## 4 Discussion
### Characterization of Collagen  
FTIR spectrum gives a good indication of the presence and general structure of the collagen; however, this data cannot be used as an exclusive test for the presence and purity of collagen. All bluefin tuna collagen samples were found to consist of two \(\alpha\) tropocollagen chains (\(\alpha\)1 and \(\alpha\)2), with an estimated molecular weight of approximately 120-130 kDa, adimer of two alpha chains that make up the \(\beta\) component (approximately 250 kDa), and a trimer that is the gamma component, representing a trimer of \(\alpha\) chains. This is similar to that reported for other fish and tuna species [36]. The absence of any other bands further down the gel in most lanes indicated that the collagen structure was intact and confirmed the absence of extraneous non-specific proteins. The pattern discussed is indicative of the type I collagen found primarily in skin and bone [37].  
To further confirm the presence of collagen in different samples, the quantity of hydroxyproline as a percentage of the total protein was assessed. Uniquely, hydroxyproline is found at high levels in collagen, where it stabilizes the triple helix and thus may be used to quantify collagen. Hyroxyproline (HYP) was found to range from 7.44% in general waste up to 12.14% in bone and 14.40% in skin. The HYP value recorded in this study for general waste was comparable to the +6.41% to 7.8% previously described for other tuna species [33; 38]. These low values may be due to the presence of muscle and viscera content, which have lower values of collagenous protein and, hence, hydroxyproline. The purified collagen obtained from skin and bone seems to be in agreement with the 12.5% of hydroxyproline content specified by [PERSON] and [PERSON] [48].  
Assessment of the heavy metals (Zn, Cr, Co, Cu, Ni, Fe, and Pb) in the collagen samples from skin, bone, and muscle, with the exception of cadmium in bone, was either negative or present at levels that did not exceed the limits of the regulations such as Regulation (EC) No. 853/2004 [43]. Likewise, the outsourced assessment of mercury was also within the permitted limits of the same regulation. The general absence or low levels of these heavy metals may be linked to the farmed origin of the fish and the nature of the (collagen) source material. [PERSON] et al. suggested that farmed tuna tend to have lower mercury and cadmium levels as the fed diet contains fewer heavy metals [49]. In general, it was found that collagen obtained from bone had a higher amount of heavy metal content when compared to the general waste. These results are in agreement with the study carried out by [PERSON] et al., where it was shown that a higher concentration of heavy metals tends to accumulate in the fish bones compared to the fleshy parts [50]. Although these results are promising, further testing on heavy metal deposition from other organs and tissues in Atlantic bluefin tuna, such as the liver, where most heavy metals would be concentrated, would be recommended if the product is to be considered for pharmaceutical or nutraceuticaltical purposes. Despite this, it should be considered that most nutraceuticaltical products and cosmetics containing collagen are very unlikely to be made primarily of collagen as they would be a part of formulations, mixtures, and creams and would therefore contain a considerable number of additives, reducing the concentration of potentially harmful heavy metals per serving/application.  
Microbiological analysis of the extracted collagen indicated the complete absence of the six common microbial species tested. This is to be expected as the acetic acid and, later, citric acid used in the extraction process are effective antimicrobial and bactericidal agents even at the low concentrations used in this study. Furthermore, the high NaCl concentrations utilized in the separation process would further reduce the risk of microbial contamination [51].