Maria Buchweitz

For flavan-3-ols, significant effects to prevent the development of diabetes mellitus are postulated. Inter alia, this is attributed to inhibitory effects on the intestinal α-amylase, in particular for high-molecular-weight procyanidins. In order to gain a deeper insight into the mode of interaction and the resulting α-amylase inhibition, the interaction between the monomers (+)-catechin (CAT) and (-)-epicatechin (EC), the dimers procyanidin (PC) B1 and PC B2, and the trimer PC C1 and their inhibition of porcine pancreatic α-amylase were investigated. Weak interactions were determined by isothermal titration calorimetry (ITC), with no clear difference between monomers and dimers and even no observable interaction with PC C1. Data from saturation transfer difference (STD)-NMR experiments supported these results with respect to reversible interactions. The detailed NMR signal assignments revealed that the formation of rotamers is solvent-dependent, which might explain the differences in the interaction strength between both diastereomers. The results for interaction were in contrast to the accumulating inhibitory strength with an increasing degree of polymerization when monitoring hydrolysis of the natural substrate starch in a novel continuous approach by ITC. By combining the data from the interaction and inhibition studies, we propose that protein aggregation occurs in the presence of flavan-3-ol oligomers, which are responsible for the inhibitory effects. This rather irreversible interaction is not susceptible to detection by ITC and STD-NMR and was also not observable by CD spectroscopy.

Interactions between phenolic compounds and the allergen Mal d 1 are discussed to be the reason for better tolerance of apple cultivars, which are rich in polyphenols. Because Mal d 1 is susceptible to proteolytic digestion and allergenic symptoms are usually restricted to the mouth and throat area, the release of native Mal d 1 during the oral phase is of particular interest. Therefore, we studied the release of Mal d 1 under different in vitro oral digestion conditions and revealed that only 6-15% of the total Mal d 1 present in apples is released. To investigate proposed polyphenol-Mal d 1 interactions, various analytical methods, e.g., isothermal titration calorimetry, ¹H-¹⁵N-HSQC NMR, and untargeted mass spectrometry, were applied. For monomeric polyphenols, only limited noncovalent interactions were observed, whereas oligomeric polyphenols and browning products caused aggregation. While covalent modifications were not detectable in apple samples, a Michael addition of epicatechin at cysteine 107 in r-Mal d 1.01 was observed.

Scope: Apples are an important polyphenol (PP) source. To compare the health benefits of traditional and commercial varieties, the phenolic contents and profiles as well as their release from the matrix (bioaccessibility) during oral digestion are determined. Furthermore, based on these data the proposed beneficial effect of PP on the variety specific allergenicity is discussed.Methods and results: Phenolics are quantified by HPLC‐DAD. Total phenolic contents (TPC) are in the range of 111–645 and 343–1950 mg 100 g⁻¹ dry weight for flesh and peel, respectively. Matrix release during oral digestion is investigated ex vivo, with centrifuged and non‐centrifuged human saliva and in vitro with simulated saliva fluid (SSF). The overall bioaccessibility is similar in all digestion media, ranging between 40–80% and 39–65% of the TPC in flesh and peel, respectively. Analyzing the correlation among Mal‐d 1‐allergen‐content, unoxidized PP, and the allergenic potential for the samples reveals a negligible effect of phenolics.Conclusion: Due to higher phenolic contents in combination with a similar release, increased PP concentrations in the oral phase and an improved uptake of PP from traditional varieties are assumed. However, the proposed beneficial effect of phenolics on allergenicity cannot be confirmed.

The allergen Mal d 1 is often responsible for adverse allergic reactions to fresh apples in northern and central Europe. The Mal d 1 content and isoallergen profile are proposed to have an impact on the allergenic potential of the fruit. Therefore, we investigated the impact of the cropping system on the Mal d 1 content and the isoallergen profile of apples by mass spectrometry for the varieties 'Jonagored' and 'Topaz'. To monitor the impact of storage time and conditions, apples of the varieties 'Santana' and 'Jonagold' were stored for up to 12 weeks under regular air (RA), under RA in combination with 1-methylcyclopropene (1-MCP) treatment, and under a controlled atmosphere (CA). The impact of the cropping system (integrated production vs organic production) was negligible. However, a significant increase in the Mal d 1 content during storage was observed, being higher when stored under CA conditions than under RA conditions. An additional treatment with 1-MCP prior to RA storage drastically reduced the level of Mal d 1 expression in the flesh of the apples by ∼50%. Furthermore, the content of isoallergens 1.03 and 1.06 increased disproportionately under CA conditions, while under RA conditions, only isoallergen 1.06 was affected. With the 1-MCP treatment, no changes in the isoallergen profile were obvious.

The apple allergy in Northern Europe is a cross-reaction to the birch pollen allergy. No correlation between the allergenicity of an apple variety and the content of the major apple allergen Mal d 1, a homologue to the Bet v 1 allergen in birch, could be found using ELISA, so far. Therefore, an impact of polyphenols and/or differences in the isoallergen profile are discussed. To allow a more detailed analysis of the Mal d 1 content and the isoallergen profile, a mass spectrometric method was applied to investigate differences in the flesh and peel of 10 traditional varieties and 10 commercial breeds. The data revealed often, but not always, lower Mal d 1 contents in traditional varieties grown in orchard meadows, which was more obvious in the flesh. Differences among the peels were less pronounced. A closer look at the individual isoallergens 1.01, 1.02, 1.03, and 1.06 reveals an increased impact of the minor isoallergens 1.03 and 1.06 on the allergenic potential, since commercial breeds like Braeburn, Santana, and Holstein Cox, which are considered to have reduced allergenic potentials, were characterized by low levels of these isoallergens.

Patients who suffer from birch pollinosis often develop adverse reactions to the consumption of fresh apples due to the structural similarity of the allergens Bet v 1 and Mal d 1 from birch and apples, respectively. A different allergenic potential for Mal d 1 isoallergens is postulated, but approaches to quantify the Mal d 1 isoallergen-specific are missing. Therefore, a bottom-up proteomics approach was developed to quantify Mal d 1 by stable isotope dilution and microHPLC-QTOF analyses. Marker peptides for individual isoallergens (Mal d 1.01-Mal d 1.03 and Mal d 1.06), combinations thereof (Mal d 1.01 + 1.02, Mal d 1.02 + 1.06, and Mal d 1.04 + 1.05), and two global marker peptides, comprising Mal d 1.01 + 1.02 + 1.04 + 1.05 and Mal d 1.03 + 1.06 + 1.07 + 1.08 + 1.09, were identified. By the use of an extraction standard (r-Mal d 1_mut), an optimized protocol for extraction and tryptic digestion of apple proteins was developed, and the variety-specific extraction efficiency was monitored for the flesh and peel of apples. The Mal d 1 contents in flesh and peel of five commercial apple breeds and four apple varieties from orchard meadows were quantified isoallergen-specific.

Health benefits of apple polyphenols for different chronic diseases are postulated. To exert bioactive properties, absorption into the body is required (bioavailability), which is strongly influenced by matrix release (bioaccessibility). For seven apple varieties, in vitro experiments with simulated saliva fluid (SSF) and ex vivo digestion with centrifuged human saliva were conducted. Polyphenol characterization (high-performance liquid chromatography-tandem mass spectrometry) and quantification (high performance liquid chromatography-diode array detection) was related to an aqueous methanolic extraction. A polyphenol release of 63–82% from flesh and 42–58% from peel was estimated. While hydroxycinnamic acid derivatives were released in total, a significant retention was observed for flavanes and flavones. In particular, procyanidins were retained with increasing molecular weight. The data reveal a considerable polyphenol release during the oral digestion; however, differences among the varieties as well as flesh and peel were obvious. Due to negligible differences between both digestion media, the data supported the use of SSF instead of human saliva in further experiments.

Phenolic structures are of great interest due to their antioxidant properties and various postulated benefits on human health. However, the quantification of these structures in fruits and vegetables, as well as in vivo or in vitro experiments, is demanding, as relevant concentrations are often low, causing problems in exactly weighing the respective amounts. Nevertheless, the determination of used concentrations is often a prerequisite for accurate results. A possibility to quantify polyphenol is the use of UV/vis spectroscopy. Therefore, the absorption coefficients of selected phenolic structures were determined in three different solvents relevant for polyphenol research (water/methanol (50/50, v/v), water, and phosphate buffer at pH 7.5). To confirm the values based on weight and to avoid errors due to impurities, hygroscopic effects, and inadequate balance care, the mass concentrations were additionally determined by quantitative NMR (q-NMR). The coefficients presented in this article can help to quickly and easily determine accurate concentrations in a laboratory routine without wasting the often-precious standard compounds.

Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production route followed by an unsophisticated purification step for Mal d 1 and respective mutants is desired to produce sufficient amounts. We describe a procedure for the transformation of the plasmid in competent E. coli cells, protein expression and rapid one-step purification. r-Mal d 1 with and without a polyhistidine-tag are purified by immobilized metal ion affinity chromatography (IMAC) and fast-protein liquid chromatography (FPLC) using a high-resolution anion-exchange column, respectively. Purity is estimated by SDS-PAGE using an image-processing program (Fiji). For both mutants an appropriate yield of r-Mal d 1 with purity higher than 85% is achieved. The allergen is characterized after tryptic in gel digestion by peptide analyses using HPLC-MS/MS. Secondary structure elements are calculated based on CD-spectroscopy and the negligible impact of the polyhistidine-tag on the folding is confirmed. The formation of dimers is proved by mass spectrometry and reduction by DTT prior to SDS-PAGE. Furthermore, the impact of the freeze and thawing process, freeze drying and storage on dimer formation is investigated.