Food allergies are on the rise and one of the main culprits is a peanut. A study published in 2002 found that about 1.5% of children across the UK was allergic to peanuts, compared to 0.5% a decade earlier. In the US, around 0.6% have peanut or tree nut allergies such as walnuts, cashews, pistachios and Brazil nuts.
Not all peanut allergies cause severe symptoms, and many people are only mildly affected – in an itchy rash or mild stomach pain and diarrhea. But, there are those who suffer the life-threatening reaction of anaphylaxis, which is a severe, whole-body reaction that can constrict the airways and cause fainting, unconsciousness or collapse. There is also a third category of the most sensitive sufferers that, in some cases, cannot remain in the same room with anyone who is eating peanut-containing foods.
The peanut allergens are a group of eight proteins, known as Ara h 1 to Ara h 8, and generally enzyme-linked immunoassays ([tag]ELISA[/tag]) is used to detect these proteins, using an antibody that responds to the presence of the allergen, but these tests have been known to produce variable results. Also, in the case of dark chocolate, some [tag]ELISA[/tag] test kits have given false negatives in up to 25% of cases, a result which endangers allergenic people if the food labels subsequently states “no peanuts.”
Immunoassays and other methods also suffer from extraction problems with dark chocolate, due to interactions of the allergenic proteins with other components, especially tannins. In light of all of these difficulties, US scientists at the Center for Food Safety and Nutrition have developed a new method for detecting the principal allergen, Ara h 1, in dark chocolate, based on proteomics.
Kevin Shefcheck, John H. Callahan, and Steven M. Musser began by digesting a sample of pure Ara h 1 with trypsin and analyzed the subsequent peptide mixture by LC/MS with electrospray ionisation, using a C18 column and an aqueous acetonitrile gradient. From the resulting mass spectrum they selected two peptides based on retention time and signal intensity that were used to detect the presence of Ara h 1 in dark chocolate.
The scientists analyzed six chocolate samples containing known amounts of peanut protein, from 0-100 ppm. Initially, they extracted all proteins with ammonium bicarbonate then digested the concentrate with trypsin. Though the peptide markers were detected by LC/MS, the sensitivity was not too good, with LOD (Limit of Detection) of 50 ppm. However, improvements in extraction techniques combined with an increase in the amount of chocolate extracted significantly lowered the LOD to 2 ppm of peanut protein.
So, the main peanut allergen can be detected in dark chocolate based on the retention time and mass spectra of two marker peptides. With the development of suitable internal standards, the method could be developed into a quantitative method for determining the actual amounts of Ara h 1 present in various samples.
The lengthy sample preparation time suggests that the method could only be used for allergen confirmation and not in QC. However, it could potentially be extended to encompass different protein allergens found in other types of foods.