Collagen Fibrils with Native D-periodicity from Fish Skin: a Simplified Fibrillogenesis Method

Abstract

Native collagen has a characteristic D-periodicity that can be reproduced in vitro by maintaining the correct conditions for fibrillogenesis. In fish collagen extraction studies, collagen fibrillogenesis is generally achieved by salting-out with sodium chloride which involves complex, expensive and time-consuming centrifugation steps to separate collagen fibrils and dialysis treatments to remove salt residues. Therefore, there is an industrial need of simple methods for fibrillogenesis. This study introduces a modified isoelectric precipitation method for this purpose by understanding the effect of initial collagen concentration, final pH of fibrillogenesis and mixing speed on fibril characteristics. The results showed that the final pH has a greater influence on D-periodicity than other two tested variables. The isoelectric point of collagen was found to be pH 7.5; however, collagen fibrils with the native D-periodicity as confirmed by the transmission electron microscopic images were formed at a pH (pH 9.3) higher than the isoelectric point. Results also revealed that a proper balance between initial collagen concentration and mixing speed is essential for D-periodicity, because the initial collagen concentration is important for molecular availability and the mixing speed is important for molecular mobility. Accordingly, in this modified method, collagen is precipitated by adding a sodium hydroxide solution to pH 9.3 with mixing, and keeping undisturbed for 24 h followed by freeze-drying. This method is novel and does not require centrifugation or dialysis techniques. Due to its simplicity, time and cost-effectiveness, this method has a high potential to be performed on an industrial scale.