Isolation And Purification Of Oat (Avena Sativa L.) Antifreeze Proteins And Their Effects On Frozen Dough

Antifreeze protein(AFP) is a class of special protein, having the abilities of thermal hysteresis activity, recrystallization inhibition activity and ice crystal morphology effect.Their unique properties of inhibiting ice crystal growth and recrystallization, which could keep the living organisms from freezing damage, make them a kind of promising additives for frozen food. In this paper, a new type of plant AFPs(AsAFP) was purified from the cold acclimated oat seeds. After sequence identification, its properties were predicted and analyzed.In order to analyze its antifreeze mechanism and provide theoretical guidance to the research of antifreeze proteins, three-dimensional structure of AsAFP was generated by homology modeling methods, and the molecular dynamics simulation was performed on the interaction of AsAFP and ice crystal surface. Then AsAFPs were applied into frozen dough and gluten protein. The physicochemical properties of frozen dough, textural characteristics of steamed bread, and the aggregation behavior, freeze-thaw characteristics, physicochemical properties of gluten protein during frozen storage were investigated.After a series of purification steps, AsAFP at electrophoretic purity was obtained from the cold acclimated oat seeds. Its thermal hysteresis activity was 1.24°C. Sequence identification was performed by MALDI-TOF-MS/MS, and the composition of amino acid,isoelectric point and relative molecular mass, secondary structure and solvability of solvents were predicted and analyzed. The results showed that, the amino acids sequence of AsAFP was VSSVISSSLF EKMLLHRGFY TYDAFIAAAK SFPAFATTGS TDVRKREVAA FLAQTSHETT GGWPTAPDGP YELGSTSDYF GRGPIQISYN YNYGAAGKAI GVDLLRNPDL VTSDNTVEFK TALWFWMTPQ SPKPSSHDVI TGRWSPSSTD KAAGRVPGYG VLTNIIDGGV ECGKGQESHV ADRIGYYKDN LDCYNQKPFA, which had the maximum homology with Endochitinase. AsAFP had a high proportion of hydrophilic amino acid residues. Its theoretical isoelectric point was 6.08, and the relative molecular mass was 21728.18. A large number of random structure and ?-helix structure were contained in the secondary structure of AsAFP, and its surface solvent accessibility was relatively high.Using amino acid sequence of AsAFP as probe, homology modeling technique was applied on AsAFP. The binding of modeled protein and different ice crystal surfaces was simulated by docking. The combination energy was calculated, and the molecular dynamics simulation was performed on the interaction between AsAFP and ice crystal surface. 4DWX was considered to be the best template of AsAFP, for their consistency was 63%, and sequence coverage reached 100%. Using the three-dimensional structure of 4DWX as atemplate, structure optimization was proceeded by modeller 9.14 single template modeling.The combining model of AsAFP and the ice layer surface was predicted by docking. Results showed that the telechelic structure of AsAFP, which was composed by helix and loop, was the major site in ice binding. Hydrogen bond and hydrophobic interaction played a key role in the stability of AsAFP and the ice layer surface combination. The amino acid residues such as Asn, Arg and Ser participated in the formation of hydrogen bond. Molecular dynamics simulation of AsAFP and the ice layer compound was carried out, and the data showed that in the process of dynamic simulation, the combination of AsAFP and the ice layer surface was highly stable. The turning radius, number of hydrogen bond, secondary structure and the solvent accessible surface were all in balance during the process of dynamic simulation,which further illustrated the stability of AsAFP-ice compound. AsAFP-Prism showed the highest antifreeze property because of the highest number of hydrogen bonds and the optimal stability in combination.AsAFPs were applied into frozen dough, and the effects of AsAFPs during frozen storage were investigated. Research showed that, the freezable water content of frozen dough was increased during the frozen process, while the supplementation with AsAFPs could absorb to the free water around them and restrict the free movement of water, thus the rise in freezable water content significantly slowed down. Since the freezable water content determined the number of ice crystals formed in frozen dough during frozen storage, this indicated that AsAFPs could effectively inhibit the ice crystal formation in frozen dough. The addition of AsAFPs could reduce water mobility, then inhibit the loss of strong bound water.The addition of AsAFPs could also slow down the decrease of G' and G'', enhance the gas productivity and holdup capacity of frozen dough, improve the yeast viability, protect the gluten network from structure damage, then eventually improve the textural characteristics of steamed bread.AsAFPs were further applied into hydrated glutens, and the effects of AsAFPs during frozen storage were investigated. The aggregation behavior of gluten, glutenin and gliadin showed that, during frozen storage, the GMP in gluten and glutenin depolymerized, and the molecular weight distribution of gliadin changed. The addition of AsAFPs could protect the gluten network from mechanical damage caused by ice crystal formation during freezing and frozen process, then slow down the depolymerization of gluten and glutenin, and lower the mechanical damage of gliadin. SDS-PAGE electrophoresis results showed that no remarkable difference could be observed for gluten and glutenin subunits with or without frozen storage.The free sulfhydryl group content of gluten, glutenin and gliadin all increased, indicating that during frozen storage the depolymerization of GMP was attributed to disulfide bondsbreakage. But the addition of AsAFPs could reduce the breaking of disulfide bonds, and lower the increase of free sulfhydryl group content. The freeze-thaw research showed that, for fresh hydrated gluten, the addition of AsAFPs could increase its melting temperature range, reduce the melting enthalpy and freezable water content, as well as increase the glass transition temperature, while for frozen hydrated gluten, the addition of AsAFPs could lower its water mobility, melting enthalpy and freezable water content. The rheological properties, secondary structure and microscopic research showed that, the supplementation with AsAFPs could slow down the decrease of G' and G'' in the frozen process, and protect the ?-helix and ?-sheet structure from mechanical damage caused by ice crystal formation, so that the gluten network was protected.