Enzyme-induced Gelation of Whey Proteins

Abstract

There is currently an array of whey protein hydrolysates on the market. The applications for these products include but are not limited to: improved heat stability; reduced allergenicity; production of bioactive peptides; tailoring amounts and size of peptides for special diets; and altering the functional properties of gelation, foaming and emulsification. With functional properties applications, hydrolyzed proteins offer advantages over unmodified proteins in increased solubility, heat-process stability, foaming and emulsification.

Customers who are buying whey protein hydrolysates for nutritional applications (sports nutrition, enteral formulas, hypoallergenic infant formulae, etc.) are looking for products with a high degree of hydrolysis (generally >10%) and a high content in short peptides. Extensive enzymatic hydrolysis is required, preferentially with an endoprotease, in order to avoid the presence of free amino acids in the final product. However, some endoproteases (e.g. Alcalase 2.4L) produce peptides that aggregate and form a gel during the course of hydrolysis. This creates a hurdle when a high degree of hydrolysis is desired. Because the array of endoproteases commercially available is not very exhaustive, and some enzymes and hydrolysis conditions cause gelation problems, it is important to understand the gelation mechanism in order to solve it and ultimately produce hydrolysates with a high degree of hydrolysis.

The first objective of this study was to compare enzyme-induced gelation of extensively hydrolyzed whey proteins by Alcalase with the plastein reaction by determining the types of interactions. The average chain length of the peptides did not increase during hydrolysis and reached a plateau after 30 min to be about 4 residues, suggesting that the gel was formed by small molecular weight peptides held together by non-covalent interactions. The enzyme-induced gel network was stable over a wide range of pH and ionic strength, and therefore showed some similarities with the plastein reaction. Disulfide bonds were not involved in the gel network. The gelation seems to be caused by physical aggregation, mainly via hydrophobic interactions with hydrogen bonding and electrostatic interactions playing a minor role.

Peptides released were characterized in order to better understand this gelation phenomenon. The apparent molecular mass distribution indicated that aggregates were formed by small molecular mass peptides (< 2,000 Da). One hundred and thirty peptides with varying lengths were identified by reversed-phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry. Alcalase was observed to have a high specificity for aromatic (Phe, Trp, Tyr), acidic (Glu), sulfur-containing (Met), aliphatic (Leu, Ala), hydroxyl (Ser), and basic (Lys) residues. Most peptides had an average hydrophobicity of 1-1.5 kcal/residue and a net charge of 0 at the pH where gelation occurs (6.0). Therefore, intermolecular attractive force such as hydrophobic interaction suggests the formation of aggregates that further leads to the formation of a gel.

The very complex peptide system identified previously for whey proteins was simplified by using beta-lactoglobulin. Dynamic rheology, aggregation measurements, isoelectrofocusing as well as chromatography and mass spectrometry were used to understand the gel formation. A transparent gel suggesting a fine-stranded network is formed above a critical concentration of peptides while non-covalently linked aggregates appear with increasing time of hydrolysis. Extensive hydrolysis is needed for gelation to occur as indicated by the small size of the peptides. Isoelectrofocusing was successful at separating the complex mixture and nineteen main peptides were identified with molecular weight ranging from 265 to 1485 Da. Only one fragment came from a beta-sheet rich region of the beta-lactoglobulin molecule and a high proportion of peptides had proline residues in their sequence.