Extraction, purification and identification of antifreeze proteins from cold acclimated malting barley (Hordeum vulgare L.)

Abstract

Antifreeze proteins from cold-acclimated malting barley were extracted by infiltration–centrifugation. The infiltration time was optimised, and its extraction effect was evaluated. The effect of cold acclimation on the accumulation of barley antifreeze proteins (BaAFPs) was assessed by comparing the thermal hysteresis activities (THA) of proteins extracted from both cold acclimated and non-cold acclimated barley grain. Ultra-filtration, ammonium precipitation and column chromatography were used successively to purify the BaAFPs, and MALDI-TOF-MS/MS was used for protein identification. The results showed that infiltration–centrifugation was more targeted than the traditional method, and 10h was the optimal infiltration time. THA was observed only after cold acclimation implied that AFPs only began to accumulate after cold acclimation. After purification, BaAFP-I was obtained at an electrophoresis level and its THA was 1.04°C (18.0mgml−1). The mass fingerprinting and sequencing results indicated the homology of the BaAFP-I to alpha-amylase inhibitor BDAI-1 (Hordeum vulgare).