From different fractions of microfiltered acid and sweet whey, we developed a method for the isolation of the proteins glycomacropeptide (GMP) and osteopontin (OPN) by ion exchange chromatography with monolithic columns (CIMmultus-QA-1mL) at preparative level (up to 150 mL). The protein was first separated by anion exchange chromatography on the basis of charge. Both proteins bound to the QA column at the pH of the original whey (sweet whey at pH 6-7, acid whey at pH 4.7) and at pH = 5.0 (in buffer with sodium acetate). This was followed by separation based on hydrophobicity by reversed-phase high-performance liquid chromatography (RP-HPLC). The method applied with guanidinium hydrochloride (GvHCl), dithiothreitol (DTT), acetonitrile (ACN) and trifluoroacetic acid (TFA) separated the proteins from other whey proteins (α- LA, β- LG, BSA and caseins). Finally, we separated the proteins based on size and sialic acid content by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Using the "stains-all" dye, we introduced a specific method for detecting sialic acid-containing proteins. This method was used to determine the content and purity of osteopontin and glycomacropeptide, as the dye stains sialic acid- containing proteins blue and the remaining proteins pink. To facilitate the interpretation of the results obtained, we also stained the proteins with silver nitrate. However, we were not able to accurately determine the purity of the isolated proteins. With the work carried out, we succeeded in characterizing the eluted proteins in three different ways, thus contributing to the valorization of whey. However, we have also shown that the production of both proteins is suitable for the industrial level, confirming their commercial interest and applicability in the food industry. The latter is also interesting from the point of view of the reuse of whey and the reduction of the negative impact of whey waste management on the environment.
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