Int Arch Allergy Immunol. 2012;157(1):41-50.

Influence of enzymatic hydrolysis on the allergenicity of roasted peanut protein extract.

Cabanillas B, Pedrosa MM, Rodríguez J, Muzquiz M, Maleki SJ, Cuadrado C, Burbano C, Crespo JF.

Servicio de Alergia, Instituto de Investigación Hospital 12 de Octubre (i+12), Avenida de Córdoba s/n, Madrid, Spain. bcabanillas.hdoc@salud.madrid.org

 

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. Copyright © 2011 S. Karger AG, Basel.

PMID: 21912172

 

Supplements:

Enzymatic hydrolysis is an efficient process for disrupting sequential and conformational IgE epitopes; therefore protein hydrolysates could be an alternative to intact proteins in the development of special formulations for food-allergic patients.

The objective of our study was to investigate the effect of the individual and sequential action of two food-grade enzymes: Alcalase (endoprotease) and Flavourzyme (exoprotease) in the allergenic properties of protein extract of roasted peanut, using sera from patients with a clinical allergy to peanuts.

Peanut protein hydrolysates were obtained as depicted in figure S1.

Figure S1
Figure S1. Plant Material, protein extract and enzymatic treatments

 

Specific anti-Ara h 1, anti-Ara h 2 and anti-Ara h 3 antibodies were used to identify Ara h 1, Ara h 2 and Ara h 3 molecules in roasted peanut before and after sequential and individual treatment with Alcalase and Flavourzyme. In figure S2, the Ara h 1 (63 kDa), Ara h 2 doublet bands (19 and 21 kDa), Ara h 3 (40 kDa) acidic subunit and Ara h 3 (23 kDa) basic subunit are indicated with arrows. There was a marked decrease in recognition of Ara h 1, Ara h 2 and Ara h 3 in roasted peanut after sequential endo- and exoprotease hydrolysis (Alcalase + Flavourzyme) and individual hydrolysis with Alcalase. Individual treatment with Flavourzyme caused a decrease of Ara h 2 and the acidic subunit of Ara h 3 levels in the first 30 min of hydrolysis; after this time, both allergens were undetected. Ara h 1 levels decreased with increased hydrolysis time with Flavourzyme; however, it was still detected after 300 min of hydrolysis. The basic subunit of Ara h 3 was not affected by Flavourzyme hydrolysis.

Figure S2Figure S2. Western blot analysis with anti-Ara h 1, anti-Ara h 2, and anti-Ara h 3 antibodies. The Ara h 1, Ara h 2 and Ara h 3 profiles were assessed in roasted peanut (RP) before and after sequential treatment with Alcalase and Flavourzyme ( panel a ), individual treatment with Alcalase ( panel b ), and individual treatment with Flavourzyme ( panel c ), at the times indicated at the bottom of each panel. A = Alcalase; F = Flavourzyme.

 

In order to assess the IgE reactivity of roasted peanut hydrolysates, an ELISA was carried out using the serum pool from the 5 patients with a clinical allergy to peanuts. The ELISA results are summarized in figure S3. Percentages of reduction in IgE reactivity are shown in the table annexed to figure S3. Hydrolysis with Alcalase/Flavourzyme sequentially and with Alcalase individually caused a higher loss of IgE reactivity in roasted peanut than hydrolysis with Flavourzyme individually. Alcalase was very effective in reducing the IgE reactivity of RP proteins since the enzyme at 0.4 and 0.2 AU/g led to a 100 and 98% reduction in IgE reactivity, respectively, in the first 30 min (DH 16 and 11%, respectively); the addition of Flavourzyme in the sequential assay caused a slight increase in IgE reactivity above the positive cutoff point. Individual treatment with Flavourzyme during 30 min caused an increase in IgE reactivity. Nevertheless, Flavourzyme led to a 65% reduction in IgE reactivity at the end of the assay (300 min).

Figure S3

Figure S3. Specific IgE to roasted peanut (RP) before and after sequential treatment with Alcalase and Flavourzyme ( a ), individual treatment with Alcalase ( b ), and individual treatment with Flavourzyme ( c ), at the times indicated at the bottom of each figure, using a serum pool from 5 peanut-allergic patients. The horizontal line indicates the cutoff point for positivity (0.085). Percentages of reduction in IgE reactivity and DHs are also shown. A = Alcalase; F = Flavourzyme.

 

Significance:

Our results show that hydrolysis with endoprotease Alcalase produces a decrease in allergenicity of roasted peanut protein extract due to a decrease in the recognition of the main peanut allergens (Ara h 1, Ara h 2 and Ara h 3) by IgE antibodies from patients with peanut allergy.

Peanut protein isolates are used where bland and highly concentrated forms of peanut protein are desired, e.g. in bread and bakery goods, thus the enzymatically treated protein hydrolysates developed in this study could constitute an alternative to intact proteins in the development of these products.

 

Full text here:

https://www.dropbox.com/s/fs1530rva3wqua1/Influence%20of%20enzymatic%20hydrolysis%20on%20roasted%20peanut%20protein%20extract.pdf

Contact information:

Dr. Beatriz Cabanillas

Department of Dermatology and Allergy,

University of Bonn, Sigmund-Freud-Str. 25,

53127 Bonn, Germany.

Tel.: +49 162 494 4655

Fax: +49 228 287 11613

Emai 1: Beatriz.Cabanillas@ukb.uni-bonn.de

Email 2: bcabanillas.hdoc@salud.madrid.org

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