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Novel Ultrasonic Technologies for monitoring of enzymatic hydrolysis of polysaccharides and proteins

2022, Papoutsidakis, Georgios, 0000-0001-7817-6186

The importance of enzymes which catalyse hydrolytic reactions, especially in the food industry, is established. These enzymes can catalyse amid, glycosidic and ester bonds, hence their names, proteases, glucosidases and esterases, respectively. Such hydrolytic enzymes are quite popular in industry and their applications range from cleaning process, proteomics, or food biotechnology processes. In particular, the application of these enzymes for the hydrolysis of food biopolymers have gained great interest due to the improvement of their physiochemical properties such as digestion, bioavailability, antioxidant improvement or reduction in their viscosity. Namely, applications of proteases and glucanases in industrial processes are constantly being introduced and with distinct advantages compared to chemical processes, by increasing the specificity of hydrolysis, purity and product preservation and reducing environmental impact. The industrial application of enzymes though, requires specific and effective control of the enzyme activity in real-time. Thus, novel techniques and approaches are needed for the real-time monitoring of the enzymatic reactions of these systems. This work describes the application of a novel method, high-resolution ultrasonic spectroscopy (HR-US) for the real-time monitoring of enzymatic reactions in various systems, protein molecules, protein particles and polysaccharides. Both the enzymes applied and the biopolymers of this work are widely used for a variety of applications in food industry. In the first part of this research work, HR-US was employed for precise, real-time and non-destructive monitoring of the hydrolysis of barley beta-glucan (BBG) by a glucanase, 1,3(4)-ß-glucanase, commercially known as Filtrase NL Fast (FNL), in buffered solutions. The degree of hydrolysis, average degree of polymerization and molar mass, throughout the hydrolysis are described. The obtained results were compared with discontinuous assay that measures the concentration of reducing sugars and high-performance liquid chromatography (HPLC). The change in apparent specific compressibility and specific volume was monitored as the glycosidic bonds were hydrolysed, and compared to literature values of smaller oligosaccharides. The reduction in viscosity of the polymer during the hydrolysis was correlated with the decrease in molar mass to obtain the Mark-Houwink parameter. Michaelis-Menten kinetic parameter was obtained from the initial reactions stage. The change in ultrasonic attenuation and the appearance of ultrasonic relaxation phenomena both in the substrate and during hydrolysis are also discussed. In the second part of the project, three different protein systems are hydrolysed by two different proteases, trypsin and a-chymotrypsin. These protein structures have different structural characteristics and this is showcased from the different calibration value of ultrasonic velocity. The change in ultrasonic velocity is translated to the concentrations of peptide bonds hydrolysed, degree of hydrolysis, average degree of polymerization and molar mass. Our ultrasonic results are compared to a discontinuous assay, trinitro-benzene sulfonic acid (TNBS) which determines quantifies the free ammino groups. The contribution of density to the change in ultrasonic velocity during the enzymatic hydrolysis is also discussed. The effect of hydrolysis to volume and compressibility of the proteins in this work are discussed and compared with literature values for smaller oligopeptides. Dynamic light scattering (DLS) is applied for monitoring the size of each protein before and during the enzymatic hydrolysis. The results show structural differences of, initially the substrates, and subsequently, the resulting hydrolysates based on their ability to form micelles. The evolution of ultrasonic attenuation before and after the enzymatic hydrolysis of the proteins is provided and possible relaxation mechanisms are discussed.