellular cytosol leakage [35]. Collectively, these results demonstrate that microencapsulated CIN significantly reduces pork loin drip loss through sustained release kinetics, thereby effectively delaying meat spoilage.

3.4.5. Peroxide Value

Lipid oxidation in fresh pork loin affects its quality during storage and transportation, typically leading to rancidity and flavor deterioration [36]. The peroxide value (PV) reflects the extent of primary lipid oxidation, indicating the initial stage of lipid degradation, and is employed to determine whether hydroperoxides constitute the principal products of lipid oxidation in meat products. When these peroxides decomposed, the resulting secondary oxidation products are assessed via the thiobarbituric acid reactive substances (TBARS) method. Throughout the storage period, as illustrated in Figure 5D, the PV of the control pork loin group increased steadily from 0.003 g/100 g on day 0 to 0.018 g/100 g by day 14. Consequently, this increase indicates that lipid hydroperoxides generated by lipid oxidation are rapidly produced during storage of fresh pork loin. The observed increase in PV over time is attributed to the formation rate of hydroperoxides exceeding their degradation rate. This phenomenon may be driven by the activity of lipases, lipoxygenases, and other enzymatic systems associated with lipid oxidation. In contrast, the CIN-loaded microcapsules, through controlled-release of CIN, resulted in a PV of only 0.006 g/100 g after 14 days of storage. This formulation significantly suppressed microbial growth, thereby reducing both lipid oxidation and hydrolysis. The observed trend is consistent with findings from other studies [37].

3.4.6. Thiobarbituric Acid Reactive Substances

The TBARS value is established as a critical indicator of lipid oxidation progression in meat systems as well. As delineated in Figure 5E, which represents mean malondialdehyde (MDA) content per unit meat mass, all experimental groups manifested progressively increasing TBARS values throughout storage. This increases likely results from hydroperoxides decomposition and subsequent conversion to secondary oxidation products, including aldehydes. The blank control group displayed the most accelerated oxidation kinetics, followed by the SPIHs/MD treatment. Maximum TBARS elevation was documented in both the blank control and SPIHs/MD groups, whereas the SPIHs/MD/CIN and free CIN formulations demonstrated significantly attenuated oxidation rates, consistent with established literature on antioxidant efficacy [38]. Food safety protocols define a TBARS value of 1.0 mg/kg as the critical threshold for chilled pork. Beyond this value, meat is determined to have developed organoleptic deterioration characterized rancidity and off-odors. The blank control cohort reached this critical spoilage index by day 8, while the SPIHs/MD group exceeded the limit approximately 2 days later, showing consistent correlation with parallel degradation patterns in pH and complementary quality parameters.

Compared to the free CIN group, the SPIHs/MD/CIN treatment provides a more comprehensive preservation strategy, concurrently mitigating increases in both PV and TBARS. The protective mechanism operates via two phases: primary protection involved sustained CIN release from intact microcapsules, effectively constraining microbial proliferation. Secondary protection is triggered by the accumulation of alkaline metabolite during advanced spoilage, inducing capsule rupture and consequent burst release of antimicrobial CIN. This coordinated release profile demonstrates the superior preservation efficacy of microencapsulated CIN in maintaining chilled pork loin quality, validating the earlier experimental observations.

Specifically, the controlled release of CIN within SPIHs/MD/CIN microcapsules primarily occurred through Fickian diffusion, driven by intermolecular interactions and the physical barrier of the composite wall. These interactions weaken the concentration gradient between the interior and exterior of the microcapsule, slowing the diffusion and volatilization rates of CIN. This sustained-release characteristic achieves effective preservation of pork loin during prolonged storage.

4. Conclusions

This study enhanced the amphiphilicity of SPI through enzymatic modification, rendering it a more suitable wall material for microencapsulation. Using SPIHs and MD as wall materials, CIN was effectively encapsulated, and microcapsules with high antibacterial activity and preservation efficacy were prepared via spray drying technology. Characterization via SEM and FTIR confirmed successful encapsulation of CIN within the SPIHs/MD microcapsule walls. Furthermore, SPIHs/MD/CIN microcapsules effectively mitigated changes in texture, pH, and lipid oxidation during the storage of pork loin. In conclusion, these findings provide technical support for fabricating microcapsules with controlled-release capability and preservation performance, offering a potential solution for extending the shelf life of reduced-salt and low-sugar food products in the future. In subsequent studies, it is planned to evaluate the antimicrobial activity of CIN-loaded microcapsules against a wider range of foodborne bacteria and fungi, as well as to investigate the specific molecular behavior underlying microcapsule formation through molecular dynamics simulations.