Molecular Weight Analysis of Blue Shark (Prionace glauca) Collagen Hydrolysates by GPC-LS; Effect of High Molecular Weight Hydrolysates on Fibroblast Cultures: mRNA Collagen Type I Expression and Synthesis
Abstract
:1. Introduction
2. Results and Discussion
2.1. Collagen Extraction
2.1.1. SDS-PAGE
2.2. Effect of Hydrolysis Conditions on BSCH Mw
2.2.1. GPC-LS Analysis
2.2.2. HPLC-SEC Analysis
2.3. Amino Acid Characterization of BSCH-C and CHC
2.4. Effect of HMW-BSCH and CHC on Cell Viability
2.5. Effect of High Mw BSCH and CHC on mRNA Collagen Synthesis of Fibroblast Culture
2.6. Effect of HMW-BSCH and CHC on Pro-Collagen I Synthesis (ELISA) of Fibroblast Culture
3. Materials and Methods
3.1. Biological Samples and Compositional Analysis
3.2. Extraction of Acid Soluble Collagen
3.2.1. Proximate Composition
3.2.2. SDS-PAGE
3.3. Enzymatic Hydrolysis of Acid Soluble Collagen
3.4. Characterization of Hydrolysates
3.4.1. Size-Exclusion Chromatography
3.4.2. GPC of Blue Shark Collagen Hydrolysates
3.4.3. Statistical Analysis
3.5. Amino Acid Characterization of BSCH and CHC
3.6. Cell Culture and Treatment with Blue Shark Collagen Hydrolysate
3.7. Cell Viability Assay
3.8. RNA Isolation and Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
3.9. Human Pro-Collagen I Quantification from Fibroblast Cell Culture Supernatants by Sandwich ELISAs
3.10. Cell Viability, RNA Expression, and Pro-Collagen I Biosynthesis Statistical Analysis
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Lyophilized BSASC | Proximate Composition (%) | ||
Moisture | Protein | Ash | |
12.93 ± 0.70 | 84.46 ± 0.65 | 1.32 ± 0.54 |
Blue Shark Collagen Hydrolysis Conditions | Mw (kDa) | |||
---|---|---|---|---|
Experiments | Enzyme | E/S Rate | Time (h) | |
A | Papain | 1/5 | 6 | 11.6 ± 5.4 |
B | Alcalase | 1/20 | 6 | 3.5 ± 0.2 |
C | Papain | 1/20 | 0.5 | 34.43 ± 10.2 |
D | Alcalase | 1/20 | 0.5 | 3.2 ± 0.7 |
E | Alcalase | 1/5 | 6 | 8 ± 5.0 |
F | Papain | 1/5 | 0.5 | 20.72 ± 4.2 |
G | Papain | 1/20 | 6 | 9.26 ± 5.3 |
H | Alcalase | 1/5 | 0.5 | 4.3 ± 0.0 |
Retention Time (min) | Mw (kDa) | % Area | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
BSCH | CHC | |||||||||
A | B | C | D | E | F | G | H | |||
20–30 | 17–3 | 37 ± 7 | 2 ± 0 | 9.33 ± 2 | 4 ± 1 | 3.5 ± 2 | 18.3 ± 7 | 30 ± 0 | 2.67 ± 2 | 50.6 ± 0.6 |
30–40 | 3–0.5 | 52 ± 9 | 84.5 ± 3 | 71.7 ± 3 | 88.6 ± 1 | 82 ± 2 | 69.3± 6 | 55 ± 13 | 89 ± 2 | 42.4 ± 0.2 |
40–50 | 0.5–0.09 | 11 ± 2 | 13 ± 4 | 19 ± 2 | 7.3 ± 2 | 14.5 ± 4 | 12.3 ± 4 | 15 ± 14 | 8.33 ± 2 | 6.9 ± 0.4 |
Amino Acid | BSCH-C | CHC |
---|---|---|
Hydroxyproline | 70.249 ± 0.05 | 89.85 ± 0.33 |
Aspartic acid | 44.761 ± 0.02 | 43.27 ± 0.07 |
Threonine | 23.567 ± 0.02 | 15.91 ± 0.06 |
Serine | 43.928 ± 0.07 | 35.31 ± 0.12 |
Glutamic acid | 76.674 ± 0.08 | 70.18 ± 0.05 |
Proline | 112.081 ± 0.08 | 115.41 ± 0.67 |
Glycine | 306.533 ± 0.32 | 296.87 ± 1.48 |
Alanine | 118.869 ± 0.04 | 106.385 ± 0.31 |
Cysteine | 3.696 ± 0.004 | 3.383 ± 0.11 |
Valine | 24.817 ± 0.030 | 20.650 ± 0.20 |
Methionine | 14.3 ± 0.006 | 6.137 ± 0.00 |
Isoleucine | 21.357 ± 0.04 | 11.158 ± 0.00 |
Leucine | 24.856 ± 0.02 | 23.058 ± 0.03 |
Tyrosine | 4.4 ± 0.01 | 2.086 ± 0.02 |
Phenylalanine | 16.699 ± 0.06 | 12.299 ± 0.05 |
Hydroxylysine | 4.518 ± 0.02 | 5.977 ± 0.04 |
Histdine | 7.543 ± 0.002 | 5.543 ± 0.016 |
Lysine | 28.206 ± 0.06 | 27.158 ± 0.08 |
Arginine | 52.948 ± 0.02 | 46.090 ± 0.16 |
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Blanco, M.; Sanz, N.; Sánzhez, A.C.; Correa, B.; Pérez-Martín, R.I.; Sotelo, C.G. Molecular Weight Analysis of Blue Shark (Prionace glauca) Collagen Hydrolysates by GPC-LS; Effect of High Molecular Weight Hydrolysates on Fibroblast Cultures: mRNA Collagen Type I Expression and Synthesis. Int. J. Mol. Sci. 2022, 23, 32. https://doi.org/10.3390/ijms23010032
Blanco M, Sanz N, Sánzhez AC, Correa B, Pérez-Martín RI, Sotelo CG. Molecular Weight Analysis of Blue Shark (Prionace glauca) Collagen Hydrolysates by GPC-LS; Effect of High Molecular Weight Hydrolysates on Fibroblast Cultures: mRNA Collagen Type I Expression and Synthesis. International Journal of Molecular Sciences. 2022; 23(1):32. https://doi.org/10.3390/ijms23010032
Chicago/Turabian StyleBlanco, María, Noelia Sanz, Ana C. Sánzhez, Begoña Correa, Ricardo I. Pérez-Martín, and Carmen G. Sotelo. 2022. "Molecular Weight Analysis of Blue Shark (Prionace glauca) Collagen Hydrolysates by GPC-LS; Effect of High Molecular Weight Hydrolysates on Fibroblast Cultures: mRNA Collagen Type I Expression and Synthesis" International Journal of Molecular Sciences 23, no. 1: 32. https://doi.org/10.3390/ijms23010032