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

Published Date

15 May 2015, Vol.175:74–81, doi:10.1016/j.foodchem.2014.11.027

Title 

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

• Author 

• Xiangli Ding ^a
• Hui Zhang ^a,,
• Haiying Chen ^b
• Li Wang ^a
• Haifeng Qian ^a
• Xiguang Qi ^a

• aState Key Laboratory of Food Science and Technology & School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, PR China
• bJiangsu Key Laboratory of Advanced Food Manufacturing Equipment & Technology & School of Mechanical Engineering, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, PR China

Received 20 July 2014. Revised 8 October 2014. Accepted 3 November 2014. Available online 11 November 2014.

Highlights

• •
  LF-NMRI was used to monitor water migration of barley grain during infiltration.
• •
  Infiltration–centrifugation was demonstrated to be targeted in BaAFPs extraction.
• •
  BaAFPs was demonstrated to be accumulate after cold-acclimation.
• •
  BaAFP-I at an electrophoresis level was obtained after a series of purification.
• •
  BaAFP-I was identified to be an homology of alpha-amylase inhibitor BDAI-1.

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 10 h 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 (18.0 mg ml⁻¹). The mass fingerprinting and sequencing results indicated the homology of the BaAFP-I to alpha-amylase inhibitor BDAI-1 (Hordeum vulgare).

Abbreviations

• AFPs, antifreeze proteins
• BaAFPs, barley antifreeze proteins
• THA, thermal hysteresis activity

Keywords

• Barley (Hordeum vulgare L.)
• Cold acclimate
• Antifreeze proteins
• Infiltration–centrifugation
• Purification
• Identification

Chemical compounds studied in this article

• Disodium phosphate dodecahydrate (PubChem CID: 61456)
• Sodium dihydrogen phosphate (PubChem CID: 23673460)
• Sodium chloride (PubChem CID: 5234)
• Tromethamine (PubChem CID: 6503)
• Hydrochloric acid (PubChem CID: 313)
• Glycerol (PubChem CID: 753)
• Bromophenol blue (PubChem CID: 8272)
• 2-Mercaptoethanol (PubChem CID: 1567)
• Ammonium sulphate (PubChem CID: 6097028)

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• ⁎ 
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