Effect of Fl : Al ratio and hydrolysis time on the DH of JBH
The JBH samples had a DH in the range of 7.09–19.31% (Table 1). The model showed a highly significant value (p < 0.0001) (Table 3). The DH was strongly influenced by the linear term of the Fl : Al ratio (X1) and hydrolysis time (X2) (p < 0.0001) and the quadratic term of the Fl : Al ratio X21 and time X22 (p < 0.001), whereas the interaction of the two factors (X1X2) was not significant (p ≥ 0.05). The statistical analysis showed a lack of fit of the model (p = 0.57), reflected in a relatively low determination coefficient (R2 = 0.96) (Table 3). The regression coefficient (β) values showed that the hydrolysis time (X2) had a greater influence on DH than the Fl : Al ratio. The response plot for DH with respect to the Fl : Al ratio and hydrolysis time is presented in Fig. 1A. The response plot shows that the Fl : Al ratio increased, the DH decreased.
This might be due to flavourzyme being less active when in combination with alcalase or hydrolyzing rice bran to a limited extent. This result is in agreement with Wang et al.6 who demonstrated a higher DH for the cleavage of protein from tree peony seeds by alcalase than by flavourzyme, due to alcalase having very broad specificity to break peptides. In addition, rice bran protein hydrolysate appears to be a more desirable substrate for alcalase given the higher DH observed using only alcalase. During the initial stage of the enzyme reaction (60 min), DH increased at a rapid rate and then decreased slightly with an increase in reaction time (Fig. 1A). This reduction of the rate of enzyme hydrolysis might be due to substrate limitation and the reduction of enzyme activity. The same has been found for hydrolysis of peptides from pecan meal using alcalase7.
Effect of Fl : Al ratio and hydrolysis time on protein content of JBH
The protein content of JBH samples was determined by the combustion method and was in the range from 34.40 to 42.36 g/100 g sample, as presented in Table 1. The response plot for protein content shows that the Fl : Al ratio (X1) had a major influence on protein content: as the ratio increased, the amount of protein increased (Fig. 1B). Alcalase is an endopeptidase, which breaks peptide bonds from C-terminal amino acids, whereas Flavourzyme is an endo- and exopeptidase that breaks the N-terminal of peptide chains5. This indicates that Flavourzyme could also increase the number of N-terminal sites for the action of the exopeptidase. However, as hydrolysis time increased, the protein content decreased slightly (Fig. 1B). This might be due to the effect of the Maillard reaction during hydrolysis.
Effect of Fl : Al ratio and hydrolysis time on MW distribution of JBH
The MW distribution of rice bran hydrolysates varied according to Fl : Al ratio and hydrolysis time, as presented in Table 1. The MW of JBH was categorized as high molecular weight (HMW, 5–10 kDa,), medium molecular weight (MMW, 3–5 kDa,) or low molecular weight (LMW, less than 3 kDa): 673.52–857.88, 413.75–505.51 and 272.42–367.24 mAU*s, respectively. The regression coefficients for MMW and LMW models of the multiple regression are presented in Table 3. As shown in Fig. 1C and D, as the Fl : Al ratio was increased, the proportion of MMW (3–5 kDa) and LMW (< 3 kDa) peptides increased. Exopeptidases can attack different active sites of a polypeptide, resulting in an increase in the content of MMW and LMW. However, increasing the hydrolysis time had no effect on MW. The MW distribution of protein is related to its antioxidant activity8, 9.
Effect of Fl : Al ratio and hydrolysis time on antioxidant activity of JBH
The antioxidant activity of rice bran protein hydrolysates was determined by DPPH, ABTS, FRAP and H2O2 assays (Table 2). According to statistical analysis, the DPPH, ABTS and FRAP values were identified as significant model terms whereas for H2O2 was found to be non-significant (Table 3). The adj. R2 for DPPH, ABTS and FRAP was 0.63, 0.76 and 0.84, respectively, with a non-significant value for lack of fit (p ≥ 0.05) for all responses, showing a signiﬁcant and good ﬁt with the experimental data and having less variation.
DPPH radical scavenging activity
The half-maximal inhibitory concentration (IC50) of DPPH radical scavenging activity in JBH samples showed variation, ranging from 5.13 to 9.90 mg/mL. The DPPH radical scavenging ability indicates that the JBH possessed the capacity to donate hydrogen atoms and electron10. The result shown in Fig. 1E demonstrates that the IC50 of DPPH scavenging activity was not affected by the Fl : Al ratio. The response plot shows that as hydrolysis time (X2) increased, the IC50 of DPPH (low activity) increased up to 300 min and after that decreased. This might be due to initial breakdown of peptide bonds in the hydrolysate, resulting in more HMW peptides than LMW peptides. Reduction in DPPH activity for prolonged hydrolysis does not tally with argument below. This result was correlated with the regression model for an increase in < 3 kDa peptides as hydrolysis time increased. The potency of the hydrolysate’s DPPH radical scavenging activity depends on the size of peptides and type of enzyme10.
ABTS radical scavenging activity
The ABTS radical scavenging assay, based on electron transfer and hydrogen atom transfer, can be performed to assess the radical scavenging activity of both the hydrophilic and hydrophobic compounds of protein hydrolysates2, 11. As shown in Fig. 1F, as hydrolysis time was increased, the IC50 of ABTS decreased and then increased with a further increase in hydrolysis time. This might be due to the donation of electrons and hydrogen atoms of peptides in the hydrolysate liberated from protein during hydrolysis, resulting in an increase in the ability to scavenge ABTS. However, after excessive hydrolysis time, peptides were broken down into free amino acids, thus decreasing ABTS activity12. The ABTS radical scavenging ability can be explained by the DH, amino acid composition of peptide chains and the MW of peptides5, 13.
The FRAP assay is used to determine a substance’s ability to donate electrons to convert ferric ions (Fe3+) to the ferrous form (Fe2+). FRAP varied for the different JBH treatments between 532.54 and 817.74 mmol FeSO4/100 g sample. Increasing the hydrolysis time significantly decreased FRAP (Fig. 1G). For a hydrolysis time of 60 min, more HMW peptides than LMW peptides or free amino acids were found, which could be responsible for hydrolysate being a poorer source of reducing electrons and protons 3. After excessive hydrolysis time, FRAP increased slightly. These results are inconsistent with the study of Olagunju et al.10 who stated that HMW peptides (>10 kDa) exhibit better FRAP than LMW peptides (< 5 kDa). In addition, Phongthai et al.3 reported that the presence of Tyr and Trp in a peptide indicates greater FRAP to reduce or donate electrons.
H2O2 scavenging activity
The H2O2 scavenging activity of JBH samples prepared using different Fl : Al ratios and hydrolysis times ranged from 192.87 to 262.11 mM Trolox/g sample (Table 1). The JBH samples hydrolyzed for 60 min had the highest H2O2 scavenging activity, compared with those hydrolyzed for longer at the same Fl : Al ratio. It was noticed that as hydrolysis time increased, more short peptide chains or free amino acids were obtained, resulting in low H2O2 scavenging activity14.
Interestingly, the Fl : Al ratio had effect on the antioxidant activities, indicating that these enzymes are more effective for obtaining both hydrophobic amino acid (e.g. Ala, Val, Leu, Iso, Pro, Phe, Try, Cys and Met) and hydrophilic amino acid (e.g. Ser, Thr, Asn, Glu, His and Tyr) sequences in protein hydrolysate samples4. These results indicate that the combination of flavourzyme and alcalase can enhance antioxidant activity, by producing peptides with different specific amino acids. Tang et al.4 demonstrated that Tenebrio molitor larvae hydrolysate obtained using a mixture of alcalase and flavourzyme exhibits the highest antioxidant activity. According to Ambigaipalan et al.5, date seed hydrolysates obtained using an alcalase and flavourzyme combination exhibited the highest antioxidant activity. However, Kumar and Roy15 reported that hydrolysates obtained using an enzyme combination showed lower values for DPPH activity and FRAP than those obtained using alcalase and flavourzyme alone.
Optimization and verification of protein rice bran hydrolysate
According to the RSM analysis, rice bran protein hydrolysate that is considered to be desirable should at least provide these properties: 1) a high protein content, 2) high antioxidant activity and 3) a high content of LMW protein. The optimal conditions were an Fl : Al ratio of 9.81 : 90.19 and hydrolysis time of 60 min; the desirability value for these conditions was 80.6% which was located in the optimal area as shown in Fig. 2.
Under the optimum conditions for producing the protein hydrolysate, obtained values were in agreement with the predicted values; the difference error ranged from 0.42% to 8.42% (Table 4). The corresponding response values obtained from the actual data and those predicted from the models were similar.
Amino acid composition of optimum rice bran protein hydrolysate
The amino acid composition for the optimum JBH conditions are presented in Table 5. It was observed that the JBH sample had a high content of Glu, Arg and Asp: 338.90, 99.10 and 91.22 mg/g protein, respectively. These results are consistent with those of Xiao et al.16 who found that Glu, Asp and Arg were the most abundant amino acids in rice bran protein hydrolysate. However, the protein hydrolysate of some plants such as mung bean is rich in Asp, Glu and Pro 17. The high content of Glu and Asp could have been due to their abundance in the plant protein. The total amount of essential amino acids in the JBH sample was 262.50 mg/g protein; the recommendation for human nutrition is approximately 277 mg/g protein, suggesting that JBH could enhance or provide suitable protein nutrition18. The sample showed a high content of flavor enhancers, especially Glu (338.90 mg/g), followed by Asp (91.22 mg/g), Ala (59.85 mg/g) and Gly (37.34 mg/g). The JBH had a hydrophobic amino acid content of 266.98 mg/g protein. Samaranayaka and Li-Chan19 obtained a protein hydrolysate containing Ala, Lys, Pro, Leu, His, Tyr and Met, suggesting that hydrophobic amino acids could contribute to high antioxidant activity. In addition, the JBH contained 52.86 mg/g protein of aromatic amino acids, which have been reported to improve the radical scavenging activity of peptides by donating electrons to electron-deficient radicals6.