Abstract
BackgroundEnzymatic treatments such as hydrolysis with proteases and polymerization using transglutaminase (TG) have been studied to reduce the immunoreactivity of β-Lactoglobulin (β-Lg). Bromelain is a cisteine protease that is not usually used for production of hypoallergenic hydrolysates. TG is an enzyme that catalyses the formation of inter and intramolecular isopeptide bonds between glutamine and lysine residues. The present study is aimed at investigating the antigenic response of β-Lg polymerized by TG pre or post hydrolysis with bromelain.Methodsβ-Lg (donated by Davisco Inc), was hydrolyzed with bromelain (3% protein w/w in distilled water, 25 U enzyme g−1 of substrate, pH 7.5, 240 min) and then polymerized by TG (7% hydrolysate, 10 or 25 U TG g−1 protein, 50°C/180 min). When polymerization was carried out pre hydrolysis, β-Lg (7% w/w) was polymerized by TG (10 U TG g−1 protein, 50°C/180 min) in 0.1 M Cys or after heat treatment (80°C/60 min), and then hydrolyzed under the same conditions previously described. The hydrolysis reaction was monitored by pH-stat method and the samples were evaluated by SDS-PAGE/tricine. Antigenicity was evaluated by Immunoblotting and ELISA assays using sera from mice sensitized with β-Lg (IgE anti-β-Lg).ResultsThe treatment with TG (10 or 25 U TG g−1), post hydrolysis, lead to formation of products with a wide molecular weight (MW) distribution (3 to 26 kDa), and other products with high MW, indicating partial polymerization. The samples obtained from polymerization pre hydrolysis showed bands with low MM (<6.5 kDa) and also products >26 kDa, indicating a partial hydrolysis of the polymers. Immunoblotting analysis showed no reaction towards specific IgE with any of the samples. ELISA assay showed that the IgE-binding response to sample polymerized by TG post or pre hydrolysis with bromelain were significantly reduced (IgE ≤ 35.21 ug mL−1) as compared to untreated β-Lg (IgE 216.20 ug mL−1 ± 27.58) (P < 0.05).ConclusionsThese results suggested that hydrolysis with bromelain combined with polymerization by TG was capable of reducing the antigenicity of β-Lg, being a potential method for modifying the antigenic properties of proteins.
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