Exploring the Use of a Modified High-Temperature, Short-Time Continuous Heat Exchanger with Extended Holding Time (HTST-EHT) for Thermal Inactivation of Trypsin Following Selective Enzymatic Hydrolysis of the β-Lactoglobulin Fraction in Whey Protein Isolate

Abstract

Tryptic hydrolysis of whey protein isolate under specific incubation conditions including a relatively high enzyme:substrate (E:S) ratio of 1:10 is known to preferentially hydrolyse β-lactoglobulin (β-LG), while retaining the other major whey protein fraction, i.e., α-lactalbumin (α-LA) mainly intact. An objective of the present work was to explore the effects of reducing E:S (1:10, 1:30, 1:50, 1:100) on the selective hydrolysis of β-LG by trypsin at pH 8.5 and 25 °C in a 5% (w/v) WPI solution during incubation periods ranging from 1 to 7 h. In addition, the use of a pilot-scale continuous high-temperature, short-time (HTST) heat exchanger with an extended holding time (EHT) of 5 min as a means of inactivating trypsin to terminate hydrolysis was compared with laboratory-based acidification to <pH 3 by the addition of HCl, and batch sample heating in a water bath at 85 °C. An E:S of 1:10 resulted in 100% and 30% of β-LG and α-LA hydrolysis, respectively, after 3 h, while an E:S reduction to 1:30 and 1:50 led >90% β-LG hydrolysis after respective incubation periods of 4 and 6 h, with <5% hydrolysis of α-LA in the case of 1:50. Continuous HTST-EHT treatment was shown to be an effective inactivation process allowing for the maintenance of substrate selectivity. However, HTST-EHT heating resulted in protein aggregation, which negatively impacts the downstream recovery of intact α-LA. An optimum E:S was determined to be 1:50, with an incubation time ranging from 3 h to 7 h leading to 90% β-LG hydrolysis and minimal degradation of α-LA. Alternative batch heating by means of a water bath to inactivate trypsin caused considerable digestion of α-LA, while acidification to <pH 3.0 restricted subsequent functional applications of the protein.

Whey Protein Hydrolysates of Sheep/Goat Origin Produced by the Action of Trypsin without pH Control: Degree of Hydrolysis, Antihypertensive Potential and Antioxidant Activities

“…Similar to our findings, enhancement of α-LA hydrolysis by trypsin has been observed under non-controlled pH “free-fall pH” conditions [ 31 ]. Moreover, trypsin preferably hydrolyzed α-LA further than β-LG during the keeping of a WPI hydrolysate under pH 5.5–6.5 [ 32 ].…”

Milk Whey Hydrolysates as High Value-Added Natural Polymers: Functional Properties and Applications

“…The most common hydrolysis treatment is enzymatic; the functionality of these hydrolysates depends on different factors such as the type of enzyme, pH, temperature, time, and enzyme/substrate ratio [ 93 , 94 ]. Compared to chemical hydrolysis, enzymatic hydrolysis usually takes place under relatively mild operating conditions (temperature 20–70 °C, pH 6.0–8.0) [ 95 ]. The most widely used enzymes to produce whey hydrolysates are proteases and are capable of promoting specific and selective protein modifications.…”

Optimization of Peroxidase Activity of Turnip (Brassica rapa) Using Response Surface Methodology

“…High temperatures will break the bonds responsible for maintaining the 3D structure of the enzyme that induces the modification of enzyme shape and consequently disrupts the active site. In this case, the enzymatic activity will be reduced or completely will be suppressed (Agostini et al, 2002;Sáez et al, 2019). The enzymatic activity usually depends on the temperature of the medium.…”