Influence of Enzymatic Hydrolysis on the Allergenicity of Roasted Peanut Protein Extract

Abstract

Background: Peanut allergy is recognized as one of the most severe food allergies. Some studies have investigated the effects of enzymatic treatments on the in vitro immunological reactivity of members of the Leguminosae family, such as the soybean, chickpea and lentil. Nevertheless, there are only a few studies carried out with sera from patients with a well-documented allergy. Methods: Roasted peanut protein extract was hydrolyzed by the sequential and individual action of 2 food-grade enzymes, an endoprotease (Alcalase) and an exoprotease (Flavourzyme). Immunoreactivity to roasted peanut extract and hydrolyzed samples was evaluated by means of IgE immunoblot, ELISA and 2-dimensional electrophoresis using sera from 5 patients with a clinical allergy to peanuts and anti-Ara h 1, anti-Ara h 2 and anti-Ara h 3 immunoblots. Results: Immunoblot and ELISA assays showed an important decrease of IgE reactivity and Ara h 1, Ara h 2 and Ara h 3 levels in the first 30 min of hydrolyzation with Alcalase. In contrast, individual treatment with Flavourzyme caused an increase in IgE reactivity detected by ELISA at 30 min and led to a 65% inhibition of IgE reactivity at the end of the assay (300 min). Ara h 1 and the basic subunit of Ara h 3 were still recognized after treatment with Flavourzyme for 300 min. Conclusion: Hydrolysis with the endoprotease Alcalase decreases IgE reactivity in the soluble protein fraction of roasted peanut better than hydrolysis with the exoprotease Flavourzyme.

Allergenic Properties of Enzymatically Hydrolyzed Peanut Flour Extracts

“…Pepsin resulted in hydrolysates with the darkest bands of peptides in the <20 kDa region, and pepsin resistant Ara h 2 fragments have been well documented [24]. Previously, Western blotting experiments suggested that Ara h 1 and Ara h 3 were more resistant than Ara h 2 to Alcalase and/or Flavourzyme [13]. Discrepancies are attributed to different extraction and hydrolysis protocols between the two studies.…”

“…Indeed, peptides of only 8 amino acids are known to bind IgE [27] and peptides as small as 30-35 amino acids that contain at least two IgE-binding epitopes are capable of cross-linking IgE molecules on mast cells or basophils [28]. Sequential hydrolysis with Alcalase and Flavourzyme is commonly applied to increase DH, and recent work with peanut protein extracts found that the DH of sequential Alcalase/Flavourzyme hydrolysates was 69% compared to individual DH values of 17 and 29% for Alcalase and Flavourzyme, respectively [13]. Increasing the ratio of enzyme/substrate with increased incubation times may also result in further hydrolysis of the peanut proteins.…”

“…Enzymatic proteolysis is a commonly utilized strategy for improving the functionality, digestibility, bioactive and immunogenic properties of food proteins, including peanut [13,14,15]. Recently, ELISA testing indicated that Ara h 1 and Ara h 2 in peanut kernel extracts were reduced up to 100 and 98%, respectively, after hydrolysis with trypsin/chymotrypsin [16].…”

“…When processing food proteins with enzymes, choices are not limited to digestive proteases, and two commonly studied proteases are: (1) Alcalase, which is as an endoprotease with broad specificity, and (2) Flavourzyme, which has both endo- and exoprotease activities, but is primarily used as an exoprotease for debittering of hydrolysates. The effects of these two proteases on the allergenicity of roasted peanut protein extracts were recently examined [13]. Western blotting and ELISA data indicated that IgE binding for the major peanut allergens (Ara h 1, Ara h 2 and Ara h 3) was significantly decreased in the first 30 min of Alcalase hydrolysis, but Ara h 1 and Ara h 3 were more persistent with Flavourzyme hydrolysis.…”

Strategies to Mitigate Peanut Allergy: Production, Processing, Utilization, and Immunotherapy Considerations

“…Enzymatic treatment with digestive and commercial proteases has been investigated as a strategy to reduce the allergenicity of peanut proteins by hydrolyzing them to smaller peptides that no longer contain IgE-binding epitopes (Hong et al 1999, Cabanillas et al 2012b, Yu et al 2011, Shi et al 2013). Individual and sequential hydrolysis with the commercial proteases Alcalase and Flavourzyme decreased the IgE-binding capacity of roasted peanut protein extracts and decreased levels of Ara h 1, Ara h 2, and Ara h 3 (Cabanillas et al 2012b). This was attributed to proteolysis of the whole proteins and breakdown of IgE-binding epitopes.…”