Structure Characterization And Physicochemical Properties Of Euryale Ferox Seed Protein Fractions

As the seed of medicinal and edible aquatic plant, Euryale ferox seed has rich nutrition and special role in nutrition and efficacy. In recent years, with the development of processing and preservation processes of Euryale ferox seed, the planting area and output of it has steadily increased. And the products of Euryale ferox seed were gradually diversification in the market. However, the protein fractions in Euryale ferox seed has been studied less in detail, in contrast to the situation with lotus seeds and other medicine food homology, especially research on structure characterization and physicochemical properties of Euryale ferox seed protein fractions.In this study, Osborne classification method was used to determine the composition of Euryale ferox seed protein fractions. The results indicated that glutelin was the highest protein fractions(38.61%) in Euryale ferox seed, followed by albumin and globulin. On the basis of a single factor, the protein fractions in Euryale ferox seed was optimized by response surface methodology. Under the optimum conditions, the extraction yield of albumin, globulin and glutelin was 80.75%, 82.13% and 84.04% respectively, and the purity(dry basis) was 80.75%, 85.31% and 90.60% respectively.Structure characterization and physicochemical properties of Euryale ferox seed protein fractions were studied. And the results indicated that each protein fraction has rich essential amino acids, the first limiting amino acids was lysine. The molecular weight of albumin, globulin and glutelin concentrated in 8~20 k Da, 52~143 k Da, 10~20 k Da respectively. Under reducing conditions, their patterns were very different from those showed in non-reducing conditions, indicating that most of protein fractions were organized in structures stabilized by disulfide bonds. Under non-reducing conditions, the molecular weight of albumin distribution is more extensive, mainly concentrated in the range of 35~116 k Da and 8~20 k Da; under reducing conditions, electrophoresis pattern changed significantly, mainly concentrated in the range of 8~14 k Da and 35~40 k Da. Under non-reducing conditions, the molecular weight of globulin is mainly concentrated in the range of 45~120 k Da; while under reducing conditions, the molecular weight is mainly concentrated in the range of 33~ 74 k Da. Under non-reducing conditions, there are seven bands of glutelin, a molecular weight distribution in the range of 15~120 k Da; under reducing conditions, there are five major bands, a molecular weight in the range of 15~ 74 k Da. Globulin has the highest surface hydrophobicity in the protein fractions, followed by albumin and glutelin. The main secondary structure of protein fractions in the Euryale ferox seed were β-sheet and β-turn, which revealed high levels. In addition, glutelin was most stable component in the protein fractions.The thermal properties of protein fractions in the Euryale ferox seed were investigated by DSC. The results showed that the protein fractions had good thermal stability. Based on their higher denaturation temperature, these protein fractions probably are suitable for an application in specific products where native forms are needed to be maintained under higher temperature during processing. As the p H increased, the denaturation temperature and denaturation enthalpy of protein fractions were reduced. At the same p H conditions, glutelin showed the highest denaturation temperature and maximum denaturation enthalpy in the protein fractions. Na Cl solution had a stabilizing effect for protein fractions. With increasing concentration of Na Cl solution, there was a significant influence on the structure of albumin and globulin, but no obvious effect on the structure of glutelin was found.The antimicrobial activity of Euryale ferox seed protein fractions had been studied. The results showed that albumin treated with SDS had a good inhibitory effect on E. coli, whereas globulin and glutelin demonstrated little inhibitory activity. The antimicrobial albumin was purified by Sephadex G-100 gel column and Hitrap Capto DEAE column, and one antimicrobial fraction was obtained, named A-32. The A-32 had good inhibitory effect on E. coli and S. aureus. The SDS-PAGE analysis of A-32 showed a single band with a molecular mass of approximately 9 k Da and non-disulfide bond. Comparing the secondary structure of A-32 with albumin, we found that there was no significant difference between the proportion of β-sheet and random coil, and a significant difference between α-helix and β-turn. The denaturation temperature of A-32 was 68.5°C, indicating a weak thermal stability. The molecular mass spectrum of A-32 was collected by matrix-assisted laser desorption/ionization-time of flight mass spectrometry(MALDI-TOF-MS). The MALDI-TOF-MS data were submitted to the search software Mascot. Protein identification was searched against the NCBI databases. The results showed A-32 protein fraction shares a significant homology with ubiquitin. A-32 treated by SDS may be associated with similar structure with ubiquicidin which has also been shown to be antibacterial.