Defatted Canary (Canarium indicum L.) as an oilcake was used as a nutraceutical due to its high protein content (36.039%). One of its applications is the preparation of protein hydrolysate using enzymes, namely, papain, flavourzyme, and pepsin. The production was performed using the Box-Behnken response surface methodology, which involves three factors and three levels, namely, concentration [E/S], hydrolysis time, and hydrolysis temperature which are all factors to be considered. The antioxidant test on the sample was used as a parameter for determining the optimal conditions. In addition, other properties, namely, protein content, degree of hydrolysis, and SDS-PAGE electropherogram profile, were also analyzed to determine the character of the product. The results showed that the optimum process for producing antioxidative protein hydrolysate with papain (A13), flavourzyme (B1), and pepsin (C14) was performed at 60°C, 50°C, and 42°C, with an enzyme concentration of 0.55%, 0.55%, and 0.1% (w/v) for 3, 1, and 3 h, respectively. Furthermore, the antioxidant activity (IC 50 ) values of each hydrolysate were 2.622 ± 0.072 (A13), 0.426 ± 0.008 (B1), and 0.195 ± 0.001 mg/mL (C14). This implies that the protein hydrolysate produced by pepsin has the highest antioxidant activity.

Quantitative analysis of bioactive peptide mostly conducted by measuring the activity. While the determination of peptide content in natural sources has been conducted using various instruments, vibrational spectroscopy remains underutilized. Here, we attempted new developed method in peptide quantification and degradation kinetic analysis using Fourier-Transform Infrared Spectroscopy. Bovine Serum Albumin was used as standard protein in method development and validation. Peptide content was estimated by converting peak area to concentration. The method was used to estimate peptide content in Canarium nut protein and its hydrolysates, which potentially hold biological activity. Kinetic study was conducted with microwave as an accelerator for hydrolysis, an apparatus rarely used in peptide study. Amide I band on wavenumber range of 1724.05-1619.91 cm-1 was selected for analysis, considering its selectivity and linearity. The method also met other validation requirement, including accuracy and precision. When applied in quantitative analysis, the method was able to calculate peptide content decrease in Canarium nut protein after hydrolysis using papain (38.24%), pepsin (33.67%) and alkaline reagent (28.53%). In kinetic study, microwave-assisted peptide degradation exhibited logarithmic profile with the equation of y=-0.148ln(x)+0.9591 and R² value of 0.963. Based on these results, FTIR is useful in estimating peptide content and in analyzing degradation kinetic profile.