Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Application of high-resolution ultrasonic spectroscopy for real-time monitoring of trypsin activity in β-casein solution Sopio Melikishvili a , Mark Dizon b , Tibor Hianik a, ⁎ a Department of Nuclear Physics and Biophysics, Comenius University, Mlynska dolina F1, 842 48 Bratislava, Slovakia b School of Chemistry and Chemical Biology, University College of Dublin, Belfield, Dublin 4, Ireland ARTICLE INFO Keywords: Trypsin β-casein Hydrolysis Ultrasonic spectroscopy Ultrasonic velocity ABSTRACT High-resolution ultrasonic spectroscopy (HR-US) was applied for real-time monitoring of β-casein hydrolysis by trypsin at various conditions for the first time. The technique is based on the precision measurement of hydration changes proportional to the number of peptide bond hydrolyzed. As HR-US exhibits ultrasonic transparency for most solution, the analysis did not require optical transparency like for 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay. Appropriate enzymatic models were fitted with degree of hydrolysis (d h ) profiles to provide kinetic and mechanistic description of proteolysis in terms of initial hydrolysis rate, r 0 , and rate constant of hydrolysis, k h , and enzyme inactivation, k d . Maximal r 0 and d h were obtained at 45 °C and pH 8. The exponential depen- dence of kinetic parameters allowed determination of the activation (E A = 50.3 ± 7 kJ/mol) and deactivation (E D = 62.23 ± 3 kJ/mol) energies of hydrolysis. The ultrasonic assay provided rapid detection of trypsin activity even at sub-nanomolar concentration. 1. Introduction Proteolysis plays an important role in various fields of bioscience and biotechnology. Technologically, there are broad applications of proteolysis in food processing (Vorob’ev, Vogel, Güler & Mäntele, 2011). For instance, the proteolytic activities in milk affect the texture and flavor of dairy products (Datta & Deeth, 2002). Milk has an average protein concentration of 3.2% in cow’s milk in which 80% of the pro- teins are caseins and 20% are whey proteins. β-casein is one of the major casein proteins (~35% of bovine caseins) of 209 amino acids per monomer with corresponding average molecular weight of 23.6 kDa. It is a non-compact globular protein which usually exists in the form of micelle associated with other caseins such as α s1 -caseins, α s2 -caseins and κ-caseins in milk. Since dominant milk protein, modification of β- casein alters the overall properties and qualities of milk yielding both positive and negative impacts. Specifically, proteolytic activities have also been linked with the release of caseinophosphopeptides (CPPs), the phosphorylated bioactive peptides from milk casein which can be used as supplements for fortifying foods, with a view to improving mineral bioavailability (Cruz-Huerta, García-Nebot, Miralles, Recio & Amigo, 2015; García-Nebot, Alegría, Barberá, Clemente & Romero, 2009). Therefore, detection and quantification of proteolytic activity in milk has important industrial impacts. Trypsin is a highly specific serine protease which selectively cleaves peptide bonds on the carboxyl-terminal side of arginine (Arg-X) and lysine (Lys-X) (Huber & Bode, 1978). Its monomeric form consists of 223 amino acids with corresponding average molecular mass of 23.3 kDa. It is an important digestive enzyme which is produced in pancreas as an inactive precursor, trypsinogen. It is commonly used as a model protease because it is inexpensive and readily available (Sato & Kato, 2016). Traditional methods for trypsin detection involve multiple clinical tests including radioimmunoassay, gelatin-based film techni- ques, enzyme-linked immunosorbent assay (ELISA) and colorimetric assay. However, these methods are time-consuming and costly (Gao, Tang, Li & Su, 2012). To overcome the limitations of traditional dis- continuous methods, a variety of novel techniques providing the real- time data of trypsin activity have been reported. Among them are fluorescence techniques based on fluorescence sensors which have the advantage of the lowest detection limit compared with the other methods (Sato & Kato, 2016; Gao, Tang, Li & Su, 2012; Zhang, Qin, Cui, Zhou & Du, 2016; Zhang et al., 2018; Song et al., 2019). However, the efficiency of these techniques is dependent on the optical transparency or transmission of medium and can be affected by light scattering in dispersions. High-resolution ultrasonic spectroscopy (HR-US) provides a poten- tial technology for real-time, non-invasive and precision monitoring of enzyme activity in a wide range of concentrations and substrates as well as medium. It does not require optical transparency or optical markers https://doi.org/10.1016/j.foodchem.2020.127759 Received 11 January 2020; Received in revised form 30 July 2020; Accepted 2 August 2020 ⁎ Corresponding author. Food Chemistry 337 (2021) 127759 Available online 04 August 2020 0308-8146/ © 2020 Elsevier Ltd. All rights reserved. T