The residue was dissolved in one hundred mL of 0.1 M HCl [22]. Then 50 on the sample solution was analyzed making use of high-performance liquid chromatography (HPLC-MS/MS, Ultimate 3000-API 4000 Q TRAP, Thermo Fisher Scientific, Dreieich, Germany). 4.6. Microscopy Characterisation The collagen remedy (50 ) devoid of acetic acid was poured into a 12-cm-diameter lyophilization dish after which freeze-dried. The morphology of the sample was imaged using SEM (S-4800, HITACHI, Tokyo, Japan), with an accelerating voltage of 5 kV. After being coated with Pd, the samples have been observed at 400and 800magnifications. four.7. Thermal Stability The thermal stability with the samples was measured making use of a differential scanning calorimeter (DSC2, Mettler-Toledo corp., Zurich, Switzerland) beneath a nitrogen atmosphere having a flow price of 100 mL min- 1 . The samples were dissolved in 0.four M acetic acid at the ratio of 1:40 (w/v) for 48 h at four C. The remedy (5 mL0 mL) was placed into aluminium crucible, and then scanned more than the range of 200 C at a heating rate of 1 C/min. The empty aluminium crucible was made use of for reference. The maximum transition temperatureMar. Drugs 2021, 19,14 of(Tmax ) was obtained from the DSC thermogram, as well as the enthalpy of denaturation (H) was calculated from the area of your corresponding endothermic peak. four.8. Solubility four.eight.1. Effect of pH The impact of pH on collagen solubility was determined using the system Bomedemstat In stock described by Chen et al. (2016) [18], with bovine serum albumin (BSA) because the protein regular. The samples were dissolved in 0.5 M acetic acid in the final concentration of 0.2 mg/mL. The pH from the sample resolution (5 mL) was adjusted from two to ten, with six M HCl or six M NaOH. Then, the sample options were mixed with distilled water on the very same pH till the option volume reached ten mL. The relative solubility was calculated via comparison together with the solubility obtained at the pH that exhibited the highest solubility. Collagen solubility was determined at different pH levels applying the system described by Chen et al. (2016) [18] with slight modifications. The samples had been dissolved in 0.5 M acetic acid at a concentration of 0.3 (w/v) with gentle stirring at 4 C for 12 h. The collagen answer (8 mL) was placed in a centrifuge tube. Then, the pH was adjusted to distinctive levels, ranging from 2 to ten, applying six M HCl or 6 M NaOH. The final volume was brought to 10 mL by distilled water previously adjusted towards the exact same pH because the collagen option tested. The options had been gently stirred at 4 C for 30 min and left overnight. Next, the supernatants had been collected following centrifugation for 30 min at 10,000g. Protein content material inside the supernatant was calculated using the Lowry ML-SA1 TRP Channel approach (1951) [52], with BSA as the protein typical. The relative solubility was determined in comparison with that obtained at the pH level that offered the highest solubility. four.8.2. Effect of NaCl The effect of NaCl on collagen solutions was measured in accordance with all the method described by Chen et al. [18], BSA was employed as regular. The samples had been dissolved in 0.five M acetic acid at a concentration of 0.two mg/mL. The sample remedy (five mL) was mixed with five mL of a series of NaCl concentrations containing 0.five M acetic acid to receive the final solutions with NaCl concentrations of 0 , 2 , four , six , eight , 10 , 12 , and 14 , w/v. The protein content was measured as described in Section four.eight.1, as well as the relative solubility was calculated working with the option with final NaCl concentrati.
