Effects of high hydrostatic pressure on immunoreactivity, digestibility and intestinal permeability of major peanut and milk allergens

Abstract

Food allergy has become an increasing public and personal health burden in the developed countries over the last decade. Peanut and cow’s milk are two of the most common triggers in Australia. In this thesis, the effects of HHP on major peanut and milk allergens on immunoreactivity, digestibility and intestinal permeability were studied in view to reduce their allergic potential. Major peanut allergens (Ara h1, Ara h2, Ara h3 and Ara h6) were purified using a combination of anion exchange chromatography and size exclusion chromatography. The Ara h2/h6 ELISA with a limit on detection of 8.4 μg L-1 was developed using the antibodies raised separately against purified Ara h2 and Ara h6. Together with previously developed three ELISAs individually detecting total protein, Ara h1 and Ara h3 ELISAs, these four ELISAs were utilised in the following peanut-related experiments. The protein solubility of four peanut cultivars (Middleton, Menzies, Sutherland and Walter) with different kernel size roasted at 160 °C for 20 to 60 min was analysed to study the effects of heat transfer during roasting on the protein solubility/extractability. Menzies with medium kernel size showed the highest protein solubility. No obvious relationship between kernel size and protein solubility had been identified. The difference in protein solubility of roasted kernels was attributed by the genotypes. The effects of roasting time on the detectability of the four ELISAs (total protein, Ara h1, Ara h2/h6 and Ara h3 ELISAs) were assessed. The ELISA detectability affected by the two extraction methods using cold and heat application was also assessed. The cold extraction method showed less variability in ELISA and the Ara h2/h6 ELISA was less affected by the roasting conditions. The immunoreactivity of total peanut protein and purified peanut allergens (Ara h1, Ara h2, Ara h3 and Ara h6) upon HHP processing were evaluated. The immunoreactivity of HHP-induced total peanut protein was reduced to zero, but with limited structure modification. Purified Ara h1, and Ara h2/h6 showed resistance to HHP processing, showing little changes in the immunoreactivity whereas the immunoreactivity of Ara h3 was diminished to zero at pressures higher than 300 MPa. With increased pressure and holding time, HHP processed peanut protein tended to be more resistant to pepsin digestion, comparable with the thermally processed peanut protein. HHP combined with thermal processing were optimised for the immunoreactivity and dimerization of β-Lg, and the immunoreactivity of α-La. A response surface model of β-Lg with an R2 of 0.81 was successfully developed, which reduced the immunoreactivity to 7.5%. The relationship between dimerization and immunoreactivity of β-Lg was determined to follow a quadratic equation with an R2 of 0.95. α-La showed no significant changes in immunoreactivity after HHP processing. The immunoreactivity of β-Lg and α-La in different milk matrices revealed that the β-Lg and α-La mixture, whey protein and raw milk reduced the immunoreactivity of β-Lg, but at different degrees. In contrast, the immunoreactivity of β-Lg in skim milk was increased after HHP processing. The immunoreactivity of α-La was least affected in whey protein, skim milk and raw milk but the aggregates formed with β-Lg in a two-protein system (β-Lg and α-La mixture) contributed to an 82.5% reduction in the immunoreactivity. The combining HHP with thermal processing showed a synergistic effect on the enhancement of enzymatic digestion of β-Lg. HHP-thermal processing accelerated both in-vitro pepsin and trypsin digestions, however, the sizes of peptides from pepsin digestion did not reduce. The in-vitro intestinal permeability of in-vitro digested HHP-thermal processed β-Lg and whey protein using the Caco-2 cell model was investigated. HHP-thermal processed β-Lg was extensively digested into smaller peptide sizes compared to the untreated form, thereby potentially reducing the risk of causing allergic reactions. The intestinal permeability of whey protein digesta using the same model was not affected by HHP-thermal processing suggesting that the presence of α-La contributed to the changes in in-vitro digestion and the intestinal permeability.