Improvement of allergenicity and functional properties of proteins from ginkgo seeds by high hydrostatic pressure treatment

Abstract: Ginkgo biloba, one of the oldest species of tree, has existed on the earth for 200 million years, and 70% is from China. The seeds of Ginkgo biloba have been used in China as traditional food and medicine source for several thousand years. Research shows that ginkgo seeds have a relatively high protein content (10%-15%), and ginkgo seed protein (GSP) has many biological activities, including anti-oxidation, anti-aging, anti-tumor and anti-bacterial properties, so it has high nutritional and medicinal value. However, eating GSP can result in allergic reactions in humans. Till now, research on how to reduce and eliminate GSP allergy remains lacking. High hydrostatic pressure (HHP) treatment can modify the tertiary and quaternary structures of proteins and alter some of the epitope structures, which can reduce the allergenicity and improve the functional properties of food proteins. The aim of the present study was to investigate the effects of HHP treatment on structure, allergenicity and functional properties of GSP. GSP was treated under the pressure of 100, 200, 300, 400, 500, 600 and 700 MPa at 20℃ for 20 min respectively, and the target pressure was achieved within 1-2 min, held for 20 min and released to atmospheric pressure within 1-2 min. After HHP treatment, the allergenicity was measured by ELISA assay, the changes in molecular weight and conformational structure (secondary structure, free sulfhydryl group (SH) content, surface hydrophobicity and ultraviolet (UV) absorbance) of GSP were characterized by sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE), circula dichroism spectrum, fluorescence spectra and UV absorption spectrum, and the functional properties were analyzed for heat stability and emulsifying properties. The results showed that HHP treatment with the pressure range from 300 to 700 MPa could significantly reduce the allergenicity of GSP, an antigenicity reduction of 95% was observed in protein samples treated at 500 MPa, and a complete loss of allergenicity was observed in protein samples treated at 600 or 700 MPa. Meanwhile, after HHP treatment, the intermolecular and intramolecular disulfide bonds that originally stabilized the native protein structure were disrupted and the molecular weights, secondary, tertiary and quaternary structures of GSP were significantly changed. The electrophoretogram of raw GSP showed that the bands with apparent molecular weights (MW) ranged from 10 to 100 kDa, and the major protein bands were approximately 5, 10, 22, 30, 39 and 46 kDa. But after HHP treatment, the GSPs were disintegrated into proteins with small molecular weights ranging from 4 to 30 kDa, and moreover, α-helices and β-sheet in its secondary structures were largely destroyed and turned into random coil, and the UV absorption intensity, surface hydrophobicity and SH content of GSP were significantly increased. The great influence of HHP, which made IgE not bind to GSP, was correlated with the significant change in the structure and epitopes of the allergenic protein. Moreover, the HHP-treated GSP markedly improved the heat stability and emulsification index values comparable to those of the untreated GSP, suggesting that the HHP-treated GSP retained the ability to efficiently form emulsions. These results suggest that HHP can act as an important technological approach for the industrial production of hypoallergenic and improved-function GSP for potential use as base ingredient in food.