Application of high-resolution ultrasonic spectroscopy for for real-time non-destructive monitoring of hydrolysis of whey proteins

Enzymatic protein hydrolysis is well-established manufacturing process in various industries. Protein hydrolysis, catalysed by proteases, is the most effective and efficient way to improve the physicochemical properties of proteins, such as digestibility, bioavailability, emulsion stability or viscosity. The advancement of protease-based technologies depends on analytical tools available for non-destructive real-time assessment of various aspects of the performance of protease in a simple liquid system, as well as in complex systems such as emulsion, suspension and gel. The majority of routine analytical techniques are limited in their applicability, especially if the media is opaque or the reactants/products do not possess optical activity. These limitations result in uncontrolled protein hydrolysis reducing the possible applications of the hydrolysate products. Thus, novel techniques and approaches are needed for the real-time monitoring of the enzymatic reactions of these systems. This thesis aims to describe the recent advancement in the application of high-resolution ultrasonic spectroscopy and demonstrate its capabilities for monitoring, controlling, and optimising the enzymatic hydrolysis of major whey proteins, β-lactoglobulin, α-lactalbumin and bovine serum albumin, by α–chymotrypsin and trypsin. Both whey proteins and the enzymes employed are widely used for a variety of applications in the food industry. The research work section is divided into four parts consolidated as stand-alone publications: Chapter 4 (1st paper) describes the inclusion of the effects of ionisation of protein functional groups into procedures for ultrasonic real-time non-destructive monitoring of enzymatic hydrolysis of proteins in aqueous mixtures. Since the reaction of hydrolysis is accompanied by a release of H+ ions in solution, affecting the ionisation state of the side chains of amino acids and the buffers present in the reaction mixture. Such effects are incorporated into the calibration characteristics and algorithms of analysis of ultrasonic reaction profiles allowing the measurement of the concentration of peptide bonds hydrolysed with very high precision. This is followed by Chapter 5 (2nd paper) with the discussion of the relaxation contribution to ultrasonic characteristics caused by the proton transfer between the functional groups of protein hydrolysates and buffers present in the reaction mixture, the determination of the kinetic constants and the volume effect of the proton transfer, and the utilisation of the relaxation effects in real-time monitoring of protein hydrolysis. Chapter 6 and Chapter 7 (3rd and 4th paper) demonstrate the application of high-resolution ultrasonic spectroscopy in assessing the effects of pH, temperature, and medium on hydrolysis of whey proteins by serine proteases. The ultrasonic measurements were verified with complementary techniques such as trinitrobenzene sulfonic acid (TNBS) assay, pH measurements and reverse-phase liquid chromatography (RP-HPLC). Importantly the detailed profiles of enzyme autolysis obtained ultrasonically are included in the interpretation of the dynamics of the hydrolysis. The physical effects described in the first part are incorporated into the ultrasonic calibration, which allows the recalculation of ultrasonic velocity to the degree of hydrolysis profile. Parallel measurements with density allowed the investigation of the effect of hydrolysis on the volume and compressibility of the proteins. The obtained ultrasonic profiles of hydrolysis, which are measured precisely over the whole course of the reaction, can be utilised in advanced analysis of protein hydrolysis, including monitoring of the evolution of the degree of polymerisation and molar mass of the hydrolysates in the hydrolysis mixture, and evolutions of reaction rates. It allows the evaluation of kinetic mechanisms, measurements of kinetic constants and activation energies of the protein hydrolysis.