Hydrolysis of β-lactoglobulin by trypsin under acidic pH and analysis of the hydrolysates with MALDI–TOF–MS/MS

Trypsin (EC 3.4.21.4) hydrolysis of food proteins are done at the optimum pH (7.8) and temperature (37 °C). Little information is available on the effect of sub-optimal conditions on hydrolysis. Bovine β-lactoglobulin (β-Lg) was hydrolysed by trypsin under acidic pH (pH 4–7) between 20 and 60 °C and the substrate concentration from 2.5% to 15% (w/v) and compared with hydrolysis at pH 7.8 and 37 °C. Aliquots were taken at different times (t = 0 up to 10 min). Samples were analysed using matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI–TOF–MS/MS) with α-cyano-4-hydroxycinnamic acid (HCCA) and 2,5-dihydroxyacetophenone (DHAP) matrices. Hydrolysis patterns of β-Lg were generally similar at pH 7.8, 7, 6 and 5 while at pH 4 fewer peptides were detected except a unique fragment f(136–141). The different cleavage sites of β-Lg showed low resistance to trypsin at optimum conditions and pH 7 while being random and simultaneous. At lower pH, some cleavage sites showed increased resistance, while hydrolysis was relatively slow and ordered. Initial attack by trypsin occurred at Arg40–Val41, Lys141–Ala142 and Arg148–Leu149 resistance was at Lys60–Trp61, Arg124–Thr125 and Lys135–Phe136. Five domains were identified based on β-Lg resistance to trypsin in the order f(1–40) < f(41–75) < f(76–91) > f(92–138) > f(139–162). Results suggest that hydrolysis away from trypsin optimum offer better hydrolysis process control and different peptides. This strategy may be used to protect target bioactive or precursor peptides, or avoid the production of unwanted peptides.

Research highlights
► Bovine β-lactoglobulin (β-Lg) hydrolysis patterns of β-Lg similar at pH 5–7.8.
► Low resistance of β-Lg to trypsin at optimum and pH 7; random and simultaneous.
► Increased resistance with low pH; slow and ordered.
► Trypsin first attack points: Arg40–Val41, Lys141–Ala142 and Arg148–Leu149.
► Five domains in β-Lg with (f(1–40) < f(41–75) < f(76–91) > f(92–138) > f(139–162)).