pubs.acs.org/JAFC Published on Web 08/04/2010 © 2010 American Chemical Society 9800 J. Agric. Food Chem. 2010, 58, 9800–9808 DOI:10.1021/jf101722t In Vitro Digestion of β-Lactoglobulin Fibrils Formed by Heat Treatment at Low pH LIBEI BATEMAN,AIQIAN YE, AND HARJINDER SINGH* Riddet Institute, Massey University, Private Bag 11 222, Palmerston North 4442, New Zealand Extensive studies have been done on β-lactoglobulin (β-Lg) fibrils in the past decade due to their potential as functional food ingredients, gelling agents, and encapsulation devices etc. (van der Goot, A. J.; Peighambardoust, S. H.; Akkermans, C.; van Oosten-Manski, J. M. Creating novel structures in food materials: The role of well-defined shear flow. Food Biophys. 2008, 3 (2), 120-125 and Loveday, S. M.; Rao, M. A.; Creamer, L. K.; Singh, H. Factors affecting rheological characteristics of fibril gels: The case of β-lactoglobulin and R-lactalbumin. J. Food Sci. 2009, 74 (3), R47-R55). However, most of the studies focus on the formation and mechanism of the fibrils. Little is known about fibril digestibility to date. In this work, in vitro pepsin digestion of bovine β-lactoglobulin (β-Lg) fibrils in simulated gastric fluid was investigated using thioflavin T fluorescence photometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, size-exclusion chromatography, matrix-assisted laser desorption/ionization mass spectrometry, and transmission electron microscopy (TEM). The fibrils were formed by heating β-Lg solutions at 80 °C and pH 2.0 for 20 h. The fibrils were found to be digested completely by pepsin within 2 min, when long, straight fibrils were no longer observed by TEM. The peptides in the fibrils (2000-8000 Da) could be digested to smaller peptides (mostly <2000 Da) by pepsin. The peptides in the fibrils were believed to be more susceptible for pepsin to access and attack because of their hydrophobic nature. For comparison purposes, solutions of β-Lg heated at neutral pH (pH 7.4) were also studied under the same conditions. KEYWORDS: β-Lactoglobulin; fibrils; in vitro pepsin digestion; heat treatment INTRODUCTION Bovine β-lactoglobulin (β-Lg) is a globular protein that con- tains 162 amino acids and has a molecular weight of 18.3 kDa. It is composed of nine β-strands and one R-helix, in which the hydrophobic sequences are mostly buried. At room temperature and neutral pH, it exists in the form of a dimer, which dissociates into monomers at acidic pH (<pH 3) ( 2 ). It is known that β-Lg is scarcely hydrolyzed by enzymes such as pepsin under gastric conditions because of its stable, globular tertiary structure at low pH (<pH 3). The highly hydrophobic β-barrel makes it very difficult for enzymes to access the target peptide bonds ( 2 -4 ). However, it has been found that under certain circumstances such as heating, high pressure, high ionic strength, or in organic solvents, denatured or hydrolyzed β-Lg can be diges- ted by enzymes more easily than the native protein ( 5 , 6 ). Under denaturing conditions, β-Lg is able to form gels and aggregates or precipitates, depending on the protein concentration and various conditions, such as pH, temperature, and heating time and speed ( 2 , 7 ). β-Lg gels formed through heating treatment are widely used in the food industry ( 8 ). This whey protein contri- butes not only to the consistency and texture of foods but also to their nutritional value. Applications for β-Lg gels in both drug and food supplements have also been reported ( 9 -12 ). Recently, it was found that β-Lg can form fibrils on heating treatment at low pH, on heat treatment under high pressure, or in the presence of organic solvents ( 1 , 13 -16 ). Many studies on the mechanism of fibril formation and on the building blocks of fibrils have been carried out ( 8 , 17 -19 ). Akkermans et al. ( 20 ) claimed that rather than denatured intact proteins, the building blocks of β-Lg fibrils are peptides obtained through acid hydro- lysis of β-Lg molecules on heat treatment. They found that after heat treatment at 85 °C and pH 2.0 for 20 h, β-Lg molecules were hydrolyzed into peptides that subsequently formed fibrils. They argued that only specific peptides with characteristics of high hydrophobicity, the ability to form β-sheets, low charge, and proper charge distribution along the peptide are able to form fibrils. This may explain the low yield (about 20% on average) of β-Lg fibril formation on heat treatment at low pH. As well as extensive studies on the mechanism of β-Lg fibril formation, some work on the use of β-Lg fibrils to improve both the texture and the nutritional value of foods has been carried out ( 7 , 21 ). However, to our knowledge, there has been little work on the digestion of β-Lg fibrils in the gastrointestinal system. This work attempts to address this issue in a designated in vitro system, in which β-Lg fibrils formed by heat treatment at pH 2.0 and 80 °C for 20 h were subjected to digestion by pepsin under simu- lated gastric conditions. The digestion processes were monitored using transmission electron microscopy (TEM), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), *To whom correspondence should be addressed. Tel: þ64-6-350- 4401. Fax: þ64-6-350-5655. E-mail: H.Singh@massey.ac.nz.