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 degrees C) Little information is available on the effect of sub-optimal conditions on hydrolysis Bovine beta-lactoglobulin (beta-Lg) was hydrolysed by trypsin under acidic pH (pH 4-7) between 20 and 60 degrees C and the substrate concentration from 2 5% to 15% (w/v) and compared with hydrolysis at pH 7 8 and 37 degrees 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 alpha-cyano-4-hydroxycinnamic acid (HCCA) and 2 5 dihydroxyacetophenone (DHAP) matrices Hydrolysis patterns of beta-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 beta-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 Arg(40)-Val(41) Lys(141)-Ala(142) and Arg(148)-Leu(149) resistance was at Lys(60)-Trp(61) Arg(124)-Thr(125) and Lys(135)-Phe(136) Five domains were identified based on beta-Lg resistance to trypsin in the order f(1-40) < f(41-75) < f(76-91) > f(92-138) > R139-162) 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 (C) 2010 Elsevier Ltd All rights reserved