Effect Of Maillard Reaction On The Structural And Immunological Properties Of Fish Parvalbumin

Fish is one of the eight prominent allergic foods that cause a series of hypersensitive reactions.In recent years,the reports about adverse reactions to fish have increased due to the rising of consumption and production in China.Parvalbumin(PV)has been proved as the major allergen in different species of fish.Thus,it is important to develop hypoallergenic fish products though food processing.The Maillard reaction(also referred as ‘glycation’)is one of the most frequent processes used in food industry.Recent studies highlighted the importance of this reaction not only in areas of food chemistry,but also in food allergy.The aim of this work was to investigate the effect of Maillard reaction on the immunoreactivity and digestibility of PV.The glycation sites and the structural modification of glycated PV were further determined to illustrate the possible mechanism of these changes.The recombinant parvalbumin was expressed using strain(BL21/pET-28a-PV),prepared previously in our laboratory.Three different conditions(sugar types,sugar to PV weight ratios,and incubating time)of Maillard reaction were compared,and the optimum conditions were determined by Tricine-SDS-PAGE and Spectrophometic methods.The optimal condition for Maillard reaction of rPV was obtained with the ratio of rPV to glucose/xylose as 1:8(weight ratio),incubating at 100 °C for 100 min.IgG-binding ability of rPV was shown to be weakened after Maillard reaction by dot blotting analysis.QE-MS/MS was applied to identify the specific site for modifications of rPV.The reactive residues were K46,K55,K84,K88,K97 and K108,respectively,and glycated K88,K97 and K108 residues overlapped with the reported epitopes,suggesting a direct blocking of IgE-binding epitopes by the glycation.Maillard reaction had a minor effect on the secondary structures of rPV with a decrease of α-helix and an increase of β-strand content as shown by circular dichroism spectroscopy.In addition to the overall structures,the effect of glycation on the surface hydrophobicity of rPV was evaluated by the ANS binding method,and the results indicated that glycation of rPV might have exposed the buried hydrophobic patches to the protein surface,thus increased the extent of hydrophobic interactions,which might contribute to the decrease of rPV immunoreactivity.We further investigated the influence of Maillard reaction on the immunoreactivity and structure of natural PV.Two PV isoforms(named as SPVI and SPVII)from shark(Mustelus griseus)were purified to homogeneity by a series of procedures including ammonium sulfate precipitation and column chromatographies of DEAE-Sepharose and Sephacryl S-200.Maillard reaction of SPVs was carried on with the ratio of SPVs to glucose as 1:8(weight ratio),incubating at 100 °C for 100 min.IgG-binding ability of SPVI was weakened and IgG-binding ability of SPVII was vanished after Maillard reaction analyzed by dot blotting.Allergenicity decrease of SPVs by Maillard reaction was further confirmed on sensitized RBL-2H3 cell with the decreasing release of β-hexosaminidase,histamine and suppressing the production of interleukin-4(IL-4)and tumor necrosis factor-α(TNF-α).Circular dichroism spectroscopy and ANS binding method were used to determine the influence of Maillard reaction on SPV’s structure,and the results showed that Maillard reaction had a more effect on the structures of SPVII than SPVI.Based on these results,Maillard reaction combined with high pressure treatment were conducted to produce dried fish floss.The IgG-binding ability of dried fish floss was vanished by dot blotting analysis.Allergenicity decrease of dried fish floss produced by Maillard reaction combined with high pressure treatment was further confirmed on Balb/c mice with alleviate allergies and rectal temperature drop,suppressing the production of Th2 type spleen lymphocytes factors including IL-4,IL-10,IL-13 and IL-17F.