Anantharamkrishnan, Vaidhyanathan2021-01-252021-01-252020-07https://hdl.handle.net/11299/218055University of Minnesota Ph.D. dissertation. July 2020. Major: Food Science. Advisor: Gary Reineccius. 1 computer file (PDF); 186 pages.Demand for high protein plant and dairy-based diets has been increasing but delivering them has become problematic for the food industry. The flavor issues are due to multifaceted interactions that occur between food proteins and flavoring components. Over 40 years of research has been done on studying temporary interactions between flavor and proteins, but very little work has been done on more permanent interactions – covalent bonding. Covalent bonding takes place between the side chains and terminal amino acids of food proteins and reactive aroma compounds that will change the flavor profile of the product in a permanent manner. β- lactoglobulin (BLG) was chosen as a model protein for this study as it is well characterized in both amino acid sequence and structure, its molecular weight is suitable for intact protein mass spectrometry and it is a major protein used in food industry. This thesis study developed a methodology using UPLC-ESI/qTOF-MS for monitoring the nature and extent of the covalent reactions based upon the change in molecular weight (Protein + flavor) that occurs after reaction. The cross linking of protein with flavor compounds was evaluated using gel electrophoresis. A proteomics approach using LC and tandem MS after enzymatic digestion was taken to identify the sites of post-translational modification between the flavor compounds and the BLG protein. The UPLC-ESI/qTOF-MS methodology in tandem with proteomics and gel electrophoresis yield a detailed view of flavor/BLG interactions that offered insights on minimizing these undesirable reactions in the future. A flavoring typically is created from a mixture of volatile chemicals that generally comprise a variety of functional groups. Some of these flavor components when added to a protein matrix form covalent adducts resulting in a change in flavor character or a loss of its potency. The end result of these reactions create an imbalanced flavor, one that is not acceptable to a consumer. This research study analyzed 47 different flavor compounds from 13 different functional groups for their covalent adduct formation with BLG. Aldehydes, sulfur-containing molecules (especially thiols), and functional group-containing furans were found to be the most reactive of the flavor components studied. Thiol-containing compounds reduced disulfide linkages in BLG to result in disulfide interchange and formation of new disulfide linkages with the free cysteine group. Ketones were generally stable, but α-diketones (e.g., diacetyl) were reactive. Some bases (e.g., pyrazines and pyridines) were non-reactive, while the nucleophilic allylamine was reactive. Hydrocarbons, alcohols, acids, esters, lactones, and pyrans did not give observable levels of adduct formation within the time period studied. Due to the varied environmental conditions present in various food systems, the nature and extent of covalent interactions would likely change. This study investigated the influence of pH, temperature and water activity on the covalent adduct formation between BLG and selected flavor molecules. Covalent adduct formation was slower in acidic pHs. The rate and extent of the reaction increased with increasing pH. The rate of formation of adducts increased with temperature. Higher temperatures (45°C) caused the formation of products that were not observed at lower temperatures (4°C and 25°C). An increase in water activity lead to an increase in formation of adducts for allyl isothiocyanate. There were no observable differences for the effect of water activity on the reaction rate for benzaldehyde, citral and dimethyl disulfide. Results will help in understanding the conditions at which flavor compounds will covalently bond with a protein and ways to potentially avoid it. Thereby, helping the food industry to develop flavor protein matrices that have a longer shelf life.enCovalent reactionsFlavorMass spectrometryProteinβ-LactoglobulinThesis or Dissertation