Purification of natural Mal d 1 and Mal d 2 allergens and monitoring of their expression levels during ripening in Golden Delicious apple

Apple (Malus domestica) is the most widely cultivated fruit crop in Europe. Consumption of fresh apples can cause allergic reaction with a variable degree of severity in susceptible individuals. Previous studies have indicated that expression pattern of Mal d allergens is affected by growing and storage circumstances. At the same time only a few data are available on purification and expression levels of the heat labile Mal d 1 apple allergen localised in peel and pulp of the fruit because this protein can be extracted in an active form only if reactions with phenolic compounds present in apple are inhibited. Our aim was to concurrently purify and monitor the expression levels of the heat labile Mal d 1 (18 kDa), a Bet v 1 related allergen and the heat resistant Mal d 2 (32 kDa), a thaumatin-like allergenic protein from Golden Delicious apple cultivar with high potential to express Mal d 1 allergens. This assumption was proven by amplification of a polymorphic pattern of PCR products with 154–174 bp fragments by using Mal d 1.06A SSR primers. The apple proteins were extracted from frozen apple pulp under acidic circumstances at pH 3.0 to prevent reaction of the allergens with phenolic compounds of apple. Under such circumstances protein-binding brown pigments can be avoided and its deleterious effect on analysis of Mal d allergens can be eliminated. The separation of Mal d allergens was performed by cation-exchange chromatography and gel filtration. Mal d allergens were quantified during ripening in Golden Delicious apple extract. Mal d 1 and Mal d 2 specific ELISAs were developed to be used for quantification of the immune reactive Mal d allergens. It was found that during the ripening period, the contents of Mal d 1 and Mal d 2 allergens were continuously, but a bit differentially increased. According to the chromatographic results the content of Mal d 1 (0.1 –0.25 mg/100 g apple) increased intensively from 144th until 152th day, then it did not change (152th–164th days), while the content of Mal d 2 (0.18–0.75 mg/100 g apple) started to increase from 144th until 158th day. ELISA results showed the same tendency.

► We developed a liquid chromatographic method to purify Mal d 1 and Mal d 2 apple allergens simultaneously.
► We found that the main advantage of this method is the inhibition of enzymatic browning at low pH during the purification process.
► The cation-exchange membrane chromatography coupled with gel filtration was proved to be suitable for monitoring the Mal d apple allergens during ripening.