Chicken Combs and Wattles as Sources of Bioactive Peptides: Optimization of Hydrolysis, Identification by LC-ESI-MS2 and Bioactivity Assessment
Abstract
:1. Introduction
2. Results and Discussion
2.1. Optimization of the Hydrolysis Process
2.2. Electrophoresis Profile
2.3. Profile of Total and Free Amino Acids
2.4. Fe2+ Chelating Capacity and Fe3+ Reductive Activity
2.5. Sequestering Activity for 2,2-Diphenyl-1-Picrylhydrazyl Radical (DPPH●) and 2,2-Azino-bis (3-Ethylbeothiazoline)-6-Sulphonic Acid (ABTS●)
2.6. Peptides Profile Identification from Chicken Comb/Wattle Protein Hydrolysate
3. Materials and Methods
3.1. Materials
3.2. Optimization of Hydrolysis of Extracts from the Chicken Comb + Wattle Mixture
3.3. Production of Enzymatic Hydrolysates
3.4. Electrophoresis
3.5. Profile of Total and Free Amino Acids
3.6. Fe2+ Chelating Capacity
3.7. Ferric Reducing Antioxidant Power (FRAP)
3.8. Sequestering Activity for the 2,2-Diphenyl-1-Picrylhydrazyl Radical (DPPH●)
3.9. Sequestering Activity for the 2,2-Azino-bis (3-Ethylbeothiazoline)-6-Sulphonic Acid Radical (ABTS●+)
3.10. Identification of Peptide Sequences by Nanoflow Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (nanoLC-ESI-MS/MS) Analysis
3.11. Statistical Analysis
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample Availability: Samples of the protein hydrolysates are available from the authors. |
Amino Acids | Total Amino Acids | Free Amino Acids | |||
---|---|---|---|---|---|
C + W | <DH | >DH | <DH | >DH | |
Hydrophobic | |||||
Glycine | 9.05 b ± 0.02 | 11.76 a ± 0.09 | 8.82 c ± 0.00 | 0.76 b ± 0.01 | 1.37 a ± 0.01 |
Alanine | 4.82 b ± 0.00 | 5.38 a ± 0.02 | 4.09 c ± 0.00 | 1.03 b ± 0.00 | 1.90 a ± 0.01 |
Leucine | 2.88 c ± 0.00 | 3.60 a ± 0.02 | 2.95 b ± 0.00 | 1.25 b ± 0.01 | 2.02 a ± 0.01 |
Methionine | 0.84 c ± 0.00 | 1.22 a ± 0.02 | 0.99 b ± 0.00 | 0.39 b ± 0.01 | 0.74 a ± 0.02 |
Isoleucine | 0.32 c ± 0.00 | 2.09 a ± 0.00 | 1.74 b ± 0.00 | 0.56 b ± 0.01 | 0.95 a ± 0.01 |
Phenylalanine | 0.90 c ± 0.03 | 1.80 a ± 0.01 | 1.49 b ± 0.00 | 0.65 b ± 0.00 | 1.02 a ± 0.01 |
Hydroxyproline | 4.18 c ± 0.01 | 6.45 a ± 0.04 | 4.70 b ± 0.00 | ND | ND |
Proline | 5.70 b ± 0.01 | 6.82 a ± 0.03 | 5.07 c ± 0.00 | 0.54 b ± 0.00 | 0.62 a ± 0.01 |
Serine | 3.96 a ± 0.00 | 2.60 b ± 0.02 | 2.12 c ± 0.00 | 0.39 b ± 0.00 | 0.79 a ± 0.01 |
Tyrosine | 0.69 c ± 0.00 | 1.17 a ± 0.00 | 0.99 b ± 0.00 | 0.33 b ± 0.00 | 0.74 a ± 0.00 |
Threonine | 1.93 c ± 0.03 | 2.57 a ± 0.02 | 2.06 b ± 0.00 | 0.54 b ± 0.01 | 1.08 a ± 0.00 |
Valine | 0.94 c ± 0.00 | 2.67 a ± 0.00 | 2.23 b ± 0.00 | 0.78 b ± 0.00 | 1.30 a ± 0.01 |
Tryptophan | ND | ND | ND | 0.59a ± 0.01 | 0.53a ± 0.05 |
Total | 36.20 c ± 0.00 | 48.12 a ± 0.02 | 37.2 4b ± 0.00 | 7.78 b ± 0.00 | 13.03 a ± 0.02 |
Hydrophilic | |||||
Aspartic acid | 7.38 a ± 0.00 | 6.03 b ± 0.02 | 4.85 c ± 0.00 | 0.64 b ± 0.00 | 1.02 a ± 0.01 |
Glutamic acid | 10.01 a ± 0.00 | 9.12 b ± 0.05 | 7.05 c ± 0.00 | 1.22 b ± 0.00 | 1.92 a ± 0.01 |
Arginine | 4.37 c ± 0.00 | 5.53 a ± 0.02 | 4.55 b ± 0.00 | 0.63 b ± 0.01 | 1.93 a ± 0.01 |
Histidine | 0.66 a ± 0.04 | 0.32 b ± 0.09 | 0.23 b ± 0.00 | 0.08 a ± 0.01 | 0.09 a ± 0.05 |
Lysine | 1.95 c ± 0.01 | 3.56 a ± 0.03 | 2.80 b ± 0.00 | 0.88 b ± 0.01 | 1.34 a ± 0.01 |
Total | 24.37 a ± 0.00 | 24.56 a ± 0.03 | 19.48 b ± 0.00 | 3.45 b ± 0.00 | 6.30 a ± 0.02 |
Peptide Sequences | Mass (Da) | Length | ppm | AUC |
---|---|---|---|---|
High Degree Hydrolysis | ||||
GLP(+15.99)GPIGP(+15.99)P(+15.99)GPR | 1161.61 | 12 | −5.9 | 5.26 × 107 |
GAP(+15.99)GDRGEP(+15.99)GPP(+15.99)GPA | 1378.61 | 15 | −7.7 | 2.94 × 107 |
RGFP(+15.99)GLP(+15.99)GPS | 1015.50 | 10 | −7.2 | 2.14 × 107 |
GDIGGP(+15.99)GFP(+15.99)GPK | 1129.54 | 12 | −9.1 | 2.05 × 107 |
GAP(+15.99)GLP(+15.99)GPR | 852.445 | 9 | −9.6 | 1.59 × 107 |
GKDGLNGLP(+15.99)GPIGP(+15.99)P(+15.99)GPR | 1745.90 | 18 | −6 | 1.31 × 107 |
GPP(+31.99)GPPGPP(+15.99)GPP(+15.99)GPPSGGF | 1685.76 | 19 | −4.6 | 1.22 × 107 |
RGPGGPP(+15.99)GTP(+15.99)GPA | 1148.55 | 13 | −7.9 | 1.18 × 107 |
N(+.98)GDAGRP(+31.99)GEPGLM | 1302.55 | 13 | −7.1 | 1.16 × 107 |
GEP(+15.99)GRSGPP(+15.99)GPAGPR | 1419.68 | 15 | −8.8 | 1.14 × 107 |
Low Degree Hydrolysis | ||||
TIGIGDIFDQPEQSETL | 1861.89 | 17 | −7 | 2.72 × 107 |
GVDTKYVPPPFNPDMFSF | 2056.96 | 18 | −7.8 | 2.46 × 107 |
GARGPAGPQGPRGDK(+15.99)GETGEQGDRGMK(+15.99)GH | 2891.34 | 29 | −9.5 | 1.24 × 107 |
GDRGDP(+15.99)GPKGADGAPGKDGLRGL | 2178.07 | 23 | −8.9 | 7.89 × 106 |
GLP(+15.99)GQPGSP(+31.99)GPAGKEGPVGFP(+15.99)GADGRVGPIGPA | 3013.48 | 33 | −7.4 | 7.23 × 106 |
GPAGPQGPRGDK(+15.99)GETGEQGDRGMK(+15.99)GH | 2607.18 | 26 | −9.8 | 7.03 × 106 |
GPAGNRGASGPVGAKGPNGDAGRP(+15.99)GEP(+15.99)GLMGPR | 3042.48 | 33 | −9.1 | 6.65 × 106 |
GFP(+15.99)GADGIAGPKGPP(+15.99)GE | 1554.73 | 17 | −9.8 | 6.33 × 106 |
P(+15.99)GADGIAGPKGPP(+31.99)GER(+31.99) | 1554.72 | 16 | −8.5 | 6.33 × 106 |
GNRGEP(+15.99)GNIGFP(+15.99)GPKGPTGEPGKP(+31.99)GEKGNVGLAGPR | 3515.75 | 36 | −9.7 | 6.20 × 106 |
Assay | Independent Variables and Variation Levels | Response Function (Y) | |
---|---|---|---|
Enzyme to Substrate Ratio (%) | Hydrolysis Time (minutes) | Degree of Hydrolysis (%) | |
1 | −(1) | −(60) | 7.51 |
2 | +(5) | −(60) | 13.55 |
3 | −(1) | +(240) | 12.19 |
4 | +(5) | +(240) | 19.34 |
5 | 0(3) | 0(150) | 14.90 |
6 | 0(3) | 0(150) | 14.29 |
7 | 0(3) | 0(150) | 14.53 |
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Bezerra, T.; Estévez, M.; Lacerda, J.T.; Dias, M.; Juliano, M.; Mendes, M.A.; Morgano, M.; Pacheco, M.T.; Madruga, M. Chicken Combs and Wattles as Sources of Bioactive Peptides: Optimization of Hydrolysis, Identification by LC-ESI-MS2 and Bioactivity Assessment. Molecules 2020, 25, 1698. https://doi.org/10.3390/molecules25071698
Bezerra T, Estévez M, Lacerda JT, Dias M, Juliano M, Mendes MA, Morgano M, Pacheco MT, Madruga M. Chicken Combs and Wattles as Sources of Bioactive Peptides: Optimization of Hydrolysis, Identification by LC-ESI-MS2 and Bioactivity Assessment. Molecules. 2020; 25(7):1698. https://doi.org/10.3390/molecules25071698
Chicago/Turabian StyleBezerra, Taliana, Mario Estévez, José Thalles Lacerda, Meriellen Dias, Maria Juliano, Maria Anita Mendes, Marcelo Morgano, Maria Teresa Pacheco, and Marta Madruga. 2020. "Chicken Combs and Wattles as Sources of Bioactive Peptides: Optimization of Hydrolysis, Identification by LC-ESI-MS2 and Bioactivity Assessment" Molecules 25, no. 7: 1698. https://doi.org/10.3390/molecules25071698