Effects of enzymatic hydrolysis of buckwheat protein on antigenicity and allergenicity
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BACKGROUND/OBJECTIVES: Due to its beneficial health effects, use of buckwheat has shown a continuous increase, and concernsregarding the allergic property of buckwheat have also increased. This study was conducted for evaluation of the hydrolyticeffects of seven commercial proteases on buckwheat allergens and its allergenicity. MATERIALS/METHODS: Extracted buckwheat protein was hydrolyzed by seven proteolytic enzymes at individual optimumtemperature and pH for four hours. Analysis was then performed using SDS-PAGE, immunoblotting, and competitive inhibitionELISA (ciELISA) with rabbit antiserum to buckwheat protein, and direct ELISA with pooled serum of 21 buckwheat-sensitivepatients. RESULTS: Alkaline protease, classified as serine peptidase, was most effective in reducing allergenicity of buckwheat protein. It caused decomposition of the whole buckwheat protein, as shown on SDS-PAGE, and results of immunoblotting showedthat the rabbit antiserum to buckwheat protein no longer recognized it as an antigen. Allergenicity showed a decrease ofmore than 50% when pooled serum of patients was used in ELISA. Two proteolytic enzymes from Aspergillus sp. could nothydrolyze buckwheat allergens effectively, and the allergenicity even appeared to increase. CONCLUSIONS: Serine-type peptidases appeared to show a relatively effective reduction of buckwheat allergenicity. However,the antigenicity measured using rabbit antiserum did not correspond to the allergenicity measured using sera from humanpatients. Production of less allergenic buckwheat protein may be possible using enzymatic hydrolysis.
Abstract
BACKGROUND/OBJECTIVES: Due to its beneficial health effects, use of buckwheat has shown a continuous increase, and concernsregarding the allergic property of buckwheat have also increased. This study was conducted for evaluation of the hydrolyticeffects of seven commercial proteases on buckwheat allergens and its allergenicity. MATERIALS/METHODS: Extracted buckwheat protein was hydrolyzed by seven proteolytic enzymes at individual optimumtemperature and pH for four hours. Analysis was then performed using SDS-PAGE, immunoblotting, and competitive inhibitionELISA (ciELISA) with rabbit antiserum to buckwheat protein, and direct ELISA with pooled serum of 21 buckwheat-sensitivepatients. RESULTS: Alkaline protease, classified as serine peptidase, was most effective in reducing allergenicity of buckwheat protein. It caused decomposition of the whole buckwheat protein, as shown on SDS-PAGE, and results of immunoblotting showedthat the rabbit antiserum to buckwheat protein no longer recognized it as an antigen. Allergenicity showed a decrease ofmore than 50% when pooled serum of patients was used in ELISA. Two proteolytic enzymes from Aspergillus sp. could nothydrolyze buckwheat allergens effectively, and the allergenicity even appeared to increase. CONCLUSIONS: Serine-type peptidases appeared to show a relatively effective reduction of buckwheat allergenicity. However,the antigenicity measured using rabbit antiserum did not correspond to the allergenicity measured using sera from humanpatients. Production of less allergenic buckwheat protein may be possible using enzymatic hydrolysis.
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