Influence of the milieu conditions on trypsin hydrolysis of β-lactoglobulin

Temperature and pH influence the conformation of the whey protein β-lactoglobulin (β-Lg) monomer, dimer and octamer formation, its solubility and denaturation. Most hydrolysis have been reported at trypsin (EC 3.4.21.4) optimum conditions (pH 7.8 and 37°C), while the hydrolysates mass spectrometry were largely limited to peptides with < 4kDa. This work investigated the influence of acidic pH (pH 4, 5, 6 and 7) and alkaline pH (pH 8.65 and 9.5) compared to the optimum pH (pH 7.8) at different temperatures (25°C, 37.5°C and 50°C) on the β-Lg (2.5% to 12.5%, w/v) hydrolysis. Sample aliquots were drawn out before the addition of trypsin (blank sample) and at various time intervals (15 s to 10 min), thereafter. Two matrices (α-cyano-4hydroxycinnamic acid (HCCA) and 2.5-dihydroxyacetophenone (DHAP)) were used for matrix-assisted laser desorption/ionisation time of flight tandem mass spectrometry (MALDI-TOF-MS/MS) analysis to monitor peptide evolution over time (t = 0, 5s, 10s, 15s, 1min, 2.5min, 5min, 7.5min and 10min). At lower pH, some cleavage sites showed increased resistance, while hydrolysis was relatively slow and ordered. The fragments f(1-8), f(1-40), f(15-40) were detected at 5 sec. Initial attack by trypsin occurred at Lys-Val, Arg-Val, Lys-Ala and Arg-Leu resistance was at Lys-Trp, Arg-Thr and Lys-Phe. Hydrolysis at the optimum conditions was random, simultaneous and unordered whereas hydrolysis away from optimum conditions were generally slow, ordered and sequential. Five domains were identified based on β-Lg resistance to trypsin at acid pH in the order f(1-40) < f(41-75) < f(76-91) > f(92-138) > f(139-162), with resistance towards its core while being easily hydrolysable at the terminals. Non-specific ‘chymotrypsin-like’ behaviour occurred more at higher, than at lower, temperatures. These results offer a platform for ‘peptide design’ using the milieu conditions to drive the hydrolysis patterns.