1. Introduction
The use of plant proteins has increased significantly in recent years. Proteins from plant sources are considered isolated macromolecules from highly renewable and abundant sources; their uses and potentization are among current market trends, and they are gaining popularity as a non-allergenic source of protein that offers a clean label to food products [
1,
2].
These proteins can be used as functional ingredients, as they have been proven to reduce the risk of cardiovascular disease and blood pressure [
3]. In addition to their nutritional contribution, the flexible, amphiphilic, amphiprotic, and dynamic protein structure according to the conditions of the environment give proteins functional properties, such as the capacity to modify the rheological properties and surface tension of the medium, to stabilize colloidal systems, as well as the absorption capacity of water and fat, the ability to form gels, and the capacity to encapsulate bioactive compounds by techniques such as spray drying, coacervation, and ionic gelation [
4,
5,
6]. Isolated soy proteins have been studied more frequently than other proteins from plant sources, possibly due to their wide commercial availability [
5]. However, some research has used protein isolates from lentils, sunflower seeds, quinoa, peas, and beans as emulsion stabilizers, gelling agents, and carrier material in the encapsulation processes of α-tocopherol [
7], flaxseed oil [
3], probiotic bacteria [
8], and vitamin D [
6,
9].
The annatto seed extract has a high content of bixin and seven polyphenol compounds, such as catechin, chlorogenic acid, chrysin, butein, hypolaetin, licochalcone A, and xanthohumol [
10]. These bioactive compounds can deactivate sensitizers’ excited triplet state, which is usually associated with photosensitization to quench singlet oxygen, sweeping free radicals, and the denaturation of proteins from cell membranes [
11,
12,
13]. These characteristics give annatto seed extracts the ability to be antioxidant and antimicrobial [
13,
14]. As stated in the literature, these bioactive compounds are not stable under conditions such as extreme pH, light, and high temperatures, which leads to the enforcement of techniques such as encapsulation to increase its stability and enhance its applications in the food, pharmaceutical, and cosmetic industries [
13].
Our recent research suggests that lentil (LP) and quinoa (QP) protein isolates represent a highly suitable carrier material for the encapsulation process of annatto seed extract by ionic gelation, with encapsulation efficiencies of 68.61% and 58.38% for LP and QP, respectively [
15]. Regarding the stability of the bioactive compounds, both proteins protect and stabilize the extract’s antioxidant and antimicrobial activities, even up to a storage temperature of 65 °C for 12 d [
15].
Encapsulation technology plays a crucial role in enhancing the stability, bioavailability, and targeted delivery of bioactive compounds in food. By entrapping these compounds within protective matrices, encapsulation shields them from adverse environmental conditions (such as temperature and oxygen) and physiological factors (such as gastric acid). Among the various coating materials used, proteins—such as whey proteins, casein, and soy proteins—stand out as effective encapsulation agents [
15]. These proteins form stable microcarriers or nanoparticles that can modulate release kinetics and protect sensitive ingredients during food processing and storage. Additionally, protein-based encapsulation allows for tailored release profiles, ensuring optimal bioactivity. Overall, understanding the impact of different encapsulation materials on bioactive compound release is essential for advancing functional foods and nutraceuticals.
However, the application of proteins from plant sources in the encapsulation processes of active compounds remains very limited, mainly due to their low solubility in water and the need for high concentrations that are required to generate a change in the viscoelastic properties of the dispersions. For this reason, in several studies, the structural modification of proteins has been proposed as a strategy to generate changes in their functional properties, allowing them to increase their solubility in water, emulsifying capacity, and capacity for gel formation [
16,
17,
18,
19,
20]. These properties can increase the encapsulation efficiencies of modified proteins to potentiate their use as coating materials in the encapsulation processes of active compounds. Some of the most outstanding research studies were carried out by Nesterenk et al. in 2012 [
21]. They evaluated the effect of enzymatic hydrolysis, the treatment of N-acylation, and N-cationization of isolated soy proteins on their functional properties, specifically, on the encapsulation capacity of α-tocopherol, using the spray drying method [
21]. For the report, the structural changes produced by the three methodologies, with a more significant effect of N-acylation, allowed for decreasing the drop size and modifying the viscoelastic properties of the emulsions produced during the encapsulation process. As a result, an increase was obtained in the encapsulation efficiency of the bioactive compounds from 80% to 87% [
21].
Likewise, other researchers have evaluated the effect of enzymatic hydrolysis, N-acylation, and N-cationization on the functional properties of proteins. Adler-Nissen and Olsen observed that the emulsifying and foaming properties of soy protein could be improved by enzymatic hydrolysis to a limited degree of hydrolysis of up to 10%, and the peptides obtained must be large enough to form a stable film around the droplets of the dispersed phase [
22]. Meanwhile, the use of modifications such as N-acylation, covalently linking a fatty chain in the protein structure, and N-cationization implies the generation of positive charges in the molecules by grafting cationic groups. Little explored methodologies have been used to increase the functional properties of proteins [
18,
23]. This study aimed to (i) evaluate the effect of structural modifications by enzymatic hydrolysis, N-acylation, and N-cationization of the LP and QP on their functional properties and (ii) compare the encapsulation efficiency of annatto seed extract by ionic gelation employed as the carrier material of LP and QP in native and modified states.
4. Discussion
Results obtained by the modification degree of the proteins by enzymatic hydrolysis, N-acylation, and N-cationization, reported in
Table 1, show that the variation in the percentage of protein modification is attributed to the disposition of NH
2 groups in the protein structure, according to its folding and the steric hindrance that nearby groups can generate in these experimental conditions [
18]. For SP isolated by acid precipitation, structural modifications such as N-acylation and N-cationization were evaluated, reporting higher degrees of modification than those obtained in this study—~60% for N-acylation and ~ 92% for N-cationization [
18,
29]. This shows that the method of obtaining the protein and the protein source influence these two chemical structural modifications. On the other hand, enzymatic hydrolysis using Alcalase 2.4L achieved percentages of plant-based proteins comparable with results reported in the literature [
29,
46].
Analysis of the secondary structure of native and modified proteins is shown in
Figure 1d. The structures of α helices, β leaves, and random coil were observed in the deconvolved spectra according to four main Gauss bands centered at ∼1654, ∼1638, and ∼1670 cm
−1 [
31]. The results obtained reported an average content between ∼5 and 10% of random coils and turns in the secondary structure of the proteins regardless of the source. Furthermore, these random coils and turn percentages held constant for chemical and enzymatic modifications for all proteins (
Figure 1d). The α-helix content is usually less constant than β-sheets’ content for native and modified proteins; however, it presents a slight decrease in the enzymatic modifications. The longer the hydrolysis time, the greater the decrease in the α-helix content in the secondary structure of the protein.
This effect was due to the breaking of the peptide bonds caused by the Alcalase 24L; when breaking these bonds, the entire secondary structure is modified. The effects of enzymatic hydrolysis on native proteins are not so marked in the analyses of the FT-IR spectra and secondary structure. The analysis is carried out on a set of proteins obtained from a specific source, with the bands being the weighted expression of all the proteins present.
Figure 1d shows that the mean proportion of β-sheets for all proteins, both native and modified, was between 20% and 30%. The results obtained for β-sheets were consistent with those reported for proteins from different plant sources, where β-sheet-like folds are predominant [
31]. Xuan Li et al., (2018) reported a percentage of ~29% β-sheets for isolated quinoa proteins [
46]. Similarly, Meng and Ma (2001) reported that 36% and 19% of the folding of the secondary structure of bean globulins were β-sheets and α-helices, respectively [
47]. On the other hand, it was reported that lentil proteins had ~47% β-sheet structures against ~25% α-helices that were quantified in samples of native lentil proteins [
48].
The differential scanning calorimetry (DSC) thermogram of plant proteins reported that the highest Td1s were those reported for LP, regardless of whether it was in its native or modified state, which is due to the close relationship between Td1 with the amino acid composition and the secondary structure of the proteins α-helices, β-sheets, random coils, and turns. The higher the amino acid chain and folding in the protein structure, the greater the energy requirement for denaturation and, therefore, the higher Td1 [
31]. This behavior is supported by the results reported in
Figure 1 and
Figure 3, where it is reported that among the studied protein sources, all presented a comparable percentage in the content of α-helices and β-sheets. Still, LP reported a higher molecular weight than QP and SP. Likewise, Carbonaro et al. (2008) studied the thermal stability of proteins extracted from lentils and black beans, reporting that lentil proteins showed greater thermal stability than bean proteins, mainly attributed to the higher content of β-sheets [
49]. The effect of the hydrolyzed proteins was due to the partial denaturation of the proteins by the enzymes used in the hydrolysis process, which cut the peptide bonds and decreased the molecular weight and folding of the proteins, entailing a lower expenditure of energy on the denaturation processes and, therefore, no endothermic peak. This is in line with Molina and Cañon’s description in their 2001 study of the thermal behavior of soy proteins with and without various degrees of hydrolysis [
50]; they determined that increasing the degree of hydrolysis, the endothermic peak, which describes the denaturation process, decreases significantly [
50].
Regarding the proteins derivatized by N-acylation and N-cationization, increases in the reported Td1 were obtained (
Table 2). Modifications by N-acylation and N-cationization affect protein conformation by promoting the deployment of the secondary structure, which favors functional properties, such as protein solubility [
29]. By increasing the solubility of the protein, the hydrogen bonds with water increase, and the proteins have more bound water than native proteins; therefore, a higher temperature is necessary for protein denaturation [
51].
In general, the percentages of solubility increased at neutral and low pHs when executing the hydrolysis process of proteins compared to native proteins. In doing so, soluble peptides and more exposed ionizable carboxyl and amino groups are obtained on the protein, improving solubility [
31]. Due to the degree of hydrolysis being so close between the 15 min and 60 min treatments for all protein sources (
Table 1), the solubility profiles are very comparable between the same protein source for both hydrolysis times. These results coincide with other reports where the hydrolysis process favors the solubility of soy, lentil, and quinoa proteins [
52,
53,
54].
The solubility profiles of the proteins modified by N-cationization and N-acylation presented variations compared to the profiles of native proteins proportional to the percentage of the degree of modifications reported in
Table 1. The proteins modified by N-cationization obtained a profile with higher solubility in the acid pH ranges than native proteins. However, these same proteins reported lower percentages of solubility close to pH 7. The cationization increased the net number of positive charges for the protein and reduced the number of NH
2 available. This structure change modified the general balance between acidic and basic groups in the native protein, resulting in a change in the solubility profile, as shown in
Figure 3. Likewise, the proteins derivatized by N-acylation presented modifications in their solubility profiles, increasing the percentages of solubility between pH 4 and 9 due to the decrease in reactive NH
2 groups that had already reacted during the modification processes.
The functional properties of the native and modified proteins are shown in
Table 3,
Table 4 and
Table 5. The type of structural modification was the independent variable that affected the studied parameters. The structural parameters, such as the z-potential (ζ), the isoelectric point (pI) of the proteins, the molecule weight (MW), and the free thiol groups (SHs), presented statistically significant differences (
p-value < 0.05) between the modified and native protein. ζ values of the native and modified protein dispersions at pH 10—the pH in which the proteins presented their highest percentage of solubility (
Figure 3)—reported a decrease for proteins modified by N-cationization and N-acylation compared to native proteins, contrary to hydrolyzed proteins. ζ values obtained for the proteins at pH 10 were negative due to the neutralization of the acid groups and the ionization of the NH
2 groups on the protein structure by the alkaline medium. The decrease in ζ after the modification processes by N-cationization and N-acylation was due to the modification of the primary amino groups that were replaced by positively charged groups from DDC and GTMAC [
30]. The proteins modified by N-cationization and N-acylation, due to having fewer amino groups to neutralize, experienced a decrease in ζ for the native proteins; therefore, the quantification of ζ confirmed the modification of the proteins, which is a relationship that is supported by the percentage of the degree of modification for each protein source reported in
Table 1. Concerning the variation in the solubility profile and the ζ of the native and modified proteins, the pI of the proteins presented an increasing trend for the modified proteins by N-cationization and N-acylation; however, they did not show statistically significant differences (
p-value > 0.05).
The breaking of these peptide bonds in the enzymatic hydrolysis process generated the exposure of the SH groups of the protein that were not free in the native structure. A statistically significant increase in this parameter was observed in the hydrolyzed proteins compared with native proteins. The modified proteins did not show a uniform behavior of increasing or decreasing SH groups; however, this variation was due to the structural modification generated by the incorporation of DDC and GTMAC, which favors the detachment of the protein tertiary structure, exposing some functional groups that were buried inside the structure in its native state [
30].
The chemical and enzymatic modifications studied reported the ability to improve the functional properties of proteins, such as solubility, emulsion, and foam formation capabilities, increased hydrophobicity, and swelling power, which are results that coincide with those obtained in this study [
18,
55]. Functional properties such as WHC, FAC, and EAI; the stability of the emulsions determined by ESI and the Z potential in the emulsion (ζe); and the gel formation temperature (Tgel) of the native and modified proteins are shown in
Table 3,
Table 4 and
Table 5. WHC and FAC were functional properties that presented variations due to the structural modifications of the proteins regardless of the source studied. WHC in the majority of cases did not present statistically significant differences (
p-value > 0.05) between the modified proteins and the native protein. However, the enzymatic hydrolysis processes did show a statistically significant decrease (
p-value ˂ 0.05) for WHC, and the N-cationization processes showed an increasing trend for WHC despite not showing statistically significant differences (
p-value > 0.05).
On the other hand, FAC reported an increase with statistically significant differences (
p-value ˂ 0.05) for proteins modified by both chemical and enzymatic processes. In the case of enzymatic hydrolysis, the decrease in WHC was probably attributed to the reduction in the length of the protein’s molecular chain, which generated the greater exposure of the hydrophobic regions of the protein that decreased WHC and, in turn, increased FAC [
17,
29]. Regarding N-cationization, the incorporation of the polar groups on the protein structure increased its amphiphilic character, which, in turn, affected the protein conformation by promoting the deployment of the tertiary structure, further exposing the embedded hydrophobic groups in the structure, favoring the WHC and FAC [
17,
29]. Finally, the binding of the fatty acid chains to the protein at the N-acylation process also increased the amphiphilic properties of the protein, which explains the increase in FAC in all the proteins modified by this chemical process [
17,
29].
The EAI, ESI, and ζe obtained for the proteins in their native and modified states presented statistically significant differences (
p-value ˂ 0.05). ζe is considered as a factor related to the stability of the emulsions since at absolute values of surface charge (ζ) greater than ±30 mV, the repulsion of the oil droplets in the colloidal system was generated and, therefore, had better stability against coalescence [
56]. This behavior of colloidal systems stabilization was related to the solubility changes, the WHC, and the FAC of the proteins attributed to the structural changes, such as the reduction in the polypeptide chain and the incorporation of the hydrophobic and polar groups explained above. In the case of soy proteins (
Table 3), the EAI did not present statistically significant differences between the modified proteins and the native protein (
p-value > 0.05); however, the ESI and ζe presented an increase with a statistically significant difference (
p-value ˂ 0.05) for ASP, CSP, and H15SP compared to native proteins (NSPs). H60SP exhibited a decrease in the parameters related to the stability of the emulsion, possibly due to the increase in the degree of hydrolysis, which, by further breaking the polypeptide chains of the protein, generated short chains of hydrolyzed proteins that could not efficiently wrap the drop of oil, and therefore, were less stable. QP showed a comparable behavior for EAI with soy proteins (
Table 4), differentiating the significant increase in EAI for H15QP compared with NQP and a decrease for H60QP compared with NQP. In this case, enzymatic hydrolysis generated a greater effect than the chemical modifications used, possibly due to the difference in the low percentages of the degree of modification (
Table 1) obtained for QP between the different modifications. The stability of the emulsions obtained with the native and modified quinoa proteins showed comparable behavior with the SP described above. However, the variation is proportional to the degree of modification. Finally, the lentil proteins presented a lower EAI (
Table 5) but better emulsion stability—ESI and ζe—than the proteins from the other two sources studied. Despite these differences, the trend in the behavior of the parameters of EAI, ESI, and ζe was the same as those previously described based on the types of modifications studied.
The reported gelation temperature (Tg) for the native and modified proteins is shown in
Table 3,
Table 4 and
Table 5. The temperature sweep used for the Tg determination uses the storage modulus (G′) and the phase change angle (δ) as parameters to define the temperature where the change in protein dispersion to a gel-like system is promoted due to the formation of disulfide bonds and electrostatic interactions [
57,
58]. Furthermore, δ determines the hardness of the gel obtained. The Tg of all the proteins modified by N-cationization and N-acylation increased regardless of the source of the protein, while for the hydrolyzed proteins, there was no gel formation (
Table 3,
Table 4 and
Table 5). The increase in Tg of the proteins modified by N-cationization and N-acylation coincides with the results obtained in the thermograms reported in
Table 2, specifically in Td1, which refers to the denaturation temperature of the proteins. The impossibility of the fractions of the hydrolyzed protein molecules to form an adequate network structure during the heat treatment does not allow the hydrolyzed proteins to form the gel. This behavior has been previously reported for proteins from other plant sources [
27].
Microencapsulation processes by ionic gelation are commonly performed using sodium alginate as the coating material. Alginate refers to a group of naturally occurring anionic polysaccharides extracted from brown algae. These linear polymers consist of 1,4-linked β-D-mannuronic acid (M) and 1,4 α-L-guluronic acid (G) chains, which can be arranged in either homogenous (poly-G, poly-M) or heterogenous (MG) block-like patterns. In this process, the COO- groups of alginates react with calcium ions present in the gel-forming solution, resulting in the formation of a hydrogel that encapsulates bioactive compounds of interest.
The encapsulation efficiency in ionic gelation processes is attributed to the material’s ability to form gels in the presence of ions through directed covalent bonds. Additionally, the affinity between the coating material and the active compound before gel formation plays a crucial role. Greater affinity and solubility between the material, the active compound, and the medium can lead to higher encapsulation efficiency [
59]. These parameters explain how protein structural modifications, protein sources, and the bioactive compound’s linear expression interact statistically to affect encapsulation efficiency, as observed in
Table 7 and
Figure 7. For instance, modifying protein structures through hydrolysis can expose negatively charged radical groups like COO-, which are abundant in aspartic and glutamic amino acids found in plant proteins. Increasing exposure to these compounds enhances gelation efficiency but may also impact encapsulation efficiency. Incorporating basic and acidic groups into protein structures similarly modifies charges and gel formation processes, potentially decreasing efficiency.
Some of the results obtained agree with those reported by other authors. Nesterenko et al., between 2013 and 2014, evaluated the structural modification of soybean and sunflower seed proteins as a strategy to increase the encapsulation efficiency of α-tocopherol by spray drying [
7,
17,
21,
29,
30]. They applied enzymatic hydrolysis, N-acylation, and N-cationization to increase both proteins’ functional properties and encapsulation efficiency. N-acylation was the modification that achieved the highest increase for both proteins, proceeding from a native state encapsulation efficiency of 79.7% and 92.6% for soy and sunflower seeds proteins, respectively, to 94.8% and 99.6% for the same proteins modified by N-acylation.
If different behaviors comparable to those reported by Nesterenko et al. were obtained regarding the modifications of the proteins’ functional properties, the encapsulation efficiencies would be much lower than those obtained in Nesterenko et al.’s study, and the variations in the modifications did not show any differences. Statistical significance obtained in our study did occur in the one reported by Nesterenko and company. This is mainly attributed to the encapsulation methods employed. While Nesterenko et al. employed spray drying, we standardized and employed the ion gelation encapsulation process. On the other hand, comparing the EE obtained in the encapsulation processes of natural extracts by ionic gelation using sodium alginate as a coating material, we found EE reports to be comparable to those obtained with proteins in their native state and modified by N-acylation and N-cationization. Belščak-Cvitanović et al., 2018, encapsulated cocoa husks, poppy, and hemp bioactive compounds, reporting 73% EE for calcium alginate hydrogel particles obtained by external ionic gelation [
59]. Likewise, the results of Moura et al. in 2018 [
60] obtained the EE of polyphenols from the extract of
Sambucus nigra L. for 74.4% and 88.5% of particles obtained by external ionic gelation.