Hydrolysis of b-lactoglobulin by trypsin under acidic pH and analysis of the hydrolysates with MALDI–TOF–MS/MS Seronei Chelulei Cheison a,b,⇑ , Ming-Yu Lai a , Elena Leeb a , Ulrich Kulozik c a Zentralinstitut für Ernährungs- und Lebensmittelforschung (ZIEL) – Junior Research Group: Bioactive Peptides and Protein Technology, Technische Universität München, Weihenstephaner Berg 1, D-85354 Freising, Germany b School of Public Health and Community Development, Maseno University, Private Bag, Kisumu, Kenya c Food Process Engineering and Dairy Technology Department, ZIEL Technology Section, Technische Universität München, Weihenstephaner Berg 1, D-85354 Freising, Germany article info Article history: Received 25 June 2010 Received in revised form 29 September 2010 Accepted 7 October 2010 Keywords: Acid pH Trypsin optimum conditions Hydrolysis resistance Matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry abstract 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 b-lac- toglobulin (b-Lg) was hydrolysed by trypsin under acidic pH (pH 4–7) between 20 and 60 °C and the sub- strate 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 a-cyano-4- hydroxycinnamic acid (HCCA) and 2,5-dihydroxyacetophenone (DHAP) matrices. Hydrolysis patterns of b-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 b-Lg showed low resistance to trypsin at opti- mum 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 b-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 pro- tect target bioactive or precursor peptides, or avoid the production of unwanted peptides. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction b-Lactoglobulin (b-Lg) is the major whey protein (3 g/L) of ruminant species and is also present in the milk of other species, but not in human milk. Therefore it is one of the main causes of cows’ milk allergy in humans. Bovine b-Lg is a small protein with 162 residues and 18.4 kDa. There are at least nine known genetic variants; A, B, C, D, E, H, I, J, and W. The three common variants are A, B and C (Hambling, McAlpine, & Sawyer, 1992). The second- ary structure of bovine b-Lg consists of an eight-stranded, flattened b-barrel, which folds into a calyx and a flanking three-turn a-helix. Two disulphide bonds (Cys 106 –Cys 119 , Cys 66 –Cys 160 ) stabilise the globular structure. It exists as a dimer in solution at physiological pH (pH 6.7) and temperature. The self-association properties of b- Lg A and B are also different. b-Lg A forms dimers and then octa- mers under increasingly acidic conditions, whereas variants B and C do not form octamers, but do form dimers (Fig. 1). Trypsin (EC 3.4.21.4) is a serine protease which is widely used in research, because of its high specificity and activity. It predom- inantly cleaves peptide bonds at carboxyl terminals of arginine and lysine, except when linked to a proline residue (Olsen, Ong, & Mann, 2004). In addition, the hydrophobic walls of the catalytic pocket create a favourable environment for the long aliphatic and unbranched parts of the basic arginine and lysine. The optimal temperature and pH for the performance of trypsin are 37 °C and pH 7.8 (Olsen et al., 2004). At 55 °C, it was observed that tryp- sin activity decreased quickly (Galvão, Silva, Custódio, Monti, & Giordano, 2001). At low temperature, its denaturing is slow. Between pH 6 and pH 4.25, trypsin is also slowly denatured but below pH 4.25 it rapidly loses its activity (Murthy, Kostman, & Dinoso, 1980). Deviations from trypsin-specificity have been reported, how- ever, where it acquired chymotrypsin-like activity which was attributed to a ‘‘chymotrypsin memory” in its activity owing to 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.10.042 ⇑ Corresponding author at: Zentralinstitut für Ernährungs- und Lebensmittelf- orschung (ZIEL) – Junior Research Group: Bioactive Peptides and Protein Technol- ogy, Technische Universität München, Weihenstephaner Berg 1, D-85354 Freising, Germany. Tel.: +49 0 816171 5056; fax: +49 0 816171 4384. E-mail address: Seronei.Cheison@wzw.tum.de (S.C. Cheison). Food Chemistry 125 (2011) 1241–1248 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem