Release of Major Peanut Allergens from Their Matrix under Various pH and Simulated Saliva Conditions—Ara h2 and Ara h6 Are Readily Bio-Accessible (1)
One source of such traces of peanut is the home environment, where peanut residues are found on floors, table surfaces, and furniture, even after standard household cleaning. Upon ingestion, the peanut is transported through the gastro-intestinal tract for digestion, but, interestingly, some digestion-resistant peanut proteins appear in the blood serum in a relatively intact form and may reappear in the saliva even several hours after ingesting peanut. Peanut is a complex biological product that consists of lipids (49%), proteins (25%), carbohydrates (16%), and some moisture and ash.
The following buffers were used for extraction: pH = 1.5, 32 mM HCl; pH = 2.6, 21.6 mM Na2HPO4 + 89.2 mM citric acid; pH = 3.0, 40.8 mM Na2HPO4 + 79.6 mM citric acid; pH = 4.0, 77.2 mM Na2HPO4 + 61.4 mM citric acid; pH = 5.0, 102.8 mM Na2HPO4 + 48.6 mM citric acid; pH = 6.0, 128.5 mM Na2HPO4 + 35.8 mM citric acid; pH 7.2 to pH 9.0, 50 mM TRIS adjusted with HCl to the target pH.
A clear efficiency in the extraction of proteins between pH 3 and pH 6 was obtained. Both at lower pH values and higher pH values, protein extractability became higher, in particular at a pH corresponding to the typical stomach conditions. The theoretical maximal protein concentration is 16.7 mg/mL. The highest protein concentration we found was about 8.5 mg/mL at pH 1.5. This concentration corresponds to a recovery of about 50%. At a high pH, we found a protein concentration of about 5 mg/mL, which corresponds to about 30% recovery.
All these bands were well visible at extremely low pHs and at moderately high pHs. In contrast, at neutral and slightly acidic pHs (3 to 7.6), the bands corresponding to Ara h3 and, to a lesser extent, Ara h1, were far less clear. The bands corresponding to Ara h2 and Ara h6 remained extractable over a wider pH range, although they were poorly visible at pH 4 and 5. Interestingly, at pH 6 the protein profile of the extract indicated a strong enrichment in Ara h2 and Ara h6. The commercial ELISA kits used essentially recognize Ara h3, the protein whose extractability appeared low at pHs from 6 to 7.2 in our experiments.
At the higher end of the range, intense bands, corresponding to Ara h1, Ara h2, Ara h3, and Ara h6, were recovered in the extracts. A gradual decrease of band intensity was observed when decreasing the pH from pH 7.5 to 6.5.
At pH 6.5, the concentrations of Ara h1, Ara h2, Ara h3, and Ara h6 were 0.24, 1.52, 1.58, and 0.40 mg/mL, respectively, and these concentrations increased with increasing extraction pH, in particular for Ara h3, but also for Ara h1and Ara h6.
In the pellet materials, no Ara h2 bands were observed, and, for Ara h6, only a vague band was seen at the lowest tested pH conditions.
The solubility difference may be determined by their molecular weight and quaternary structure. Ara h2 and Ara h6 are small (15–20 kDa) monomeric proteins, while Ara h1 and Ara h3 are larger and form complexes up to 180–700 kDa and 360–380 kDa, respectively. Upon roasting, Ara h1 and Ara h3 may aggregate, further limiting their solubility, while this is not the case for Ara h2 and Ara h6].