Physicochemical characteristics and antioxidant stability of spray‐dried soy peptide fractions

Abstract The direct addition of health‐promoting peptides to food products is limited due to their physicochemical instability and bitter taste as well as their bio‐functionality may be influenced by M W . In this study, SPI hydrolysate (SPIH) was Alcalase‐prepared, size‐fractionated (<10, 10–30, and 30–100 kD), and the amino acid composition of peptide fractions determined. The physicochemical properties, morphology, and antioxidant stability of the fractions were also investigated after spray‐drying encapsulation in maltodextrin‐WPC carrier. The two low M W peptide fractions (especially, PF < 10) were more active than intact SPI, SPIH, and high M W peptide fraction in scavenging free radicals and chelating transition metal ions. As compared to the particles containing SPIH, those containing the smallest peptide fraction (PF < 10) had higher solubility and hygroscopicity, lower production yield and wettability, and more wrinkles, indentations and surface roughness. The highest antioxidant stability during spray‐drying was observed for the two low M W peptide fractions, which examined by scavenging of free radicals of DPPH (88%), ABTS (97%), OH (93%) and NO (80%), chelating of iron (88%) and copper (87–90%) ions, reducing power (93%), and total antioxidant activity (90%). This finding reflects more structural and biological stability of the low M W fractions to shear stress and dehydration during spray‐drying, as compared with SPIH. The spray‐drying encapsulated soy peptide fractions may be used as nutraceuticals for the development of functional foods.

anti-cariogenic, antidiabetic, anti-hypertensive, anti-microbial, antioxidative stress, anti-inflammatory, cholesterol lowering, growth enhancing, immunomodulatory, mineral binding, radical scavenging, and regulation of glucose-insulin homeostasis and satiety (Wang & Selomulya, 2020). The fortification of foods with peptides is also valuable for populations with special nutritional needs (e.g., infants, athletes, and the elderly) owing to their ease of digestion (Michaelidou, 2008). Moreover, some peptides may have superior techno-functional characteristics. Nevertheless, several challenges, such as high hygroscopicity, physicochemical instability, perishability, and cross-reactivity with other compounds, limit the direct use of peptides in food and dietary supplement formulations. Another disadvantage of peptides is their unpleasant bitter taste, which reduces the sensory acceptability of fortified products (Mohan et al., 2015;Udenigwe, 2014). Therefore, it is necessary to use techniques to minimize these challenges, and microencapsulation is the most important technology for this purpose.
The microencapsulation or delivery technology itself includes several methods, of which spray-drying is the most common and economical, and is commercially available on a large scale. Spraydrying converts various water-based feeds containing bioactive compounds (e.g., extracts, solutions, and dispersions) into flowable powders , and fixes the core materials within a particulate carrier matrix in solid state. Therefore, considerable research efforts have been conducted on the spray-drying microencapsulation of bioactive peptides and protein hydrolysates.
However, the complex structure and specific physicochemical characteristics of peptides/proteins make them sensitive to the environmental stresses encountered during spray-drying. For example, the mechanical stresses created by atomization and by binding these surface-active components to air-liquid interface, as well as the thermal and dehydration stresses during spray-drying, lead to conformational changes, denaturation, aggregation, and thereby inactivation of them (Ajmera & Scherließ, 2014). That is why different strategies, such as modifying the composition of feed formulation and using various carriers (e.g., proteins, polysaccharides, sugars, amino acids, and surfactants), have been applied to protect peptides during spray-drying (Mohan et al., 2015).
Soybeans as an excellent and inexpensive source for protein and health-promoting compounds have a high world annual production (more than 353 million metric tons in 2020; FAOSTAT, 2023) and a special value in diets. Soybean peptides have important functional characteristics, such as good solubility, emulsifying ability, and biological activity (Liu et al., 2012). It has been reported that spray-drying is more effective than freeze-drying for microencapsulation of non-fractionated soybean hydrolysates in soy protein isolate-maltodextrin mixture, and yields a final product of better quality (e.g., solubility, flowability, and bitterness-masking) .
The biological activity and techno-functional characteristics of peptides show a complex dependence upon their molecular weight (M W ) distribution, amino acid composition and sequence, degree of hydrophobicity, and solubility (Phongthai et al., 2018). The M W of the peptides to be spray-dried can influence on the quality characteristics of final powder due to having different surface activity and behavior at air-liquid interface during drying. Since there is sparse information about this issue in literature, this study aimed to investigate the amino acid composition, antioxidant capacity, and spraydrying encapsulation of size-fractionated soy protein hydrolysates, and to assess the remaining antioxidant activity, and physicochemical and morphological characteristics of the spray-dried peptides.

| Enzymolysis of SPI
SPI (5% w/v) was dissolved in phosphate buffer solution (PBS, 0.01 M, pH 8) under magnetic stirring at 50°C for 30 min. Then, Alcalase (2% v/v) was added to it, and stirred at 50°C and pH 8, for 120 min. Afterward, the enzyme was inactivated by immersing the reaction flask in a hot water bath (95°C) for 15 min. Finally, the undi-

| Amino acid composition
SPI, SPIH, and peptide fractions were hydrolyzed in 6 N HCl at 110°C for 24 h, then their amino acid composition assessed by an HPLC system equipped with reversed-phased column (Novapack C18, 4 μm, Waters, Milford, MA) and reported as mg/g dry sample (You et al., 2010). The tryptophan content of samples was determined after alkaline hydrolysis.

| Spray-drying encapsulation of peptide fractions
The SPI peptide fractions were microencapsulated by spray-drying technique as following. A 2 g portion of each freeze-dried peptide fraction was dissolved in 20 mL PBS (0.1 M, pH = 7.4) and mixed with 80 mL of carrier solution (20% w/v of maltodextrin and WPC in equal ratio). The final solution was then microencapsulated by feeding into a lab-scale spray dryer (Büchi B-191; Büchi Laboratoriums-Tecnik, Switzerland) under the following process parameters (Sarabandi et al., 2022): inlet-air temperature (130 ± 1°C), outlet-air temperature (82 ± 2°C), feed rate (5 mL/min), drying air flow rate (0.54 m 3 h −1 ), nozzle diameter (0.7 mm), and air pressure (5.6 bar). The spray-dried powders were packed in airtight bags and kept in refrigerator until use.
The powder production yield (% w/w) during spray-drying was calculated by dividing the weight of the obtained powder by the total weight of the primary solid matter and then multiplying by 100.

| Antioxidant characterization
The activity of SPI and freeze-dried SPIH and peptide fractions in scavenging radicals of DPPH, ABTS, hydroxyl and nitric oxide, and chelating prooxidant transition metal ions of iron and copper, their reducing power, and their total antioxidant activity (TAA) were measured in the same manners as described by Sarabandi and Jafari (2020). The retention percentage (%R) of each of the abovementioned antioxidant activity indices during spray-drying was calculated from equation: %R = (A 2 /A 1 ) × 100, where A 1 and A 2 are the values of that index in the spray dryer feed and in the corresponding reconstituted powder, respectively.

| Physicochemical properties of spraydried powders
The moisture content, water activity (A W ), bulk and tapped densities, solubility, wettability, and hygroscopicity of spray-dried pow-

| Morphology of spray-dried powders
The morphology of spray-dried powders was assessed using a Hitachi PS-230 scanning electron microscope (SEM) under 25 kV accelerating voltage after coating with a gold film.

| Statistical analysis
The experiments were performed in triplicate, and the data were reported as mean ± SD and analyzed by one-way ANOVA using SPSS software ver. 19.0 (SPSS Inc., Chicago, IL). The Duncan's test was employed to determine statistical differences (p < .05) between selected treatments.

| Amino acid composition
The amino acid composition is a key factor influencing biological activity and functional characteristics of peptides. Table 1 shows the amino acid composition of intact-SPI, SPIH, and SPI peptide fractions with different M W distribution (PF < 10, PF-10-30, PF-30-100). No obvious difference was observed in amino acid composition of these samples. Considering that Alcalase is a nonspecific protease (Yu & Mikiashvili, 2020), it is the difference in M W of the hydrolysates fractions that mainly affects their characteristics in this study. Glutamic acid, aspartic acid, arginine, leucine, and lysine were the predominant residues in SPI and its hydrolysates, and their total hydrophobic and antioxidant amino acids (HAA and AAA) were found to be about 34% and 16%, respectively. In another study, different endopeptidases (Alcalase, Flavourzyme, Thermolysin Proteinase K, and Pepsin + Pancreatin) were employed to hydrolyze rapeseed protein isolate, and it was found that Alcalase produces the highest yield of protein hydrolysate and its hydrolysate contains significant levels of HAA (28%) and AAA (6.7%) (He et al., 2013).

| DPPH and ABTS radicals scavenging
The capacity of intact-SPI, unfractionated hydrolysate (i.e., SPIH), and peptide fractions with different M W distribution in inhibiting DPPH and ABTS free radicals were examined (Figure 1a). The SPIH was significantly more efficient than intact-SPI in scavenging free radicals of DPPH (65% vs. 33%) and ABTS (57% vs. 32%). Also, the peptide fractions PF < 10 and PF-10-30 had more DPPH and ABTS scavenging activities than fraction PF-30-100 ( Figure 1). The M W of peptides plays a decisive role in their antioxidant activity (Islam et al., 2021), because the free amino acid content, structure flexibility, and accessibility of reactive side chains increase with decreasing M W of hydrolysate. TA B L E 1 Amino acid composition of SPI, SPIH, and peptide fractions (mg amino acid/g dry sample).

F I G U R E 1
The antioxidant activity of SPI, SPIH, and soy peptide fractions (PF < 10, PF-10-30, and PF-30-100), in terms of scavenging of DPPH, ABTS (a), OH and NO (b) radicals, chelating of Fe and Cu ions (c), reducing power and total antioxidant activity (TAA; d).
Moreover, the presence of special free amino acids with ability to donate electrons (like hydrophobic and antioxidant ones) and thereby to convert DPPH free radicals into stable diamagnetic molecule can be another reason (Xie et al., 2019). For the same reasons, the peptide fractions with low M W had high capacities for inhibiting ABTS radicals (Moghadam et al., 2020). In a similar research work, the enzymatic hydrolysate of cod protein was size-fractionated, and it was found that the low M W fraction (<3 kD) has higher levels of free and antioxidant amino acids as compared to intact protein, unfractionated hydrolysate, and the high M W fraction (5 < kD) (Farvin et al., 2016).
In another study, the Flavourzyme-hydrolyzed spent hen meat was size-fractionated, and it was found that the smallest peptide fraction (<5 kD) has the highest antioxidant activity (Kumar et al., 2022).

| HO · and NO · radicals scavenging
The reactive oxygen species (ROS) of hydroxyl and nitric oxide radicals have damaging effects on the vital biomolecules, it is therefore very important to quench them in biological systems. The capacities of intact-SPI, SPIH, and peptide fractions with different M W distribution in scavenging HO · and NO · free radicals were investigated (Figure 1b).
Enzymatic hydrolysis increased SPI capacity to scavenge HO · radicals (from ~25 to ~50%). Moreover, fractions PF < 10 and PF-10-30 had higher HO · -inhibitory activity than SPIH and fraction PF- Excessive levels of NO · free radicals lead to the production of pro-inflammatory cytokines and occurrence of several diseases such as atherosclerosis, chronic inflammation, rheumatoid arthritis, Parkinson's disease, and cancer (Ahn et al., 2012). In this research, the enzymatic hydrolysis of SPI significantly increased its ability in the inhibition of NO · radicals (from ~22 to ~45%). Moreover, fractions PF < 10 and PF-10-30 showed the highest NO · -inhibitory activity (~52%) (Figure 1b). This can be attributed to the release of hydrophobic (e.g., leucine, isoleucine, alanine, phenylalanine, and valine) and

| Iron and copper ions chelating
The use of natural chelating agents in food formulations can have a great influence on preventing the destructive reactions accelerated/mediated with metal ions, especially lipid oxidation. In this research, enzymolysis of SPI and fractionation of the hydrolysate had a significant effect on the activity of peptide materials in chelating iron and copper ions (Figure 1c). The iron chelation percentage by SPIH and fractionated peptides was much higher than that by intact-SPI, and fraction PF < 10 was most effective in this regard (p < .05). In the same way, the copper chelating percentage by SPIH and peptide fractions was much higher than that by the original protein, but the peptide fractions had the same effectiveness. The higher chelating activity of SPIH and peptide fractions can be attributed to the release of acidic and basic amino acids containing an extra carboxylic or amino group (Shahidi & Zhong, 2015) together with the release of more of these groups by hydrolysis of peptide bonds, which can coordinate or form complexes with iron and copper ions through their lone pair electrons (Lindsay, 2017). In similar studies, it has been observed that the metal ion chelating activity of proteins of grass turtle (Islam et al., 2021), rice bran (Phongthai et al., 2018), and calabash nut-

| Characterization of spray-dried peptides
The effect of core material type (i.e., SPIH and peptide fractions with different M W distribution) on some physical, techno-functional, and stability characteristics/indices of the spray-dried powders was investigated (Tables 2 and 3).
Powder production yield is a measure of process economic efficiency; its maximum (~74%) and minimum (~59%) levels were observed for the feeds containing SPIH and fraction PF < 10, respectively (Table 2). This finding reflects that small peptides increase the particle adhesion during spray-drying, probably due to their reduced glass transition temperature (Zhou et al., 2014). The moisture con-  (Table 2). In agreement with the solubility data, the hygroscopicity increased with decreasing M W of peptides. This finding can be a result of a more increase in the exposed net charge density in low M W fractions (Netto et al., 1998) and in the number of their low M W components which may affect the water sorption pattern through colligative effects (Zhou et al., 2014). The bulk density, tapped density, angle of repose, Hausner ratio, and compressibility index (Carr's index) of the spray-dried powders were found in the ranges of 0.55-0.55 g/mL, 0.62-0.67 g/mL, 32-37°, 1.21-1.25, and 0.17-0.2 respectively, and M W of the encapsulated peptide fraction had no significant effect on these values (Table 3).
Anyway, these data indicate a proper flowability for the powders particles (Akbarbaglu et al., 2019).
In another study, the hydrolysates of stripped weakfish were microencapsulated in maltodextrin by spray-drying, and it was found that the type of hydrolysate (with Alcalase or Protamex) influences the production efficiency (73-76%), moisture content ( Sarabandi & Jafari, 2020).

| Morphological characterization
The effect of core material type (SPIH or PF < 10) on the morphological characteristics of spray-dried powder particles was investigated  F I G U R E 2 SEM images of microparticles containing SPIH (a, b) or the smallest soy peptide fraction (PF < 10; c, d).

F I G U R E 3
The retention percentage of antioxidant activity in SPI, SPIH, and soy peptide fractions (PF < 10, PF-10-30, and PF-30-100) after spray-drying encapsulation, in terms of scavenging of DPPH, ABTS (a), OH and NO (b) radicals, chelating of Fe and Cu ions (c), reducing power and total antioxidant activity (TAA; d).
surface of spray-dried powders to slow film formation during the drying and cooling phases of the process (Kurozawa et al., 2009).

| Retention of antioxidant activity in spraydried peptides
One of the most important goals of microencapsulation is to stabilize and preserve the biological activity of bioactive substances.
In this study, the antioxidant activity retention of the peptide samples (SPIH, PF < 10, PF-10-30 and PF-30-100) after spray-drying encapsulation was investigated. Figure 3 shows the retention percentage of antioxidant activity in the spray-dried peptides. With all antioxidant activity assays, 75-97% of initial antioxidant activity of free peptides preserved after spray-drying, and the fractionated peptides generally had more antioxidant stability than SPIH.
Regardless of the type of core material, the highest and lowest antioxidant stability was related to ABTS (97%) and NO · (75%) radical scavenging, respectively. The microcapsule powders of PF < 10 and PF-10-30 had the same antioxidant stability, except in copper ion chelating assay; their antioxidant stability was also higher than that  (Sarabandi & Jafari, 2020) have been different after spray-drying. The drying method type (oven-, spray-, or freeze-drying) have also had a significant effect on the retention of antioxidant/biological activity in the hydrolysates of Perinereis aibuhitensis (Liu et al., 2022) and edible bird's nest (Gan et al., 2020).

| CON CLUS ION
The direct fortification of food formulations and diets with protein hydrolysates is limited due to their physicochemical instability and bitter taste, as well as, their bioactivity may be influenced by M W .
In this research, SPI hydrolysates were prepared by alcalase, then to improve their utilization, as lower concentration of them will be required for the development of functional foods and thereby little change seen in the finished products. resources (equal).

ACK N OWLED G M ENTS
The authors are thankful to the financial support of the University of Kurdistan (Sanandaj, Iran) under the project no. 99/11/11908.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed in this article are included within it.