## 4 Discussion
As we all understand, the smaller the size of the molecule, the less the steric hindrance the compound has, making it easier to react with other compounds. Thus, the peptides in the peptide fraction <3 kDa were easier to react with the DPPH radicals, compared to the peptides in the larger peptide fraction. Interestingly, the hydrolyses gave a better effect on the resulting antioxidant activities compared to the peptide fractions 3-10 kDa and the peptide fractions >10 kDa. This may be due to the different types of peptides in the hydrolyses. However, for the semi-purified peptide fraction, the small peptide fraction (the peptide fraction <3 kDa) gave better antioxidant activity than the large peptide fraction (the peptide fractions 3-10 and >10 kDa). In general, antioxidant activity was found to be inversely related to molecular size. For the peptide fraction <3 kDa, the order of antioxidant activity was PBN > PPS > PPN > PPA > PBS > PBA. A comprehensive study on the effect of these factors on the antioxidant activity produced, especially for the antioxidant activity of hydrolyses and peptide fractions from collagen by-products of the fishery industry, has not been reported. However, our results are consistent with previous studies [32; 33] that reported that small peptides provided better antioxidant activity than larger peptides. The peptides in the PBN and PPS fractions of <3 kDa as molecules with the two highest antioxidant activities had a length range of amino acids belonging to the range of bioactive peptides, which has also been reported by previous researchers [17; 71]. The combination of in vitro and in silico methods can increase the discovery of bioactive peptides for further applications in food and health [36]. This combination of methods quickens, simplifies, and reduces research costs. The amino acid sequences of the peptides in the PBN and PPS fractions of <3 kDa were analyzed by LC-MS/MS (in vitro study with the DPPH method), then used for in silico analysis to predict their bioactive properties and to study their characteristics. In the PBN fractions of <3 kDa, one peptide with a residue length of 23 amino acids was identified according to the results of the LC-MS/MS analysis. From the in silico results with the BIOPEP program, 16 dipeptides from this peptide sequence were indicated to possess bioactive properties. The sixteen dipeptides were PW, FP, LF, VG, GR, DA, GS, HG, DY, YV, AD, HF, SL, WC, TV, VH. These 16 dipeptides were grouped as antidiabetic peptides 81.25%, antihypertensive peptides 56.25% and antioxidant peptides 6.25%. Among these peptides, there were those predicted as multifunctional peptides, namely 25% as antidiabetic and antihypertensive (FP, VG, DA, YV); and 6.25% as antidiabetic and antioxidant (PW). The identified antidiabetic peptides were alpha-glucosidase inhibitors 6.25%, dipeptidyl peptidase III inhibitors 12.50%, and dipeptidyl peptidase IV inhibitors 62.50%. Antihypertensive peptides were identified as ACE inhibitors. In the PPS fractions of <3 kDa, 12 peptides with residue lengths of 10-20 amino acids were identified according to the results of LC-MS/MS analysis. From the in silico results with the BIOPEP program, there were 83 peptides from the peptide sequences that have bioactive properties. The eighty-three peptides were KD, IY, GPP, LK, GAA, PGP, PP, EA, AD, WM, SM, PF, YG, GE, PR, GPRG, GPRGP, GPRGP, GPR, GP, PG, GPGG, LSP, IG, GS, NY, MW, VF, FP, NK, GEP, KG, GT, TP, RG, ILP, IL, AP, GA, VG, GL, MG, QG, GD, EV, PQ, VGP, DM, MGP, AG, GG, TG, GRP, RP, GR, NG, DG, GPA, AA, AGP, QGP, GV, GAGP, SP, SL, WC, DR, PN, SW, YD, KK, EP, NP, PPG, PS, LP, DN, NV, PK, QL, VI, PA, GPAG. These 83 peptides were grouped as 73.49% antidiabetic peptides, 61.45% antihypertensive peptides, 6.02% antioxidant peptides and 15.66% peptides with other bioactive properties (i.e., chemotactic, antithrombotic, antiamnestic).