Isolation And Purification Of Collagen Peptides From Cod Skins By Deep Eutectic Solvents(DESs)

Collagen peptides have recently attracted much attention owing to their important biological functions.It is reported that two separate operations have been required to extract the collagen peptides in traditional methods.Firstly,gelatin is obtained from raw materials,and then the gelatin is hydrolyzed by enzymes.However,the method has the shortcomings of low extraction efficiency,complicated and time-consuming,and it needs to be further improved.Deep eutectic solvents(DESs)are a kind of new,sustainable and green solvents with many advantages,such as non-toxic,biodegradable and atomic utilization of 100%.It is reported that DESs have been employed in various areas of chemistry.This paper proposed a method for extraction of collagen peptides from cod skins with high extraction efficiency and high purity by deep eutectic solvents.Initially,a series of DESs were designed and synthesized by directly mixing choline chloride with hydrogen-bond donors-urea,ethylene glycol,glycerol,acid A,acid B,and acid C,and the leaching system of cod skins was explored with the prepared DESs.It is obvious that there were almost no collagen peptides leached into Ch Cl/Urea.The leaching solutions from other DESs were purified by organic solvents to obtain collagen or collagen peptides.The precipitations were subjected to purity and molecular weight analysis by UV-vis,HPLC and SDS-PAGE.It is clear that the acidic deep eutectic solvents can get higher purity of the targets.Among them,the products obtained from Ch Cl/acid A and Ch Cl/acid B possessed the molecular weight of more than 100 KDa and the yields were lower.The products obtained from Ch Cl/acid C possessed the molecular weight of below 11 KDa and the yield was higher.Therefore,Ch Cl/acid C was selected as the optimal DESs for extraction of collagen peptidesThe single factor experiments were carried out to prove that the extraction efficiency and purity of collagen peptides were influenced by the molar ratios of Ch Cl/acid C,extraction temperature,reaction time and solvent-to-solid ratio,determining the factors and levels required to respond to the surface methodology.The reaction conditions of different molecular weight collagen peptides were optimized by 3-factor,3-level Box-Behnken design.It is obvious that the optimum extraction conditions of the higher molecular weight collagen peptides were Ch Cl/acid C(1:1.0)/cod skins(80:1,m L/g)at 65°C for 2.2 h.The corresponding yield and purity were 91.57% and 92.85%.While the optimum extraction conditions of the lower molecular weight collagen peptides were Ch Cl/acid C(1:1.0)/cod skins(120:1,m L/g)at 66°C for 6 h.The corresponding yield and purity were 96.36% and 100%.UV-vis and FT-IR were utilized to study the leaching mechanism of neutral and acidic deep eutectic solvents.From the results,it can be concluded that the formation of intermolecular hydrogen bonds between Cl-and the amino or carboxyl groups at the end of the collagen peptides would promote the dissolution of collagen in the neutral deep eutectic solvents.In the acidic deep eutectic solvents,the free hydrogen ions are specifically bound to the imino groups in the collagen,resulting in the high purity.In addition,the formation of the new DESs between acidic deep eutectic solvents and collagen would further promote the leaching of collagen.