Pontastacus leptodactylus (Eschscholtz, 1823) and Faxonius limosus (Rafinesque, 1817) as New, Alternative Sources of Chitin and Chitosan
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.1.1. Biomass
2.1.2. Reagents
2.2. Chitin and Chitosan Isolation
2.2.1. Standard Chitin Extraction
Demineralization
Deproteinization
2.2.2. Standard Chitosan Extraction
Deacetylation
2.2.3. NADES Chitin Extraction
- The material was kept with NADES at 100 °C under mechanical stirring for 2 h. After filtration under vacuum, the obtained material was washed seven times with 25 mL of distilled water in a centrifuge at 4000 rpm (7 times for 5 min) until the neutral pH was obtained and dried in the oven at 80 °C for 48 h. Then, the extracted material was additionally treated with 20 mL of H2O2 at 10% for 2 h at 80 °C, washed with 25 mL of distilled water in a centrifuge at 4000 rpm (7 times for 5 min) until the neutral pH was obtained, and dried in the oven at 80 °C for 48 h.
- The material was pre-treated with 10 mL of C6H8O7 (10%, w/v) for 1 h at room temperature and then treated following the point above.
2.3. Fourier Transform Infrared Spectroscopy (FTIR)
2.4. Thermogravimetric Analysis (TG)
2.5. Scanning Electron Microscopy (SEM)
2.6. Molar Mass Distribution
2.7. Antimicrobial Assays
2.7.1. Chitosan Salification
2.7.2. Bacteria Culture
2.7.3. Evaluation of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
2.8. Cytotoxicity Assay (MTT)
2.8.1. Cell Culture
2.8.2. Assessment of metabolic activity
2.9. Statistical Analysis
3. Results
3.1. Chitin and Chitosan Recovery
3.1.1. Chitin and Chitosan Obtained Using Standard Chemical Procedures
3.1.2. Preliminary Extraction with NADES Mixture
3.2. Fourier Transform Infrared Spectroscopy (FTIR)
Chitin and Chitosan Obtained Using Standard Extraction Characterisation
3.3. Thermogravimetric Analysis (TG) of Chitin and Chitosan Obtained Using Standard Extraction Characterisation
3.4. Scanning Electron Microscopy (SEM)
3.5. Molecular Weight (MW)
3.6. Antimicrobial Activity
3.7. Determination of Cytotoxicity
4. Discussion
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Species | Treatment | Chitin Recovery [%] | Chitosan Recovery from Chitin [%] | Chitosan Recovery from Shells [%] |
---|---|---|---|---|
P. leptodactylus | standard extraction | 22 ± 2.7 a | 70 ± 13.0 b | 15 ± 0.3 b |
P. leptodactylus | NADES | 24 c | N/D | N/D |
P. leptodactylus | NADES with citric acid pretreatment | 29 c | N/D | N/D |
F. limosus | standard extraction | 20 ± 3.4 d | 76 ± 9.0 b | 14 ± 4.2 b |
F. limosus | NADES | 31 c | N/D | N/D |
F. limosus | NADES with citric acid pretreatment | 25 c | N/D | N/D |
Functional Group | Commercial Chitin Wavenumber [cm−1] | P. leptodactylus Wavenumber [cm−1] | F. limosus Wavenumber [cm−1] | P. leptodactylus (NADES) Wavenumber [cm−1] | F. limosus (NADES) Wavenumber [cm−1] |
---|---|---|---|---|---|
Amide I | 1622 | 1633 | 1628 | 1622 | 1635 |
Amide II | 1551 | 1553 | 1552 | 1551 | 1549 |
-C-H | 2875 | 2879 | 2890 | 2883 | 2874 |
-C-O-C- | 1070 | 1063 | 1067 | 1071 | 1068 |
1026 | 1026 | 1026 | 1011 | 1027 |
Functional Group | Commercial Chitosan Wavenumber [cm−1] | P. leptodactylus Wavenumber [cm−1] | F. limosus Wavenumber [cm−1] |
---|---|---|---|
Amide I | 1647 | 1654 | 1650 |
Amide II | 1564 | 1590 | 1590 |
-C-H | 2872 | 2874 | 2864 |
-C-O-C- | 1063 | 1054 | 1059 |
1027 | 1027 | 1026 |
Species | MIC (E. coli) | MIC (S. aureus) | MBC (E. coli) | MBC (S. aureus) |
---|---|---|---|---|
P. leptodactylus | 1.19 | 1.59 | 1.58 | 2.48 |
F. limosus | 2.48 | 1.96 | 4.90 | 4.90 |
Chitosan s.* | 1.15 | 2.30 | >9.20 | >9.20 |
Ampicilin | 5 × 10−3 | 0.5 × 10−3 | 10 × 10−3 | 2 × 10−3 |
Species | IC50 (3 h) | IC50 (24 h) | IC50 (48 h) |
---|---|---|---|
P. leptodactylus | 0.51 | 0.24 | 0.14 |
F. limosus | 0.52 | 0.49 | 0.34 |
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Nuc, Z.; Brusotti, G.; Catenacci, L.; Grenha, A.; Pontes, J.F.; Pinto da Silva, J.; Rosa da Costa, A.M.; Moro, P.; Milanese, C.; Grisoli, P.; et al. Pontastacus leptodactylus (Eschscholtz, 1823) and Faxonius limosus (Rafinesque, 1817) as New, Alternative Sources of Chitin and Chitosan. Water 2023, 15, 3024. https://doi.org/10.3390/w15173024
Nuc Z, Brusotti G, Catenacci L, Grenha A, Pontes JF, Pinto da Silva J, Rosa da Costa AM, Moro P, Milanese C, Grisoli P, et al. Pontastacus leptodactylus (Eschscholtz, 1823) and Faxonius limosus (Rafinesque, 1817) as New, Alternative Sources of Chitin and Chitosan. Water. 2023; 15(17):3024. https://doi.org/10.3390/w15173024
Chicago/Turabian StyleNuc, Zofia, Gloria Brusotti, Laura Catenacci, Ana Grenha, Jorge F. Pontes, Joana Pinto da Silva, Ana Maria Rosa da Costa, Paola Moro, Chiara Milanese, Pietro Grisoli, and et al. 2023. "Pontastacus leptodactylus (Eschscholtz, 1823) and Faxonius limosus (Rafinesque, 1817) as New, Alternative Sources of Chitin and Chitosan" Water 15, no. 17: 3024. https://doi.org/10.3390/w15173024