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Separation of proteins from the milk fat globule membrane with minimal losses
Components of the milk fat globule membrane (MFGM) are reported to have functional properties. However, determination of the specific properties of individual MFGM components is hardly possible, since the MFGM is a complex three-layered structure consisting of phospholipids and proteins. Therefore, to assess the MFGM proteins´ functionalities, firstly an isolation of the MFGM from the bulk milk proteins is required. Secondly, single MFGM proteins must be separated from the phospholipid phase. The aim of this project was to compare established and novel isolation methods in terms of the capacity to obtain MFGM proteins at high yield. First, the method of washing the cream for several times was used. After each washing step the cream sample was churned and the buttermilk obtained was analyzed regarding MFGM proteins using SDS-PAGE. The intensities of the different proteins (XO/XDH, BTN PAS6/7) were compared to a non-washed sample. Beside the removal of caseins and whey proteins it was found that a significant loss of MFGM proteins up to 90% occurs, which has been neglected in previous studies. Particle size measurements showed that the fat globules increased during the washing process. This is due to coalescence of the fat globules and consequently results in a loss of MFGM material including membrane proteins. Second, filtration experiments with buttermilk were done. Because of an overlap in size between the casein micelles and the MFGM fragments a pre-treatment of the buttermilk was necessary to realize an isolation of the MFGM. In this study, a new approach based on the coagulation of casein micelles by adding rennet was developed. The supernatant (buttermilk-sweet whey) obtained was used for a subsequent diafiltration to remove the residual whey proteins. All permeates and retentates of each diafiltration step were again analyzed for the remaining MFGM proteins, caseins and whey proteins by SDS-PAGE to evaluate the isolation procedure. However, significantly higher amounts (~70%) of the total MFGM proteins were recovered when the newly developed MFGM isolation method was used in comparison to the washing method. Concluding, both separation methods are applicable to realize MFGM isolates but a simultaneous loss of MFGM proteins is hardly avoidable. As far as we oversee the literature, this fact was not sufficiently considered in previous studies. Finally, the results of this work show that different MFGM proteins are enriched in the particular isolates depending on their location in the MFGM and the applied extraction method.
Keywords: MFGM proteins