Actinopyga lecanora (A. lecanora) is classified among the edible species of sea cucumber, known to be rich in protein. Its hydrolysates were reported to contain relatively high antioxidant activity. Antioxidants are one of the essential properties in cosmeceutical products especially to alleviate skin aging. In the present study, pH, reaction temperature, reaction time and enzyme/substrate ratio (E/S) have been identified as the parameters in the papain enzymatic hydrolysis of A. lecanora. The degree of hydrolysis (DH) with antioxidant activities of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power (FRAP) assays were used as the responses in the optimization. Analysis of variance (ANOVA), normal plot of residuals and 3D contour plots were evaluated to study the effects and interactions between parameters. The best conditions selected from the optimization were at pH 5.00, 70 °C of reaction temperature, 9 h of hydrolysis time and 1.00% enzyme/substrate (E/S) ratio, with the hydrolysates having 51.90% of DH, 42.70% of DPPH activity and 109.90 Fe2+μg/mL of FRAP activity. The A. lecanora hydrolysates (ALH) showed a high amount of hydrophobic amino acids (286.40 mg/g sample) that might be responsible for antioxidant and antityrosinase activities. Scanning electron microscopy (SEM) image of ALH shows smooth structures with pores. Antityrosinase activity of ALH exhibited inhibition of 31.50% for L-tyrosine substrate and 25.40% for L-DOPA substrate. This condition suggests that the optimized ALH acquired has the potential to be used as a bioactive ingredient for cosmeceutical applications.
Enzyme hydrolysates (trypsin, papain, pepsin, α-chymotrypsin, and pepsin-pancreatin) of Tinospora cordifolia stem proteins were analyzed for antioxidant efficacy by measuring (1) 1,1-diphenyl-2-picrylhydrazyl (DPPH•) radical scavenging activity, (2) 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) radical scavenging capacity, and (3) Fe2+ chelation. Trypsin hydrolysate showed the strongest DPPH• scavenging, while α-chymotrypsin hydrolysate exhibited the highest ABTS+ scavenging and Fe2+ chelation. Undigested protein strongly inhibited the gastrointestinal enzymes, trypsin (50% inhibition at enzyme/substrate ratio = 1:6.9) and α-chymotrypsin (50% inhibition at enzyme/substrate ratio = 1:1.82), indicating the prolonged antioxidant effect after ingestion. Furthermore, gel filtration purified peptide fractions of papain hydrolysates exhibited a significantly higher ABTS+ and superoxide radical scavenging as compared to non-purified digests. Active fraction 9 showing the highest radical scavenging ability was further purified and confirmed by MALDI-TOF MS followed by MS/MS with probable dominant peptide sequences identified are VLYSTPVKMWEPGR, VITVVATAGSETMR, and HIGININSR. The obtained results revealed that free radical scavenging capacity of papain hydrolysates might be related to its consistently low molecular weight hydrophobic peptides.
Palm kernel cake protein was hydrolyzed with different proteases namely papain, bromelain, subtilisin, flavourzyme, trypsin, chymotrypsin, and pepsin to generate different protein hydrolysates. Peptide content and iron-chelating activity of each hydrolysate were evaluated using O-phthaldialdehyde-based spectrophotometric method and ferrozine-based colorimetric assay, respectively. The results revealed a positive correlation between peptide contents and iron-chelating activities of the protein hydrolysates. Protein hydrolysate generated by papain exhibited the highest peptide content of 10.5 mM and highest iron-chelating activity of 64.8% compared with the other hydrolysates. Profiling of the papain-generated hydrolysate by reverse phase high performance liquid chromatography fractionation indicated a direct association between peptide content and iron-chelating activity in most of the fractions. Further fractionation using isoelectric focusing also revealed that protein hydrolysate with basic and neutral isoelectric point (pI) had the highest iron-chelating activity, although a few fractions in the acidic range also exhibited good metal chelating potential. After identification and synthesis of papain-generated peptides, GGIF and YLLLK showed among the highest iron-chelating activities of 56% and 53%, whereas their IC50 were 1.4 and 0.2 μM, respectively.