The Effect of Limited Enzymatic Hydrolysis and Maillard-Induced Glycation on Sodium Caseinate Allergenicity, Bioactivity, and Functionality

University of Minnesota M.S. thesis. June 2016. Major: Food Science. Advisor: Baraem Ismail. 1 computer file (PDF); xiii, 129 pages.With superior functional properties, especially emulsification properties, high nutritional value, and excellent physiological benefits, sodium caseinate has been widely used in the formulation of many food and beverage products. However, the major drawback of the expanded utilization of sodium caseinate is its allergenicity. Therefore, in order to prevent a decrease in the market value of casein ingredients, different protein modification techniques have been explored to reduce the allergenicity of casein. Extensive enzymatic hydrolysis (> 25% DH) can reduce the allergenicity of a protein. However, this technique is detrimental to both flavor and functionality. Limited enzymatic hydrolysis, on the other hand, can maintain the functionality, but may only reduce the immunoreactivity to a certain extent. Another protein modification technique that may reduce the allergenicity of proteins is Maillard-induced glycation. Glycation, however, should be properly controlled to prevent the formation of mutagenic compounds and brown pigments with adverse effects on functionality, digestibility, and bioactivity. It was hypothesized that combining the two mechanisms, proteolysis and Maillard glycation, under controlled and mild conditions, will have a dual effect on reducing allergenicity by altering potential epitopes, while maintaining nutritional quality, bioactivity and functionality. Thus, the objectives of this study were twofold: (1) Determine the effect of several proteolysis conditions and Maillard-induced glycation conditions on the allergenicity of sodium caseinate. (2) Determine the effect of the dual modification approach on the bioactivity and emulsification properties of sodium caseinate. Sodium caseinate isolate (SCI) was hydrolyzed using three enzymes separately, alcalase, neutrase and trypsin, following the pH-stat method, while maintaining limited degree of hydrolysis (DH 3.6-17 %). Different enzyme hydrolysis conditions, including enzyme to substrate ratio, incubation temperature, pH, and time were investigated. The sodium caseinate hydrolysate (SCH) with the highest emulsifying properties (emulsification capacity, stability and activity) and ACE-inhibitory activity was tested for remaining allergenicity. The same SCH was further glycated with maltodextrins at 60°C, water activity (aw) of 0.49, 0.63, or 0.79, and a 2:1 ratio of protein to maltodextrin over a period of 24-120 h. Sodium caseinate isolate was also glycated to determine the effect of glcation alone on modifying emulsification properties and allergenicity. Loss in free amine groups along with the formation of Amadori compounds and browning were monitored. Flouresence compound formation was monitored to determine progression of the reaction into intermediate stages before formation of brown pigments. Urea-PAGE gel was utilized to confirm the glycation formation. Select glycated samples were tested for allergenicity, emulsifying properties, and ACE-inhibitory bioactivity. ELISA was performed to determine the immunoreactivity of the samples using sera from five allergenic donors following ELISA. Trypsin was better than Alcalase and Neutrase in producing hydrolysates that retained emulsification properties. This might be due to trypsin’s narrow and unique specificity, where large molecular weight peptides with better amphiphilic properties were generated compared to those generated by Alcalase and Neutrase. Limited hydrolysis (5% DH) resulted in pronounced ACE inhibitory activity and reduced immunoreactivity (6%-45%). Differences in the reduced immunoreactivity were attributed to the sensitivity of sera used and the variation in milk-protein-specific IgE among the different sera that could react with the epitopes present in casein proteins samples and newly generated epitopes upon hydrolysis. Among all the glycation conditions studied, optimum conditions for the desired objectives were determined to be 96 h of incubation at 0.63 aw, in a 2:1 ratio of casein protein to maltodextrin, due to a moderate Amadori compounds formation, as noted by adsorbace at 304 nm, limited % amino group blockage (9.7%), minimal progression to intermediate stages, as noted by % fluorescence intensity, and limited progression to advances stages of the Maillard reaction, as noted by minimal browning measured at 420 nm. No significant (P < 0.05) effect on ACE inhibitory activity, emulsification properties and immunoreactivity was observed post glycation at at 0.63 aw. This is likely due to insufficient Amadori compounds formation and the interference from free maltodextrin and unadsorbed intact sodium caseinate, which can destabilize the emulsion through depletion flocculation. Results from this work, for the first time, provided valuable insights into the effect of proteolysis conditions and Maillard-induced glycation conditions on the allergenicity, emulsification properties, and ACE-inhibitory activity of sodium caseinate. Combining two different protein modification techniques under controlled and mild conditions could maintain the emulsification properties and ACE-inhibitory activity, but had no dual effect on reduction of immunoreactivity. Therefore, further optimization of glycation conditions needs to be conducted for the development of hypoallergenic casein ingredient with acceptable functionality and bioactivity