| The thermal stability of milk is the ability of milk protein to resist high temperature heat treatment without forming visible solidification and gel.Casein micelles are stable aqueous polymeric colloid.The mechanism of the thermo-coagulation is the reaction among denaturation whey proteins and casein micelle,accompanied with changes in the surface charges and structure of casein micelles,resulting the visible protein sidemen(coagulate)duo to the micelle thermal dissociation and association.In the milk,the degree of thermal stability/ thermal denaturation is the important factor to determine the heat stability of milk and it is also an important index to evaluate the heat processing characteristics of milk.Based on the structure and properties of yak ?-lactalbumin(?-La)and ?-lactoglobulin(?-Lg),the thesis systematically studied the thermal properties,thermal denaturation mechanism of yak ?-La and ?-Lg,the thermal aggregation of ?-La and ?-Lg,and the effect of casein micelles deaggregation/aggregation on the thermal stability of milk.Based on HPLC-MS-MS and bioinformatics,the structure and biological functions of yak ?-La and ?-Lg were analyzed.Compared with cow milk,yak milk had higher contents of total protein and ?-Lg.The ratio of casein and whey protein in yak milk was higher than that in cow milk.Yak ?-La genetic variant was the B type.The ?-La was composed of 123 amino acids.There was a calcium binding site in the 79~89 region of amino acid sequence.There were four disulfide bonds in ?-La molecule structure.Yak ?-Lg was composed of 162 amino acids.?-Lg had three genetic variants which were ?-Lg A/B and ?-Lg E.There were two disulfide bonds and a free sulfhydryl group in ?-Lg molecule.The ?-helix??-fold and random coil were founded in the structure of ?-La and ?-Lg.The tertiary structure of ?-La and ?-Lg was globulin.The ?-La and ?-Lg were hydrophilic.The thermal properties of pure yak ?-La and ?-Lg under different environmental conditions were studied by DSC,and the mechanism of thermal denaturation of ?-La and ?-Lg was also discussed.Under natural p H of milk,the denaturation temperature of ?-La and ?-Lg was 52.1 °C and 81.4 °C,and the denaturation temperature of ?-Lg negatively correlated with its concentration.In the p H 4~10 range,the ?-La had a better stability and the denaturation temperature of ?-Lg in the acidic p H(4~7)was higher than that in the alkaline p H(7~10).Na+ can increase the thermal stability of ?-Lg by inhibiting the folding of ?-Lg.Ca2+ can improve the thermal stability of ?-La and ?-Lg.Glucose,sucrose and lactose can increase the thermal denaturation temperature of ?-Lg to 4.5~6.3 °C,The stability of ?-Lg were positively related to the concentration of sugars.It has been demonstrated that the thermal denaturation of ?-Lg was the result of intramolecular and intermolecular interaction among sulfhydryl-disulfide bonds,the sulfhydryl oxidation and the hydrogen bond in the peptide chain.The thermal denaturation of the ?-La was caused by the dissociation of Ca2+ in the molecule.The thermal aggregation of ?-La,?-Lg and ?-La/ ?-Lg mixture in different p H,heat treatment strength,Ca2+concentration,concentration of phosphate and lactose were studied by size exclusion chromatography and transmission electron microscopy.The thermal aggregation began to occur when ?-La and ?-Lg were heated at 80 °C,and the thermal aggregation positively correlated with the heat treatment strength.During the aggregation of ?-Lg,it firstly formed oligomer(dimer,tetramer,octamer)and then a soluble aggregate,and finally a large molecular insoluble coagulate.Under high temperature and low p H(<6.0),the aggregation degrees of ?-La and ?-Lg were larger,however,the aggregation degrees of ?-La and ?-Lg decreased under alkaline p H(>7.0).During heat treatment of(>20min),High concentration Ca2+(>50 mmol/L)and long heating time resulted in the increase of ?-La and ?-Lg aggregation degrees(even to form protein gel).The proper concentrations of phosphate(30~60 mmol/L)and lactose(4.0%~6.0%)could significantly reduced the thermal aggregation of ?-La and ?-Lg and improved their thermal stability.The relationship between the disassociation / association of casein micelles and the thermal stability of milk was studied by HPLC.The thermal aggregation of whey protein-casein micelles and the disassociation of casein micelles occurred simultaneously.The disassociation of ?-CN from the micelle surface positively correlated with the heat treatment intensity.When heating temperature was lower than 100 °C,the integrity of casein micelle maintained by the interaction of whey protein aggregation and ?-CN disassociation,so the milk protein keep in a relatively stable state.The denatured whey protein and ?-CN of casein micelle surface mainly formed a complex of whey protein /?-CN at p H<6.7.The whey protein /?-CN complex and ?-CN mainly associated from casein micelles at p H>6.7.The particle sizes of casein micelle positively correlated with the amount of whey protein binding to casein micelle and milk turbidity.The association and disassociation of casein micelles changed the surface and internal structural properties of micelles and reduced the stability of casein micelles. |
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