Advancing Analytical Methodologies to Study Oligosaccharides, Bioactive peptides, and Allergenic Proteins in Plant-based Beverages and Related Products
As the global consumption of plant-based beverages and related food products made from various crops has been rapidly growing, the contribution of plants as a source of various nutrients needs to be assessed and compared with traditional sources such as dairy milk. Proteins and carbohydrates are two classes of macronutrients; besides providing energy and building blocks for biosynthesis, they may exert additional biological activities in the human body. Several peptides derived from food digestion (or food processing) have been reported to exert specific bioactivities, thereby beneficially affecting human health. Indigestible carbohydrates, especially oligosaccharides that are low in molecular weight, hold the potential to reshape the gut microbiome via prebiotic activity and lead to various health-promoting effects. The bioactivities of peptides and oligosaccharides are governed by their structures, such as the amino acid sequences, constituent monomers, glycosidic linkages, and molecular sizes. On the other hand, some plant proteins are known food allergens; yet, their allergenicity might be reduced during food processing by altering allergenic proteins’ structures. Characterizing the structures of these molecules at different processing stages can improve our understanding of the potential bioactivities of specific foods and guide the development of optimized processing conditions. Analytical approaches based on liquid chromatography and mass spectrometry are useful to achieve in-depth characterization of all these compounds. This dissertation presents seven case studies about the analysis of bioactive molecules and protein allergens in plant-based products using glycomics, peptidomics, and proteomics techniques.Chapter I reviews the current knowledge on the nutritional and bioactive properties of protein and carbohydrate components in plant-based beverages. Chapters II to IV focus on the development and optimization of analytical methods for performing high-quality glycomics and peptidomics in complex food matrices, and provides solutions to overcome current challenges. Chapter IV presents a workflow for discovering small bioactive peptides in plant-based foods by LC-MS/MS. The innovative application of the dimethyl labeling technique in food peptidomics effectively facilitated full-length sequencing of small peptides with two to four amino acid residues that are believed to exert more potent in vivo bioactivities than larger peptides. Chapters V to VII presents further applications of the optimized glycomics and peptidomics analytical methods on plant-based beverages and related products generated by various processing approaches. Chapter V describes a comprehensive analysis of oligosaccharides in almond milk, soy milk, and soy flour. Chapter VI demonstrates the application of the optimized glycomics method to identify naturally occurring oligosaccharides in chickpeas as well as new structures generated from polysaccharides breakdown operated by enzymes. Chapter VII describes the discovery of potentially bioactive oligosaccharides and peptides in the cooking water of chickpeas and common beans, which is known as aquafaba, with our optimized glycomics and peptidomics approaches. It was found for the first time that when peptides were dimethyl labeled, α- and γ-glutamyl peptides could be easily differentiated with the uniquely significant a1 and b1 fragment ions. Based on that, many γ-glutamyl peptides with potential kokumi and anti-inflammatory activities were identified in aquafaba. Chapter VIII investigates the effect of enzymatic treatments for decreasing almond protein allergenicity, using proteomics analysis and immunoassay. Enzymatic extraction using neutral protease significantly reduced immunoglobulin E- and immunoglobulin G-reactivities, as evidenced by immunoblotting using human sera from patients allergic to almonds. The results were supported by proteomics, which revealed that a majority of almond proteins were hydrolyzed by neutral protease during the enzymatic extraction; however, the β-subunit regions in prunin 1 and prunin 2, which are constituents of a major almond allergen—amandin, showed resistance to proteolysis by neutral protease. Proteomics analysis also confirmed that the linear epitopes in the β-subunit regions in prunin 1 and prunin 2 largely kept their integrity. This work provides innovative and optimized analytical approaches for characterizing food-derived oligosaccharides and bioactive peptides as well as demonstrating practical applications of the optimized methodologies to various plant-based foods. The in-depth characterization of oligosaccharides, peptides, and allergenic proteins offers key insights into strategies to optimize processing conditions, valorize low-cost streams, and enhance the nutritional values of food products.