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)
Although peanut is consumed raw or boiled in some cultures, roasting is a preferred processing step because it improves microbial stability, provides a longer shelf life, and also improves the organoleptic properties. 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; peanut allergens may not be immediately bio-accessible when peanut-containing food is consumed.
An amount of 0.5 g of peanut flour (PF) was suspended in 15 mL of buffer and mixed for 90 min at room temperature. Subsequently, the mixture was centrifuged at 1700xg at room temperature for 5 min, and the supernatant was collected and centrifuged again at 2660xg for 10 min. The supernatants were stored for analysis. 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.
Both at lower pH values, protein extractability became higher, in particular at a pH corresponding to the typical stomach conditions. Using the extraction ratio (1:30 w/v) and taking into account the protein content of the peanut flour (50%), the theoretical maximal protein concentration is 16.7 mg/ml.
At extremely low pHs and at moderately high pHs, the typical protein band pattern of peanut extracts was observed. ]. 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. At pH 6 the protein profile of the extract indicated a strong enrichment in Ara h2 and Ara h6.
Using dissolution in artificial saliva, protein bands of all allergens (Ara h1, Ara h2, Ara h3, and Ara h6) were clearly visible, even though the extraction time was short.
For buffers with pH < 7, a small acidifying effect was observed (less than 0.2 pH units). However, for buffers at higher pHs, the acidifying effect was stronger, up to 0.8 pH units. The amounts of hydroxide needed to re-adjust the pH increased as a consequence of the increasing pH of the extraction medium.
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. The recoveries for Ara h1 and Ara h3 were low at pH 6.5, i.e., 5% and 6% of the theoretical maximal values, respectively.
The treatment of boiling the non-dissolved residues remaining after extraction in buffer containing the strong detergent SDS and a reducing agent to break disulfide bonds could recover proteins from the residues left over after the dissolution experiments.
Ara h2 and Ara h6 are highly bio-accessible in the mouth (and esophagus) suggest that these allergens have the unique opportunity to interact with the mucosal immune system before reaching the stomach, which may lead to different sensitizing potential in comparison to other peanut allergens.