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Precision Plus Proteinв„ў Prestained Standards (BioRad В®, Hercules, CA, USA) ranging from 2 kDa to 250 kDa (161-0377), 40% acrylamide/bis solution (37.5:1) (161-0148), 2X Laemmli sample buffer (161-0737), 10x tris/glycine/SDS running buffer (161-0772), Tris (161-0719) were obtained from BioRad. ОІ-Lg A and ОІ-Lg B from bovine milk, О±-La from bovine milk, sodium dodecyl sulfate (SDS), 2-mercaptoethanol, goat anti-rabbit IgG-horseradish peroxidase (IgG-HRP), 3,3вЂІ,5,5вЂІ-Tetramethylbenzidine (TMB), Tween 20 (polyoxyethylene sorbitan monolaurate), sodium acetate (CH 3COONa), beta-cyclodextrin and urea hydrogen peroxide, dimethylsulfoxide (DMSO)-99.9% molecular biology grade, sodium periodate, horseradish peroxidase (HRP), Bicinchoninic protein assay kit (BCA-1) containing bicinchoninic acid, sodium carbonate, sodium tartarate, sodium bicarbonate in 0.1 M NaOH (pH 11.25), copper (II) sulfate pentahydrate 4% ( w/ v) solution and a protein standard (i.e., bovine serum albumin(BSA)) with a concentration of 1.0 mg mL в€’1 were purchased from Sigma-Aldrich (St. Whey protein and skim milk were prepared from raw milk, which was collected and delivered from the Lidcombe laboratory of Parmalat (Sydney, Australia). The typical pressure needed for the unfolding is around 500 MPa, however, it varies from protein to protein. Pressures between 200 and 500 MPa induce non-native monomers, dimers, trimers, and tetramers, while pressures >500 MPa induce higher molecular weight aggregates. For example, dissociation of oligomeric proteins and multi-protein complexes could be induced by the moderately high pressure of 100вЂ“200 MPa, whereas pressures of 400вЂ“800 MPa are sufficient to induce denaturation of small monomeric proteins. High hydrostatic pressure (HHP) processing could modify the molecular structure and functional properties of proteins and other associated macromolecules without affecting color, flavor and micronutrient contents.

The ОІ-Lg dimers also resulted in the undetectable human IgE-binding.ДЇood processing has the potential to alter the allergenic properties of proteins by either masking, destroying or disclosing allergenic epitopes through cleavage of linear structures or inducing conformational changes, or by improving the digestibility of proteins.

The molecular change of ОІ-Lg was directly correlated with HHP-induced dimerization in this study, which followed a quadratic equation. The validation carried at the optimal condition and its surrounding region showed that the model to be underestimating the ОІ-Lg structure modification. The optimal condition defined as minimum (ОІ-Lg specific) IgG-binding derived from the model was 505 MPa at 56 вЊ with a holding time of 102 min (R 2 of 0.81 and p-value of 0.01). The RSM model derived for HHP-induced molecular changes of ОІ-Lg determined immunochemically showed that temperature (temp), pressure (p 2) and the interaction between temperature and time ( t) had statistically significant effects ( p < 0.05).

The response surface methodology (RSM) and central composite design (CCD) technique were used to optimize the three key process parameters (i.e., pressure, temperature and holding time) of the high-hydrostatic-pressure (HHP) processing either standalone or combined with moderate thermal processing to modulate molecular structures of ОІ-lactoglobulin (ОІ-Lg) and О±-lactalbumin (О±-La) with reduced human IgE-reactivity.

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