# Effects of Temperature on the FT NIR Raman Spectra of Fish Skin Collagen
###### Abstract  
The development of regenerative medicine turns attention toward native collagen as a biocompatible material. Particularly interesting is fish skin collagen, which is relatively easy to extract comparing mammalian tissues and free of some pathogens that are dangerous to humans. The paper presents results of IR Raman spectroscopy studies of silver carp (_Hypophthalmichtthys molitrixy_ skin collagen. As collagen properties result from its structure and conformation, both sensitive to temperature, FT NIR Raman spectroscopy is an excellent tool to characterize the molecular structure of fish skin collagen, particularly in temperature range typical for the manufacturing processes of biomedical products. Therefore, the Raman spectra were recorded in a temperature range of 300 to 403 K. The analysis of Raman spectra of prepared collagen films, particularly in the range of the bands related to amide I and amide III entities, showed a high content of \(\alpha\)-helix and \(\alpha\)-helix type molecular organization in fish skin collagen. Additionally, the secondary structure of the studied fish skin collagen is stable up to -358 K. Heating to 403 K leads to irreversible changes in the molecular structure of fish skin collagen. It was found that the Raman spectrum of fish skin collagen preheated in this manner becomes similar to the spectrum of the collagen obtained from bovine Achilles tendon, whose secondary structure does not change up to 403 K.  
Raman spectroscopy; fish skin collagen; molecular structure; thermal stability +
Footnote †: journal: Applied Sciences  
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and its ability to bond with other polymers, both natural and synthetic [13; 14]. Collagen, including that of animal origin, which is recognized by the organism as its component and not a foreign matter [11; 15; 16; 17; 18], is characterized by a high degree of biocompatibility [15]. The biocompatibility of collagen is connected with its structure and conformation of molecules. This fact justifies the importance of research into its structure and thermal stability. Fish collagen, contrary to mammalian collagen, is usually free of some pathogens dangerous to humans. The advantages of fish collagen involve ethical issues related to customs and religions and economic considerations, which are equally important, as fish waste products are a cheap source of collagen [19]. Thus, attention was turned towards fish collagen due to its native structure maintained during the extraction process [20; 21; 22]. Collagen originating from different fish species differ in their denaturation temperature. Generally, the lower the temperature of the feeding ground (water) for fish, the lower the denaturation temperature of collagen macromolecules. Particularly interesting, from the point of view of economy, is silver carp (_Hypophthalmichtluys molitrix_), a freshwater fish. The denaturation temperature of silver carp skin collagen is reported to be either 302 K [1] or 307 K [3; 23; 24] depending on the experimental technique. The closer the denaturation temperature of fish skin collagen to mammalian collagen, the higher the potential in supplementing/replacing the skin of land vertebrates as a collagen source and substituting mammalian collagen. Moreover, fish skin collagen extraction is easier and has a higher yield comparing the extraction from mammalian skin [17; 25; 26]. The basic structural unit of collagen and collagen structure is described elsewhere [27; 28; 29; 30; 31; 32]. Collagen polypeptide chains are rich in glycine (Gly), proline (Pro) and 3 hdcryproline (Hyp) (Figure 1), linked together in the characteristic pattern XPro-YHyp-Gly [29]. Other amino acids residues, such as alanine, lysosome and tyrosine, may occur in the X or Y locations of the polypeptide chain. In the \(\alpha\)-chain of fibrous collagen, about 25% of amino acids are proline and hydroxyproline. They stabilize the triple-helical structure and effectively block internal rotations of collagen chains at these sites [33].