2.5.1 DPPH^• scavenging activity

The DPPH^• scavenging activity of lyophilized PH and AH and their fractions was estimated using the method described by Jemil et al. (2014) with minor modifications. Protein hydrolysate solution (2 mL; 1, 2, 3, 4, and 5 mg/mL) and ultrafiltration fractions (1 mL; 1, 2, 3, 4, and 5 mg/mL) were added separately to 95 % ethanol-based DPPH^• solution (3 mL, 0.1 mM).These combinations were shaken and maintained for 30 min in the dark at ambient temperature. The decrease in DPPH^• was detected at 517 nm using UV-spectrophotometer (Mapada Instruments Co., ltd., Shanghai, China). As a control sample, deionized water was utilized in place of the sturgeon hydrolysate samples. According to hydrolysate concentration (mg/mL) that achieves 50 % inhibition of DPPH, IC₅₀ was calculated according to the antioxidant activity of samples. Butylated hydroxytoluene (BHT) was utilized as a positive control. To identify the DPPH^• scavenging activity, the following equation was used:

2.5.2 ABTS^•+ scavenging activity

The ABTS^•+ scavenging activity of lyophilized PH and AH, their fractions and purified peptides was evaluated using a modified procedure reported by Cai et al. (2015). The ABTS^•+ working solution (7.4 mM) and potassium persulfate (K₂S₂O₈) solution (2.6 mM) were prepared, and equal volumes were mixed to prepare the working solution, which was then allowed to react in a dark place at 25 ± 2 °C for nearly 16 h. Before measuring the activity of the samples, the absorbance of this solution was adjusted to 0.70 ± 0.02 at 734 nm by diluting 1 mL in 50 mL ethanol (95 %). To estimate the antiradical activity, 2 mL of hydrolysates solution, 1 mL of peptide factions or 200 µL purified peptides solution (1–5 mg/mL) were combined with 3.5 mL of ABTS^•+ working solution and kept in the absence of light at ambient temperature for 10 min. The control sample was prepared using the same procedure, but instead of protein hydrolysate, deionized water was used. As a positive control, BHT was used. UV- spectrophotometer (Mapada Instruments Co., ltd., Shanghai, China) was utilized to detect the samples absorbance at 734 nm and determine the ABTS^•+ scavenging activity using the following formula:

2.6.1 Ultrafiltration of protein hydrolysate

Accurately 50 mg/mL of enzymatically hydrolyzed protein (PH and AH) were subjected to ultrafiltration (Millipore Minitan unit, Millipore, Bedford, MA) by three different molecular weight cut-off (MWCO, 3000, 2000, and 1000 Da) membranes. To dissolve each hydrolysate, 60 mg/mL deionized water was used, and four fractions were isolated and collected (fraction, >3000 Da), (fraction, 2000–3000 Da), (fraction, 1000–2000 Da), and (fraction, <1000 Da). The resultant peptide fractions were freeze-dried using the same procedure as described above, and their antioxidant capacity was identified using DPPH and ABTS assays.

2.6.2 Purification of peptides using RP-HPLC

Using ultrafiltration membranes, the most active fraction (fraction, <1000 Da) derived from PH and AH was purified using an RP-HPLC unit (Agilent 1260, Santa Clara, CA, USA). In brief, these fractions were diluted (50 mg/mL) in 0.1 % trifluoroacetic acid (TFA), and 20 μL of each sample was loaded into an Agilent Zorbax, SB C-18 column (4.6 mm 250 mm, 5 µm particle size). TFA (0.1 %, v/v) and acetonitrile (30 %, v/v) with 0.1 % (v/v) TFA were the mobile phases A and B, respectively. With a flow rate of 0.8 mL/min, the gradient program of 0 % B, 0–9 min; 10 % B, 10–20 min; 20 % B, 20.5–31.5 min; 40 % B, 32–41 min; and 50 % B, 41.5–43 min was used. The eluate was measured at 280 nm, and nine peptides (P1-P9) were separated from each sample, gathered, and lyophilized. The antioxidant capability of these peptides was estimated in order to verify the most active peptide.

3.3.1 Antioxidant activity of protein hydrolysates

The scavenging activity of pH and AH was identified against DPPH^• at 517 nm. There are two mechanisms of the antiradical action of antioxidants include single electron transfer and hydrogen atom transfer; hence, these two mechanisms produce the same end products (Osipova et al., 2021). The compounds that contribute to hydrogen (H⁺) act as radical scavengers, causing the purple sample mixture to turn yellow, resulting in reduced absorption (Ktari et al., 2012). As illustrated in Fig. 1A, the DPPH^• scavenging activity of pH and AH obtained from Chinese sturgeon was demonstrated at concentrations ranging from 1 to 5 mg/mL. The radical scavenging activity of both hydrolysates significantly increased (p < 0.05) as their concentration raised from 1 to 5 mg/mL, which is consistent with previous reports showing that protein hydrolysates activity against radicals increased as their concentration increased (Bougatef et al., 2009; Ktari et al., 2012). At a concentration of 5 mg/mL, the highest capacity was found to be 62.45 % for PH and 67.42 % for AH. Bahari et al. (2020) reported that leucine, valine, methionine, and alanine play a fundamental role in peptide activity to scavenge DPPH^•. The amino acid profiles of pH and AH indicated the existence of these amino acids with appropriate levels; however, the content of alanine and valine was higher in AH, as shown in Table 1. According to the current results, AH was more active (IC₅₀ of 3.15 mg/mL) than PH (IC₅₀ of 3.64 mg/mL). Both results were within the IC₅₀ range (1.63–4.95 mg/mL) of salmon byproduct protein hydrolysate obtained by (Ahn et al., 2014) using different enzymes. However, it was less than IC₅₀ of standard BHT (IC₅₀ = 0.24 ± 0.01 mg/mL). The difference between synthetic scavengers and protein hydrolysate can be attributed to the fact that synthetic scavengers are pure compounds, whereas hydrolysate mostly is a mixture of protein/peptide and non-protein ingredients. This type of protein activity is generally associated with the composition and amino acid sequence of peptides, as well as their MW and the hydrophobic property caused by various DH (Bougatef et al., 2010). According to Phongthai et al. (2016), the amino acid profile released in the hydrolysis process could affect the antioxidant activities, especially phenylalanine tyrosine, histidine, and methionine, which were shown to be higher proton donors compared to other amino acids.

Close

Fig. 1

Antioxidant activities of protein hydrolysates powder obtained by using two different enzymes (PH; papain hydrolysate and AH; alcalase 2.4L hydrolysate), (A) DPPH^• scavenging activity; (B) ABTS^•+ scavenging activity. Data are expressed as mean ± standard deviation. Different lower-case letters (a and b) indicate significant differences (p < 0.05) between the different concentrations in each hydrolysate, and upper-case letters (A and B) indicate significant differences at the same concentration with the different hydrolysates.

Export to PPT

When an antioxidant substance or active sample is included in the mixture, ABTS^•+ is scavenged, which is proportional to the sample's antioxidant activity. This assay depends on the formation of a blue/green ABTS radical, which can be limited by antioxidants capable of donating hydrogen atoms to scavenge aqueous phase radicals or lipid peroxyl radicals (Wang et al., 2021), and the color changes can be detected by reading the absorbance at 734 nm. Fig. 1B depicts the PH and AH ABTS^•+ scavenging activity. When the concentration of pH was raised from 1 mg/mL to 5 mg/mL, the radical scavenging capacity ranged from 31.43 % to 71.32 % and from 34.19 % to 73.07 % for AH. According to the findings in Fig. 1B, both hydrolysates contained peptides that may differ in amino acid content and sequence, and thus their antioxidant activity differed. The AH activity was higher (IC₅₀ of 1.58 mg/mL) than the PH activity (IC₅₀ of 1.92 mg/mL). Because the size of the peptides released during the hydrolysis process is determined by enzyme specificity, the differences in DH could explain the difference in antioxidant activity between PH and AH (Noman et al., 2022). Both hydrolysates had lower activity compared to standard BHT (IC₅₀ = 0.19 ± 0.02 mg/mL). According to Sarmadi and Ismail (2010), peptides with MW in the range from 500 to 1500 Da had higher antioxidant activity than peptides with MW > 1500 Da and <500 Da. Noman et al. (2018), found that the MW of pH peptides (<500 Da) obtained with papain enzyme was >87 %, while it was significantly lower for AH peptides (Noman et al., 2019). Chalamaiah et al. (2015), on the other hand, mentioned that the histidine, phenylalanine, tyrosine, and methionine, may lead to increased ABTS^•+ scavenging activities.

3.3.2 Antioxidant activity of hydrolysate fractions

The ultrafiltration procedure is frequently applied to separate fractions of protein hydrolysate with specific biological properties based on their MW (Chi et al., 2014). The Chinese sturgeon hydrolysates were separated by ultrafiltration membranes, and four fractions (>3000, 2000–3000, 1000–2000, and <1000 Da) were collected from PH and AH (Fig. 2). Fig. 2A depicts the PH and AH peptide fractions activities against DPPH^•. The findings show that the peptide fractions exhibited varying levels of antioxidant activity, but the peptide fractions of AH were more active than PH peptide fractions, which could be related to the distribution of amino acids in each fraction's peptides. The fractions of pH and AH with MW < 1000 Da displayed the highest antioxidant activity compared to other fractions, which was 80.30 and 84.81 % with IC₅₀ of 2.59 and 2.31 mg/mL, respectively. According to these findings, it can be concluded that this fraction (<1000 Da) from both hydrolysates may have contained more active antiradical peptides, which could efficiently transform DPPH^• into more stable compounds, preventing the free radical chain reaction. The IC₅₀ values of pH and AH fractions to scavenge DPPH^• decreased with decreasing MW compared to pre-fractionation values. Previous studies found that protein hydrolysates or low MW peptide molecules interact more effectively with radicals and exhibit higher antioxidant capability (Wang et al., 2012; He et al., 2013; Onuh et al., 2014; Cai et al., 2015). Aromatic amino acids including tyrosine, histidine, and phenylalanine, as well as the hydrophobic acids, such as valine, methionine leucine and alanine are abundant in the PH and AH and play a remarkable role in bioactive peptide antioxidant activities (Bahari et al., 2020). However, because DH affects the amino acid profile, as well as the sizes and structures of the bioactive peptides released during the enzymolysis process, hence, the antioxidant activity of these fractions is linked to DH (Jang et al., 2016).

Close

Fig. 2

Radical-scavenging activity of ultrafiltration fractions of protein hydrolysates obtained using two different enzymes (PH; papain hydrolysate and AH; alcalase 2.4L hydrolysate), (A) DPPH; (B) ABTS^•+. Data are expressed as mean ± standard deviation. Different lower-case letters (a and b) indicate significant differences (p < 0.05) between the different fractions in each hydrolysate, and upper-case letters (A and B) indicate significant differences at the same fraction with the different hydrolysates.

Export to PPT

The ABTS^•+ scavenging activity assay is a common method for determining the antioxidant capacity of hydrophilic and lipophilic compounds (You et al., 2009). In the presence of proton-donating compounds, such as antioxidants, the ABTS^•+ are scavenged and the absorption is reduced by changing the solution color. As shown in Fig. 2B, based on MW of both protein hydrolysates, peptidic fractions exhibited significantly different antioxidant activities at p < 0.05, whereas peptide fractions with a MW of <1000 Da demonstrated significantly stronger activity compared to the other fractions in both hydrolysates. Most AH fractions showed higher antioxidant activities against ABTS^•+ than those of PH, particularly the fraction with MW of <1000 Da, which were 82.87 % and 87.46 % (IC₅₀ of 1.54 and 1.36 mg/mL), respectively. Reduced scavenging activity in papain peptide fractions samples may be attributed to an increased DH, as Intarasirisawat et al. (2012) found that protein hydrolysates with DH > 20 % have a lower ability to scavenge ABTS^•+. Moreover, the distribution of MW of peptides within the fraction is critical in determining the antioxidant activity (Sarmadi and Ismail, 2010).

3.3.3 Antioxidant activity of purified peptides

The <1000 Da fraction of pH and AH was the most active in scavenging free radicals, particularly the ABTS^•+. Therefore, this fraction was separated further by RP-HPLC, and chromatogram shows nine peaks corresponding to the P1-P9 peptides as shown in Figs. 3 and 4. Among these nine fractions, the peak corresponding to P5 peptide from PH and P3 peptide from AH demonstrated higher (p < 0.05) than the other isolated peptides, with 87.78 and 93.34 %, respectively. According to this result, the purification process increased the activity of the purified peptides compared to the previous stages, where the IC₅₀ values were 0.89 and 0.72 mg/mL, sequentially. Although these values were higher than the IC₅₀ of standard BHT (0.19 ± 0.02 mg/mL), they were remarkably close to BHT value compared to hydrolysates and their fractions, enhancing their potential as natural substitutes for synthetic antioxidant compounds. On the other hand, the result in Table 2 revealed that the purified peptide (P5) of pH contained valine and glycine, whereas the purified peptide (P3) of AH contained glycine and alanine. These acids are classified as hydrophobic amino acids that exhibited strong antioxidant capacity (Cai et al., 2015). Furthermore, the peptide isolated from AH (P3) contains histidine (aromatic acid) residue, which has the potential to donate electrons to radicals and convert them to stable molecules. The difference in activity between the two peptides can thus be traced to the peptides' varied distribution of these amino acids.

Close

Fig. 3

RP-HLPC chromatogram on a Zorbax, SB C-18 column of the papain hydrolysate fraction < 1000 Da obtained using ultrafiltration process (A), and ABTS^•+ scavenging activity of the separated peptides (B). Data are expressed as mean ± standard deviation. Different letters above bars indicate significant differences at p < 0.05.

Export to PPT

Close

Fig. 4

RP-HLPC chromatogram on a Zorbax, SB C-18 column of the alcalase 2.4L hydrolysate fraction < 1000 Da obtained using ultrafiltration (A), and ABTS^•+ scavenging activity of the separated peptides (B). Data are expressed as mean ± standard deviation. Different letters above bars indicate significant differences at p < 0.05.

Export to PPT

Protein structural properties such as amino acid profile, sequences, hydrophobicities, and MW highly affect the antioxidant activity of peptides purified prepared from various protein sources (Zou et al., 2016). Furthermore, hydrophobic amino acids can increase peptide solubility in lipids, improving interaction with free radicals, which in turn might improve the antioxidant properties of peptides (Sarbon et al., 2018). In contrast, the purified peptides (P3 from PH and P5 from AH) obtained in this study have low MW as shown in Table 2. Although amino acids content in the peptide structure are responsible for antioxidant activity, the MW of peptides was found to be critical in peptide efficiency, where Kumar et al. (2012) documented that peptides with smaller MW can pass the intestinal barrier and perform a biological activity.