Production of β‐Lactoglobulin hydrolysates by monolith based immobilized trypsin reactors

Abstract

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 Lys -Phe was resistant against the immobilized trypsin in N1.

Flow‐through enzymatic reactors using polymer monoliths: From motivation to application

“…We were not able to find a study directly comparing axial flow and radial flow guided monolithic catalysts. In a study of Naldi et al [41], 4.5 mg trypsin was reported to be immobilized on per milliliters CIM ® ‐ALD monolith disk (axial flow), and around 5.0 mg trypsin was immobilized on per milliliters CIM ® ‐ALD tubular monolith column (radial flow) in our previous work [44]. This slight difference in the amount of immobilized trypsin is more attributed to the varied immobilization protocols or calculation methods, rather than the choice between disk and tubular columns.…”

“…In addition, another study [40] directly correlated the sequence recovery rate to the enzymatic activity of trypsin‐IMER, and indicated that improving digestion flow rates up to 0.4 mL/min increased the reaction rate, which is quite disputable. Here, we try to sort out these references and give a critical discussion including our previous works [42,44].…”

“…In our previous works [42,44], where trypsin was immobilized on CIM ® ‐ALD monoliths, the increase in flow rate contributed to a higher V max value. Analogous results were demonstrated for chymotrypsin, ribonuclease immobilized on various polymer monoliths [7].…”

“…When large macromolecules are substrates, a following concern is the potential blockage problem in IMERs. In our previous work [44], trypsin was immobilized on polymer monolith with pore size 6 μm, which is less prone to pore blockage than that with 2 μm pore size. This trypsin‐IMER showed great activity toward β‐lactoglobulin, and it was upscaled from a bed volume of 1 to 8 mL in our following work [42], which was able to selectively hydrolyze β‐lactoglobulin in whey proteins.…”

Transforming food waste: how immobilized enzymes can valorize waste streams into revenue streams

“…Importantly, the peptide analysis differed between the immobilized trypsin and the free trypsin, demonstrating the impact of immobilization methods on enzyme selectivity. 111 Trypsin has also been immobilized on renewable supports for whey protein hydrolysis, including spent grain and lignocellulose. 112,113 Multipoint covalent attachment was used to tether trypsin (from porcine pancreas) to spent grain, and retained greater than 90% activity after 5 cycles of reuse, as measured by the synthetic substrate, N -α-benzoyl- dl -arginine- p -nitroanilide.…”