Effect Of Processing On Peanut Allergens And Its Mechanisms

The peanut is one of the biggest eight food allergens, which has a extremely low dose to stimulate allergy reaction and caused high rate of death. Currently, due to one-sided results or very high test demand, these detection methods of allergenicity could not be widely used in scientific research and industry production, which hindered the development of technique to reduce food allergenicity. Therefore, in this study, used peanut and its major allergen, Ara h1 as tested materials; and applied of animal and other bioactive materials, and molecular or immunological detection technologies to study the effects of autoclaving on peanut allergens, and revealed the mechanisms of a allergenicity drop caused by processing from the resistance of proteins to digestion, sensitization capacity, triggering potential of allergenic proteins, polarization of Th2 cells and the changes of structural characteristics of allergenic proteins in multi-level and multi-perspective. The main results are as follows:1. Rosting, frying, boiling, autoclaving and germination significantly reduced the immunoreactive of peanut allergens, which were only 92%、73%、92% and 28% of control, and with the effect of autoclaving is best; the inhibition rate of Ara h1 treated by autoclaving at 135 °C for 20 and 60 min were only 1/3 and 1/6 of the control respectively; at the same time, in the mixed system of peanut crude proteins, the inhibition of it treated by same condition were only 1/4 and 1/10 of the control, which decreased more markedly.2. After short-term germination, the bands of ~65 and 40 kDa became weak gradually, which caused by degradation of proteins in peanut seed. Simultaneously, the 19-17 k Da band has become more clearly; the inhibition rate of Ara h1 at 84 and 132 h decreased by 6.6% and 11.5% respectively compared to that at 36 h; Aware of plant disease and quality, 25°C and dark surroundings were suitable conditions under which peanut sprouts were processed, and neither of them significantly affected the allergenicity of Ara h1.3. Ara h1 could be degraded to smaller molecular weight protein fragments in SGF and SIF for 60 min, and those fragments were mainly with the molecular weight of ~10 and less than 15 kDa, which no longer had immunoreactive; after autoclaving treatment, the resistance of Ara h1 to SGF digestion was decreased, but no effect to SIF, and some high stability protein fragments with molecular weight of 25 and 15 kDa appeared in these samples treated at 121°C for 60 min and 135°C for 10 min; Ara h1 in the mixed system of peanut crude proteins had stronger resistance to SGF and SIF digestion.4. Animal model of peanut anaphylaxis was successfully established with C3H/HeJ mice and these mice with systemic reactions similar to human anaphylactic symptoms, and with higher levels of specific IgE and MCPT-1 than that of CT control; after autoclaving treatment of Ara h1, in addition to some extremely sensitive mice, most of them were not apparent systemic reactions, and contents of specific IgE and MCPT-1 were significantly lower than that of positive control.5. Proliferative responses of splenocyte and tryptase contents of mast cell from mice gavaged with autoclaved Ara h1 were distinctly slighter and lower than that of the positive control, and the effect of treatment at 135 °C for 60 min is more obvious. Concomitantly, there was a significant dependent relationship between concentration of treated Ara h1 and its capacity to stimulate mast cell degranulation, but not the relationship for the proliferation of splenocyte.6. The ratio of T to B and CD4 to CD8 of spleen cell from allergic mice were evidently lower and higher than CT control respectively, but these values in mice gavaged with autoclaved Ara h1 were not significantly different with CT control; the levels of IFN-γ and IL-4, IL-10, TGF-β in serum and culture supernatant of splenocytes from allergic mice decreased and increased respectively, but there was a obvious rise and fall of these value in mice gavaged with autoclaved Ara h1, and no significant difference between mice gavaged with autoclaved Ara h1 at 135 °C for 60 min and negative control mice.7. Under high temperature and pressure treatment, Ara h1 formed aggregates mainly caused by electrostatic and hydrogen bonding, soluble proteins were decomposed to smaller fragments with molecular weight of ~15 kDa; shape of the molecular became to short rod from global, hight was smaller and size was different; the protein molecular unfolded, increasing of surface hydrophobicity, exposure of tryptophan residues on the molecular surface, and the proportion of random coil and α-helix increased markedly.All the above results demonstrated that roasting, frying, boiling, autoclaving and germination can significantly reduce the immunoreactivity of peanut allergens, and the effect of autoclaving is the most significant. The Ara h1 treated by autoclaving have lost the potential of triggering anaphylaxis, duo to the clear alteration of the structure of it, which caused the decrease in resistance to digestion, sensitization capacity and immunoreactivity. This removal is closely associated with the faint in polarization of Th2 caused by it.