Study On The Preparation And The Antifreeze Mechanism Of Antifreeze Protein From Barley(Hordeum Vulgare)

Antifreeze proteins(AFPs), is kind of proteins which could lower the freezing point of solution, modificate the ice morphology and inhibit the ice recrystallization. Their unique properties makes them good candidate for frozen food additives. In this paper, AFPs was extracted from cold acclimated barley grain using the infiltration-centrifugation method. Then purification and identification was proceeded on it and properties of purified Ba AFP-1 was predicted and analyzed. Ba AFP-1 was added into dough samples and hydrated gluten, its effect on the thermal properties, water status and other physical properties during freezing and freeze-thaw treatment was investigated to explore its cryoprotection mechanism in frozen food. The three-dimensional structure of Ba AFP-1 was modelled, docked with ice crystal surface, and their interaction was analysed using molecular dynamics simulations in order to reveal the optimal binding site and consequently interpret the cryopreservant mechanism of Ba AFP-1.Infiltration-centrifugation method was adopted in the extraction of barley grain proteins extraction. Solution type, the ratio of material and solvent, p H, ion strength and the infiltration time of extracton was optimized. The spectral character, molecular weight distribution, and the thermal hysteresis activity of the extracts were analyzed. A series of purification was carried out on the extracts, the purity and activity of purified protein was identified. The optimal extraction condition was achieved using Na2HPO4-Na H2PO4 buffer(50 m M, p H 7.6, 0.05 M Na Cl) when the ratio of material and solvent was 1:5 and infiltrated for 10 h. Results also showed that, infiltratin-centrifugation was targetted, as the composition of the extracts is simple and its thermal hysteresis is relatively high. AFPs was existed in the cold acclimated barley grain, and existed after cold acclimation. Ba AFP-1 at electrophoretic purity was obtained after purification, its THA was 1.04 °C.The relative molecular weight of Ba AFP-1 was determined, its amino acid sequence was identified using MALDI-TOF-MS/MS, and its amino acid composition and sequence homology was analyzed. Its isoelectric point, relative molecular weight, secondary structure composition, and solvent accessibility was predicted according to its amino acid sequence. Its spectral character, secondary structure composition, hydrophilcity and hydrophobicity, effect of ice fraction on its THA and the most effective concentration of it were also determined. Results showed that, the relative molecular weight of Ba AFP-1 was 13370, its amino acid sequence was SGPWMWCDPE MGHKVSPLTR CRALVKLECV GNRVPEDVLR DCCQEVANIS NEWCRCGDLG SMLRSVYAAL GVGGGPEEVF PGCQKDVMKL LVAGVPALCN VPIPNEAAGT RGVCYWSAST DT; it has high homology with 12 genus of the Triticeaein Poaceae, and it has the highest match score with pathogenesis-related proteins in the Triticum, it has low homology with AFPs from rice and A. thaliana. Its isoelectric point and relative molecular weight was predicted to be 5.04 and 13101.11, respectively. α-helix was riched in its secondary structure, and the solvent accessibility of its amino acid residue was relatively high. The characteristic absorption wavelength of it was 280 nm, α-helix and random coil were the main components of its secondary structure. It is not an antifreeze glycoprotein, its most effective concentration was 20 mg/m L and the ice fraction content of it could influent its THA. It has relatively high hydrophilcity and low hydrophobicity, the thermal denaturation temperature of it was 65.60 °C.Ba AFP-1 was added into doughs, its effect on the thermal properties, water status, rheology, microstructure, fermentation rheology, baking porperties and bread quality during freezing and freeze-thaw treatment was investigated. Results showed that, Ba AFP-1 could affect the apparent specific heat of fresh dough during phase transition. During freezing, it could influence the supercooling phenomenon of dough samples, increase the melting temperature and enlarge the range of melting temperature, increase the glass transition temperature, lower the melting enthalpy and consequently decrease the freezable water content. During freeze-thaw treatment, the cryoprotective effect of Ba AFP-1 on dough was achieved by prevent the shift of melting temperature toward the higher region, slow down the increasement of freezable water content, that is influent the ice recrystallization caused by temperature fluctuation. Study on the change of water status revealed that, the addition of Ba AFP-1 could lower the weight loss of dough samples during freezing and frozen storage, lower water mobility, retard the transformation of water to higher mobility and the migration of water from the centre of dough to the surface. The addition of Ba AFP-1 could slow down the decrease of the elasticity modulus and the viscous modulus of dough caused by freezing and freeze-thaw treatment, protect the micro-structure of dough; It could also protect the gas productivity of the yeast, the integrity and the gas holding capacity of the gluten network to protect the fermentation and baking properties of dough.Ba AFP-1 was added into hydrated glutens, and its effect on the thermal properties, water status, rheology, microstructure, SDS-sloluable protein composition, secondary structure composition and hydrophobic index during freezing and freeze-thaw treatment was investigated. Results showed that, the effect of Ba AFP-1 on the apparent specific heat of hydrated gluten was the same as that of dough samples. Significant supercooling phenomenon was appeared in both hydrated gluten samples. Unlike that of dough samples, the cryoprotect effect of Ba AFP-1 in hydrated gluten was manifested by lower the freezing temperature. Ba AFP-1 could lower the melting enphalpy, the freezable water content and increase the glass transition temperature of hydrated gluten. The capillary bound water of hydrated gluten was damaged and the water content of the hydrated gluten was decreased during the freeze-thaw treatment. The addition of Ba AFP-1 could influence the water mobility of hydrated gluten, weaken the increase effect of freeze-thaw treatment on water mobility. Water migration on the hydrated gluten surface during freeze-thaw treatment was not significant as that of dough samples, but Ba AFP-1 play its role in ice recrystallization inhibition and consequently influence the water distribution in the internal of hydrated gluten. The addition of Ba AFP-1 could slow down the decreasement of elasticity modulus and the viscous modulus of hydrated gluten caused by freeze-thaw treatment, protect the microstructure of hydrated gluten by influencing the size and the shape of ice crystals during freezing and inhibiting ice recrystallization during freeze-thaw treatment. During freezing and early freeze-thaw treatment, Ba AFP-1 have little effect on the molecular weight distribution of SDS-soluable protein while latterly it could slow down the destructive effect of freeze-thaw treat on the polymerization degree of hydrated gluten. It could protect the α-helix from the damage of ice and enlarge the intramolecular and intermolecular hydrogen-bond of gluten. During freeze-thaw treatment, it could enhance the intramolecular hydrogen-bond of gluten, promote the reformation of damaged intermolecular hydrogen-bond, keep the secondary structure of gluten in a relatively stable state and consequently reduce the exposure of the hydrophobic residues caused by structure damage and slow the decline of hydrophobic index.The three-dimensional structure of Ba AFP-1 was simulated by molecular simulation using homology modeling technique, the binding of Ba AFP-1 to ice crystal surface was simulated by docking, and the binding mode between them was analyzed by dynamic simulation in order to optimal their binding surface and explore its binding mechanism. 1HSS was chosen as the optimal templete of Ba AFP-1, its query covery was 0.89, E value was 9e-28, and their sequence identity was 50%; homologous modeling was conducted, optimization on builded models was proceeded and the rationality of the optimal structure was assessed, a well-constructed with high accuracy structure was modelled. Ba AFP-1 was docked with different ice crystal planes with different angles, the interaction between each docking system at the lowest energy conformation was analyzed. Results showed that the plane composed by αX and the bilateral loop of it interacted with ice crystal planes, this plane may play an important role in ice binding. Except for Thr, Glu and Asn also participated in the binding of Ba AFP-1 to ice. Molecular dynamic simulation results showed that Ba AFP-1 could combined with three typical ice surface and form effective conformation, and the combination was implemented through hydrogen bonding and hydrophobic interaction. Ba AFP-1 has the strongest combination with the Prism ice crystal, and the hydrogen bonding interaction is the first approximationof the binding intensity. The high flexible residues in the combination region may go against the binding between the Ba AFP-1 and the secondary-prism crystal.