Identification of apple cultivars hypoallergenic for birch pollen‐allergic individuals by a multidisciplinary in vitro and in vivo approach

Abstract

Background: Birch pollen-related apple allergy is the most frequent IgE-mediated food allergy in Central-Northern Europe with Mal d 1 as major allergen. Its concentration in apples varies with the cultivar and storage time. Year-round appealing, hypoallergenic cultivars still are needed to satisfy the nutritional needs of affected individuals. We characterized three promising cultivars by multidisciplinary in vitro assays including long-term storage and by clinical challenges of allergic individuals before and after the birch pollen season. Methods: Proteins were extracted from fruits of 'Santana', 'Golden Delicious' (GD), and three genuine cultivars in November 2018 and April 2019. Mal d 1-levels were analysed by mass spectrometry, SDS-PAGE, immunoblotting, competitive ELISA, and basophil activation tests. Twenty-eight allergic individuals underwent singleblinded open food challenges and skin testing with the cultivars and birch pollen in November 2018 and May 2019. Allergen-specific IgE-levels were determined. Results: After storage all cultivars except 'Santana' were of appealing appearance and taste. Their Mal d 1 content had increased, also reflected by significantly amplified basophil activation and stronger reactions in clinical challenges. Besides, individuals showed boosted reactivity after pollen exposure indicated by enhanced allergen-specific IgE-levels and skin reactions to birch pollen. Still, all cultivars remained significantly less allergenic than GD and comparable to Santana in November 2018 in all assessments except for skin testing.

Impact of Cultivation and Storage Conditions on Total Mal d 1 Content and Isoallergen Profile in Apples

“…26 The highest increase in Mal d 1 was observed during CA storage and the lowest if the apples were treated with 1-MCP. The huge decline of Mal d 1 in 'Santana' flesh, by approximately 50% between weeks 8 and 12, was surprising and might be due to the poor storability of 'Santana', 24 is well-established that proteins can be degraded during senescence. 39 In two studies by Kiewning et al, 12 different varieties were stored under CA (1.5 kPa O 2 , 1.5 kPa CO 2 , 2 °C) and RA (2 °C) conditions for up to 44 weeks.…”

“…The increase during storage is in agreement with literature data, since all previous studies also reported rising Mal d 1 contents. [12][13][14]24,29,30 The analyzed apples were kept under the optimal storage conditions for each variety. Therefore, different parameters for the gas composition in CA storage for 'Jonagold' (1.0 kPa O 2 , 2.5 kPa CO 2 ) and 'Santana' (1.0 kPa O 2 , <0.7 kPa CO 2 ) were applied.…”

“…The dominance of isoallergens 1.01 and 1.02 in the flesh and the peel is in accordance with previously published results by us and other literature. 17,18,24,37,38 After harvest, in the peel, isoallergens 1.03 and 1.06 were detected in small amounts, while in the flesh only traces were present.…”

“…Furthermore, biological replicates experience individual stressors and therefore might differ in their allergen content . Differences among individual fruit are often not taken under consideration when quantifying the allergen content, since usually homogenized and pooled samples are used. , For oral provocation challenges, often information about the sample size is missing, but a low number of individual apples must be assumed since in most cases, whole fruit pieces are given to the patients. , In a standard procedure for oral provocation tests , often the complete challange is conducted with a single apple per patient . This might be responsible for the discrepancies between the results of oral provocation studies and the analytical data.…”

“…13,23 For oral provocation challenges, often information about the sample size is missing, but a low number of individual apples must be assumed since in most cases, whole fruit pieces are given to the patients. 24,25 In a standard procedure for oral provocation tests , often the complete challange is conducted with a single apple per patient. 1 This might be responsible for the discrepancies between the results of oral provocation studies and the analytical data.…”