Figure 1.
Concentration of peptides, <3000 Da (left) and >3000 Da (right) (equivalents of collagen, mg/L) from young and old bovine femur and tibia collagen solutions hydrolysed through the enzymes alcalase (A), collagenase (C), esperase (E), neutrase (N), papain (P), savinase (S) (mean values ± standard deviation (SD), n = 3). For each enzyme, significant differences among bones (p < 0.05) are indicated by different lowercase letters (only for the left Figure). FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 1.
Concentration of peptides, <3000 Da (left) and >3000 Da (right) (equivalents of collagen, mg/L) from young and old bovine femur and tibia collagen solutions hydrolysed through the enzymes alcalase (A), collagenase (C), esperase (E), neutrase (N), papain (P), savinase (S) (mean values ± standard deviation (SD), n = 3). For each enzyme, significant differences among bones (p < 0.05) are indicated by different lowercase letters (only for the left Figure). FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 2.
Principal component analysis (PCA) on collagen peptides concentration considering enzymes and particle size as variables (A: loading plot) and bone age and anatomy as individuals (B: score plot). FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 2.
Principal component analysis (PCA) on collagen peptides concentration considering enzymes and particle size as variables (A: loading plot) and bone age and anatomy as individuals (B: score plot). FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 3.
Highest (A) and lowest (B) FRAP values (µM Fe2+) for collagen peptides solutions (<3000 Da) obtained through papain and esperase, respectively, at increasing temperatures. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 3.
Highest (A) and lowest (B) FRAP values (µM Fe2+) for collagen peptides solutions (<3000 Da) obtained through papain and esperase, respectively, at increasing temperatures. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 4.
Highest and lowest DPPH• inhibition (%) for collagen peptides solutions (<3000 Da) obtained through neutrase (A) and esperase (B), respectively, and at increasing temperatures. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 4.
Highest and lowest DPPH• inhibition (%) for collagen peptides solutions (<3000 Da) obtained through neutrase (A) and esperase (B), respectively, and at increasing temperatures. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 5.
ABTS•+ inhibition (%) by collagen peptides solutions (<3000 Da) obtained through the most effective enzyme, i.e., papain, for 10 µL (A) and 100 µL peptides solutions (B), at ambient temperature and 80 °C, and after 30 ant 180 min of incubation. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 5.
ABTS•+ inhibition (%) by collagen peptides solutions (<3000 Da) obtained through the most effective enzyme, i.e., papain, for 10 µL (A) and 100 µL peptides solutions (B), at ambient temperature and 80 °C, and after 30 ant 180 min of incubation. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 6.
Reduction of carbonyls value (%) in bovine meat (semimembranosus muscle) at ambient temperature and at 80 °C through the addition of collagen peptides solutions (<3000 Da) from young and old bovine femur and tibia obtained through the enzymes savinase (A) and alcalase (B). FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 6.
Reduction of carbonyls value (%) in bovine meat (semimembranosus muscle) at ambient temperature and at 80 °C through the addition of collagen peptides solutions (<3000 Da) from young and old bovine femur and tibia obtained through the enzymes savinase (A) and alcalase (B). FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 7.
Reduction of TBArs (%) in bovine meat (semimembranosus muscle) at ambient temperature and at 80°C through the addiction of collagen peptides solutions (<3000 Da) from young and old bovine femur and tibia obtained through the enzymes papain (A) and collagenase (B). FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 7.
Reduction of TBArs (%) in bovine meat (semimembranosus muscle) at ambient temperature and at 80°C through the addiction of collagen peptides solutions (<3000 Da) from young and old bovine femur and tibia obtained through the enzymes papain (A) and collagenase (B). FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 8.
PCA loading plot of antioxidants activity and peptides concentration for alcalase (A), collagenase (B) and papain (C).
Figure 8.
PCA loading plot of antioxidants activity and peptides concentration for alcalase (A), collagenase (B) and papain (C).
Figure 9.
(A) Hierarchical clustering analysis (HCA) of enzymes on the basis of the antioxidant activities and for all the bones (FO, TO, FY and TY); (B) HCA of bones based on antioxidants activities and amount of peptides released by each enzyme. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 9.
(A) Hierarchical clustering analysis (HCA) of enzymes on the basis of the antioxidant activities and for all the bones (FO, TO, FY and TY); (B) HCA of bones based on antioxidants activities and amount of peptides released by each enzyme. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Figure 10.
Overview of the antioxidant activity of bovine bone collagen peptides (at 80 °C) by considering bones of different ages and anatomies (femur old, 10 and; femur young, 4.5 years; tibia old, 10 years; tibia young, 4.5 years), and six enzymes (alcalase, collagenase, esperase, neutrase, papain, savinase).
Figure 10.
Overview of the antioxidant activity of bovine bone collagen peptides (at 80 °C) by considering bones of different ages and anatomies (femur old, 10 and; femur young, 4.5 years; tibia old, 10 years; tibia young, 4.5 years), and six enzymes (alcalase, collagenase, esperase, neutrase, papain, savinase).
Table 1.
FRAP index (mean values) at 80 °C for each enzyme, expressed as mmol of electrons donated per 100 g of peptides solution. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Table 1.
FRAP index (mean values) at 80 °C for each enzyme, expressed as mmol of electrons donated per 100 g of peptides solution. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
| Alcalase | Collagenase | Esperase | Neutrase | Papain | Savinase |
---|
FO | 0.0050 a,A | 0.0035 a,B | 0.0077 a,C | 0.0076 a,C | 0.0096 a,D | 0.0029 a,E |
TO | 0.0051 b,A | 0.0073 b,B | 0.0084 b,C | 0.0075 b,D | 0.0140 b,E | 0.0083 b,F |
FY | 0.0092 c,A | 0.0102 c,B | 0.0094 c,C | 0.0064 c,D | 0.0138 c,E | 0.0076 c,F |
TY | 0.0136 d,A | 0.0130 d,B | 0.0090 d,C | 0.0110 d,D | 0.0178 d,E | 0.0104 d,F |
Table 2.
FRAP index at 80 °C for each enzyme, expressed as mmol of electrons donated per 100 g of peptides. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Table 2.
FRAP index at 80 °C for each enzyme, expressed as mmol of electrons donated per 100 g of peptides. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
| Alcalase | Collagenase | Esperase | Neutrase | Papain | Savinase |
---|
FO | 16.1 ± 0.8 a,A | 7.7 ± 0.5 a,B | 25.3 ± 0.9 a,C | 23.4 ± 2.1 a,D | 40.4 ± 0.9 a,E | 10.5 ± 1.3 a,F |
TO | 11.2 ± 1.1 b,A | 15.0 ± 0.3 b,B | 29.3 ± 1.5 b,C | 17.9 ± 1.8 b,D | 25.9 ± 0.8 b,E | 18.3 ± 0.9 b,D |
FY | 29.6 ± 1.3 c,A | 30.8 ± 1.2 c,A | 33.5 ± 1.7 c,B | 21.3 ± 1.6 c,C | 20.2 ± 1.4 c,D | 23.6 ± 1.2 c,E |
TY | 40.2 ± 0.9 d,A | 37.0 ± 0.9 d,B | 32.9 ± 1.3 c,C | 30.1 ± 2.0 d,D | 25.2 ± 1.6 b,E | 23.0 ± 1.5 c,F |
Table 3.
DPPH• inhibition (%) per mg of peptides, at 80 °C. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Table 3.
DPPH• inhibition (%) per mg of peptides, at 80 °C. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
| Alcalase | Collagenase | Esperase | Neutrase | Papain | Savinase |
---|
FO | 69.0 ± 2.3 a,A | 35.8 ± 0.8 a,B | 57.3 ± 1.1 a,C | 72.8 ± 1.3 a,D | 80.2 ± 0.9 a,E | 60.6 ± 2.2 a,F |
TO | 41.3 ± 1.5 b,A | 38.8 ± 1.6 b,B | 56.6 ± 0.6 a,C | 57.6 ± 0.8 b,C | 35.3 ± 2.3 b,D | 36.7 ± 1.4 b,E |
FY | 48.0 ± 1.8 c,A | 57.3 ± 1.7 c,B | 66.7 ± 1.6 b,C | 78.5 ±1.6 c,D | 25.7 ± 1.4 c,D | 50.5 ± 1.3 c,E |
TY | 47.3 ± 1.6 c,A | 52.1 ± 2.1 d,B | 59.5 ± 1.5 c,C | 66.1 ±1.4 d,D | 24.2 ± 1.8 d,E | 34.9 ± 1.8 d,F |
Table 4.
Reduction of carbonyls values (%) in 210 mg of meat protein (i.e., 1 g of raw meat), per mg of collagen peptides, at ambient temperature. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Table 4.
Reduction of carbonyls values (%) in 210 mg of meat protein (i.e., 1 g of raw meat), per mg of collagen peptides, at ambient temperature. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
| Alcalase | Collagenase | Esperase | Neutrase | Papain | Savinase |
---|
FO | 15.1 ± 2.1 a,A | 10.3 ± 0.7 a,B | 14.1 ± 1.3 a,C | 14.4 ± 0.6 a,C | 20.5 ± 1.1 a,E | 18.1 ± 0.7 a,F |
TO | 9.6 ± 1.8 b,A | 9.4 ± 1.2 b,A | 15.6 ± 1.8 b,C | 9.5 ± 0.9 b,A | 7.7 ± 1.2 b,B | 9.9 ± 0.9 b,C |
FY | 12.8 ± 1.6 c,A | 10.5 ± 2.0 a,B | 15.2 ± 0.9 b,C | 12.7 ±1.1 c,A | 6.0 ± 0.3 c,D | 11.2 ± 1.1 c,E |
TY | 14.1 ± 1.3 d,A | 12.1 ± 0.9 c,B | 18.5 ± 1.3 c,C | 11.1 ±0.4 d,D | 7.1 ± 0.8 b,E | 8.6 ± 1.2 d,F |
Table 5.
Reduction of TBArs (%) in 22.5 mg of meat lipids (i.e., 1 g of raw meat), per mg of collagen peptides, at ambient temperature. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Table 5.
Reduction of TBArs (%) in 22.5 mg of meat lipids (i.e., 1 g of raw meat), per mg of collagen peptides, at ambient temperature. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
| Alcalase | Collagenase | Esperase | Neutrase | Papain | Savinase |
---|
FO | 55.2 ± 1.8 a,A | 30.3 ± 0.9 a,B | 63.1 ± 2.2 a,C | 56.3 ± 2.1 a,A | 76.2 ± 1.2 a,D | 65.1 ± 1.4 a,E |
TO | 33.4 ± 1.1 b,A | 22.5 ± 1.3 b,B | 66.2 ± 1.7 b,C | 39.4 ± 1.6 b,D | 34.1 ± 0.9 b,A | 38.2 ± 1.3 b,E |
FY | 58.2 ± 1.6 c,A | 52.4 ± 1.6 a,B | 55.1 ± 1.4 b,C | 58.1 ± 1.4 c,A | 27.2 ± 0.8 c,D | 52.2 ± 1.2 c,B |
TY | 47.1 ± 1.3 d,A | 43.3 ± 1.5 c,B | 53.2 ± 1.9 c,C | 50.2 ± 0.9 d,D | 25.3 ± 1.1 b,E | 44.1 ± 1.5 d,F |
Table 6.
Kinetics parameter for the iron reducing activity models (Equation (1a) and (1b)) and models adjustment (Adj. χ2) for papain and esperase peptides. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Table 6.
Kinetics parameter for the iron reducing activity models (Equation (1a) and (1b)) and models adjustment (Adj. χ2) for papain and esperase peptides. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Peptides (FRAP) | y0 (µM Fe 2+) | T0 (°C) | A (µM Fe 2+) | t (°C −1) | Adj. χ2 |
---|
Papain FO (ambient–70 °C) (Equation (1a)) | 7.9 | 0 | 4.07 | 21.52 | 0.97 |
Papain TO (Equation (1a)) | 6.22 | 0 | 5.77 | 23.96 | 0.98 |
Papain FY (Equation (1a)) | 3.97 | 0 | 5.15 | 23.23 | 0.98 |
Papain TY (Equation (1b)) | 16.94 | 17.25 | 14.76 | 24.24 | 0.96 |
Esperase FO (50–80 °C) (Equation (1a)) | 50.37 | 0 | 4.07 | 17.58 | 0.95 |
Esperase TO (Equation (1a)) | 20.12 | 0 | 1.57 | 20.28 | 0.99 |
Esperase FY (Equation (1a)) | 20.28 | 0 | 1.96 | 20.71 | 0.96 |
Esperase TY (Equation (1a)) | 13.05 | 0 | 4.56 | 26.31 | 1 |
| a (µM Fe 2+) | b (µM Fe 2+/°C) | | | |
Papain FO (70–80 °C) (Equation (2)) | 99 | 0.2 | | | |
Esperase FO (ambient–50 °C) (Equation (3)) | −1.53 | 6.23 | | | |
Table 7.
Kinetic parameter for the DPPH• quenching models and model adjustment (Adj. χ2) for neutrase and esperase. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Table 7.
Kinetic parameter for the DPPH• quenching models and model adjustment (Adj. χ2) for neutrase and esperase. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Peptides (DPPH) | y0 (µM Fe 2+) | A (µM Fe 2+) | t (°C −1) | Adj. χ2 |
---|
Esperase TY (Equation (1a)) | 10.42 | 0.57 | 19.02 | 0.98 |
Neutrase TO (Equation (1a)) | 1.99 | 8.14 | 36.97 | 0.99 |
Neutrase FY (Equation (1a)) | 9.86 | 3.42 | 27.70 | 0.96 |
Neutrase TY (Equation (1a)) | 10.10 | 5.38 | 32.81 | 0.99 |
Peptides (DPPH) | a | b | c | Adj. χ2 |
Esperase FO (amb.–50 °C) (Equation (4)) | 0.83 | −4.88 | −16.93 | 0.97 |
Esperase FO (50–80 °C) (Equation (5)) | 340 | −0.27 | | 0.99 |
Esperase TO (amb.–50 °C) (Equation (2)) | 0.80 | 0.47 | | 1 |
Esperase TO (50–80 °C) (Equation (2)) | −17.83 | 0.85 | | 0.96 |
Esperase FY (amb.–50 °C) (Equation (2)) | −1.58 | 0.48 | | 0.98 |
Esperase FY (50–80 °C) (Equation (2)) | −39.33 | 1.20 | | 0.99 |
Neutrase FO (amb.–50 °C) (Equation (2)) | 5.54 | 0.47 | | 0.98 |
Neutrase FO (50–80 °C) (Equation (5)) | 458.37 | −0.27 | | 0.99 |
Table 8.
Composition of collagen powders. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Table 8.
Composition of collagen powders. FO = old femur, 10 years; TO = old tibia, 10 years; FY = young femur, 4.5 years; TY = young tibia, 4.5 years.
Sample | Organic Matter (%) | Collagen (%) | Non-Collagen (%) | Minerals (%) | Calcium (%) |
---|
FO | 35.3 | 34.5 | 0.83 | 64.7 | 20.5 |
TO | 36 | 35.1 | 0.93 | 64 | 20 |
FY | 39 | 38.4 | 0.64 | 61 | 19 |
TY | 40 | 39.2 | 0.74 | 60 | 18 |
Table 9.
Molecular weight, specificity, cutting sites, and optimal pH and temperature of hydrolysis for the 6 enzymes (endopeptidases) tested to hydrolyse bovine bone collagen (AA = amino acid).
Table 9.
Molecular weight, specificity, cutting sites, and optimal pH and temperature of hydrolysis for the 6 enzymes (endopeptidases) tested to hydrolyse bovine bone collagen (AA = amino acid).
Enzyme | MW (kDa) | Specificity/Cutting Sites | Temperature (°C) | pH |
---|
Alcalase | 20–45 | non-specific/larger and uncharged AA | 60 | 7 |
Collagenase B | 68–130 | specific/Gly-AA | 37 | 7 |
Esperase | 20–30 | non-specific/larger and uncharged AA | 55 | 10 |
Neutrase | 37 | non-specific/larger and uncharged AA | 55 | 6 |
Papain | 23.4 | specific/Ala, Val, Leu, Ile, Phe, Tyr | 65 | 6.5 |
Savinase | 20–30 | non-specific/larger and uncharged AA | 55 | 10 |