Effect of maillard-induced glycosylation on the molecular configuration of whey protein and its solubility, thermal stability, and overall quality for beverage applications

University of Minnesota Ph.D. dissertsation. Major: Food science. Advisor: Baraem Ismail. 1 coputer file (PDF); xi, 115 pages, appendices A-E.Whey protein has numerous nutritional, physiological and functional benefits. It is therefore a perfect candidate for high protein beverage applications. Whey protein beverages are gaining popularity among athletes, bodybuilders and health conscientious individuals. In spite of the reasonable solubility of whey proteins in acidic beverages (pH 3.4-4.5), thermal processing and prolonged ambient storage can result in protein aggregation via hydrophobic and disulfide molecular interactions between denatured proteins, especially at relatively high protein concentration (>4.2%, w/v). Currently, whey protein acidic beverages available in the market contain at most 4% whey protein. To better deliver the nutritional quality and expand the commercial value, it is desirable to develop shelf stable high whey protein acidic beverages (>4.2% protein). It is also desirable to produce a less acidic beverage, to reduce undesirable sourness and astringency. We hypothesized that glycosylation of whey protein via early stage Maillard reaction results in higher net charge, enhanced thermal stability, increased structural rigidity, and reduced buffering capacity. Partially glycosylated whey protein (PGWP) can thus be used to produce high protein acidic beverages that are shelf stable with acceptable sensory quality. Therefore, our long term goal was to produce a partially glycosylated whey protein with enhanced solubility and thermal stability over a wide pH range, while maintaining nutritional and sensory quality. Glycosylation conditions were optimized to promote glycosylation of whey protein, while minimizing browning and maintaining nutritional quality. Unreacted dextran was removed using liquid chromatography, and the protein fractions, both non-reacted protein and glycosylated protein were collected and labeled as PGWP. Extent of glycosylation, browning, % lysine blockage, % loss in free amine, and in-vitro digestibility were monitored. Solubility and thermal stability of PGWP were compared to whey protein isolate (WPI), at 5-10% (w/v) protein concentration, pH 3.4, 4.5, 5.5 and/or 7.0, and pre and post thermal treatments. Differential scanning calorimetry (DSC) and SDS-PAGE were employed to monitor onset of denaturation and polymerization, respectively. Exposure of sulfhydryl groups and surface hydrophobicity were monitored during heat treatment of 5% protein solutions at 75 °C for 60 min. The pI of PGWP was as well. For structural characterization mass spectrometry (MS) analysis and surface enhanced Raman spectroscopy (SERS) were used. The glycosylation site was determined using matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS). Solutions (5% protein, w/v) of WPI and PGWP were analyzed by SERS to determine secondary and tertiary structural changes of the protein as affected by glycosylation, pH and heating. The sourness and astringency of protein acidic solutions/beverages formulated with PGWP and WPI at 5% protein and at different pHs were determined. The storage stability of the formulated solutions/beverages over 6-8 weeks was determined, by measuring solubility, turbidity, and protein aggregation using capillary electrophoresis (EC). Overall, results proved that the solubility and thermal stability of whey protein, at relative high protein concentrations, can be enhanced over a broad range of pH upon partial and limited Maillard-induced glycosylation. The enhanced solubility and thermal stability of PGWP was attributed to structural rigidity, unique glycosylation sites, resistance to denaturation, and shift to a more acidic pI, all of which contributed to reduced protein/protein interactions. The nutritional quality was maintained and advanced stages of Maillard reaction were not detected. Partial glycosylation of whey protein reduced its buffering capacity, and consequently less amount of acid was required to reach the targeted pH of the protein fortified solution/beverage. The reduced acidity of the PGWP fortified beverages, especially at pH 4, resulted in lower perceived sourness and astringency compared to the WPI fortified beverages. In addition, the solubility and turbidity of PGWP solution prepared at pH 4.5 were maintained fairly well during storage at or below 25 °C. Our findings demonstrate the possibility of using PGWP in the production of shelf stable high protein acidic beverages (> 4.2% protein) that can be less sour and astringent than what is currently present in the market. Our results also provided information that is essential to understand the structure/function relationship upon Maillard-induced glycosylation of whey proteins. This basic information is useful for directed structural modification research aiming at improving whey protein functionality