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Analysis of the Effect of Temperature Changes Combined with Different Alkaline pH on the β-Lactoglobulin Trypsin Hydrolysis Pattern Using MALDI-TOF-MS/MS

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Seronei Chelulei Cheison*‡†, Janina Brand†, Elena Leeb†, and Ulrich Kulozik§

^† Zentralinstitut für Ernährungs- und Lebensmittelforschung (ZIEL)-Junior Research Group: Bioactive Peptides and Protein Technology, Technische Universität München, Weihenstephaner Berg 1, D-85354 Freising, Germany

^‡ School of Public Health and Community Development, Maseno University, Private Bag, Kisumu, Kenya

^§ Chair for Food Process Engineering and Dairy Technology Department, ZIEL Technology Section, Technische Universität München, Weihenstephaner Berg 1, D-85354 Freising, Germany

J. Agric. Food Chem., 2011, 59 (5), pp 1572–1581

DOI: 10.1021/jf1039876

Publication Date (Web): February 14, 2011

*Tel. +49 +49 (0) 816171-5056. Fax: +49 (0) 816171-4384. E-mail: Seronei.Cheison@wzw.tum.de.

Abstract

Temperature and pH influence the conformation of the whey protein β-lactoglobulin (β-Lg) monomer, dimer, and octamer formation, its denaturation, and solubility. Most hydrolyses have been reported at trypsin (EC 3.4.21.4) optimum conditions (pH 7.8 and 37 °C), while the hydrolysate mass spectrometry was largely limited to peptides with <4 kDa. There are few reports on trypsin peptide release patterns away from optimum. This work investigated the influence of alkaline (8.65 and 9.5) and optimum (7.8) pH at different temperatures (25, 37.5, and 50 °C) on β-Lg (7.5%, 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. Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) was used to monitor peptide evolution over time with the use of two matrixes: α-cyano-4-hydroxycinnamic acid (HCCA) and 2.5-dihydroxyacetophenone (DHAP). Mass analysis showed that the N- and C-terminals (Lys₈-Gly₉, Lys₁₀₀-Lys₁₀₁, Arg₁₂₄-Thr₁₂₅, Lys₁₄₁-Ala₁₄₂, and Arg₁₄₈-Leu₁₄₉) of β-Lg were cleaved early (15 s) implying the ease of trypsinolysis at the exposed terminals. Hydrolyses at 25 °C and pH 7.8 as well as at 50 °C and pH 9.5 were slowed down and ordered. Nonspecific chymotrypsin-like behavior occurred more at higher temperatures (50 °C) than at lower ones (25 and 37.5 °C). In addition to our earlier work in the acid pH region, it can be concluded that there is potential for controlled hydrolysis outside the trypsin optimum, where different target peptides with predictable biofunctionalities could be produced.