International Dairy Journal 16 (2006) 831–839 Effects of combined high pressure and enzymatic treatments on the hydrolysis and immunoreactivity of dairy whey proteins Elena Pen˜as a,b , Guadalupe Pre´stamo a , Maria Luisa Baeza c , Maria Isabel Martı´nez-Molero c , Rosario Gomez b,Ã a Departamento de Ciencia y Tecnologı´a, de Productos Vegetales, Instituto del Frı´o, Ciudad Universitaria, 28040-Madrid, Espan˜a b Departamento de Ciencia y Tecnologı´a, de Productos La´cteos, Instituto del Frı´o, Ciudad Universitaria, 28040-Madrid, Espan˜a c Seccio´n de Alergia Infantil, Hospital Gregorio Maran˜o ´n Received 1 December 2004; accepted 31 August 2005 Abstract The effect of high-pressure (HP) treatment on the hydrolysis of dairy whey proteins by trypsin, chymotrypsin and pepsin was analysed. Isostatic pressure (100–300 MPa for 15 min at 37 1C) was applied to the protein substrate prior to its enzymatic hydrolysis. Digestion was also conducted at atmospheric pressure (0.1 MPa) and under high pressure. The extent of hydrolysis was measured by the o-phthaldialdehyde method, the peptide profile was analysed by reverse-phase high performance liquid chromatography (RP-HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the residual immunochemical reactivity was assessed by an ELISA test using a pool of seven sera from children allergic to bovine milk, an individual serum also positive (positive control) and two sera from non-allergic children (negative controls). The high pressure increased the degree of hydrolysis by the three enzymes used. Chymotrypsin and trypsin showed the highest proteolysis at 100 and 200 MPa followed by pepsin at 300 MPa. The b-lactoglobulin was hydrolysed by trypsin and chymotrypsin at atmospheric and at high pressures, whereas the pepsin only hydrolysed this protein under high pressure. Pepsin and trypsin hydrolysed a-lactalbumin in all cases. In contrast, this protein was not digested by chymotrypsin, irrespective of the pressure applied. An important decrease of immunochemical reactivity was found for pepsin and trypsin hydrolysates obtained under high pressure. The pool of seven sera detected immunoreactivity in the products of chymotrypsin hydrolysis under high pressure, which was not detected when the serum of one patient was used. The results suggest that dairy whey hydrolysates obtained by pepsin and trypsin in combination with HP treatment could be used as a source of peptides in hypo-allergenic infant formulae. r 2005 Elsevier Ltd. All rights reserved. Keywords: Dairy whey proteins; High pressure; Hydrolysis; Trypsin; Pepsin; Chymotrypsin 1. Introduction The majority constituents of bovine whey proteins are b-lactoglobulin (55–60%) and a-lactalbumin (15–20%), but they also contain other minor proteins such as bovine serum albumin, immunoglobulins, lactoferrin, phospholi- poproteins and bioactive factors and enzymes (Smithers et al., 1996). The use of protein hydrolysates by the food industry is increasing because they have properties that make them attractive as a source of essential amino acids in human nutrition (Pen˜as, Pre´stamo, & Go´mez, 2004). The hydrolysates are physiologically better than intact proteins because their intestinal absorption appears to be more effective, and they also present advantages with respect to the elementary diets in which nitrogen components consist exclusively of a mixture of free amino acids. The absorption of amino acids from short chain peptides is more efficient than the equivalent amount of free amino acids (Siemensma, Weijer, & Bak, 1993). On the other hand, peptides are less hypertonic than free amino acid mixtures, favouring the absorption of other dietary components (Adibi, 1989; Parrado, Bautista, & Machado, ARTICLE IN PRESS www.elsevier.com/locate/idairyj 0958-6946/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2005.08.009 Ã Corresponding author. Tel.: +34 91 549 23 00x294; fax: +34 91 549 36 27. E-mail address: rgomez@if.csic.es (R. Gomez).