Rheological and Bioactive Profile of Gelatin—Hemp Protein Hydrogels
1Adaptive Food Systems Accelerator-Science Centre, Wroclaw University of Economics and Business, Komandorska 118/120, 53-345 Wroclaw, Poland
2Department of Biotechnology and Food Analysis, Wroclaw University of Economics and Business, Komandorska 118/120, 53-345 Wroclaw, Poland
Abstract
The aim of this study was to investigate the effect of hemp protein addition on the structural, rheological, textural, color, and bioactive properties of gelatin hydrogels. Composite systems containing 0–20% hemp protein were analyzed to clarify the mechanism of interaction with the gelatin matrix and to determine whether hemp protein acts as a passive filler or an active structure-forming component. In all formulations, the gelatin concentration was kept constant at 5% (w/w), while hemp protein was added at increasing levels without replacing the gelatin phase, resulting in systems with increasing total solid content. The addition of hemp protein significantly enhanced water-holding capacity and gel strength, as confirmed by rheological measurements and texture profile analysis. Thermorheological analysis revealed a gradual transition from a classic thermoreversible gelatin gel to reinforced composite networks, with the viscoelastic response increasingly governed by the hemp protein structure at higher concentrations (15–20%). Frequency- and amplitude-sweep tests demonstrated improved mechanical stability and reduced frequency dependence. FTIR analysis indicated reorganization of hydrogen bonding and an increasing contribution of hydrophobic interactions related to the lipid fraction of hemp protein. Furthermore, the addition of hemp protein led to a marked increase in antioxidant activity (ABTS and FRAP) and significant changes in color parameters. These results demonstrate that hemp protein functions as an active structural and functional component in gelatin hydrogels, enabling the development of materials with tailored mechanical properties and enhanced bioactivity.
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Keywords: gelatin, hemp protein, hydrogel, rheology, thermorheology, FTIR, bioactive compounds
Article notes
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Received 2026 Jan 31; Revised 2026 Feb 21; Accepted 2026 Mar 2; Collection date 2026 Mar.
1. Introduction
Protein hydrogels are one of the most important classes of structural materials used in food technology, biomaterials, and functional systems with controlled mechanical properties. Their ability to form three-dimensional spatial networks capable of retaining large amounts of water results from non-covalent interactions, such as hydrogen bonds, hydrophobic interactions, and electrostatic interactions [1,2]. In recent years, there has been growing interest in composite hydrogels, in which classic protein matrices are modified by the addition of other biopolymers, including plant proteins, to obtain new rheological, structural, and functional properties [3,4].
Gelatin, a product of partial collagen hydrolysis, is one of the most used gelling proteins. Its ability to form thermoreversible gels is based on the mechanism of renaturation of collagen triple helix fragments during cooling of the solution after prior heat treatment [5,6]. The resulting network of physical nodes gives gelatin gels their characteristic elastic properties, good water-binding capacity, and predictable rheological behavior as a function of temperature. At the same time, gelatin has a relatively neutral sensory profile and high compatibility with other ingredients, making it an attractive carrier matrix for bioactive compounds [7,8]. Despite their numerous advantages, gelatin hydrogels have certain functional limitations, including moderate mechanical resistance, temperature sensitivity, and a limited ability to stabilize multiphase systems [9]. In response to these limitations, attempts are increasingly being made to modify gelatin by creating mixed systems with other biopolymers, in particular plant proteins, which can play a reinforcing, structure-forming, or functional role [10,11].
Commercially available hemp protein isolate (HPI) is an interesting plant-based protein with a complex composition and high application potential. In addition to the protein fraction, which mainly consists of edestin and albumins, hemp protein preparations also contain naturally occurring lipids, phospholipids, and bioactive compounds such as phenols and peptides with antioxidant activity [12,13]. This chemical composition gives HPI amphiphilic properties that promote adsorption at the oil–water interface and stabilize emulsion systems [14,15,16].
From a structural perspective, HPI’s ability to simultaneously form a protein network and interact with the lipid fraction is important. In hydrogel systems, this can lead to the formation of emulsion-like structures in which the continuous phase is gel-like, while the dispersed phase comprises retained lipid domains [17]. Emulgels combine the characteristics of classic hydrogels and emulsions, exhibiting increased mechanical stability, enhanced water-holding capacity, and the ability to simultaneously transport hydrophilic and hydrophobic components [18,19].
Previous research on gelatin-plant protein composite systems has focused mainly on globular proteins, such as soy, pea, or whey protein, indicating that their addition can either strengthen or weaken the gelatin structure depending on environmental conditions, concentration, and molecular compatibility [20,21]. Compared to these raw materials, commercial hemp protein is distinguished by a significant lipid fraction and greater structural diversity, which affects the mechanisms of interaction with the gelatin matrix and the nature of the resulting spatial networks [22,23]. The incorporation of hemp protein into a gelatin matrix can enrich the system with antioxidant compounds, significantly expanding the potential applications of these materials in functional foods, nutraceuticals, and delivery systems. At the same time, the presence of the lipid fraction may affect the availability and mechanism of action of these compounds, especially within emulsifying structures [12,24,25].
The aim of this study was to comprehensively determine the effect of HPI addition on the structural, rheological, textural, color, and bioactive properties of gelatin hydrogels. Particular attention was paid to identifying the mechanisms of interaction between gelatin and HPI. In addition, the study sought to determine whether HPI acts as a passive filler or an active structure-forming component capable of assuming a dominant role in shaping the system’s mechanical and functional responses.
2. Results
2.1. Water Absorption Capacity of Gelatin–HPI Gels
The base (HP0) reference sample (5% gelatin) was characterized by high water absorption capacity (WAC-95.0%) (Figure 1). The gradual increase in hemp protein isolate (HPI) in the matrix resulted in a systematic increase in WAC. The addition of 5% HPI was already sufficient to significantly enhance water-holding capacity to 97.4%, demonstrating the high efficiency of hemp protein even at low incorporation levels. Further increases in HPI share led to a gradual, less noticeable, but significantly different rise. The highest values were recorded for samples HP15 and HP20 (98.19% and 98.31%, respectively), indicating very effective water capture in the gel structure.
The water absorption capacity increases with increasing protein content in the matrix, an expected behavior that slows down with further increases due to competition for water. Fang et al. (2023) showed that increased hemp protein concentration in thermally induced gels promotes the formation of a more homogeneous, compact spatial network, thereby improving water-holding capacity [26]. The increased protein–protein bonds and, therefore, enhanced hydrophobic interactions are responsible for more effective water retention in the gel matrix. Guidi et al. (2022) indicated that the presence of hemp proteins in mixed systems significantly modified water retention properties, with this effect potentially being non-linear as a function of plant protein content [27].
The almost non-significant differences in WAC values between samples HP10–HP20 in our studies suggest that the capacity for further water absorption was hindered by a gel structure saturation, in which further increases in the proportion of hemp protein no longer result in a significant improvement in water-binding capacity.
2.2. Thermorheological Behavior of Gelatin–HPI Gels
The rheological behavior in all samples was characteristic of gelatin-based systems, with the storage modulus (G′) exceeding the loss modulus (G″) at low temperatures, confirming their elastic, gel-like nature (Figure 2). Upon heating, both moduli decreased sharply, corresponding to the gel–sol transition and the disruption of physical junction zones within the gelatin network. During cooling, G′ and G″ increased again; however, a clear hysteresis between heating and cooling curves was observed, reflecting kinetic limitations in network reconstruction.
Thermorheograms of composite gels showed that HPI incorporation specifically altered the thermorheological response, indicating that HPI did not simply dilute the gelatin but rather reinforced multiphase networks. The pure gelatin sample (HP0) displayed a distinct gel–sol transition at approximately 30–35 °C during heating, associated with the melting of triple helices and the breakdown of physical crosslinks. During cooling, network recovery occurred at lower temperatures, resulting in pronounced thermal hysteresis due to the kinetics of helix renaturation.
The 5% and 10% HPI concentrations share increased moduli in the gel state, particularly during cooling, showing a reinforcing effect of the hemp component (Figure 2). The gel–sol transition became less abrupt and extended over a broader temperature range, suggesting increased structural heterogeneity and restricted gelatin chain mobility.
The HP10 sample exhibited a wide temperature interval with very low modulus values, which may suggest a predominance of viscous behavior at intermediate temperatures and the absence of a well-defined elastic plateau beyond the low-temperature region. A marked change in thermorheological behavior was observed for HP15. Although heating still induced a reduction in moduli within the gelatin melting range, G′ did not decrease to zero, indicating partial preservation of mechanical continuity. During cooling, elevated moduli were maintained over a broad temperature range, followed by further structural reorganization. This response suggests the coexistence of a thermoreversible gelatin network with a more temperature-stable structure associated with hemp protein aggregation or filler–matrix interactions.
The HP20 system (Figure 2) exhibited the highest moduli across the entire temperature range and the lowest sensitivity to the classical gelatin gel–sol transition. Although some decrease in G′ and G″ occurred during heating, complete liquefaction was not observed. During cooling, high G′ values persisted, indicating that the viscoelastic response was dominated by a hemp-protein-based network, with gelatin primarily acting as a binding and hydrating phase.
In all the analyzed systems, hysteresis was observed between heating and cooling cycles, with its intensity and mechanism depending on the proportion of hemp protein in the gel structure. In the HP0 reference sample, hysteresis was mainly related to the kinetics of the breakdown and reformation of gelatin triple helices; the gel–sol transition occurred during heating in the range of 30–35 °C, while gelation during cooling occurred at 4–8 °C lower temperatures [28,29].
In composite systems, the addition of hemp protein led to an increase in rheological modules and an extension of the transition temperature range. Hysteresis included not only gelatin helical transformations but also modified water distribution and additional protein–protein interactions. In HP5–HP10 samples, the gel–sol transition was less abrupt and extended over a 10–15 °C range, indicating increased structural heterogeneity. Similar effects have been described in mixed gelatin–konjac and gelatin–alginate gels [28,29].
In samples with the highest hemp protein content (HP15 and HP20), the storage modulus did not decrease to values close to zero, even above the classical melting point of gelatin, indicating that the mechanical continuity of the system was maintained. This behavior suggests that the hemp protein fraction has taken over the dominant load-bearing role while reducing the importance of reversible gelatin helical structures. The progressive increase in G′ (from 72 Pa in HP0 to 2534 Pa in HP20), the maintenance of mechanical continuity above the classical gelatin melting temperature (HP15–HP20), and the systematic shift of the G′ = G″ crossover point toward higher stress values clearly indicate that hemp protein isolate does not act as a passive filler. Instead, at higher concentrations, HPI functions as an active structure-forming component that progressively assumes the dominant load-bearing role within the hydrogel network. It cannot be excluded that at higher HPI concentrations, partial phase heterogeneity or localized accumulation of hemp protein domains may occur, contributing to the broadened transition range and altered thermorheological response. Similar phenomena have been observed in composite hydrogels, in which the second biopolymer phase stabilized the network and determined the rheological response of the system [28,29]. The results indicate a gradual transition from a classic, thermoreversible gelatin gel to reinforced composite systems, in which the rheological behavior is increasingly controlled by hemp protein.
2.3. Structural Response of Gelatin–HPI Gels to Frequency and Amplitude Sweeps
At 18 °C, all samples exhibited a pronounced gel-like response, as evidenced by the clear dominance of the storage modulus over the loss modulus (G′ ≫ G″) and low values of the loss tangent (tan δ < 0.15) (Table 1). The HP0 reference gel already displayed a stable elastic network, with G′ of approximately 72 Pa. However, increasing the hemp protein content resulted in a strong, monotonic increase in elastic stiffness, with G′ reaching approximately 2534 Pa for the HP20 sample.
| 18 °C | |||||
|---|---|---|---|---|---|
| Sample | HP_0 | HP_5 | HP_10 | HP_15 | HP_20 |
| G′ | 72.38 ± 22.17 d | 205.6 ± 4.17 d | 540.64 ± 76.57 c | 1419.75 ± 98.15 b | 2534.35 ± 99.65 a |
| a | 0.21 ± 0.04 a | 0.22 ± 0 a | 0.19 ± 0.02 ab | 0.15 ± 0.005 b | 0.15 ± 0.004 b |
| G″ | 2.06 ± 0.34 d | 17.65 ± 0.16 d | 49.6 ± 10.63 c | 160.23 ± 12.93 b | 334.21 ± 12.58 a |
| b | 0.52 ± 0.03 a | 0.3 ± 0.01 b | 0.25 ± 0.02 b | 0.19 ± 0.01 c | 0.18 ± 0.004 c |
| tan δ | 0.03 ± 0.005 d | 0.08 ± 0.004 c | 0.09 ± 0.01 c | 0.11 ± 0.001 b | 0.13 ± 0 a |
| c | 0.31 ± 0.01 a | 0.08 ± 0.01 b | 0.07 ± 0.01 bc | 0.04 ± 0.01 cd | 0.03 ± 0.001 d |
| G′ = G″ | 612 ± 0.8 d | 101 ± 16.91 cd | 1512 ± 585.86 bc | 1892 ± 88.31 ab | 2454 ± 5.29 a |
| 25 °C | |||||
| G′ | 112.6 ± 22.7 a | 17 ± 6.9 d | 37.9 ± 17.3 cd | 70.2 ± 2.1 bc | 94.1 ± 13.5 ab |
| a | 0.12 ± 0.01 c | 0.35 ± 0.07 a | 0.37 ± 0.05 b | 0.25 ± 0.01 c | 0.26 ± 0.01 c |
| G″ | 5.09 ± 1.04 c | 3.21 ± 0.84 c | 16.31 ± 7.98 b | 13.96 ± 1.2 bc | 29.69 ± 4.12 a |
| b | 0.41 ± 0.03 abc | 0.48 ± 0.02 a | 0.46 ± 0.06 ab | 0.38 ± 0.02 bc | 0.34 ± 0.01 c |
| tan δ | 0.05 ± 0.0003 c | 0.2 ± 0.06 b | 0.4 ± 0.04 a | 0.2 ± 0.02 b | 0.32 ± 0.003 a |
| c | 0.29 ± 0.01 a | 0.13 ± 0.06 b | 0.09 ± 0.02 b | 0.13 ± 0.02 b | 0.09 ± 0.002 b |
| G′ = G″ | 206.9 ± 27.6 a | 78.1 ± 12.9 b | 148.1 ± 41.8 a | 183.1 ± 17.6 a | 146 ± 0.3 a |
Although loss modulus also increased with hemp protein addition (Figure 3), this increase was markedly less pronounced than that observed for storage modulus. Consequently, tan δ decreased progressively, together with a reduction in the frequency dependence of both moduli, reflected by lower power-law exponents (a for G′ and b for G″). This suggests that higher HPI contents promote the formation of a stiffer, more deformation-resistant network, characteristic of systems enriched in water-binding and structurally reinforcing components. Also, the systematic shift in the G′ = G″ crossover point toward higher stress values with increasing hemp protein concentration (Table 1) indicates an increased resistance of the gel network to applied deformation and a delayed transition from predominantly elastic to viscous behavior.
At 25 °C, the rheological response of the systems changed markedly, particularly for samples with low to intermediate hemp protein content (Figure 3). The HP0 reference sample retained the characteristics of a physical gelatin gel, with the storage modulus remaining higher than the loss modulus and a low loss tangent (tan δ ≈ 0.05), indicating preserved continuity of the gelatin network.
In contrast to the characteristic observed at 18 °C, the HP5 and HP10 samples at 25 °C exhibited a pronounced reduction in G′, accompanied by a substantial increase in tan δ (to approximately 0.2–0.4). This response reflects a partial relaxation of the gel structure and a greater contribution of viscous dissipation. Concurrently, both frequency dependence exponents (a for G′ and b for G″) increased, indicating an enhanced sensitivity of the moduli to frequency changes and a weakening of the solid-like character of the systems.
For the HP15 and HP20 samples, a different trend was observed (Table 1). Despite the elevated temperature, these systems showed a renewed increase in G′ and a relative decrease in tan δ compared to HP10. At higher hemp protein concentrations, the composite structure seemed to maintain its load-bearing capacity even as the gelatin network substantially weakened. Moreover, the G′ = G″ crossover points for HP15 and HP20 occurred at higher stress values than those observed for HP5 and HP10, confirming greater mechanical resistance and improved structural stability in the high-hemp-protein gels.
At 18 °C, all samples exhibited gel-like properties, as evidenced by the dominance of the storage modulus over the loss modulus (G′ ≫ G″) and low tan δ values (<0.15), typical of gelatin gels stabilized by helical structures. For the pure gelatin sample, the value of G′ ≈ 72 Pa was within the range reported for gelatin gels with low and moderate concentrations (approx. 10–100 Pa) [6,30]. With an increase in HPI share, a rise in network stiffness was observed, with the HP20 sample reaching a storage modulus of 2534 Pa, which corresponds to a more than 35-fold increase compared to the control sample. The scale of this effect is comparable to observations for other gelatin-based composite gels [30,31].
The observed increase in tan δ at 25 °C for HP5–HP10 correlates with a partial change in gelatin triple helices, as confirmed by thermorheological measurements. However, the recovery of G′ in HP15–HP20 indicates the formation of a secondary support network dominated by hemp protein aggregates and hydrophobic interactions, as indicated by FTIR analysis.
The simultaneous, significantly weaker increase in the loss modulus and the systematic decrease in tan δ indicate a transition towards a more elastic and deformation-resistant structure, characteristic of gels in which the rheological response is less dependent on deformation frequency [29,31]. Increasing the temperature to 25 °C led to a gradual weakening of the gelatin network, especially in samples containing low and moderate amounts of hemp protein (HP5–HP10), which manifested itself in an increase in tan δ to values in the range of 0.2–0.4 and is consistent with the partial breakdown of the gelatin structure in the range of 20–30 °C [30,32].
Conversely, for samples with a high HPI share (HP15–HP20), relatively high values of the conservative modulus were maintained, and the intersection point G′ = G″ shifted towards higher stresses. This behavior confirms the increasing contribution of the composite reinforcement mechanism in shaping the system’s mechanical response, in which the hemp protein fraction assumes a dominant load-bearing role [31,33].
2.4. Texture Characteristics of Gelatin–HPI Gels
The HP0 reference sample had the lowest hardness (4.79 N), typical of a gelatin gel with moderate stiffness (Figure 4).
The addition of hemp protein led to a statistically significant increase in hardness, with this effect clearly dependent on its proportion. For samples HP10, HP15, and HP20, an increase in this parameter was observed, with HP20 reaching a maximum (17.79 N). Similarly, gumminess increased from 3.52 N (HP0) to 10.55 N (HP20) (Figure 4). Cohesion, however, decreased with increasing HPI share, with the pure gelatin sample (HP0) being the highest and the maximum HPI load (HP20) the lowest (0.59). The decrease in cohesion suggests a reduction in the structure’s ability to maintain its integrity during successive deformation cycles, indicating that the increase in HPI shares makes the structure more brittle and less homogeneous, despite an increase in its stiffness.
The increase in hardness and viscosity is attributed to higher total solids content, enhanced protein–protein and hydrophobic interactions, and the formation of rigid hemp protein aggregates, resulting in a stiffer network that resists initial compression. However, the emergence of local inhomogeneities and rigid domains reduces the structure’s ability to reorganize after deformation, leading to increased energy dissipation and, consequently, reduced cohesion and resilience.
The springiness (Figure 4) did not differ significantly between the tested samples, confirming that the addition of hemp protein did not affect the gels’ ability to recover after deformation. This means that, despite significant changes in hardness and gumminess, the mechanism of the structure’s elastic recovery is preserved, as observed in amplitude and frequency sweeps. Elastic resistance (resilience) decreased with increasing hemp protein content. The highest value of this parameter was recorded for HP0 (0.88), while the lowest was for HP20 (0.64). The decrease in elastic resistance indicates increased energy losses during the first compression cycle, suggesting a more viscous, less ideally elastic deformation in gels with a high hemp protein content.
A similar trade-off between hardness and cohesion was observed in gels based on gelatin, gellan gum, and inulin, where an increase in hardness from 1.54 to 12.3 N was accompanied by a decrease in cohesion from 0.91 to 0.61 and resilience from 0.85 to 0.36 [34]. These results indicate that the increase in hardness in the HP10–HP20 range is not solely due to the increased solid-phase content but due to the formation of a more rigid composite structure characterized by a weakened elastic response. Similar trends have been described in gelatin and furcellaran gels, in which a moderate addition of polysaccharide increased hardness (from 14.4 to 18.42 N), while its high content led to a marked decrease in cohesion (to 0.49) and deterioration of elasticity, which the authors attributed to excessive crosslinking and/or phase separation [35].
At the same time, the literature indicates that adding plant protein does not always result in stiffening of the gel structure. In gelatin–soy or whey protein systems, a 3.5-fold decrease in hardness was observed when gelatin was partially replaced with globular proteins, which was attributed to changes in electrostatic conditions (pH) and weakening of effective gelatin crosslinking [32]. Interestingly, in our study, the results indicate that hemp protein acts as an active structure-forming and strengthening component in the tested systems, rather than a passive filler, which is confirmed by the simultaneous increase in hardness and gumminess with a decrease in cohesion and resilience parameters.
2.5. External Appearance of the Gelatin–HPI Gels
The HP0 reference sample (5% gelatin) exhibited the highest L* value (74.72) (Figure 5), naturally, indicating a nearly colorless appearance typical of pure gelatin gels. L* represents lightness in the CIELAB color space, ranging from 0 (black) to 100 (white). The incorporation of HPI resulted in a statistically significant decrease in brightness in all composite systems. The strongest fall in L* values was recorded for HP5 and HP10 (47.34 and 46.92, respectively), demonstrating pronounced darkening, but further increases in HPI content only led to a slight increase in L* (49.82–50.44). This trend indicates that, for HPI additions at higher concentrations, the effect becomes partially saturated and does not continue to increase. Similar relationships were observed after the introduction of hemp protein into bread, where the color of the product shifted towards darker tones because of the presence of natural pigments and phenolic compounds [36].
As samples were visibly greenish, the a* values were negative (Figure 5), confirming the shift toward the green region. The most negative a* value was observed for HP0 (−2.60). The introduction of hemp protein resulted in a noticeable progression of green hue, with the a* values ranging from −1.58 to −2.04 in the composite gels. Definitely, the increase in a* among samples HP5–HP20 suggests the HPI modifies the color balance of the gels, most likely due to the presence of natural plant pigments and microstructural changes that affect selective light absorption. Consequently, the b* parameter increased significantly. The HP0 sample showed a low b* value (3.51), characteristic of an almost colorless, slightly yellow gelatin gel. In the composite systems, b* values increased several-fold, reaching maximum values for HP10 and HP15 (14.52–14.58), indicating a strong intensification of yellow-brown coloration. For HP20, a slight decrease in b* (13.06) was observed compared to HP10 and HP15. This behavior may reflect changes in light scattering within a denser composite network or partial masking of color due to increased opacity at the highest hemp protein concentration.
Negative values of the a* parameter indicate a predominance of green colors, but with increasing hemp protein content, the color gradually shifted towards more yellow tones. This phenomenon is attributed to microstructural changes affecting selective light absorption, which was also observed in studies of the color of meat analogues enriched with plant proteins [36]. At the same time, an increase in the b* parameter value indicated an intensification of the yellow-brown color. Similar trends have been described in studies on the enrichment of food products with various plant proteins, where changes in the L*, a*, and b* parameters were closely related to the type and natural color of the incorporated protein raw material. It has been pointed out that the color of the protein raw material is one of the main factors determining the final color of the product [37].
2.6. Antioxidant Properties of Gelatin–HPI Gels
The antioxidant activity of gelatin–hemp gels was assessed using three complementary methods: ABTS radical scavenging capacity (expressed as mg Trolox/kg dry weight), reducing capacity using the FRAP method (mg FeSO4/kg dry weight), and DPPH radical scavenging capacity (mg Trolox/kg dry weight).
The HP0 reference sample showed the lowest ABTS radical scavenging capacity (291 mg Trolox/kg dry weight) (Figure 6), indicating limited antioxidant activity of pure gelatin gel. The addition of hemp protein increased antioxidant activity as ABTS values increased systematically from 473 mg Trolox/kg d.w. for HP5 to 979 mg Trolox/kg d.w. for HP20. The obtained trend clearly confirms that the hemp fraction is the main source of compounds responsible for the ability to neutralize free radicals in the tested systems.
The reducing capacity, assessed by the FRAP method, was undetectable in the HP0 sample (Figure 6). In samples containing hemp protein, a clear increase in FRAP values was observed. The lowest level of reducing capacity was observed for HP5 (0.10 mg FeSO4/kg d.w.), while the highest was for HP20 (13.04 mg FeSO4/kg d.w.). This pattern indicates the presence of electron-transferring compounds in the hemp fraction, such as bioactive peptides, phenols, or other accompanying components.
In contrast to the ABTS and FRAP tests, the values obtained by the DPPH method were similar for all samples tested and fell within a narrow range of 30.6–31.8 mg Trolox/kg DM. The lack of a clear upward trend with the addition of hemp protein indicates that the reaction mechanism with the DPPH radical is less sensitive to the components present in the tested gels or that the availability of these compounds in the gel matrix is limited.
According to the literature, gelatin’s significant antioxidant activity is mainly observed after enzymatic hydrolysis, whereas the unhydrolyzed gel matrix exhibits only weak antioxidant properties [38]. With increasing hemp protein addition, a systematic increase in ABTS values was observed, reaching nearly 1000 mg Trolox/kg dry weight in the HP20 sample. The values obtained are within the lower range of activity reported for raw materials and extracts of Cannabis sativa L., whose antioxidant potential is mainly attributed to the presence of phenolic compounds and lignanamides associated with the protein fraction [39]. A similar trend was observed in the FRAP assay, where an increase in the proportion of HPI corresponded to a higher reducing capacity of the gel. Although the values obtained were significantly lower than those reported for concentrated hemp extracts, this difference is explained by the dilution of bioactive compounds within the gel matrix and by diffusion limitations [40].
In contrast to the ABTS and FRAP results, the DPPH method did not show significant changes with the addition of hemp protein. This phenomenon is consistent with reports indicating that the DPPH test is less sensitive to hydrophilic antioxidants and compounds trapped in biopolymer structures [41].
2.7. Conformational Changes in Composite Gels
FTIR spectra of samples (Figure 7) containing gelatin (G), hemp protein (HP), and gelatin–hemp mixtures (HP0–HP20) show the amide bands (Amide I, II, and III) typical for protein systems, but with increasing HP content, there is a growing contribution from bands characteristic of the plant fraction, in particular signals from aliphatic C–H groups and ester groups [42,43].
All samples show a band in the range of 3280–3304 cm−1 (Table S1), corresponding to N–H (Amide A) and, to some extent, O–H stretching vibrations. In gelatin (G), this band has low intensity (3292.4 cm−1; ABS = 0.072), while in HP, it is much stronger (3280 cm−1; ABS = 0.325), which can be associated with a greater number of polar groups and a greater potential for hydrogen bonding in the plant system. Changes in the intensity of the Amide A band are commonly regarded as a direct indicator of the reorganization of the hydrogen bond network in protein hydrogels [1,44].
The manufacturing process of the gelatin gel (HP0) results in a significant increase in the intensity of this band (3295.3 cm−1; ABS = 0.179), suggesting a strengthening of hydrogen interactions after thermal processing and cooling. In the mixed samples, a clear dependence on HP concentration is observed: for HP10 and HP20, the intensity of the ~3300 cm−1 band is high (ABS = 0.233 and 0.219, respectively), indicating that with larger HP additions, the hydrogel network is stabilized by a greater number of hydrogen bonds between the components (gelatin–HP) [44].
A key element differentiating the spectra is the presence in HP of very strong bands at 2924 cm−1 (ABS = 0.415) and 2853.6 cm−1 (ABS = 0.295) (Table S1), corresponding to the C–H stretching of CH2/CH3 groups, and a band at 3010.3 cm−1 (ABS = 0.209), attributed to =C–H (unsaturated lipids). The 1743.8 cm−1 band (ABS = 0.175) also confirms the presence of C=O ester groups. The increase in the 2924/2854 cm−1 bands indicates the presence of a lipid fraction and the contribution of hydrophobic interactions to the stabilization of the hydrogel structure [45]. The transition from a homogeneous gelatin network to a heterogeneous composite architecture involving hydrophobic interactions and lipid-related domains derived from the hemp protein fraction shows the following changes: (i) an increase in the intensity of the ~3300 cm−1 band (reorganization of hydrogen bonding), (ii) a shift in Amide I towards 1632–1636 cm−1, (iii) an increase in lipid-related C–H bands (2924/2854 cm−1), and (iv) the presence of ester carbonyl groups (~1743 cm−1).
In the composite samples (Figure 7), these signals increase with the addition of HP: from a very weak contribution at HP5 (1744 cm−1; ABS = 0.028) to clear signals at HP10–HP20 (e.g., HP20: 2923.6 cm−1, ABS = 0.259; 2853.7 cm−1, ABS = 0.195; 1743.3 cm−1, ABS = 0.100). In practice, this means that as the HP content increases, a lipid fraction is introduced into the hydrogel matrix, which can interact with protein components through hydrophobic interactions [42,46].
The amide range confirms the dominance of protein components in the tested materials. Gelatin (G) exhibits an Amide I band at 1628 cm−1 (ABS = 0.083), while HP exhibits one at 1633.4 cm−1 (ABS = 0.368). For HP0, the intensity of Amide I increases significantly (1629.1 cm−1; ABS = 0.250), indicating that the gelation process leads to a clear structural reorganization of the protein system [44,47,48].
With increasing HP addition, a shift in Amide I towards 1632–1636 cm−1 (HP10–HP20) and an increase in intensity (e.g., HP10: ABS = 0.311) are observed. This trend can be interpreted as a change in the environment of C=O carbonyl groups, driven by the increasing proportion of interactions between the two protein types. This is consistent with the literature, which treats shifts in the Amide I/II region as indicators of conformational changes and the intensification of interchain interactions in hydrogels [44,48,49].
3. Materials and Methods
3.1. Materials
Hemp protein isolate (HPI) powder was purchased from K&L FOOD Sp. z o.o. (Dębica, Poland). The raw material came from Libya (batch number: G262P1/KUKL/0067). According to the manufacturer’s declaration, the product contained only hemp protein powder, without any functional additives. The nutritional value was 52.7 g of protein/100 g of product, with a fat content of 10.6 g/100 g and carbohydrates of 0.5 g/100 g.
Edible gelatin with a gel strength of 180 Bloom was purchased from AGNEX Sp. z o.o. (Białystok, Poland). The raw material came from the Netherlands (batch number: 0711/6). The product was supplied in powder form and did not contain any technological additives.
3.2. Preparation of Gelatin–HPI Hydrogels
Gelatin–HPI hydrogels were prepared in several stages. First, gelatin (5% w/w) was hydrated in a portion of water and then dissolved at 50 °C until a homogeneous solution was obtained. At the same time, HPI in the appropriate amount (5, 10, 15, or 20% (w/w)) was hydrated in water for 3 h at room temperature to ensure its complete hydration. After the hydration stage was completed, the hemp protein was added to the gelatin solution, and the resulting mixture was heated at 80 °C for 10 min to uniform the system and activate the interactions between the components. The samples were cooled under static conditions in a refrigerator at 4 °C (convective cooling) for 16 h to ensure complete gelation, after which the hydrogels were subjected to further analyses.
3.3. Analytical Methods
The water absorption capacity (WAC) of the gels was determined according to the procedure described by [50], with minor modifications. Each experimental variant was performed in at least three replicates. The WAC value percentage was calculated using the following equation:
where m0 is the mass of the empty centrifuge tube, m1 is the total mass of the tube with the gelatin or gelatin–hemp protein gel before centrifugation, and m2 corresponds to the mass of the sample after centrifugation (4000 rpm, 4 °C, 10 min).
A temperature ramp test was performed to evaluate the thermo-induced viscoelastic behavior of the samples, according to [50], with modifications related to the measuring system. The oscillatory measurements were carried out using the same MCR 102 rotational rheometer (Anton Paar, Graz, Austria) equipped with a plate–plate geometry (25 mm diameter, 1 mm gap). Temperature control was ensured by a Peltier temperature control system. To prevent sample dehydration during heating, the exposed edges of the sample were coated with paraffin oil. The test was conducted under oscillatory conditions at a constant frequency of 0.1 Hz and constant stress of 100 Pa, within the linear viscoelastic region. The samples were first heated from 25 °C to 90 °C, then cooled from 90 °C to 5 °C at 1 °C/min. During the thermal treatment, changes in the storage modulus (G′) and loss modulus (G″) were continuously recorded. Each measurement was performed in triplicate.
Frequency- and amplitude-sweep characterization of the samples was performed using the same setup equipped with a plate–plate (diameter 25 mm, gap 1 mm), at 18 and 25 °C. The sample was then positioned on the lower measuring plate and allowed to reach equilibrium for 5 min. To limit material dehydration, the sample edges were protected with paraffin oil. Subsequently, a frequency-sweep test was conducted at a constant strain of 0.5% over the range of 0.1 to 10 Hz. This was followed by an amplitude-sweep test, which examined the response at varying strain levels (0.01–100%) at a constant frequency of 1 Hz. The modulus intersection point, i.e., the moment at which G′ = G″, was also determined. The storage modulus (G′), loss modulus (G″), and loss angle tangent (tan δ) values were recorded. All determinations were performed in triplicate using RheoCompass v. 1.24 software.
Gels were subjected to texture profile analysis (TPA) using an FC200STAV500/300 texture analyzer (AXIS, Gdańsk, Poland). A cylindrical polyethylene flat-ended probe (Φ = 35 mm) was used. Samples were compressed to 50% of their original height in a double compression cycle test. The pre-test, test, and post-test speeds were set at 200 mm/s. The time interval between compressions was 10 s. Measurements were performed at 20 °C. Data recording and processing were performed using AXIS FM software version v.2_18.
The color of gelatin–hemp hydrogels was assessed immediately after the gelation process. Measurements were performed using a CR-310 colorimeter (Konica Minolta, Ramsey, NJ, USA). Before each series of measurements, the device was calibrated using standard black-and-white reference plates. The following color parameters were recorded: lightness (L*), red–green component (a*), and yellow–blue component (b*). Each value was determined as the average of three independent readings for a given sample.
Antioxidant activity was determined according to the protocol [51]; prior to analysis, the hydrogel samples were freeze-dried. Methanol extracts were prepared by weighing 0.5 g of dry sample and adding 10 mL of methanol. The resulting suspensions were thoroughly homogenized, then extracted for one hour and centrifuged at 10,000 rpm/min, after which they were used for further determination of antioxidant properties.
Antiradical activity was also determined using the ABTS method. To 0.0816 mL of the extract, 4.0 mL of a previously prepared stable ABTS+∙ cation radical solution was added. Absorbance was measured at 734 nm 10 s after mixing the reagents. A Trolox standard in the concentration range of 100–800 μmol/L was used for calibration, and the results were expressed as mg TE/kg of dry sample weight.
The reducing capacity of the samples was determined using the FRAP method. To 0.138 mL of the extract, 3.992 mL of freshly prepared FRAP working solution was added. After 15 min of incubation at 38 °C, the absorbance was measured at 593 nm. The calibration curve was prepared using iron (II) sulfate (FeSO4) in the concentration range of 0.001–0.008 μg/L. The results are presented as milligrams of FeSO4 equivalent per kilogram of dry sample weight.
Antiradical activity was determined using the DPPH method. To 0.138 mL of the extract, 4.0 mL of a working solution of DPPH∙ radical in methanol at a concentration of 0.1 mM was added. After 20 min of incubation in the dark, the absorbance was measured at a wavelength of 517 nm. The antioxidant capacity of the samples was determined using a Trolox calibration curve (100–800 μmol/L), and the results are expressed as milligrams of Trolox equivalent (TE) per gram of dry sample weight.
The FTIR spectra of the samples were recorded using a Nicolet 6700 FT-IR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a diamond crystal cell for attenuated total reflection (ATR) mode. Each sample was scanned 64 times within the wavelength range of 400–4000 cm−1 at a resolution of 4 cm−1. To ensure precise measurements, background correction was applied using the air spectrum. The analysis was conducted on gelatin gels containing hemp protein (5, 10, 15, and 20%) and on gelatin gel alone (5% gelatin w/w). Prior to the measurement process, the gels were subjected to a freezing temperature of −70 °C and subsequently underwent a lyophilization phase spanning 23 h, employing an Alpha 1–4 LSC plus lyophilizer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany).
Statistical analyses were carried out using the Statistica 13.3 software (StatSoft, Tulsa, OK, USA). The data were analyzed by one-way analysis of variance (ANOVA), followed by Tukey multiple range test to determine statistically significant differences between mean values, indicated by different letters (a–d). Results are reported as mean values for each experimental variant. Differences were considered statistically significant at p < 0.05.
4. Conclusions
The obtained results indicate that the addition of hemp protein significantly modifies the structure and functional properties of gelatin hydrogels. As its proportion increased, there was a transition from a classic, thermoreversible gelatin gel to reinforced composite systems characterized by significantly increased stiffness, water-binding capacity, and resistance to mechanical deformation. Rheological analyses showed that at low hemp protein content, the gelatin network played a dominant role, whereas at higher concentrations (≥15%), the viscoelastic response was increasingly determined by the hemp protein structure, which maintained mechanical continuity even above the melting point of gelatin.
Texture profile analysis confirmed a significant increase in the hardness and gumminess of the gels, accompanied by a decrease in cohesion and elasticity, indicating the formation of stiffer structures that were less susceptible to repeated deformation. FTIR spectroscopy results provided evidence at the molecular level, confirming the intensification of hydrogen bonding and the increased contribution of hydrophobic interactions associated with the lipid fraction of hemp protein. At the same time, a significant increase in the antioxidant potential of hydrogels, assessed by ABTS and FRAP methods, as well as clear changes in color parameters with an increase in the proportion of the plant fraction, were demonstrated.
The experiment proved that hemp protein not only acts as a passive filler but also as an active structural and functional component in gelatin hydrogels. The results confirm the possibility of consciously shaping the mechanical, thermal, and bioactive properties of such systems by controlling the proportion of hemp protein, opening prospects for their use in functional foods, nutraceuticals, and carrier systems for bioactive ingredients.
Acknowledgments
The authors thank each other for an inspiring experimental topic.
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules31050885/s1. Table S1: FTIR peak positions (wavenumber, cm−1) and absorbance for samples G, HP, and HP0–HP20.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Data are available upon request.
Conflicts of Interest
The authors declare no conflicts of interest.
Funding Statement
This research received no external funding. APC was covered by the internal project of Wroclaw University of Economics and Business—Otwarta Nauka (Open Science).
Footnotes
Footnote Group
References
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Associated Data
Supplementary Materials
Data Availability Statement
Data are available upon request.