# Changes in Collagen across Pork Tenderloin during Marination with Rosehip Nanocapsules
## 3 Results
### Histological Test and Collagen Quantification  
Connective tissue is the main factor affecting the meat's texture, and its main component is collagen; changes in collagen content and characteristics are essential markers affecting connective tissue strength. Therefore, muscle texture is significantly affected by collagen characteristics and the degree of muscle fiber fragmentation [36; 37; 38]. Figure 5 shows the images obtained in the histological test and the different applied treatments. The control samples were only immersed in a surfactant solution, and the other samples were applied with NC-RH, both conditions at different pH levels (6.5 and 4.0) and temperatures (25 \({}^{\circ}\)C and 4 \({}^{\circ}\)C) after 48 h. In the control (Figure 5a) at pH = 6.5 and 25 \({}^{\circ}\)C, the tissue structure is organized and characterized by the presence of muscle fibers delimited by the connective tissue surrounding the muscle fiber that makes up the endomysium (Figure 5a, black arrows). This makes the separations visible, generating a well-organized tissue structure [39]. Figure 5: Changes in collagen after 48 h at pH = 6.5: (**a**) control at 25 \({}^{\circ}\)C without NC-RH; (**b**) at 25 \({}^{\circ}\)C with NC-RH; (**c**) control at 4 \({}^{\circ}\)C without NC-RH; (**d**) at 4 \({}^{\circ}\)C with NC-RH. Changes in collagen after 48 h at pH = 4.0; (**e**) control at 25 \({}^{\circ}\)C without NC-RH; (**f**) at 25 \({}^{\circ}\)C with NC-RH; (**g**) control at 4 \({}^{\circ}\)C without NC-RH; (**h**) at 4 \({}^{\circ}\)C with NC-RH. H&E (40\(\times\)) TVG (10\(\times\)), bar 50 \(\mu\)m.  
The application of the NC-RH to pork tenderloin generated a tissue change characterized by the swelling of the tissue at both temperatures; the muscle fibers presented greater separation (Figure 5, yellow arrows) with the presence of disordered and lax collagen fibers (Figure 5d, white arrows). The connective tissue at the level of the endomysium and perimysium (Figure 5, yellow and blue arrows) showed a more significant loosening of the arrangements, which increased the distance between the muscle fibers. The above may occur due to the denaturation and contraction of collagen fibers during marinating [40; 41]. In addition, it is reported that the distance between cells increased with a pH decrease (to approximately pH 4.3), and the surface hydrophobicity was inversely proportional to the pH. This was due to conformational changes, i.e., the unfolding of myofibrillar proteins and the exposure of non-polar amino acids to the surface of proteins [40]; thus, it is evident that collagen is principally affected by the pH. It has been reported that collagen's isoelectric point is pH 7.0-7.5 [5], but the charge varies little from pH 5 to 9 [5; 41]. A pH below the isoelectric point indicates that charge interactions are involved in collagen aggregation and stabilization. Additionally, each collagen molecule possesses 216 hydrophobic residues essential to its stability [5]. Collagen is insoluble in neutral solutions and does not decompose in an aqueous solution, while it can dissolve in the presence of a strong acid or alkali. The muscle fiber proteins started swelling at pH 4.5 [41], with maximum degradation in the region of pH 3.5, the optimal pH range for cathepsins. The maximum degradation was at approximately pH 2.5 [42]. Acid marination improves meat tenderness over a pH range of 4.6 to 4.1 [42], which could be an effect of loosening the structure in collagen connective tissue. The acid solution breaks the transversal bounds of collagen, leading to the unstable structure loss of this connective tissue protein. The effects of acid marinades provoke swelling and the enhanced extraction of myofibrillar proteins due to decreased pH and increased ionic strength, allowing water to penetrate into the muscle [43].