Enzymatic hydrolysis of heat-denatured whey proteins

The heat-treatment of whey proteins in conjunction with enzymatic hydrolysis has potential in the development of new, or modification of existing, processes for technoand bio-functional whey protein ingredient production. Selective heat-treatment of fractions enriched in individual whey proteins and whey protein isolate (WPI) demonstrated differences in both their aggregation behaviour and subsequent susceptibility to enzymatic hydrolysis. This was examined at both a sub-molecular and macro-molecular level where whey protein samples were hydrolysed to varying degrees of hydrolysis (DH). The individual whey protein components were susceptible to denaturation on thermal treatment in the following order; β-lg A > β-lg B > α-la > CMP. The heat pre-treated substrates, which exhibited increased viscosity and surface hydrophobicity, demonstrated significantly increased (P < 0.001) rates of hydrolysis with the pancreatic enzymatic preparation, Corolase® PP. The proteinaceous components were hydrolysed in the order; CMP > β-lg A > β-lg B > α- la. The hydrolysates (5 %DH) had an increase in soluble molecular weight (Mw) material greater than 30 kDa which was shown to be peptides mainly derived from the 1Leu-Arg40, 70Lys-Phe82 and 140Leu-Met145 regions of β-lg and the 1Met-Ile20 region of CMP. Hydrolysis of heat denatured WPI favoured the generation of higher levels of free essential amino acids; lysine, phenylalanine and arginine compared to the unheated substrate. Specific peptides released from the heat-treated proteins were mapped to their parent molecules and theoretically attributed to the action of specific endo-proteinase activities. Selected whey protein hydrolysis processes were successfully scaled-up to pilot-scale. Cascade membrane fractionation of the resulting hydrolysates yielded spray dried fractions with altered Mw profiles and bio-functional characteristics. This enabled partition of both the iron chelating and the angiotensin-Iconverting enzyme (ACE) inhibitory properties in both control and heat-treated systems. A positive correlation (P < 0.01) was established between the average Mw of fractions and ferrous (Fe2+) chelating capability. Solid phase extraction of these samples yielded fractions possessing high concentrations of the basic amino acids and possessed a ferrous chelation ability equivalent to 84.4 μM EDTA. The most potent ACE inhibitory fractions were the 1 kDa permeates of both the control and heattreated WPI process streams (ACE IC50 = 0.17 g L-1). Isoelectric focussing (IEF) of these hydrolysate fractions further increased inhibitory activity in fractions collected between pH 6.1 – 6.6. From this study it was shown that enzymatic hydrolysis of whey protein substrates can be optimised through structural modification of the substrate proteins. This allowed for the development of hydrolysate products with altered characteristics / functionalities.