## 3 Results  
Bromelain, collagenase and cathepsin had a significantly different effect on the concentration of peptides and of free amino acids in the <3000 Da fraction; the effect of the enzyme/collagen ratio (E:C) was significant too (Figure 1, Table 3). Highest amount of <3000 Da peptides was found by hydrolysis with cathepsin B at the E:C 1/20, followed by the EC 1/50. If considering as the degree of hydrolysis to 3000 Da the amount of <3000 Da peptides on the total amount of peptides (<3000 and >3000 Da), at the 1/20 ratio it was 82% when using cathepsin, 80% when using bromelain and 73% when using collagenase; at the ratio 1/50, it was 82% with collagenase, 75% with bromelain and 73% with cathepsin. \begin{table}
\begin{tabular}{c c c c c c} \hline
**Enzyme** & **Test** & **E-C Ratio** & **Incubation Time** & **T** (\({}^{\circ}\)**C**) & \multicolumn{2}{c}{**Treatment**} \\  & & **(_tw/w_)** & **(min)** & **(**)** & **(for Each Test)** \\ \hline  & ABTS & 1/20, 1/50 & 30, 60, 90, 120, 180 & Amb. & (a) & No treatment \\ Bromelain & DPPH & 1/20, 1/50 & 30, 60, 90, 120, 180 & Amb. & (b) & Sterilisation \\ Cathepsin B & FRAP & 1/20, 1/50 & 30, 60, 90, 120, 180 & Amb. & (c) & Concentration/Volume \\ Collagenase B & ORAC & 1/20, 1/50 & 260 \({}^{\circ}\) & 37 \({}^{\circ}\) & (d) & Complexes mixing \\  & TBArs & 1/20, 1/50 & 5, 15, 30, 45, 60, 90, 120 & Amb., 70 & \\ \hline \end{tabular} (\({}^{\circ}\)) according to the method [22,23].  
\end{table} 
Table 2: Summary of the tests carried out (E:C = enzyme:collagen ratio). \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & \multicolumn{4}{c}{**Ec 1/20**} & \multicolumn{4}{c}{**Ec 1/50**} \\ \hline
**Amino Acid** & **Bro** & **Cat** & **Col** & **Bro** & **Cat** & **Col** \\
**(mg/L)** & & & & & & \\ \hline Alanine & 0.92 (13) & 0.46 (6.4) & 0.82 (11.5) & 0.88 (12.4) & 0.29 (4.1) & 1.4 (19.8) \\ Arginine & 1.03 (6.6) & 0.65 (4.2) & 1.04 (6.6) & 2.25 (14.4) & 0.88 (5.6) & 1.2 (7.7) \\ Asparagine & 0.17 (1.5) & 0.05 (0.4) & 0.13 (1.2) & 0.02 (0.18) & 0.21 (1.8) & 0.1 (0.9) \\ Aspartic acid & 0.3 (2.5) & 0.11 (1) & 0.28 (2.4) & 0.71 (6.1) & 0.20 (1.7) & 0.35 (3) \\ Glutamine & 11.1 (76) & 12.5 (85.5) & 14.2 (97.2) & 10.5 (71.7) & 11 (75.3) & 10.5 (72) \\ Glutamic acid & 3.4 (23) & 1.7 (11.4) & 2.7 (18.2) & 2.9 (19.4) & 1.9 (13) & 3.2 (21.8) \\ Glycine & 3.4 (46) & 1.9 (25.3) & 3.3 (43.4) & 3.8 (50.4) & 3.7 (49.3) & 4.1 (54) \\ Hydroxylysine & 0.28 (1.7) & 0.18 (1.1) & 0.3 (1.9) & 2.7 (16.7) & 1.35 (8.3) & 8.1 (50) \\ Isoleucine & 0.30 (2.3) & 0.13 (1) & 0.24 (1.8) & 0.37 (2.8) & 0.08 (0.6) & 0.35 (2.7) \\ Leucine & 0.63 (4.8) & 0.31 (2.4) & 0.58 (4.4) & 1.2 (9) & 0.67 (5.1) & 0.68 (5.2) \\ Lysine & 1.61 (11) & 0.34 (2.3) & 0.65 (4.4) & 3 (20.6) & 2.33 (16) & 2 (13.4) \\ Methionine & 2.8 (18.6) & 0.10 (0.6) & 0.23 (1.5) & 1 (7) & 0.19 (1.3) & 6.9 (46.3) \\ Phenylalanine & 0.51 (3.1) & 0.20 (1.2) & 0.35 (2.1) & 0.93 (5.6) & 0.18 (1.1) & 0.4 (2.5) \\ Proline & 1.1 (9.1) & 0.25 (2.1) & 0.51 (4.4) & 1.4 (12.5) & 0.21 (1.8) & 3 (25.5) \\ Serine & 0.7 (6.7) & 0.14 (1.3) & 0.60 (5.7) & 0.64 (6.1) & 0.12 (1.15) & 0.66 (6.3) \\ Threonine & 0.8 (6.6) & 0.37 (3.1) & 0.67 (5.6) & 0.70 (5.9) & 0.55 (4.6) & 0.7 (5.8) \\ Tyrosine & 3 (16) & - & - & - & - & - \\ Valine & 0.94 (8) & 0.31 (2.6) & 0.57 (4.8) & 1.3 (10.8) & 0.21 (1.8) & 1.2 (10) \\ \hline Total (mg/L) & 33 & 19.7 & 27.2 & 34.3 & 24.1 & 44.8 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Free amino acids composition (mg/L) for the <3000 Da peptides-minerals complexes fractions obtained by bromelain (Bro), cathepsin B (Cat) and collagenase B (Col) (values in brackets express concentration in \(\upmu\)M). Figure 1: Collagen peptides concentration according to the enzyme, to the ratio enzyme/collagen and to the molecular weight of the fraction (mean \(\pm\) standard deviation).