•Microbial degradation of caffeoylisocitric acid from amaranth was characterized.•Metabolites of caffeoylisocitric acid and chlorogenic acid fermentations are very similar.•Composition of the microbiota was not affected.The almost forgotten crop amaranth has gained renewed interest in recent years due to its immense nutritive potential. Health beneficial effects of certain plants are often attributed to secondary plant metabolites such as phenolic compounds. As these compounds undergo significant metabolism after consumption and are in most cases not absorbed very well, it is important to gain knowledge about absorption, biotransformation, and further metabolism in the human body.Whilst being hardly found in other edible plants, caffeoylisocitric acid represents the most abundant low molecular weight phenolic compound in many leafy amaranth species. Given that this may be a potentially bioactive compound, gastrointestinal microbial degradation of this substance was investigated in the present study by performing in vitro fermentation tests using three different fecal samples as inocula. The (phenolic) metabolites were analyzed using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Furthermore, quantitative polymerase chain reaction (qPCR) analyses were carried out to study the influence on the microbiome and its composition. The in vitro fermentations led to different metabolite profiles depending on the specific donor. For example, the metabolite 3-(4-hydroxyphenyl)propionic acid was observed in one fermentation as the main metabolite, whereas 3-(3-hydroxyphenyl)propionic acid was identified in the other fermentations as important. A significant change in selected microorganisms of the gut microbiota however was not detected.In conclusion, caffeoylisocitric acid from amaranth, which is a source of several esterified phenolic acids in addition to chlorogenic acid, can be metabolized by the human gut microbiota, but the metabolites produced vary between individuals.Download high-res image (256KB)Download full-size image

•Immunological detection following HPTLC of food proteins and peptides thereof.•The possibility of a semi-quantitative estimation was demonstrated.•Newly developed method might be applicable to analyze cross-reactivity of antibodies.The chromatographic analysis of intact proteins is still challenging, especially when biological functions as antigenicity of proteins or peptides are in the focus. Traditional immunoassays provide information about the entirety of antigenic proteins/peptides, e.g., in ELISA assays. On the other hand, when focusing on the investigation of (cross) reactivity of antibodies, Western blot following gel-electrophoresis represents the method of choice. However, gel-electrophoresis is limited by the molecular weight and therefore, not suitable for peptides ≤3 kDa or proteins ≥250 kDa. Furthermore, for gaining detailed information about the protein sequence (e.g., via mass spectrometric analysis), a so called in-gel digest needs to be performed following electrophoretic separation and is therefore elaborate and accompanied by a significant loss of structural, and even more severe, conformational information. Here, protein analysis using HPTLC seems to be a promising alternative due to the high level of variability regarding the chromatographic system (multiple mobile and stationary phases, even mixed) and manifold detection as well as hyphenation possibilities.This study exemplarily focused on the immunological investigation of proteins in milk following thin-layer chromatographic separation. The detection of these antigens is mandatory, as they might trigger allergenic reactions in sensitized people. Besides the proof of its applicability on different stationary phase materials, the newly developed immunoassay can be used as an approach for semi-quantitative estimation of antigenic proteins. In addition to the analysis of intact food allergens, also analyzing peptides thereof is worth considering which can be realized using HPTLC-immunostaining as well.

Bile acids are important signaling molecules that regulate cholesterol, glucose, and energy homoeostasis and have thus been implicated in the development of metabolic disorders. Their bioavailability is strongly modulated by the gut microbiota, which contributes to generation of complex individual-specific bile acid profiles. Hence, it is important to have accurate methods at hand for precise measurement of these important metabolites. Here, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous identification and quantitation of primary and secondary bile acids as well as their taurine and glycine conjugates was developed and validated. Applicability of the method was demonstrated for mammalian tissues, biofluids, and cell culture media. The analytical approach mainly consists of a simple and rapid liquid-liquid extraction procedure in presence of deuterium-labeled internal standards. Baseline separation of all isobaric bile acid species was achieved and a linear correlation over a broad concentration range was observed. The method showed acceptable accuracy and precision on intra-day (1.42–11.07 %) and inter-day (2.11–12.71 %) analyses and achieved good recovery rates for representative analytes (83.7–107.1 %). As a proof of concept, the analytical method was applied to mouse tissues and biofluids, but especially to samples from in vitro fermentations with gut bacteria of the family Coriobacteriaceae. The developed method revealed that the species Eggerthella lenta and Collinsella aerofaciens possess bile salt hydrolase activity, and for the first time that the species Enterorhabdus mucosicola is able to deconjugate and dehydrogenate primary bile acids in vitro.

•Alamethicin affects glucuronidation of phenolic substrates.•Different alamethicin concentrations lead to different depletion rates.•A maximal glucuronidation activity could be assigned to different but distinct alamethicin concentrations.•Alamethicin application is a crucial factor to deduce enzyme kinetic parameter from in vitro metabolic assays.•Correlations between the substrate lipophilicity and alamethicin effect were not detected.A major pathway for the elimination of drugs is the biliary and renal excretion following the formation of more hydrophilic secondary metabolites such as glucuronides. For in vitro investigations of the phase II metabolism, hepatic microsomes are commonly used in the combination with the pore-forming peptide alamethicin, also to give estimates for the in vivo situation. Thus, alamethicin may represent a neglected parameter in the characterization of microsomal in vitro assays. In the present study, the influence of varying alamethicin concentrations on glucuronide formation of selected phenolic compounds was investigated systematically. A correlation between the alamethicin impact and the lipophilicity of the investigated substrates was analyzed as well. Lipophilicity was determined by the logarithm of the octanol-water partition coefficient.For every substrate, a distinct alamethicin concentration could be detected leading to a maximal glucuronidation activity. Further increase of the alamethicin application led to negative effects. The differences between the maximum depletion rates with and without alamethicin addition varied between 2.7% and 18.2% depending on the substrate. A dependence on the lipophilicity could not be confirmed. Calculation of the apparent intrinsic clearance led to a more than 2-fold increase using the most effective alamethicin concentration compared to the alamethicin free control.

•Opuntia dillenii juice possesses high bioactivity and promising characteristics.•O. dillenii juice is a viscous non-Newtonian pseudoplastic fluid.•HHP, PEF, and thermal pasteurization achieved a similar microbial inactivation.•HHP and PEF preserved the physicochemical properties of the fresh juice.•HHP and PEF were effective in preserving bioactive compounds such as ascorbic acid.Besides its bioactivity, Opuntia dillenii cactus juice exhibits desirable physicochemical properties. In the present study, impact of innovative pasteurization techniques – pulsed electric fields (PEF) and high hydrostatic pressure (HHP) – on the microbial inactivation, selected physicochemical properties and rheological characteristics, ascorbic acid, flavonols, betacyanins, and the antioxidant activity was evaluated in comparison to the non-pasteurized fresh juice. Results showed that PEF and HHP, as well as thermal pasteurization (TP), reduced the microbial populations found in juice from 103 to less than 10 cfu/mL. The non-Newtonian pseudoplastic flow behaviour of juice was maintained by all three methods. PEF and HHP resulted in a better retention of ascorbic acid, while TP reduced 22% of the ascorbic acid content. In contrast, TP caused a slight transformation of isorhamnetin glycosides. PEF and HHP maintained antioxidant activity of juice comparatively better than TP.

•Leaves, stems, and fruits of Schisandra chinensis were analysed by LC-ESI-ToF-MS.•Twelve phenolic compounds were identified for the first time in Schisandra leaves.•Individual antioxidant capacities were determined by HPLC-online TEAC.•Chlorogenic acids and quercetin glycosides contribute over 80% of the total TEAC.•Leaves and stems of Schisandra can be considered as functional ingredients.Schisandra chinensis is a widely-consumed nutraceutical providing advantageous nutritional and bioactive properties. This study aimed at characterizing the chemical composition and antioxidant capacity of S. chinensis fruits as well as the underestimated leaves and stems. A special aim of this study was to characterize the contribution of single constituents (lignans and further phenolic compounds) to the overall antioxidant activity using an HPLC-online TEAC approach. By using LC-DAD/ESI-ToF-MS, twenty-eight lignans and twenty phenolic compounds were identified. Among the 48 characterized components, twelve phenolic compounds were identified for the first time in Schisandra. Gomisin D was the only lignan being able to scavenge ABTS+ radicals, suggesting that not its amount but rather its chemical structure is responsible. Overall, clorogenic acid isomers and quercetin glycosides contribute to over 80% of the total antioxidant activity. This study indicates that leaves and stems of S. chinensis may be used for obtaining bioactive formulations and functional foods.

•An immunological detection of phosphopeptides after HPTLC was developed.•Phosphopeptides were detected applying monoclonal antibodies.•Via HPTLC-MALDI-TOF a simultaneous characterization of the peptides was realized.This study aimed at developing a rapid chromatographic assay to monitor phosphorylation sites in peptides. For the analysis of nociceptive signal transduction pathways, the detection of phosphorylated proteins/peptides plays a fundamental role. To get further insights in the phosphorylation mechanism of protein kinase C-ε (PKC-ε) and protein kinase A (PKA), potential targets were divided into subsections resulting in peptides that contain only one possible phospho-binding site. The use of high-performance thin-layer chromatography (HPTLC) offers the possibility of a high throughput of samples and the advantage of a quick sample clean-up. A combined strategy of an effect-directed overlay procedure on the TLC plate using specific antibodies (immunostaining, HPTLC-IS) as well as a parallel, direct mass spectrometric methodology by HPTLC-MALDI-TOF-MS was developed. With regard to HPTLC-IS, validation of the data exhibited a lower limit of detection than the traditionally used protein derivatization reagent fluorescamine. Besides the identification of the phosphorylated peptides, a semi-quantitative estimation can be performed with HPTLC-IS.

•An HPTLC separation system for proteins was established.•Several layers and solvent systems have been evaluated.•The simplified ‘four-level scheme’ is a good tool for a systematic optimization.•The PRISMA model can be used for ‘fine-tuning’ of the eluents.In recent years, protein chemistry tends inexorably toward the analysis of more complex proteins, proteoforms, and posttranslational protein modifications. Although mass spectrometry developed quite fast correspondingly, sample preparation and separation of these analytes is still a major issue and quite challenging. For many years, electrophoresis seemed to be the method of choice; nonetheless its variance is limited to parameters such as size and charge. When taking a look at traditional (thin-layer) chromatography, further parameters such as polarity and different mobile and stationary phases can be utilized. Further, possibilities of detection are manifold compared to electrophoresis. Similarly, two-dimensional separation can be also performed with thin-layer chromatography (TLC). As the revival of TLC developed enormously in the last decade, it seems to be also an alternative to use high performance thin-layer chromatography (HPTLC) for the separation of proteins. The aim of this study was to establish an HPTLC separation system that allows a separation of protein mixtures over a broad polarity range, or if necessary allowing to modify the separation with only few steps to improve the separation for a specific scope. Several layers and solvent systems have been evaluated to reach a fully utilized and optimized separation system.

Die Migration von Weichmachern aus Kunststoffen, die zur Verpackung von Lebensmitteln verwendet werden, in die entsprechenden Lebensmittel, ist ein lange bekanntes und kontrovers diskutiertes Thema. Die Weichmacher werden den Kunststoffen zugesetzt, damit sie geschmeidig und formbar werden. Zwar ist der als Migration bezeichnete Übergang von Verpackungsbestandteilen auf das Lebensmittel aufgrund möglicher gesundheitlicher Risiken durch Richtlinien, Gesetze und Verordnungen reglementiert, eine Diskriminierung von Substanzen aus dem Lebensmittel durch einen gleichzeitig ablaufenden Übergang in den Kunststoff, wurde bisher jedoch nicht hinreichend untersucht und berücksichtigt. Auf diesem Wege können jedoch auch wertgebende Substanzen in ihrem verzehrbaren Gehalt gemindert werden. In dieser Studie wurde die Migration des Weichmachers DEHP (Bis(2-ethylhexyl)phthalat) aus einer PVC-Folie in unterschiedliche Simulanzlösungen sowie die Migration von verschiedenen pflanzlichen Polyphenolen in die PVC-Folie untersucht. Solchen Polyphenolen werden gesundheitsfördernde Wirkungen nachgesagt. Daher sollte die Migration dieser Substanzen als Modell für viele andere, möglicherweise bioaktive Lebensmittelinhaltsstoffe untersucht werden. Die Migrationsprüfungen wurden mit Migrationszellen durchgeführt. Die Kontaktdauer betrug 24 Stunden, die Kontakttemperatur 40 °C. Weiterhin wurde eine Versuchsreihe über 3, 5 und 10 Tage durchgeführt. Die Migration des DEHP wurde mittels GC/MS bestimmt. Mit der HPLC wurde die Polyphenol-Migration ermittelt. Zusätzlich wurden eine Gesamtphenolbestimmung in der aufgelösten Folie durchgeführt und IR-Spektren der Folien aufgenommen. Zwar konnte eine signifikante Migration der Polyphenole in die PVC-Folie nicht quantifiziert werden, da die instrumentellen Messergebnisse nicht eindeutig, d.h unterhalb der Nachweisgrenze waren, allerdings war eine Migration der Polyphenole bei einigen Versuchen visuell erkennbar, denn die Folien waren nach der Inkubation entsprechend den verwendeten Polyphenolen (gelb, bräunlich oder rot) gefärbt.

•Innovative, vegetarian broccoli-based bars (BBBs) have been developed.•The BBBs are organoleptically well accepted.•Different cooking methods affected the phytochemicals content.•Frying caused the most adverse effects on BBBs.•Microwaving and baking seem to be best for retaining the bioactive compounds.Recently, trend toward vegetarianism and veganism has globally increased. Effect of domestic cooking methods including microwaving, frying, frying/microwaving, steaming and baking on bioactive compounds of broccoli-based bars (BBBs) was investigated. Total phenolic content (TPC), antioxidant activity and phytochemicals were investigated. Subsequently, identification and quantification of dominant phenolics and glucosinolates were carried out. A sensory evaluation of cooked BBBs was performed, as well. Results indicate that health-promoting compounds in BBBs were significantly affected by cooking methods. Negligible change was found in TPC, whereas antioxidant activity was significantly affected. Cooking treatments except steaming and baking caused significant losses of chlorophylls, carotenoids, and flavonoids in the range of 20–51, 15–58, and 25–33%. Frying and frying/microwaving caused a loss of single phenolic compounds. Total glucosinolates content decreased significantly during frying, frying/microwaving, steaming, and baking, while microwaving did not. The presented data might be helpful for selecting the optimum processing conditions for innovative BBBs.

A variety of Cistus incanus products and thereof a majority of herbal teas are offered by manufacturers despite a classification as Novel Food. For a re-evaluation of this legal status, a characterization of bioactive ingredients will provide data. These teas consist of various compositions of plant parts and particle sizes. Whereas some include high leaf contents with a small particle size, others mainly consist of woody stem parts. For the consumer it is of interest which product yields the highest concentrations of bioactive phenolic compounds in the final infusions. In this study, four commercially available samples were divided into leaves and stems. Additionally, one sample was reconstituted in three mixtures of these plant parts. The amount of wood was determined by cellulose concentration. The aim was to estimate the influence of the plant parts on the concentration of phenolic compounds, which were identified by LC-DAD-ESI-MS/MS and quantitated by LC-DAD. Furthermore, one herbal tea was separated into six fractions with different particle sizes to investigate the influence of particle size on the extractability of phenolic compounds. On basis of the results, the highest concentrations of bioactive compounds in the infusions were yielded when leafy parts with a small particle size were used for brewing.

The applicability of pectinases and cellulases as soft hydrolysing agents on flavonol glycosides was investigated for identification and quantification of flavonol aglycons in cactus pear fruit. Freeze-dried samples of cactus pear fruit’s peel (cactus pear peels) and onions were treated with commercial pectinase and cellulase preparations at 50 °C for different time periods (up to 16 h). Additionally isorhamnetin-3-O-rutinoside and quercetin-3,4′-O-diglucoside were used as model compounds. In parallel, samples of cactus pear peels and onions were treated by the traditional acidic hydrolysis using HCl. Following hydrolysis, flavonols were characterised using HPLC–DAD. Enzymatically, all model compounds and plant material were highly hydrolysable. Flavonol glycosides were completely hydrolysed after 16 h (cactus pear) and 4 h (onion), respectively. While the acidic hydrolysis caused degradation of the flavonols and produced protocatechuic acid as a degradation product, the enzymatic hydrolysis gave gentle effects and did not produce any protocatechuic acid at all.

Flavonoids have gained much attention because of their proposed positive effects for human health. Onions are a rich source of flavonoids, consisting mainly of the major flavonols quercetin-3,4′-O-diglucoside (QDG) and quercetin-4′-O-monoglucoside (QMG) in the bulb and the aglycone quercetin in the outer scales. In this study, distribution of these three flavonoids was determined in 16 onion cultivars (Allium cepa) using HPLC–DAD. Three different parts of the onion bulb, the inner layers, the middle layers and the outer scales were analysed. The analysis showed varietal differences in the selected onion cultivars. The cultivars with the highest total flavonoid content were the red skinned ‘Red Baron’ and the yellow skinned cultivars ‘Ailsa Craig’ and ‘Prilep’. The distribution of the total flavonoid content in the different parts of the onion bulb showed the following order: middle layers > outer scales > inner layers. In the inner layers QDG was the major flavonoid, while in the middle layers QDG and QMG were in equal amounts. In the outer scales quercetin was the major flavonoid prior to QMG.

The influence of genotype and climatic factors, e.g. mean temperature and mean global radiation level, on the antioxidant activity of kale was investigated. Therefore, eight kale cultivars, hybrid and traditional, old cultivars, were grown in a field experiment and harvested at four different times. In addition to the investigation of the total phenolic content, the overall antioxidant activity was determined by TEAC assay and electron spin resonance spectrometry. A special aim was to characterize the contribution of single flavonoids to the overall antioxidant activity using an HPLC−online TEAC approach. The antioxidant activity and the total phenolic content were influenced by the genotype and the eco-physiological factors. The HPLC−online TEAC results showed that not all flavonol glycosides contribute to the overall antioxidant activity in the same manner. Taking the results of the structural analysis obtained by HPLC−ESI−MSn into account, distinct structure−antioxidant relationships have been observed.