Rapid Purification of Salmon Nasal Cartilage Proteoglycan by High-Salt Ethanol Precipitation

Proteoglycan (PG) from salmon nasal cartilage has received increasing attention owing to its potential health benefits such as improving joint function and muscle strength. However, conventional purification methods involve multiple fractionation steps using chromatography, making the process time-consuming and unsuitable for routine analytical applications. This study aimed to establish a rapid, simple, and efficient method for PG purification using ethanol precipitation under high-salt conditions. The ethanol concentration, salt concentration, and centrifugation parameters were optimized, and the optimal condition was found to be 40% ethanol with 2.0 M sodium chloride, which selectively precipitated PG while minimizing collagen contamination. Structural analyses using dot blotting, gel filtration, HPLC, NMR, and unsaturated disaccharide profiling confirmed that the purified PG retained the aggrecan core protein and characteristic chondroitin sulfate chains, with an estimated molecular weight of approximately 1,990,000 and a purity exceeding 95%. This optimized ethanol precipitation method was shown to rapidly recover high-purity PG without requiring fractionation steps using chromatography, as in conventional methods. The method demonstrated high accuracy and reproducibility in spike-and-recovery tests, with recovery rates ranging from 101.5% to 106.9%. In addition, it was successfully applied to the evaluation of PG contents in various commercial dietary supplements. This optimized ethanol precipitation (under 40% ethanol with 2.0 M sodium chloride) method not only is useful as a pretreatment for the PG analysis of functional food but also contributes to research on the biological functions of PG by serving as a practical purification technique.