Differential Expression Analysis Of Whey Protein And Milk Fat Globule Membrane Protein In Bovine Milk Under Different Heat Treatments

The object of this study was to investigate changes of whey proteome and milk fatglouble membrance proteome in bovine milk under different heat treatmentscommonly used in dairy processing. This study contains three major parts:The first part of this study was to investigate the effect of extraction methods ontwo-dimensional electrophoresis (2-DE) maps of whey proteome in cow milk andexplore an optimal eatraction method. Samples were extracted from cow milk bydifferent isoelectric precipitations and ultracentrifugation methods,2-DE maps wereanalysised by PDQuest8.0software. The results of2-DE maps showed that milkwhey protein could be effectively extracted by the methods above with lessbackground, no significant strips and good repeatability. However, there were stillsome residual caseins appearing in each map.2-DE maps of whey protein refined byisoelectric precipitations were relatively informative compared with the gel mapsobtained by ultracentrifugation. Moreover, the richness of different whey proteins invarious maps extracted by the method adjusting pH to4.6as isoelectric point isslightly higher than adjusting pH to4.8. The results indicated that adjusting pH to4.6as isoelectric point to extract whey protein has some advantages than adjusting pH to4.8and ultracentrifugation. However, all the methods used in this research couldeffectively remove high abundant casein to improve the detection sensitivity of lowabundance proteins, which could provide some useful information for the futherresearch on whey protein proteomic of cow milk.The second part of this study was to obtain protein expression profiles of bovinewhey proteome and examin their dynamic changes during different conditions (rawmilk,85℃/15s,125℃/4s,138℃/4s, and145℃/4s). Extracted whey proteins wereseparated by two-dimensional electrophoresis (2-DE), and then differential expressedprotein identifications were performed by Matrix-Assisted Laser Desorption/Ionization Tandem Time-of-Flight Mass Spectrometry (MALDI-TOF/TOF MS)which was validated by enzyme-linked immunosorbent assay (ELISA). Eight wheyprotein spots and whey protein regions with significant difference were identified tobe seven kinds of proteins, including β-casein, α-lactoalbumin (α-La), β-lactoglobulin(β-Lg), transferrin, albumin, IgG heavy chain precursor, and an uncharacterized protein, which mainly referred to immune activity and biological function in cow milk.The differential expressed proteins were all down-regulated at different levels whenmilk was heated at85℃for15s or125℃for4s. However, their contents weremarkedly reduced and tended towards stability gradually when heated at138℃and145℃for4s. It is concluded that whey proteome of cow milk are extremely sensitiveto thermal treatment. Profiles of bovine whey proteome can be used for assessingextent of heat treatment and guiding application of bovine whey proteins.The third part of this study was to investigate the impact of heat treatment on theproteome of bovine milk fat globule membrane (MFGM) by applying differentthermal procedures commonly used in dairy industry. Four heating methods, T1(85℃,15s), T2(125℃,4s), T3(138℃,4s), and T4(150℃,4s), were chosen. Bovine milkfat globule membrane proteins (MFGMPs) were extracted and characterized bytwo-dimensional polyacrylamide gel electrophoresis (2-DE), and reference profileswere established. Matrix-Assisted Laser Desorption/Ionization TandemTime-of-Flight Mass Spectrometry (MALDI-TOF/TOF MS) was performed toanalysis altered expression proteins. The results showed that spots of Xanthinedehydrogenase/oxidase, Glycoprotein2and Butyrophilin subfamily1member wereup-regulated after homogenization compared with raw milk, and then down-regulatedafter various thermal treatments. Expression of Lactadherin, Guaninenucleotide-binding protein, Cathelicidin-1and Heart fatty acid-binding protein weredown-regulated in homogeneous milk and kept on decreasing with increasingtemperature. Lysozyme was degraded during homogenization, while remainedconstant after different heat treatments. The combination of casein and whey proteinwith MFGM were increased with increasing temperature. Results suggest MFGMproteins are sensitive to homogenization and thermal treatment, which results incharacteristic changes of MFGM protein under certain conditions.