Recent advances and challenges in the analysis of natural toxins.

Natural toxins (NTs) are poisonous secondary metabolites produced by living organisms developed to ward off predators. Especially low molecular weight NTs (MW<∼1 kDa), such as mycotoxins, phycotoxins, and plant toxins, are considered an important and growing food safety concern. Therefore, accurate risk assessment of food and feed for the presence of NTs is crucial. Currently, the analysis of NTs is predominantly performed with targeted high pressure liquid chromatography tandem mass spectrometry (HPLC-MS/MS) methods. Although these methods are highly sensitive and accurate, they are relatively expensive and time-consuming, while unknown or unexpected NTs will be missed. To overcome this, novel on-site screening methods and non-targeted HPLC high resolution mass spectrometry (HRMS) methods have been developed. On-site screening methods can give non-specialists the possibility for broad "scanning" of potential geographical regions of interest, while also providing sensitive and specific analysis at the point-of-need. Non-targeted chromatography-HRMS methods can detect unexpected as well as unknown NTs and their metabolites in a lab-based approach. The aim of this chapter is to provide an insight in the recent advances, challenges, and perspectives in the field of NTs analysis both from the on-site and the laboratory perspective.

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

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.

Characterization and Quantification of Mal d 1 Isoallergen Profiles and Contents in Traditional and Commercial Apple Varieties by Mass Spectrometry.

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.

New Mass Spectrometric Approach to Quantify the Major Isoallergens of the Apple Allergen Mal d 1.

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.

Bioaccessibility of Apple Polyphenols from Peel and Flesh during Oral Digestion.

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.