Sea buckthorn pomace - enhanced SPI bio-composite films: Microstructure, thermal stability, barrier properties, and their applications in meat preservation

2.1. Materials

SPI with a protein content of >90% (dry basis) was purchased from Sigma-Aldrich (St. Louis, MO, USA). SBP was obtained as a byproduct from a local organic juice producer (Valley Organics Ltd., Hereford, UK) after the pressing of sea buckthorn berries (Hippophae rhamnoides L.). Glycerol (analytical grade, ≥99.5% purity) used as a plasticizer was supplied by Fisher Scientific (Loughborough, UK). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), Folin-Ciocalteu phenol reagent, gallic acid, 2-thiobarbituric acid (TBA), trichloroacetic acid (TCA), and 1,1,3,3-tetraethoxypropane (TEP) were procured from Sigma-Aldrich. All other solvents and reagents used were of analytical grade. Plate Count Agar (PCA) was obtained from Oxoid Ltd. (Basingstoke, UK). Fresh pork loin (Musculus longissimus dorsi) was purchased from a local certified butcher within 12 h post-mortem, transported to the laboratory on ice, stored at 4 ± 1°C, and used for experiments within 24 h of purchase.

2.2. Preparation of SBP powder

The wet SBP received from the supplier was spread thinly on trays and dried in a convection oven (Memmert GmbH, Schwabach, Germany) at 50°C for 24 h until a constant weight was achieved (moisture content <10%). The dried SBP was then ground into a fine powder using a laboratory-scale grinder (IKA A11 basic, IKA-Werke GmbH & Co. KG, Staufen, Germany) and sieved through a 150 µm mesh sieve (Endecotts Ltd., London, UK) to obtain a powder with a uniform particle size. The SBP powder was stored in airtight containers at 4°C in the dark until further use. This standardized preparation ensures consistency in SBP characteristics, which can otherwise vary based on processing.

2.3. Preparation of SPI-SBP biocomposite films

SPI-SBP biocomposite films were prepared using the solution casting method. Initially, SPI (5 g) was dispersed in 100 mL of distilled water and stirred continuously at 500 rpm at 70°C for 30 min using a magnetic stirrer hotplate to facilitate protein denaturation and dispersion. Glycerol was then added as a plasticizer at a concentration of 30% (w/w, based on the dry weight of SPI), and stirring continued for another 15 min. Subsequently, SBP powder was incorporated into the SPI-glycerol solution at different concentrations: 0% (control SPI film), 1%, 2%, 3%, and 5% (w/w, based on the dry weight of SPI). The mixture was homogenized using a high-speed homogenizer (Ultra-Turrax T25, IKA, Germany) at 10,000 rpm for 5 min to ensure uniform dispersion of SBP particles. The film-forming solutions were then degassed by sonication in an ultrasonic bath (Grant Instruments, Cambridge, UK) for 15 min to remove air bubbles.

A fixed volume (30 mL) of each film-forming solution was cast onto 90 mm diameter polystyrene Petri dishes and dried in a controlled environment chamber (Binder GmbH, Tuttlingen, Germany) at 25 ± 1°C and 50 ± 5% relative humidity (RH) for approximately 48 h, or until the films could be easily peeled off. The dried films were carefully peeled from the Petri dishes and conditioned at 25 ± 1°C and 50 ± 5% RH for at least 48 h in a desiccator containing a saturated solution of magnesium nitrate prior to characterization. The selection of SBP concentrations (0-5%) was intended to identify an optimal level, as excessive filler content can sometimes lead to particle agglomeration and negatively impact film properties, a common observation in composite materials. Scheme 1 shows the fabrication and characterization of SPI-based biocomposite films.

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Scheme 1.

Scheme for the fabrication and characterization of SPI-based biocomposite films.

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The process begins with the preparation of the SPI solution and the incorporation of SBP, followed by solution casting to form biocomposite films. These films are then subjected to physicochemical and functional characterization, including measurements of thickness, mechanical properties, WVP, and antioxidant activity. The final application involves evaluating the film’s efficacy in preserving pork loin during refrigerated storage.

2.4. Characterization of SPI-SBP films

The thickness of each film sample was measured at a minimum of 10 random positions using a digital micrometer (Mitutoyo, Kawasaki, Japan) with an accuracy of ±0.001 mm. The average thickness value was used for subsequent calculations of mechanical and barrier properties.

The transparency of the films was determined by measuring the absorbance at 600 nm using a UV-Vis spectrophotometer (Jenway 7315, Bibby Scientific, UK). Transparency was calculated as A600​/x, where A600​ is the absorbance at 600 nm and x is the film thickness in mm.

The color of the films was measured using a HunterLab ColorFlex EZ spectrophotometer (Hunter Associates Laboratory, Inc., Reston, VA, USA) with D65 illuminant and 10° standard observer.

The surface and cryo-fractured cross-sectional morphology of the films were examined using a scanning electron microscope (SEM) (JEOL JSM-6010LA, JEOL Ltd., Tokyo, Japan). For cross-sectional imaging, film samples were fractured in liquid nitrogen. All samples were mounted on aluminum stubs using double-sided adhesive carbon tape and sputter-coated with a thin layer of gold (Quorum Q150R S, Quorum Technologies, UK) to enhance conductivity. Micrographs were obtained at an accelerating voltage of 10 kV.

Fourier-transform infrared (FTIR) spectra of the SBP powder and film samples were obtained using a Nicolet iS5 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an attenuated total reflectance (ATR) accessory (diamond crystal).

X-ray diffraction (XRD) analysis was performed using a Bruker D8 Advance diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) with Cu Kα radiation (λ=0.15406 nm) generated at 40 kV and 40 mA. Samples were scanned over a 2θ range of 5° to 60° at a scan speed of 2°/min.

Thermogravimetric analysis (TGA) was conducted using a TGA Q5000 analyzer (TA Instruments, New Castle, DE, USA). Approximately 5-10 mg of each film sample was heated from 30°C to 600°C at a heating rate of 10°C/min under a nitrogen atmosphere (flow rate 50 mL/min).28 Derivative thermogravimetry (DTG) curves were obtained from the TGA data.

Differential scanning calorimetry (DSC) was performed using a DSC Q2000 (TA Instruments). Samples (5-10 mg) were sealed in aluminum pans and heated from -50°C to 200°C at a heating rate of 10°C/min under a nitrogen atmosphere (flow rate 50 mL/min) to determine the glass transition temperature (Tg).

TS, elongation at break (EAB), and Young’s Modulus (YM) were determined according to the ASTM D882-18 standard method using a universal testing machine (Instron 5967, Instron, Norwood, MA, USA) equipped with a 50 N load cell. Film specimens were cut into rectangular strips (10 mm width × 50 mm length). The initial grip separation was 30 mm, and the crosshead speed was set at 10 mm/min. At least five replicates were tested for each film formulation.

Water solubility (WS) was determined by cutting film samples (20 mm × 20 mm) and drying them at 105°C for 24 h to obtain the initial dry weight (Wi​). The dried samples were then immersed in 50 mL of distilled water and gently agitated at 25°C for 24 h. After immersion, the samples were removed, excess surface water was blotted with filter paper, and the samples were dried again at 105°C for 24 h to obtain the final dry weight (Wf​). WS was calculated as: WS(%)=((Wi​−Wf​)/Wi​)×100.34

Swelling ratio (SR) was determined by immersing pre-weighed dry film samples (Wdry​) (20 mm × 20 mm) in distilled water at 25°C for 2 h. After immersion, samples were removed, surface water was gently blotted, and the swollen weight (Wswollen​) was recorded. SR was calculated as: SR(%)=((Wswollen​−Wdry​)/Wdry​)×100.

Water contact angle (WCA) measurements were performed using an optical contact angle goniometer (OCA 15EC, DataPhysics Instruments GmbH, Filderstadt, Germany) with the sessile drop method. A 5 µL droplet of deionized water was placed on the film surface, and the contact angle was measured within 5 seconds of droplet deposition at ambient temperature (25°C). At least five measurements were taken at different locations on each film sample.

WVP was determined gravimetrically using the ASTM E96/E96M-16 standard cup method (desiccant method). Film samples were sealed over the circular opening (area 15.9 cm²) of permeation cups containing 10 g of anhydrous calcium chloride (0% RH). The cups were placed in a controlled environmental chamber at 25°C and 75% RH (maintained using a saturated NaCl solution). The cups were weighed periodically over 48 hrs. WVP (g·m/m²·s·Pa) was calculated using the following equation: WVP=(Δm×x)/(A×t×ΔP), where Δm is the weight gain of the cup (g), x is the film thickness (m), A is the permeation area (m²), t is the time (s), and ΔP is the water vapor partial pressure difference across the film (Pa).

Oxygen permeability (OP) was measured using an oxygen permeation analyzer (MOCON OX-TRAN Model 2/22 L, MOCON Inc., Minneapolis, MN, USA) at 23°C and 0% RH, according to the ASTM D3985-17 standard. Film samples were clamped in the diffusion cell, with pure oxygen (99.9%) on one side and nitrogen carrier gas on the other. Results were expressed as cm²·m/m²·d·atm.

DPPH radical scavenging activity: Film samples (25 mg) were extracted with 5 mL of methanol by shaking for 2 h at 25°C in the dark. The extract (0.1 mL) was mixed with 3.9 mL of 0.1 mM DPPH methanolic solution. The mixture was incubated for 30 min in the dark, and the absorbance was measured at 517 nm using a UV-Vis spectrophotometer. The scavenging activity was calculated as: DPPH scavenging (%)=((Acontrol​−Asample​)/Acontrol​)×100, where Acontrol​ is the absorbance of the DPPH solution without film extract and Asample​ is the absorbance of the DPPH solution with film extract.

ABTS Radical Scavenging Activity: The ABTS radical cation (ABTS·⁺) was produced by reacting 7 mM ABTS stock solution with 2.45 mM potassium persulfate and allowing the mixture to stand in the dark at room temperature for 12-16 h before use. The ABTS·⁺ solution was diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm. Film extract (0.1 mL, prepared as for DPPH assay) was added to 3.9 mL of diluted ABTS·⁺ solution, and the absorbance was measured at 734 nm after 6 min. The scavenging activity was calculated similarly to the DPPH assay.

Total phenolic content (TPC) Release: Film samples (100 mg) were immersed in 10 mL of 50% (v/v) ethanol (food simulant for fatty foods) and incubated at 25°C with gentle shaking. Aliquots (0.5 mL) were withdrawn at different time intervals (0, 1, 3, 6, 12, 24 h). The TPC in the simulant was determined using the Folin-Ciocalteu method.10 Briefly, 0.5 mL of the simulant was mixed with 2.5 mL of Folin-Ciocalteu reagent (diluted 1:10 with water) and 2 mL of 7.5% (w/v) sodium carbonate solution. After incubation for 1 h at room temperature in the dark, the absorbance was measured at 765 nm. TPC was expressed as mg gallic acid equivalents (GAE)/g of film.

2.5. Application in pork loin preservation

Fresh pork loin was aseptically cut into uniform slices of approximately 50 ± 5 g (dimensions approx. 5 cm × 5 cm × 1 cm). These dimensions were chosen to ensure homogeneity and reproducibility across all treatment groups, allowing for consistent film application and reliable physicochemical analysis. The size was also based on commonly used meat cut formats in prior packaging studies involving pork or beef slices, which typically range from 40-60 g to simulate retail-scale packaging scenarios [20]. Additionally, a thickness of ∼1 cm ensured sufficient exposure to external oxygen and moisture while avoiding excessive diffusion resistance from within the sample, thereby facilitating a realistic simulation of spoilage processes in fresh meat packaging applications. The slices were randomly assigned to five treatment groups: (1) Control (unwrapped), (2) PE (wrapped with commercial polyethylene film), (3) SPI (wrapped with control SPI film, 0% SBP), (4) SPI-SBP1% (wrapped with SPI film containing 1% SBP), (5) SPI-SBP3% (wrapped with SPI film containing 3% SBP), and (6) SPI-SBP5% (wrapped with SPI film containing 5% SBP). These groups were chosen based on the dual need to include both negative and positive packaging controls and focus the preservation study on SPI-based formulations that demonstrated superior mechanical, barrier, and antioxidant properties in prior characterization. The SPI-SBP 5% film, while characterized, exhibited particle agglomeration and compromised mechanical integrity, and was therefore excluded from meat preservation trials to ensure data reliability and practical relevance. Each meat sample was individually wrapped with the respective film (film size approx. 15 cm × 15 cm), ensuring complete coverage. The packaged samples were placed on sterile polystyrene trays and stored in a refrigerator at 4 ± 1°C for 15 days, which is a representative duration for evaluating spoilage in fresh pork loin under refrigerated aerobic conditions, as significant microbial and oxidative degradation typically becomes evident between 10-15 days [21,22]. Samples were withdrawn for analysis on days 0, 3, 6, 9, 12, and 15.

pH: 10 g of the meat sample were homogenized with 90 mL of distilled water for 1 min using a stomacher. The pH of the homogenate was measured using a calibrated digital pH meter (Mettler Toledo SevenCompact S220, Switzerland) equipped with a meat penetration probe.

Color (Lab*): Color measurements were taken directly on the surface of the pork loin samples at three different locations using the HunterLab ColorFlex EZ spectrophotometer.

Thiobarbituric acid reactive substances (TBARS): Lipid oxidation was assessed by the TBARS method as described by SØRENSEN AND JØRGENSEN with minor modifications. Then, 5 g of the minced meat sample were homogenized with 15 mL of 10% (w/v) TCA solution. The homogenate was filtered, and 2 mL of the filtrate was mixed with 2 mL of 0.02 M TBA solution. The mixture was heated in a boiling water bath for 30 min, cooled, and the absorbance was measured at 532 nm against a blank. TBARS values were calculated using a standard curve prepared with 1,1,3,3-tetraethoxypropane (TEP) and expressed as mg malondialdehyde (MDA)/kg of meat.

Total viable count (TVC): 10 g of the meat sample were aseptically transferred to a sterile stomacher bag with 90 mL of sterile 0.1% peptone water and homogenized for 2 min. Serial decimal dilutions were prepared, and 0.1 mL aliquots of appropriate dilutions were spread-plated onto plate count agar (PCA). Plates were incubated aerobically at 37°C for 48 h. Colonies were counted, and results were expressed as the logarithm of colony-forming units per gram (log CFU/g) of meat.

Weight Loss: The weight of each packaged meat sample was recorded at each sampling interval. Weight loss was calculated as: Weight loss (%)=((Winitial​−Wfinal​)/Winitial​)×100, where Winitial​ is the initial weight of the meat sample and Wfinal​ is the weight at the specific storage day.

3.1. Characterization of SBP-enhanced SPI films

The visual appearance of the SPI-based films changed progressively with the incorporation of SBP. The control SPI film (0% SBP) was light yellowish, smooth, and relatively transparent. As the concentration of SBP increased from 1% to 5%, the films became darker, exhibiting a more brownish-yellow hue, and their homogeneity appeared to decrease slightly at the highest SBP concentration (5%), where some fine SBP particles were discernible. This change in color and homogeneity is attributed to the natural pigments and particulate nature of SBP. This change in color and homogeneity is attributed to the presence of natural pigments, primarily carotenoids such as β-carotene and zeaxanthin, and flavonoids including rutin and isorhamnetin, present in SBP, which impart a characteristic brownish-yellow hue to the films [23]. Additionally, the particulate nature of SBP, comprising irregularly shaped lignocellulosic fragments from berry skin and seeds, contributes to increased light scattering and microstructural heterogeneity, particularly at higher SBP concentrations. Photographs of the prepared films have been shown in Figure 1.

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Figure 1.

Photographs of SPI-SBP films with varying SBP content: (a) SPI control (0% SBP), (b) SPI-SBP 1%, (c) SPI-SBP 2%, (d) SPI-SBP 3%, (e) SPI-SBP 5%.

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The thickness of the films, along with their optical properties (L*, a*, b*, ΔE*, and transparency at 600 nm), have been presented in Table 1. Film thickness ranged from 0.112 ± 0.005 mm for the control SPI film to 0.135 ± 0.007 mm for the SPI-SBP 5% film. A slight but statistically significant (p < 0.05) increase in film thickness was observed with increasing SBP content, which is expected due to the addition of solid SBP particles to the film-forming solution.

The L* value (lightness) decreased significantly (p < 0.05) with increasing SBP content, indicating darker films. Conversely, the a* value shifted from negative (greenish tint) towards positive (reddish tint), and the b* value (yellowness) increased significantly, reflecting the inherent color of SBP. Consequently, the total color difference (ΔE*) increased markedly with higher SBP concentrations. Film transparency, measured as A600​/mm, increased (meaning opacity increased) with SBP addition, from 0.95 ± 0.04 for the control to 2.53 ± 0.09 for the SPI-SBP 5% film. This reduction in transparency is attributed to light scattering by the SBP particles and absorption by SBP pigments. While high transparency is often preferred in food packaging, a slight increase in opacity and color, as observed here, might be acceptable for applications like meat packaging, especially if these changes are accompanied by significant improvements in functional properties such as antioxidant activity or shelf-life extension.

SEM micrographs of the surface and cryo-fractured cross-sections of the SPI control and SPI-SBP composite films are shown in Figure 2 and Figure 3, respectively. The control SPI film (Figures 2a and 3a) exhibited a relatively smooth and homogeneous surface and a dense, compact cross-section, typical of protein films plasticized with glycerol, although some minor inherent protein aggregates were visible. Upon incorporation of SBP, changes in morphology were evident. At lower SBP concentrations (1% and 2%, e.g., Figures 2b and 3b), SBP particles appeared to be relatively well-dispersed within the SPI matrix. The particles were mostly embedded, suggesting reasonable interfacial adhesion between the SBP and the SPI matrix. The cross-sections showed a more heterogeneous structure compared to the control, but still maintained good cohesion.

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Figure 2.

SEM micrographs of the surface of (a) SPI control, (b) SPI-SBP 1%, (c) SPI-SBP 3%, (d) SPI-SBP 5% films. (Scale bar = 10 µm.)

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Figure 3.

SEM micrographs of the cryo-fractured cross-section of (a) SPI control, (b) SPI-SBP 1%, (c) SPI-SBP 3%, (d) SPI-SBP 5% films. (Scale bar = 10 µm.)

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As the SBP concentration increased to 3% (Figures 2c and 3c), the particles were still fairly well distributed, though some larger particles or small clusters became more apparent. The interface between SBP particles and the SPI matrix generally appeared continuous, indicating that SBP was well integrated into the film structure. This good interfacial adhesion is crucial, as it allows for effective stress transfer from the matrix to the filler and minimizes the formation of voids or pathways for gas and vapor permeation, thereby positively influencing the composite’s mechanical and barrier properties [24].

At the highest SBP concentration of 5% (Figures 2d and 3d), the surface became noticeably rougher, and the cross-section revealed a higher density of SBP particles. Some evidence of particle agglomeration and the presence of small voids or discontinuities at the particle-matrix interface was observed. This suggests that at 5% loading, the dispersion of SBP may be less optimal, potentially leading to stress concentration points and a weakening of the film structure, which can affect mechanical properties [25].

FTIR spectroscopy was employed to investigate the chemical interactions between SPI and SBP components in the composite films. The FTIR spectra of SBP powder, control SPI film, and SPI-SBP 3% film are presented in Figure 4(a). The spectrum of SBP powder showed a broad band around 3350 cm⁻¹ (O-H stretching from cellulose, lignin, and phenolics), peaks around 2920 cm⁻¹ (C-H stretching), ∼1735 cm⁻¹ (C=O stretching from esters/carboxylic acids in hemicellulose or pectin), ∼1620 cm⁻¹ (aromatic C=C stretching in lignin/phenolics and adsorbed water), ∼1515 cm⁻¹ (aromatic skeletal vibrations in lignin), and a series of peaks between 1200-1000 cm⁻¹ (C-O stretching in cellulose, hemicellulose, and lignin) [26]. The control SPI film exhibited characteristic protein amide bands: Amide A around 3275 cm⁻¹ (N-H stretching, often overlapping with O-H stretching), Amide I around 1635 cm⁻¹ (C=O stretching of peptide bonds), and Amide II around 1538 cm⁻¹ (N-H bending and C-N stretching) [27]. Peaks attributable to glycerol (e.g., C-O stretching around 1030-1100 cm⁻¹) were also present [28,29].

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Figure 4.

(a) FTIR spectra of (1) SBP powder, (2) SPI control film, and (3) SPI-SBP 3% film. (b) XRD patterns of (1) SBP powder, (2) SPI control film, (3) SPI-SBP 1% film, (4) SPI-SBP 3% film, and (5) SPI-SBP 5% film.

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In the SPI-SBP 3% film spectrum, several changes were observed compared to the control SPI film. The broad O-H/N-H stretching band (Amide A region) appeared slightly broadened and shifted to a lower wavenumber (e.g., from 3275 cm⁻¹ to ∼3270 cm⁻¹), suggesting an increase in hydrogen bonding interactions. The Amide I and Amide II bands showed subtle shifts and changes in relative intensity, which can also be indicative of conformational changes in the protein structure due to interactions with SBP components. Furthermore, some characteristic peaks of SBP, such as those related to C-O stretching of polysaccharides (around 1000-1100 cm⁻¹), became more pronounced, confirming the successful incorporation of SBP into the SPI matrix. No new distinct peaks indicating covalent bond formation were observed, suggesting that the interactions between SPI and SBP are predominantly non-covalent, mainly hydrogen bonds. The formation of extensive hydrogen bonds between the functional groups of SPI (e.g., -NH, -CO, -OH from amino acid residues) and the abundant hydroxyl groups present in SBP’s polyphenolic compounds and lignocellulosic fractions (cellulose, hemicellulose) is considered the primary mechanism responsible for the improved compatibility and formation of a more cohesive network structure in the composite films [30]. These interactions play a crucial role in modulating the film’s physical and mechanical properties.

XRD patterns were recorded to evaluate the changes in the crystalline structure of SPI films upon SBP incorporation. Figure 4(b) shows the XRD patterns for SBP powder, control SPI film, and SPI-SBP composite films with 1%, 3%, and 5% SBP. The SBP powder exhibited distinct diffraction peaks at 2θ values of approximately 15.5°, 17.0°, and 22.5°, which are characteristic of the crystalline structure of cellulose (Type I), a major component of lignocellulosic materials.28 The control SPI film displayed two broad diffraction humps centered around 2θ ≈ 9° and 2θ ≈ 20°. These peaks are attributed to the α-helix and β-sheet secondary structures of soy protein, respectively, indicating a predominantly amorphous nature with some degree of ordered protein conformation.30

Upon incorporation of SBP into the SPI matrix, the XRD patterns of the composite films showed a superposition of the patterns of SPI and SBP. The broad peaks characteristic of SPI were still present, although their intensity appeared to decrease slightly, and the peak around 20° became less defined with increasing SBP content. This might suggest some disruption of the ordered protein secondary structures due to interactions with SBP components. Concurrently, the crystalline peaks corresponding to cellulose from SBP (especially the one around 22.5°) became more apparent, and their intensity increased progressively with higher SBP concentrations. This indicates the introduction of crystalline domains from SBP’s cellulosic fraction into the largely amorphous SPI matrix. The presence of these crystalline regions can influence the film’s mechanical and barrier properties by creating a more heterogeneous structure [31]. While a certain level of crystallinity can enhance strength and reduce permeability by creating a more tortuous path for permeant molecules, excessive crystallinity or poorly dispersed crystalline domains could potentially lead to increased brittleness. The observed changes suggest a complex interplay between the amorphous SPI matrix and the semi-crystalline SBP filler.

The thermal stability of the SPI-SBP composite films was investigated using TGA and DTG, and the results have been presented in Figure 5(a) (TGA) and Figure 5(b) (DTG). Key thermal parameters have been summarized in Table 2. All films exhibited an initial weight loss step below 120°C, attributed to the evaporation of absorbed moisture and possibly some residual volatile compounds from glycerol. The main thermal degradation of the control SPI film occurred in a major stage between approximately 150°C and 400°C, with a maximum degradation rate temperature (T_max) around 290°C, corresponding to the pyrolysis of peptide bonds and protein chains [32].

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Figure 5.

(a) TGA curves and (b) DTG curves of (1) SPI control and SPI-SBP films with (2) 1%, (3) 3%, and (4) 5% SBP.

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With the incorporation of SBP, the thermal stability of the composite films was enhanced. The onset degradation temperature (T_onset, defined as the temperature at 5% weight loss after moisture removal) and T_max for the main degradation peak generally increased with SBP content (see Table 2). For instance, T_onset increased from ∼145°C for the control SPI film to ∼165°C for the SPI-SBP 5% film. Similarly, T_max shifted to higher temperatures. The DTG curves (Figure 5b) for SPI-SBP films also showed a slight shoulder or a broader main peak, suggesting that SBP components (lignin, cellulose), which degrade at different or higher temperature ranges, contribute to the overall thermal profile [33]. Furthermore, the percentage of char residue at 600°C increased with SBP concentration, from ∼22% for the control film to ∼28% for the SPI-SBP 5% film. This is likely due to the higher char-forming tendency of lignocellulosic materials in SBP. The enhanced thermal stability observed in SPI-SBP films suggests that strong interactions, such as hydrogen bonding and physical entanglement provided by SBP fibers, restrict the thermal motion of SPI chains and require more energy for decomposition. The inherent thermal stability of SBP’s lignocellulosic components also contributes to this improvement.

DSC analysis (Table 2) revealed that the control SPI film exhibited a glass transition temperature (T_g) around 62.5°C. The incorporation of SBP led to a slight increase in T_g. For example, the SPI-SBP 3% film showed a Tg of approximately 65.8°C. This increase in T_g indicates a reduction in the mobility of SPI polymer chains in the amorphous regions, likely due to the reinforcing effect of SBP particles and the formation of a more rigid network structure through SPI-SBP interactions [34]. When compared with similar SPI-based systems reported in the literature, the thermal performance of SPI-SBP films is either comparable or slightly superior. For instance, Kadota et al. [35] reported Tonset and Tmax values of 152°C and 287°C, respectively, for SPI films reinforced with jaboticaba peel powder, parameters closely matching those of our SPI-SBP 3% and 5% films. Like wise, Zuwanna et al. [34] found Tg values of 63-66°C in SPI-lignocellulose systems, corroborating our observed Tg increase from 62.5°C (control) to 66.7°C (5% SBP). These consistent trends across studies reinforce the interpretation that bioactive lignocellulosic fillers, such as SBP, effectively enhance the thermal stability of protein-based biocomposites through polymer-filler hydrogen bonding and the integration of thermally stable fiber domains.

The mechanical properties, including TS, EAB, and YM, are critical indicators of a film’s suitability for packaging applications. The results for SPI-SBP films have been presented in Figure 6 and summarized in Table 3.

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Figure 6.

Mechanical properties of SPI-SBP films as a function of SBP content: (a) TS, (b) EAB, and (c) YM. Error bars represent standard deviation (n=5).

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The control SPI film exhibited a TS of 3.8 ± 0.3 MPa, EAB of 125.5 ± 8.2%, and YM of 45.2 ± 3.5 MPa. The incorporation of SBP significantly influenced these properties. Both TS and YM increased with SBP addition up to 3% (w/w). The SPI-SBP 3% film showed the highest TS (5.5 ± 0.4 MPa) and YM (85.7 ± 6.2 MPa), representing improvements of approximately 45% and 90%, respectively, compared to the control SPI film. This enhancement in strength and stiffness can be attributed to the reinforcing effect of SBP particles, which are relatively well-dispersed at these concentrations, and the strong interfacial interactions between SBP and the SPI matrix, allowing for efficient stress transfer.

However, when the SBP concentration was increased to 5%, both TS and YM decreased (to 4.2 ± 0.5 MPa and 65.4 ± 5.8 MPa, respectively), although they remained higher than the control film values for YM. This decline at 5% SBP is likely due to particle agglomeration and the formation of stress concentration points within the film matrix, which can initiate premature failure [36]. This behavior indicates that an optimum SBP concentration exists (around 3% in this study) where its reinforcing effect is maximized. Beyond this optimal level, the negative effects of poor dispersion and increased heterogeneity outweigh the benefits of filler addition.

Conversely, the EAB of the films consistently decreased with increasing SBP content, from 125.5% for the control film to 65.1% for the SPI-SBP 5% film. This reduction in flexibility and ductility is a common phenomenon when rigid fillers are incorporated into a polymer matrix, as the filler particles restrict the mobility of polymer chains and reduce the film’s ability to deform before fracturing.

The interaction of the films with water was assessed by measuring WS, SR, and WCA. The results are depicted in Figure 7 and summarized in Table 4. The control SPI film, being hydrophilic, exhibited high WS (35.8 ± 1.5%) and SR (285.2 ± 10.5%). The incorporation of SBP led to a significant reduction in both WS and SR up to 3% SBP content. The SPI-SBP 3% film showed the lowest WS (26.7 ± 0.9%) and SR (215.3 ± 7.7%). This improvement in water resistance can be attributed to several factors. Firstly, the formation of a more compact and denser network structure due to strong hydrogen bonding interactions between SPI and SBP components (as suggested by FTIR) can reduce the accessibility of water molecules to the hydrophilic groups of SPI [37]. Secondly, the lignocellulosic components of SBP are generally less soluble and less prone to swelling than SPI. Thirdly, SBP particles may fill voids within the SPI matrix, further hindering water penetration and absorption. At 5% SBP, a slight increase in WS and SR was observed compared to 3% SBP, possibly due to the aforementioned particle agglomeration and increased heterogeneity, creating more pathways for water ingress.

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Figure 7.

Water resistance properties of SPI-SBP films: (a) WS, (b) SR, and (c) WCA. Error bars represent standard deviation (n=3 for WS and SR, n=5 for WCA).

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The WCA measurements (Figure 7c) showed an increase in surface hydrophobicity with SBP addition. The WCA increased from 65.3 ± 2.1° for the control SPI film to a maximum of 79.8 ± 2.3° for the SPI-SBP 3% film. This indicates that the surface of the composite films became less wettable [38,39]. This could be due to the exposure of less polar components from SBP (e.g., lignin moieties) on the film surface or the formation of a denser surface structure that reduces water spreading [40]. The slight decrease in WCA for the SPI-SBP 5% film might be related to increased surface roughness caused by SBP agglomerates. The overall reduction in water sensitivity (lower WS and SR, higher WCA) is a significant improvement for SPI-based films, enhancing their stability and applicability for packaging moist food products [41].

The WVP and oxygen permeability (OP) of the films are critical parameters for food packaging applications, as they determine the extent of moisture and oxygen exchange between the food and the external environment. The WVP and OP values for SPI-SBP films have been shown in Figure 8 and summarized in Table 5. The control SPI film exhibited a WVP of 2.15±0.12×10⁻¹⁰ g·m/m²·s·Pa, which is relatively high due to the hydrophilic nature of soy protein. The incorporation of SBP significantly reduced the WVP of the films. The lowest WVP value (1.50±0.08×10⁻¹⁰ g·m/m²·s·Pa) was achieved with the SPI-SBP 3% film, representing a reduction of approximately 30% compared to the control. This improvement in water vapor barrier properties can be attributed to several factors: (i) the SBP particles, particularly their lignocellulosic components, act as impermeable fillers, creating a more tortuous path for water vapor molecules diffusing through the film; (ii) the strong interactions between SPI and SBP (e.g., hydrogen bonding) lead to a more compact film structure with reduced free volume, thus hindering molecular transport [42]; and (iii) potential changes in crystallinity (as suggested by XRD) may also contribute. The slight increase in WVP for the SPI-SBP 5% film compared to the 3% film could be due to the formation of micro-voids or channels at the interface of agglomerated SBP particles, providing easier pathways for water vapor.

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Figure 8.

Barrier properties of SPI-SBP films: (a) WVP and (b) OP. Error bars represent standard deviation (n=3).

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The OP of the control SPI film was 5.8±0.4×10⁻¹⁴ cm³·m/m²·s·Pa (at 23°C, 0% RH). Protein-based films are generally known to possess good oxygen barrier properties at low relative humidity. The addition of SBP further improved the oxygen barrier, with the SPI-SBP 3% film exhibiting the lowest OP value of 3.9±0.2×10⁻¹⁴ cm³·m/m²·s·Pa. This reduction in OP is likely due to similar mechanisms as those responsible for WVP reduction, i.e., increased tortuosity and reduced free volume due to SBP incorporation and enhanced SPI-SBP interactions. The improved barrier properties are highly desirable for food packaging, as they can help to extend the shelf-life of oxygen- and moisture-sensitive products [43].

The antioxidant potential of the SPI-SBP films was evaluated by DPPH and ABTS radical scavenging assays, and the release of total phenolic compounds (TPC) from the films into a food simulant was monitored. The results have been shown in Figure 9 and Figure 10. The control SPI film exhibited negligible DPPH and ABTS radical scavenging activity [44]. In contrast, the SPI-SBP composite films showed significant antioxidant activity, which increased in a dose-dependent manner with increasing SBP concentration. For instance, the SPI-SBP 5% film displayed DPPH scavenging activity of 65.8 ± 3.2% and ABTS scavenging activity of 75.2 ± 3.8%. This pronounced antioxidant capacity is directly attributed to the presence of phenolic compounds (flavonoids, phenolic acids) in SBP, which are known for their potent radical scavenging abilities [45]. These results confirm that SBP successfully imparts antioxidant functionality to the SPI films.

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Figure 9.

Antioxidant activity of SPI-SBP films: (a) DPPH radical scavenging activity and (b) ABTS radical scavenging activity. Error bars represent standard deviation (n=3).

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Figure 10.

PC release from SPI-SBP 3% film into 50% ethanol food simulant over 24 h at 25°C. Error bars represent standard deviation (n=3).

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The release of TPC from the SPI-SBP 3% film into a 50% ethanol food simulant has been shown in Figure 10. A sustained release of phenolic compounds was observed over the 24-h period, with an initial rapid release phase followed by a slower, more gradual release. After 24 h, approximately 4.5 mg GAE/g film had been released. This demonstrates that the bioactive phenolic compounds from SBP are not only incorporated into the film but can also migrate out of the film matrix, making them available to exert their antioxidant effects on the packaged food product. The sustained release characteristic is particularly important for active packaging, as it implies that the film can provide ongoing protection to the food throughout its storage period, rather than just an initial surface effect [46]. The release mechanism is likely diffusion-controlled, possibly facilitated by the swelling of the film matrix upon contact with the simulant, which increases the mobility of the entrapped phenolic compounds.

3.2. Efficacy of SPI-SBP films in pork loin preservation (during 15 days at 4°C)

The effectiveness of the developed SPI-SBP films in preserving the quality of fresh pork loin during refrigerated storage (4°C) for 15 days was evaluated by monitoring pH, color, lipid oxidation (TBARS), microbial growth (TVC), and weight loss. The SPI-SBP 3% film was selected for detailed comparison alongside control, PE, SPI, SPI-SBP1%, and SPI-SBP5% due to its overall superior physicochemical properties.

The pH of fresh meat is an important indicator of its quality and freshness. Changes in pH during storage are often associated with microbial activity and biochemical processes. The pH values of pork loin samples packaged with different films have been presented in Figure 11(a). The initial pH of the fresh pork loin was 5.65 ± 0.05. In the unwrapped control and PE-wrapped samples, the pH gradually increased over the storage period, reaching 6.45 ± 0.08 and 6.32 ± 0.07, respectively, by day 15. This increase is primarily due to the accumulation of alkaline compounds, such as ammonia and amines, produced by the metabolic activity of spoilage bacteria degrading proteins. Samples wrapped with the control SPI film also showed a similar increasing trend. However, pork loin samples packaged with SPI-SBP films, particularly SPI-SBP 3% and SPI-SBP 5%, exhibited a significantly slower rate of pH increase. On day 15, the pH of samples wrapped in SPI-SBP 3% film was 5.95 ± 0.06, significantly lower (p < 0.05) than the control and PE groups. This retardation in pH increase suggests that the SBP-containing films were able to inhibit or slow down the growth and metabolic activity of spoilage microorganisms, likely due to the antimicrobial properties of SBP’s phenolic compounds defined in section 3.2 [47].

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Figure 11.

Quality changes in pork loin samples during 15 days of refrigerated storage (4°C): (a) pH, (b) L value (lightness), (c) a* value (redness), (d) b* value (yellowness). Error bars represent standard deviation (n=3). Different letters on bars for the same day indicate significant differences (p < 0.05) among treatments.

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Color is a primary sensory attribute that influences consumer perception and acceptance of fresh meat. The changes in L* (lightness), a* (redness), and b* (yellowness) values of pork loin samples have been shown in Figures 11 (b-d), respectively. A summary of key quality parameters including total color difference (ΔE∗) at day 0 and day 15 has been provided in Table 6. The L* values (Figure 11b) generally showed a slight increase in most samples over time, indicating some lightening, possibly due to surface moisture changes or protein denaturation. The b* values (Figure 11d) showed minor fluctuations without consistent trends among treatments.

The most critical color parameter for fresh red meat is the a* value (redness), which is related to the state of myoglobin. A decrease in a* value indicates a loss of the desirable bright red color due to the oxidation of oxymyoglobin (red) to metmyoglobin (brownish). As shown in Figure 11(c), the a* value of the unwrapped control and PE-wrapped samples decreased significantly (p < 0.05) during storage, from an initial value of 15.2 ± 0.5 to 9.8 ± 0.6 and 10.5 ± 0.7, respectively, by day 15. Samples wrapped in the control SPI film also showed a considerable decrease in redness. In contrast, pork loin packaged with SPI-SBP films, especially SPI-SBP 3% and SPI-SBP 5%, maintained significantly higher a* values throughout the storage period. On day 15, the a* value for the SPI-SBP 3% sample was 13.5 ± 0.4, indicating much better color retention. This preservation of redness is a direct consequence of the antioxidant activity of SBP components released from the film, which inhibit the oxidation of myoglobin. The maintenance of redness is a crucial quality attribute for consumer appeal [48].

Lipid oxidation is a major cause of quality deterioration in meat, leading to the development of rancid off-flavors and odors. TBARS values, which measure secondary lipid oxidation products like malondialdehyde (MDA), are commonly used to assess the extent of lipid oxidation. The changes in TBARS values of pork loin samples during storage have been shown in Figure 12(a). The initial TBARS value of fresh pork loin was 0.22 ± 0.02 mg MDA/kg. TBARS values increased in all samples throughout the storage period, indicating ongoing lipid oxidation. However, the rate of increase varied significantly among treatments. The unwrapped control and PE-wrapped samples showed the highest TBARS values, reaching 1.85 ± 0.10 and 1.68 ± 0.09 mg MDA/kg, respectively, by day 15. Samples wrapped in the control SPI film had slightly lower TBARS values.

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Figure 12.

Spoilage indicators in pork loin samples during 15 days of refrigerated storage (4°C): (a) TBARS values, (b) TVC. Error bars represent standard deviation (n=3). Different letters on bars for the same day indicate significant differences (p < 0.05) among treatments.

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Significantly (p < 0.05), pork loin samples packaged with SPI-SBP films exhibited much lower TBARS values. The inhibitory effect on lipid oxidation was more pronounced with increasing SBP concentration. On day 15, the TBARS values for samples wrapped in SPI-SBP 1%, SPI-SBP 3%, and SPI-SBP 5% films were 1.10 ± 0.08, 0.85 ± 0.07, and 0.78 ± 0.06 mg MDA/kg, respectively. The SPI-SBP 3% and 5% films reduced TBARS by more than 50% compared to the unwrapped control at the end of storage. This marked reduction in lipid oxidation directly correlates with the intrinsic antioxidant activity of the SPI-SBP films and the sustained release of SBP’s phenolic compounds into the meat environment, which effectively scavenge free radicals and interrupt the oxidation chain reactions.

Microbial growth is a primary factor limiting the shelf-life of fresh meat. The changes in Total Viable Count (TVC) in pork loin samples are presented in Figure 12(b). The initial TVC of fresh pork loin was approximately 3.5 ± 0.2 log CFU/g. In the unwrapped control and PE-wrapped samples, TVC increased rapidly, exceeding 7.0 log CFU/g (a common spoilage threshold for fresh meat) by day 9 and reaching 8.2 ± 0.3 and 7.9 ± 0.2 log CFU/g, respectively, by day 15. Samples packaged with the control SPI film showed a slightly slower microbial growth rate. Pork loin samples wrapped in SPI-SBP films demonstrated significantly (p < 0.05) lower TVC throughout the storage period compared to the control and PE groups. The antimicrobial effect was generally dose-dependent with SBP concentration. By day 15, the TVC for samples in SPI-SBP 1%, SPI-SBP 3%, and SPI-SBP 5% films were 6.8 ± 0.2, 6.1 ± 0.3, and 5.8 ± 0.2 log CFU/g, respectively. The SPI-SBP 3% and 5% films kept the TVC below or near the 7.0 log CFU/g limit for up to 12-15 days, effectively extending the microbial shelf-life. This antimicrobial activity is attributed to the phenolic compounds present in SBP, which are known to disrupt microbial cell membranes, interfere with cellular metabolism, or inhibit essential enzymes, thereby retarding the growth of spoilage bacteria.4 This effect contributes significantly to shelf-life extension, working in concert with the antioxidant properties.

Weight loss in fresh meat during storage is primarily due to moisture evaporation, which can affect its juiciness, appearance, and saleable weight. The weight loss of pork loin samples is shown in Figure 13. The unwrapped control samples exhibited the highest weight loss, reaching 12.5 ± 0.8% by day 15. Samples wrapped in PE film showed significantly lower weight loss (4.2 ± 0.3% on day 15) due to PE’s good moisture barrier. The control SPI film, with its relatively high WVP, resulted in a weight loss of 8.5 ± 0.6% on day 15.

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Figure 13.

Weight loss of pork loin samples packaged with different films during 15 days of refrigerated storage (4°C). Error bars represent standard deviation (n=3). Different letters on bars for the same day indicate significant differences (p < 0.05) among treatments.

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The incorporation of SBP into SPI films led to a reduction in meat weight loss compared to the control SPI film. Samples wrapped in SPI-SBP 3% film showed a weight loss of 6.1 ± 0.4% by day 15, which was significantly lower than the control SPI film, reflecting the improved water vapor barrier properties (lower WVP, Figure 11a) of the SPI-SBP 3% film. While not as effective as PE in preventing moisture loss, the SPI-SBP films demonstrated a notable improvement over neat SPI films.

3.3. Overall discussion and correlations

The results of this study demonstrate that SBP incorporation into SPI films establishes a direct component–property–performance relationship. Specifically, SBP contributes phenolic antioxidants (e.g., rutin, quercetin) and lignocellulosic fiber components (cellulose, lignin), which interact with SPI via hydrogen bonding (as shown by FTIR) and form a more compact network (as visualized in SEM). This structural integration enhances the films’ mechanical properties (increased TS and YM), thermal stability (higher Tonset and char yield), and barrier function (lower WVP and OP), especially at the optimal 3% SBP loading. These physicochemical improvements are directly linked to better functional outcomes in meat preservation: reduced TBARS and TVC values, and improved color and weight retention in pork loin samples. This structure–function correlation can be better understood through the lens of polymer–filler interactions. Polyphenols in SBP are known to interact with protein matrices through hydrogen bonding and π–π stacking, thereby reducing polymer chain mobility and water uptake [49]. Concurrently, the lignocellulosic structure of SBP introduces micro-barriers that obstruct the diffusion pathways for oxygen and water vapor, a phenomenon consistent with the tortuous path model [50]. These effects collectively lower permeability and improve mechanical integrity. The improved packaging functionality, evidenced by meat shelf-life extension and reduced oxidation, is therefore rooted in the synergistic contributions of SBP’s bioactive and structural components. The SPI-SBP 3% film, for example, achieved a ∼30% reduction in WVP and a >50% reduction in TBARS values, illustrating this component-property-performance alignment.

The improved mechanical strength (TS, YM) and thermal stability of the SPI-SBP films can be attributed to the reinforcing effect of SBP’s lignocellulosic components and the formation of a more robust and interactive network structure through hydrogen bonding between SPI and SBP’s various functional groups (phenolic hydroxyls, polysaccharide hydroxyls, protein amides/carboxyls), as suggested by FTIR and SEM analyses. These interactions also contributed to the enhanced water resistance (lower WS and SR, higher WCA) and barrier properties (lower WVP and OP) by creating a more compact structure with increased tortuosity for permeant molecules.

The effectiveness of SBP as a functional additive stems from its dual-action capability: its lignocellulosic fraction provides structural reinforcement, while its rich polyphenolic content imparts active preservative properties through antioxidant and antimicrobial mechanisms. This multi-functional characteristic positions SPI-SBP films as advanced packaging materials, potentially superior to those relying on single-function additives. The sustained release of phenolic compounds from the film matrix is crucial for providing continuous protection to the packaged meat.

Furthermore, the successful utilization of SBP, an unrefined agro-industrial pomace, suggests that complex natural extracts or minimally processed byproducts can be highly effective, possibly due to synergistic interactions between the multiple bioactive components present. This approach not only adds value to agricultural waste but also offers a pathway to developing cost-effective and potent active packaging materials. While this study demonstrates promising results, limitations include the lab-scale preparation and the use of a specific batch of SBP, whose composition can vary. Future work should focus on optimizing SBP processing, exploring long-term stability, conducting sensory evaluations of the packaged meat, and investigating the scalability of film production.