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Monolith based immobilized trypsin reactors (MITRs) were developed using different immobilization protocols. Compared with the use of reducing agent NaCNBH3, the application of 2-PB led to around 33% decrease in immobilization yield, while improved the specific activity of immobilized trypsin. The developed flow-through MITR was used to hydrolyze β-Lactoglobulin (β-Lg), and the feasibility of integrating ethanol in reaction media was evaluated. Raising ethanol concentrations from 0 to 50% increased the size of β-Lg molecules from 1.74 ± 0.32 nm to 5.67 ± 2.78 nm, and doubled the viscosity of β-Lg solutions. To avoid high backpressure and potential clog on MITR, β-Lg was hydrolyzed with the addition of ethanol up to 20%, where the activity of immobilized trypsin increased by 20% than that in aqueous media. Similar to free trypsin, the addition of ethanol contributed to an increase in DH and a faster depletion of intact protein for MITR. The presence of ethanol decreased the releases of peptides locating on C- termini, i.e., f(149–162) and f(142–148) for free and immobilized trypsin. However, the release of peptides locating inner β-Lg increased, e.g., a 5 times higher amount of f(125–138) was detected for MITR in 20% ethanol.

Immobilised trypsin is an alternative to free trypsin for producing protein hydrolysates with increased functionalities. However, the influence of hydrolytic conditions on this process remains unclear. The influence of salts on β-lactoglobulin (β-Lg) hydrolysis by free and immobilised trypsin was compared. For both forms of trypsin, 0.1 m Tris accelerated the release of most final peptides except f (71–75), and had no significant effects on the hydrolysis of intact β-Lg. Increasing NaCl concentrations from 0 to 0.02 m increased the degree of hydrolysis (DH) by 22.4% for free trypsin versus 62.1% for immobilised trypsin. The presence of 0.1 or 0.5 m NaCl hindered the release of peptides associated with the breakdown of intact protein. This led to 2–4 fold decreases in depleting intact β-Lg and DH, except immobilised trypsin at 0.1 m NaCl (DH increased by 44.3% versus without NaCl). Potential mechanisms underlying the effects of salts are discussed.

Tryptic hydrolysis of β-Lactoglobulin (β-Lg) is attracting more and more attention due to the reduced allergenicity and the functionality of resulting hydrolysates. To produce hydrolysates in an economically viable way, immobilized trypsin reactors (IMTRs), based on polymethacrylate monolith with pore size 2.1 μm (N1) and 6 μm (N2), were developed and used in a flow-through system. IMTRs were characterized in terms of permeability and enzymatic activity during extensive usage. N1 showed twice the activity compared with N2, correlating well with its almost two times higher amount of immobilized trypsin. N2 showed high stability over 18 cycles, as well as over more than 30 weeks during storage. The efficiency of IMTRs on hydrolyzing β-Lg was compared with free trypsin, and the resulting hydrolysates were analyzed by MALDI-TOF/MS. The final hydrolysis degree by N1 reached 9.68% (86.58% cleavage sites) within 4 h, while only around 6% (53.67% cleavage sites) by 1.5 mg of free trypsin. Peptides analysis showed the different preference between immobilized trypsin and free trypsin. Under the experimental conditions used in this study, the potential cleavage site Lys135-Phe136 was resistant against the immobilized trypsin in N1. This article is protected by copyright. All rights reserved

Although immobilized trypsin is a viable alternative to the free one in solution for producing protein hydrolysates, the change of selectivity introduced by immobilization is unclear. In this study, we compared the selectivity of free and immobilized trypsin towards different cleavage sites of β-lactoglobulin (β-Lg) with a focus on the impact of environmental pH. Both free and immobilized trypsin exhibited greater accessibility to native β-Lg at elevated pH (from pH 7.2 to 8.7). Additionally, free trypsin preferred to attack cleavage sites located at the C-terminus at pH 7.8, whereas an opposite preference for the N-terminus was observed at pH 8.7. Regarding the immobilized trypsin, the pH did not significantly influence its preference for the C- or N-terminus. Generally, immobilization of trypsin resulted in more focused cleavage within its specificity during the initial stage of hydrolysis, and some peptides were formed more rapidly by the immobilized trypsin.

Velocity field data were acquired for Taylor-Couette flow in the annulus gap between a rotating inner cylinder and a fixed concentric outer cylinder by using particle image velocimetry. The flocculation efficiencies were also obtained in the same Taylor-Couette flow under the conditions corresponding to the velocity field measurement. The flocculation efficiencies reached the maximum values due to the closed vortices, their contraction and expansion with time in wavy vortex flow. But out of the wavy vortex flow range, the comparatively low flocculation efficiencies were obtained due to the no-closed vortices which connected with each other.

Particle image velocimetry (PIV) was applied to characterize the morphological changes of flocs and to acquired velocity field data in the coagulation process in Taylor-Couette reactor. By use of PIV,the morphological of the flocs in the coagulation process can be characterized and described with good performance. It was shown that the flocculation efficiencies reached the maximum values and the size of the generated flocs in the coagulation process was the biggest when the roating speed was in the range between 20∼60 rpm. It was demonstrated that PIV can be exploited as a useful tool in the in-situ observation flocs during coagulation processes.