Maillard-Induced Glycation of Whey Protein Hydrolysate to Increase Solubility and Thermal Stability and Reduce Allergenicity in Acidified Protein Beverages

Abstract

Interest in whey protein is continually increasing globally as a result of its excellent nutritive value, unique physiological benefits, and diverse functionality. More specifically, whey protein hydrolysates (WPH) are value-added ingredients that are experiencing a rapid increase in usage and market volume in part due to their enhanced functional properties and physiological benefits. However, maintaining quality and shelf-life stability as well as increasing concern over whey protein allergenicity are major hurdles that hinder the use of biologically active WPH in beverages. Maillard-induced glycation has been shown to be a novel protein modification technique with potential to address these challenges. It is hypothesized that a low degree of hydrolysis in addition to limited and controlled Maillard-induced glycation will enhance solubility and thermal stability while maintaining nutritional and physiological quality, and synergistically reducing allergenicity of whey protein. Thus, the objectives of this study were twofold: (1) to produce and assess solubility and thermal stability of a partially-glycated whey protein hydrolysate product using controlled and limited Maillard conditions, and (2) to assess the effects on nutritional quality, bioactivity (anti-hypertensive activity) and allergenicity. Whey protein hydrolysate was reacted with dextran over 12-120 h of incubation at 60�C, 0.49 water activity (aw), and a 4:1 ratio of dextran to protein to produce partially-glycated WPH (PGWPH). Extent of glycation was monitored via estimation of Amadori compound formation, fluorescent compound formation, browning, free amino group loss, and visualization of protein/peptide molecular weight distribution following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Glycation was initiated as early as 12 h of incubation. Partial glycation and minimal browning were maintained through 120 h of incubation, and loss of free amino groups was between 5-30%. Based on the results coupled with feasibility purposes, a 48 h incubation time period was selected for further analysis, as this time point resulted in modest Amadori compound formation and % amino group blockage (21.8%), with minimal progression to intermediate and advanced stages of the Maillard reaction. Free, unreacted dextran was then removed from the 48 h incubated sample using ultrafiltration and hydrophobic interaction chromatography (HIC) to produce purified PGWPH. The final composition of purified PGWPH was approximately 88% protein and 12% carbohydrate. Solubility and thermal stability of PGWPH were assessed at 5% protein concentration prior and post heat treatment at 80�C for 30 min. Lysine blockage was assessed using a furosine assay, and digestibility was determined using a sequential pepsin-trypsin in-vitro digestibility assay. The antihypertensive activity was determined by measuring the angiotensin converting enzyme (ACE) inhibitory activity. Allergenicity was determined following an indirect ELISA using sera from milk sensitive donors. Partial glycation of WPH resulted in enhanced solubility and thermal stability, particularly near the isoelectric point (pI), where PGWPH remained soluble after heating while WPH lost over 50% of its solubility. Changes in surface hydrophobicity and free sulfhydryl groups were minimal upon heating. The enhanced solubility and thermal stability of PGWPH, even when the pH was close to the pI of the whey protein, was attributed to the resistance to denaturation and structural modifications. Nutritional quality and bioactivity of WPH was minimally impacted upon partial glycation, as lysine blockage was only ~2%, and digestibility (58.7%) and antihypertensive activity of PGWPH (IC50 =0.249) were similar to that of WPH. However, allergenicity of WPH was not further reduced upon partial glycation. Overall, this work has shown for the first time that partial Maillard-induced glycation can be induced and controlled to low-levels in WPH, producing a value-added product with enhanced solubility and thermal stability, as well as maintained nutritional quality and bioactivity. Acidified whey protein beverages formulated with PGWPH in place of WPH or WPI may have a longer shelf life, a more acceptable flavor, and protein content greater than 4.2%, allowing for a "high protein"� beverage claim to be made. In turn, the utilization of biologically active WPH in acidified protein beverages would greatly increase, and consumer demands for a functional, high protein beverage could be fulfilled.