Sustainable dissolution of collagen and the formation of polypeptides in deep eutectic solvents for application as antibacterial agents

Abstract

Collagen is a protein that is hard to dissolve in water and many other solvents, which limits its applications. Herein, deep eutectic solvents (DESs), i.e. choline chloride : lactic acid (ChCl : LA) = 1 : 1 and ethylene glycol : zinc chloride (EG : ZnCl₂) = 4 : 1, are effectively used to dissolve type I collagen under different conditions. Type I collagen is readily soluble at a concentration of 9.5–22.5 w/v% in DESs, and the solubility is governed by the nature of the DES, temperature (45 °C, 70 °C and 90 °C) and the absence or presence of HCl_(aq.) (5 × 10⁻⁵ M). The dissolved material is regenerated by employing ethanol as an anti-solvent at 4 °C and investigated for alteration in the polymeric structure using Fourier-transform infrared spectroscopy (FTIR), circular dichroism (CD), UV-vis spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA), and SDS-PAGE techniques. The increase in temperature and the presence of dilute HCl_(aq.) result in a relatively greater disruption of the H-bonded structure of collagen, causing the unwinding of its triple-helical structure coupled with reduction in the helical content of polyproline type-II helices, which exposed vital amino acid residues in the regenerated material. Such an unwinding is accompanied by the formation of low molecular weight polypeptides, which are readily soluble in water and show antimicrobial activity comparable to or more than that exhibited by a model antibiotic Kanamycin towards both Gram-negative and Gram-positive bacteria. DESs are reused for at least 3 cycles for collagen solubilization without alteration in their inherent structure and collagen solubilizing ability, whereas the material regenerated from reused DESs shows properties similar to that shown by the material regenerated from virgin DESs. In this manner, a new sustainable strategy for solubilizing collagen and the direct preparation of essential and active low molecular weight collagen peptides directly from collagen in a single step is established. An inventive approach to using collagen is made possible by the observation that lower molecular weight peptides formed from the sustainable dissolution of collagen with exposed aromatic amino acid residues can demonstrate antibacterial activity.