Академический Документы
Профессиональный Документы
Культура Документы
23
The Key Laboratory of Leather Chemistry and Engineering of Ministry of Education, Sichuan University, Chengdu, P. R.
China, 610065
Abstract
Collagen, gelatin and collagen hydrolysate were prepared from bovine limed split wastes by different preparative
processes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the
molecular weight distribution of collagen was very narrow (about 200 and 100kDa for and chains
respectively) compared with those of gelatin (less than 300kDa and wide distribution) and collagen hydrolysate
(less than 50kDa and wide distribution ).
The isoelectric points of collagen, gelatin and collagen hydrolysate were 8.26, 4.88 and 4.54 respectively
determined by Zeta potential titration. Circular dichroism (CD) spectra revealed that there were two peaks, a
positive peak around 221nm and a negative peak around 192nm for collagen, which are the characteristics of
collagen triple helix. However, gelatin and collagen hydrolysate lacked any positive peaks around 220nm,
suggesting random coils. The denaturation temperature of collagen was about 37.5C determined by the viscosity
method, the helix-coil transitions for gelatin and collagen hydrolysate were not present in the heating process.
Collagen reaggregated to fibrils at 35C monitored at 313nm. In contrast, gelatin and collagen hydrolysate lost
the ability of fibril formation. Collagen was more resistant to trypsin hydrolysis compared with gelatin and
collagen hydrolysate. In addition, the collagen membrane exhibited superior features such as higher enthalpy,
greater network structure and better physical/mechanical properties compared with those of the gelatin
membrane.
Therefore, collagen isolated from limed split wastes can be a high value product due to its special characteristics
and has many potential future applications in biomaterials, functional additives, cosmetics and pharmaceutical
industries.
Introduction
Experimental methods
Extraction of pepsin-digested collagen
Bovine limed split waste pieces were fleshed and then
delimed with 2% NH4Cl and 0.5% HCl for 60 min. The
delimed pieces were neutralized to pH6.0-7.0 with 0.5%
HCl followed by rinsing with distilled water. Then they
were cut into smaller pieces and pulverized with a mill
(Fritsch Pulverisette 14, Germany). The powders were
extracted with 30 volumes of 0.5 M acetic acid containing
1% pepsin (1:10 000, calculated on the dry weight of limed
split wastes) at 4C for 48 hrs. The supernatants of the
extracted solutions were collected by centrifugation at 10
000g for 15 min at 4C, and then salted out by adding NaCl
to a final concentration of 3 M followed by centrifugation
under the same conditions. The precipitate was dissolved in
0.5 M acetic acid and salted out in 0.7 M NaCl solution.
The precipitate was again dissolved in 0.5 M acetic acid,
and then dialyzed against 0.1 M acetic acid. Collagen
concentration was determined indirectly from the
hydroxyproline concentration, which was analyzed by the
method of Bergman and Loxyley.13
Preparation of gelatin and collagen hydrolysate
Bovine limed split wastes were relimed with 3-4% lime
and 0.5% NaOH for two weeks. The relimed split wastes
23
Wavelength (nm)
Figure 2. CD spectra of collagen, gelatin and collagen hydrolysate.
25
26
Conclusions
Acknowledgement
References
1. Lee, C. H., Singla, A. and Lee, Y., Biomedical applications of
collagen. Int. J. Pharm., 2001, 221, 1-22.
2. Friess, W., Collagen - biomaterial for drug delivery. Eur. J. Pharm.
Biopharm., 1998, 45, 113-136.
3. Pachence, J. M., Collagen-Based Devices for Soft Tissue Repair.
J. Biomed. Res. (Applied Biomaterials), 1996, 33, 35-40.
4. Holmdahl, R., Bockermann, R., Backlund, J. et al., The molecular
pathogenesis of collagen-induced arthritis in mice - a model for
rheumatoid arthritis. Ageing Research Reviews, 2002, 1, 135-147.
5. Tamby, M. C., Chanseaud, Y., Guillevin, L. et al., New insights into
the pathogenesis of systemic sclerosis. Autoimmunity Reviews, 2003,
2, 152-157.
6. Einersona, N. J., Stevensa, K. R. and Kao, W. J., Synthesis and
physicochemical analysis of gelatin-based hydrogels for drug carrier
matrices. Biomaterials , 2002, 24, 509-523.
7.
8.
9.
10.
11.
12.
13.
14.
28