Emerging interest among consumers as to the health benefits of soy and a shifting preference toward more economical sources of protein has led to the increased global interest in soy protein. However, the main drawback to the expanded use of soy protein is its allergenicity, as soy is listed among the "Big 8" food allergens by the Food and Drug Administration (FDA). Therefore, in order to prevent a decrease in the momentum behind the usage of soy protein ingredients, different protein modification techniques must be explored in order to reduce the allergenicity of soy. Extensive enzymatic hydrolysis can reduce the allergenicity of a protein. However, this technique has negative consequences such as decreased protein functionality and bitter flavor. In order to limit enzymatic hydrolysis, response surface methodology (RSM) can be used to determine the optimized combination of factors, such as time, temperature and enzyme to substrate ratio, that results in the maximum reduction in immunoreactivity of the modified protein, while maintaining quality. Enzymatic hydrolysis, however, when limited, may only reduce the immunoreactivity to a certain extent. Thus a second technique, such as Maillard-induced glycosylation may further reduce the allergenicity of the minimally hydrolyzed soy protein. The first objective was to determine optimal hydrolysis conditions for a maximum reduction of soy protein allergenicity using response surface methodology. The second objective was therefore to determine the effect of limited and controlled Maillard-induced glycosylation on the allergenicity of soy protein hydrolysate produced under optimized conditions from objective 1. A central composite design (CCD) was used to produce different soy protein hydrolysate (SPH) samples using Alcalase 2.4L with 3 independent variables; temperature (x1), time (x2), and enzyme to substrate ratio (E:S) (x3), with the degree of hydrolysis (DH) and the % reduction in immunoreactivity as the response variables. Western blots and ELISA were used to evaluate the immunoreactivity of the different hydroylsates using sera from different soy allergenic individuals. The design included 20 experimental units with 6 center points to assess experimental variability, 8 factorial points, and 8 alpha points to assess potential curvature in the data. The effects of the different independent variables on the responses were assessed by fitting the data to the appropriate model. Once a model was selected, 3D plots were generated. Once all of the responses were fit to the appropriate models, constraints were then added onto the levels of the variables that resulted in a DH greater than 8%. The predicted % reductions in immunoreactivity were then determined for each individual sera with the added constraints. Due to overlap in the predicted optimum response among the sera, one set of conditions was chosen to produce one optimized hydrolysate. Maillard-induced glycosylation of the optimized hydrolysate was conducted using conditions that were carefully selected to control the reaction. Several different measurements were done to assess the progression of the reaction, including Amadori compound formation, browning, lysine blockage, and fluorescence, all of which confirmed limited progression of the reaction. The immunoreactivity of SPH, partially glycosylated soy protein hydrolysate (PGSPH), and partially glycosylated soy protein (PGSP) was determined using ELISA and Western blots. Significant reductions in immunoreactivity (up to 58%) were observed for the different hydrolysates produced using RSM. Variability among sera was observed for the different hydrolysates, which was attributed to the resulting protein and protein/peptide profile upon hydrolysis. Different hydrolysis conditions may have resulted in the exposure or elimination of IgE binding regions (epitopes). Using RSM modeling, the optimized hydrolysis conditions were determined and the resulting SPH had a DH of 7.8% and a % reduction in immunoreactivity ranging from 20-52%. When the produced SPH was subjected to controlled Maillard-induced glycosylation, a significantly (P ≤ 0.05) higher % reduction in immunoreactivity (up to 83%) was observed when using serum that had the highest soy specific IgE (45.30 kU/L). Results from this work, for the first time, showed that two different protein modification techniques can be utilized in combination to substantially reduce the allergenicity of soy protein while maintaining quality. Therefore, limited enzymatic hydrolysis combined with controlled Maillard-induced glycosylation has great potential for the development of hypoallergenic protein ingredients.
University of Minnesota M.S. thesis. Major: Food science. Advisor: Baraem Ismail. 1 computer file (PDF); xx, 146 pages, appendices A-J.
Walter, Jordan Kay.
Hypoallergenization of soy protein using a combination of limited enzymatic hydroylsis and controlled Maillard-induced glycosylation.
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