Institut für Lebensmittelchemie

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Effect of the Mediterranean diet on the faecal long-chain fatty acid composition and intestinal barrier integrity: An exploratory analysis of the randomised controlled LIBRE trial
(2024) Seethaler, Benjamin; Basrai, Maryam; Neyrinck, Audrey M.; Vetter, Walter; Delzenne, Nathalie M.; Kiechle, Marion; Bischoff, Stephan C.
We recently showed that adherence to the Mediterranean diet increased the proportion of plasma n-3 PUFA, which was associated with an improved intestinal barrier integrity. In the present exploratory analysis, we assessed faecal fatty acids in the same cohort, aiming to investigate possible associations with intestinal barrier integrity. Women from the Lifestyle Intervention Study in Women with Hereditary Breast and Ovarian Cancer (LIBRE) randomised controlled trial, characterised by an impaired intestinal barrier integrity, followed either a Mediterranean diet (intervention group, n 33) or a standard diet (control group, n 35). At baseline (BL), month 3 (V1) and month 12 (V2), plasma lipopolysaccharide-binding protein, faecal zonulin and faecal fatty acids were measured. In the intervention group, faecal proportions of palmitoleic acid (16:1, n-7) and arachidonic acid (20:4, n-6) decreased, while the proportion of linoleic acid (18:2, n-6) and α linoleic acid (18:3, n-3) increased (BL-V1 and BL-V2, all P < 0·08). In the control group, faecal proportions of palmitic acid and arachidic acid increased, while the proportion of linoleic acid decreased (BL-V1, all P < 0·05). The decrease in the proportion of palmitoleic acid correlated with the decrease in plasma lipopolysaccharide-binding protein (ΔV1-BL r = 0·72, P < 0·001; ΔV2-BL r = 0·39, P < 0·05) and correlated inversely with adherence to the Mediterranean diet (Mediterranean diet score; ΔV1-BL r = –0·42, P = 0·03; ΔV2-BL r = -0·53, P = 0·005) in the intervention group. Our data show that adherence to the Mediterranean diet induces distinct changes in the faecal fatty acid composition. Furthermore, our data indicate that the faecal proportion of palmitoleic acid, but not faecal n-3 PUFA, is associated with intestinal barrier integrity in the intervention group.
Effect of wort boiling on volatiles formation and sensory properties of mead
(2022) Starowicz, Małgorzata; Granvogl, Michael
Mead is an alcoholic beverage based on bee honey, which can be prepared in different variations such as modified honey-water compositions, the addition of spices, and the use of different yeast strains. Moreover, the technological process of mead production such as the step of wort preparation (with or without boiling of wort before fermentation) can be modified. All these factors might have a significant impact on the formation of aroma-active compounds, and therefore, sensory acceptance by consumers. High vacuum distillation, using the so-called solvent assisted flavor evaporation (SAFE) technique, or headspace-solid phase microextraction (HS-SPME) were applied for the isolation of the odorants. A sensory profile was used to monitor the changes in the aroma of the mead samples. Twenty-eight aroma-active compounds were detected during aroma extract dilution analysis (AEDA) based on gas chromatography-olfactometry (GC-O) and were finally identified by gas chromatography-mass spectrometry (GC-MS) using authentic reference compounds, including methyl propanoate, methyl 3-(methylthio)propanoate, and methional, all of them were identified for the first time in mead. Compounds with high flavor dilution (FD) factors were quantitated via stable isotope dilution analysis (SIDA) and revealed ethyl acetate (16.4 mg/L) to be the most abundant volatile compound, increasing to 57 mg/L after wort boiling, followed by ethyl hexanoate (both 1.2 mg/L). Furthermore, key aroma compounds were esters such as ethyl hexanoate, ethyl octanoate, and ethyl 3-methylbutanoate. The sensory panel evaluated ethanolic, honey-like, clove-like, sweet, and fruity notes as the main aroma descriptors of mead. The significant change in sensory evaluation was noted in the sweet odor of the heat-treated mead.
Risk assessment of caffeine and epigallocatechin gallate in coffee leaf tea
(2022) Tritsch, Nadine; Steger, Marc C.; Segatz, Valerie; Blumenthal, Patrik; Rigling, Marina; Schwarz, Steffen; Zhang, Yanyan; Franke, Heike; Lachenmeier, Dirk W.
Coffee leaf tea is prepared as an infusion of dried leaves of Coffea spp. in hot water. It is a traditional beverage in some coffee-producing countries and has been authorized in 2020 within the European Union (EU) according to its novel food regulation. This article reviews current knowledge on the safety of coffee leaf tea. From the various ingredients contained in coffee leaves, only two were highlighted as possibly hazardous to human health, namely, caffeine and epigallocatechin gallate (EGCG), with maximum limits implemented in EU legislation, which is why this article focuses on these two substances. While the caffeine content is comparable to that of roasted coffee beans and subject to strong fluctuations in relation to the age of the leaves, climate, coffee species, and variety, a maximum of 1–3 cups per day may be recommended. The EGCG content is typically absent or below the intake of 800 mg/day classified as hepatotoxic by the European Food Safety Authority (EFSA), so this compound is suggested as toxicologically uncritical. Depending on selection and processing (age of the leaves, drying, fermentation, roasting, etc.), coffee leaf tea may exhibit a wide variety of flavors, and its full potential is currently almost unexplored. As a coffee by-product, it is certainly interesting to increase the income of coffee farmers. Our review has shown that coffee leaf tea is not assumed to exhibit risks for the consumer, apart from the well-known risk of caffeine inherent to all coffee-related beverages. This conclusion is corroborated by the history of its safe use in several countries around the world.
Exploration of surfactin production by newly isolated Bacillus and Lysinibacillus strains from food‐related sources
(2022) Akintayo, Stephen Olusanmi; Treinen, Chantal; Vahidinasab, Maliheh; Pfannstiel, Jens; Bertsche, Ute; Fadahunsi, I.; Oellig, Claudia; Granvogl, Michael; Henkel, Marius; Lilge, Lars; Hausmann, Rudolf
As a lipopeptide (LP), surfactin exhibits properties, such as emulsifying and dispersing ability, which are useful in food industry. Discovery of new LP‐producing strains from food sources is an important step towards possible application of surfactin in foods. A total of 211 spore‐forming, Gram‐positive, and catalase‐positive bacterial strains were isolated from fermented African locust beans (iru) and palm oil mill effluents in a screening process and examined for their ability to produce surfactin. This was achieved by a combination of methods, which included microbiological and molecular classification of strains, along with chemical analysis of surfactin production. Altogether, 29 isolates, positive for oil spreading and emulsification assays, were further identified with 16S rDNA analysis. The strains belonged to nine species including less commonly reported strains of Lysinibacillus, Bacillus flexus, B. tequilensis, and B. aryabhattai. The surfactin production was quantitatively and qualitatively analysed by high‐performance thin‐layer chromatography and liquid chromatography‐mass spectrometry (LC–MS). Confirmation of surfactin by MS was achieved in all the 29 strains. Highest surfactin production capability was found in B. subtilis IRB2‐A1 with a titre of 1444·1 mg L−1.
A fast gas chromatography coupled with electron capture negative ion mass spectrometry in selected ion monitoring mode screening method for short‐chain and medium‐chain chlorinated paraffins
(2022) Schweizer, Sina; Schulz, Tobias; Vetter, Walter
Rationale Chlorinated paraffins (CPs) are a group of anthropogenic pollutants that consist of complex mixtures of polychlorinated n-alkanes of different chain lengths (~C10 to C30). Persistence, bioaccumulation, toxicity, and long-range transport of short-chain chlorinated paraffins (SCCPs, C10- to C13-CPs) have prompted their classification as persistent organic pollutants (POPs) by the Stockholm Convention in 2017. Due to the varying chain lengths and chlorination degrees, quantification of SCCPs and medium-chain chlorinated paraffins (MCCPs, C14- to C17) using gas chromatography coupled with electron capture negative ion mass spectrometry in selected ion monitoring mode (GC/ECNI-MS-SIM) is not only challenging but also very time consuming. In particular, up to eight GC runs per sample are required for the comprehensive GC/ECNI-MS-SIM quantification of SCCPs and MCCPs. These efforts are high especially if the samples do not contain CPs above the limit of detection (LOD), subsequently. Methods We developed a semi-quantitative and sensitive method for the examination of SCCPs and MCCPs in one GC run. This GC/ECNI-MS-SIM screening method was based on the recording of Cl− (m/z 35 and 37), Cl2− (m/z 70 and 72), and HCl2− (m/z 71 and 73) isotope ions and evaluation of the ratios between them. Results Correctness of the results of the screening method was verified by analysis of edible oils with and without CPs, CP standards, as well as a technical CP mixture. Polychlorinated biphenyls (PCBs) and other polyhalogenated aromatic compounds, as well as brominated flame retardants, do not form all of the fragment ions analyzed by the screening method. Conclusions After the screening, only CP-positive samples may need to be measured in detail. Measurement time will already be gained in the case of ~10% samples without CPs.
High levels of halogenated natural products in large pelagic fish from the Western Indian Ocean
(2021) Wu, Qiong; Munschy, Catherine; Aminot, Yann; Bodin, Nathalie; Vetter, Walter
Concentrations, profiles and muscle-liver distribution of halogenated natural products (HNPs) and anthropogenic persistent organic pollutants (POPs) were investigated in five large pelagic fish species and one smaller planktivore fish species from the Western Indian Ocean. Analysis of swordfish muscle from the Seychelles revealed the predominance of HNPs, with the highest concentrations found for 2′-methoxy-2,3′,4,5′- tetraBDE (2′-MeO-BDE 68 or BC-2), 6-methoxy-2,2′,4,4′- tetraBDE (6-MeO-BDE 47 or BC-3) and 2,3,3′,4,4′,5,5′-heptachloro-1′-methyl-1,2′-bipyrrole (Q1), along with varied contributions of further HNPs. The mean concentration of ∑HNPs (330 ng/g lw) was one or two orders of magnitude higher than ∑DDTs (60 ng/g lw) and ∑PCBs (6.8 ng/g lw). HNPs (BC-2, BC-3 and Q1) were also predominant in individual samples of three tropical tuna species from the Seychelles and from other regions of the Western Indian Ocean (Mozambique Channel, off Somalia and Chagos Archipelago). Non-targeted gas chromatography coupled with electron capture negative ion mass spectrometry operated in the selected ion monitoring mode (GC/ECNI-MS-SIM) analysis of one swordfish sample indicated low abundance of rarely reported HNPs (three hexachloro-1′-methyl-1,2′-bipyrrole (Cl6-MBP) isomers and pentabromo-1,1′-dimethyl-2,2′-bipyrroles (Br5-DBP)) but no further abundant unscreened polyhalogenated compounds.
Microbial incubations of 8‐phenyloctanoic acid and furan fatty acids in rumen fluid
(2022) Wiedmaier‐Czerny, Nina; Blumberg, Olga; Schulz, Tobias; Kemmler, Franziska; Titze, Natascha; Wild, Katharina; Vetter, Walter
Aims: The digestive tract of ruminants is specialized in the digestion of various plant components. One of the largest parts of the stomach is the so-called rumen, which contains a large number of micro-organisms that may degrade or modify fatty acids, for example by β-oxidation, chain elongation and/or hydrogenation. Methods and Results: Here we performed incubation experiments with less com- mon fatty acids by in vitro incubations with rumen fluid of fistulated cows for 24 h. Sample extracts were analysed by gas chromatography with mass spectrometry. As substrates, we selected one phenyl fatty acid and four furan fatty acids (FuFAs). All studied fatty acids were degraded by β-oxidation (two or three chain-shortening steps) while chain elongation or saturation of the aromatic part (terminal phenyl or central furan moiety) was not observed in any case. Conclusions: The percentage of β-oxidation products was low, especially in the case of the FuFAs. This could be due to the rather long carbon number of FuFAs (19–22 carbon atoms). In addition, compound-specific differences in the degradation rates were observed in our experiments. Significance and Impact of the Study: Our results produce evidence that FuFAs, which are valuable antioxidants that are known to be present in various feed items of the cow, can be effectively passed on the rumen into the milk.
Geometrical and positional isomers of unsaturated furan fatty acids in food
(2022) Müller, Franziska; Hammerschick, Tim; Vetter, Walter
Furan fatty acids (FuFA) are important antioxidants found in low concentrations in many types of food. In addition to conventional FuFA which normally feature saturated carboxyalkyl and alkyl chains, a few previous studies indicated the FuFA co‐occurrence of low shares of unsaturated furan fatty acids (uFuFA). For their detailed analysis, the potential uFuFA were enriched by centrifugal partition chromatography (CPC) or countercurrent chromatography (CCC) followed by silver ion chromatography from a 4,7,10,13,16,19‐docosahexaenoic acid ethyl ester oil, a 5,8,11,14,17‐eicosapentaenoic acid ethyl ester oil and a latex glove extract. Subsequent gas chromatography with mass spectrometry (GC/MS) analysis enabled the detection of 16 individual uFuFA isomers with a double bond in conjugation with the central furan moiety. In either case, four instead of two uFuFA isomers previously reported in food, respectively, were detected by GC/MS. These isomers showed characteristic elution and abundance patterns in GC/MS chromatograms which indicated the presence of two pairs of cis/trans‐isomers (geometrical isomers).
Acrocomia aculeata fruits from three regions in Costa Rica: An assessment of biometric parameters, oil content and oil fatty acid composition to evaluate industrial potential
(2020) Alfaro-Solís, Jose David; Montoya-Arroyo, Alexander; Jiménez, Víctor M.; Arnáez-Serrano, Elizabeth; Pérez, Jason; Vetter, Walter; Frank, Jan; Lewandowski, Iris
Due to increased global demand for vegetable oils, diversification of the supply chain with sustainable sources is necessary. Acrocomia aculeata has recently gained attention as a multi-purpose, sustainable crop for oil production. However, the information necessary for effective selection of promising varieties for agricultural production is lacking. The aim of this study was to assess variability in fruit morphology and oil composition of individual Acrocomia aculeata plants growing wild in different climatic regions of Costa Rica. Fruits at the same ripening stage were collected at three locations, and biometric features, oil content, fatty acid composition of oils from kernels and pulp, as well as fiber composition of husks were determined. Biometric parameters showed high variability among the regions assessed. Moreover, oil content and relative proportions of unsaturated fatty acids were higher at the most tropical location, whereas lauric acid content was lowest under these conditions, indicating a potential environmental effect on oil composition. Pulp oil content correlated positively with annual precipitation and relative humidity, but no clear relation to temperature was observed. The oil chemical composition was similar to that reported for Elaeis guineensis, suggesting that Acrocomia aculeata from Costa Rica may be a suitable alternative for industrial applications currently based on African palm oil. Analysis of husks as a coproduct revealed the possibility of obtaining materials with high lignin and low water and ash contents that could be used as a solid bioenergy source. In conclusion, Acrocomia aculeata oil is a promising alternative for industrial applications currently based on African palm oil and byproducts of its oil production could find additional use as a renewable energy source.
LC‐Orbitrap‐HRMS determination of two novel plastochromanol homologues
(2023) Hammerschick, Tim; Graf, Jana; Vetter, Walter
Scope: The antioxidant plastochromanol-8 (PC-8) is a tocochromanol which differs from γ-tocotrienol in having an unsaturated side chain of eight instead of three isoprene units. The recent isolation of PC-8 from flaxseed oil indicates the additional presence of lower shares of two previously unknown homologues, plastochromanol-7 (PC-7) and plastochromanol-9 (PC-9), which feature seven and nine isoprenoid units respectively on the γ-chromanol backbone. Here, a fast LC-Orbitrap-HRMS method is applied for the determination of PC-7 and PC-9 in seven plant oils and a plant extract. Methods and results: The presence of PC-7, PC-8, and PC-9 is confirmed in all eight investigated samples by LC-Orbitrap-HRMS analysis after saponification. PC-8 amounts of ≈315–350 mg kg−1 in two flaxseed oils, ≈75 mg kg−1 in rapeseed oil, ≈38 mg kg−1 in camelina oil, ≈80–120 mg kg−1 in two mustard oils, ≈90 mg kg−1 in candle nut oil, and ≈900 mg kg−1 dry weight in Cecropia leaves are determined by quantification. Semi-quantification of PC-7 and PC-9 indicated the presence of ≈0.1–1% of PC-7 and PC-9 in varied relative ratios. Conclusion: The novel plastochromanol homologues are of particular interest to researchers with focus on vitamin E and other tocochromanols because of their unexplored bioactivity.
Silver ion chromatography enables the separation of 2‐methylalkylresorcinols from alkylresorcinols
(2023) Hammerschick, Tim; Vetter, Walter
Alkylresorcinols (∑ARs) is the generic term for a highly varied class of lipids found mainly in cereals. These bioactive compounds consist mainly of 5‐alkylresorcinols (ARs), which differ in length, unsaturation, and substituents on the alkyl side chain on C‐5. In addition, 2‐methyl‐5‐alkylresorcinols (mARs) are scarcely studied minor compounds that are supposed to exist with the same structural diversity. In the first step, ∑ARs were enriched by solid‐phase extraction from wheat grain and quinoa seed extracts. The subsequent application of silver ion chromatography (SIC), silica gel, coated with 20% AgNO3, then deactivated with 1% water) enabled an unprecedented full separation of saturated mARs from conventional ARs. Specifically, saturated mARs were eluted with n‐hexane/ethyl acetate (92:8, v/v), and conventional ARs with n‐hexane/ethyl acetate (80:20, v/v). The unpreceded separation indicated that the SIC method could be useful not only for separations according to the degree of unsaturation, but also in the case of steric hindrance by additional (alkyl) substituents. Continued fractionation enabled the collection of unsaturated ARs in wheat and quinoa extracts. In this way, 35 ∑ARs (including five mARs) were detected by gas chromatography/mass spectrometry analysis in wheat and 45 ∑ARs (including 21 mARs) in quinoa. These included several low abundant and partly unknown ∑ARs such as 1,3‐dihydroxy‐5‐tricosadienylbenzene.
Furan fatty acid amounts and their occurrence in triacylglycerols of white asparagus (Asparagus officinalis) from the German market
(2023) Müller, Franziska; Bauer, Vanessa; Vetter, Walter
Vegetables including asparagus contain a wide range of fatty acids, mainly stored in triacylglycerols. One class of interesting minor fatty acids is the family of furan fatty acids (FuFAs) because of their antioxidant properties. Since FuFAs have not been studied previously in asparagus (Asparagus officinalis L.), we developed and applied a simplified method for their analysis in 20 fresh and three preserved samples. Four FuFAs were detected with clear dominance of the dimethyl‐substituted FuFAs (D‐FuFAs) 11D5 and 9D5 as well as small amounts of the monomethyl‐substituted FuFAs (M‐FuFAs) 11M5 and 9M5. The total amounts of FuFAs in fresh white asparagus ranged from 1.4 to 4.6 mg/100 g dry weight (mean 3.0 mg/100 g dry weight). Subsequent LC‐Q‐Orbitrap‐HRMS measurements enabled the detection of 22 different FuFA‐containing TAGs. These were predominantly found together with one or two polyunsaturated fatty acid.
Previtamin D2, vitamin D2, and vitamin D4 amounts in different mushroom species irradiated with ultraviolet (UV) light and occurrence of structurally related photoproducts
(2024) Sommer, Katrin; Hillinger, Marissa; Vetter, Walter
Mushrooms are rich in ergosterol and ergosta‐5,7‐dienol, which can be partly converted into vitamin D2 and D4 through ultraviolet (UV) light exposure. Typically, mushrooms have very low vitamin D contents, but it can be increased by UV irradiation. This process generates additional photoisomers scarcely studied in mushrooms due to analytical challenges. Here, we developed a new solid phase extraction (SPE) method to separate vitamin D2, vitamin D4, and other tri‐ and pentacyclic photoisomers from the much higher abundant ergosterol. Subsequent GC/MS analysis enabled the detection of ten photoisomers in eight UV‐treated mushroom species, including vitamin D2 (previtamin D2, tachysterol2, two suprasterol2 and trans‐vitamin D2 isomers) and vitamin D4 (previtamin D4). Quantitated vitamin D2 contents of 10–540 µg/100 g dry weight agreed well with the sparse literature data available for the investigated mushroom species. In addition, previtamin D2 (nd–1950 µg/100 g dry weight) and vitamin D4 (10–140 µg/100 g dw) were quantified in the samples. The content and photoproduct compositions varied considerably between different mushroom species. Practical applications: The novel SPE method can be applied to study the vitamin D and photoisomer content of mushrooms.
Investigations into heat- and light-induced terpene modifications in essential oils
(2023) Bitterling, Hannes; Vetter, Walter
Essential oils belong to secondary plant metabolites, with terpenoids and phenylpropanoids being among the main constituents in terms of quantity. Due to their lipophilic character and high volatility, they are mainly obtained by steam distillation. Citrus essential oils (agrumen oils) are an exception , since they are usually extracted from the peels by means of pressing, whereby less volatile components such as coumarins and furocoumarins are also introduced. Due to their odor and taste-giving properties, essential oils are used in the food, beverage, and cosmetics industries. In addition, due to a wide range of pharmacological properties, they are used in phytotherapy as well as in aromatherapy. However, most essential oils are highly susceptible to oxidation, polymerization, dehydrogenation, and isomerization reactions in the presence of atmospheric oxygen, light, and at high temperatures. The resulting organoleptic changes usually lead to a significant quality reduction. The formation of terpene hydroperoxides is another problem, as these are suspected of causing intolerances such as redness and itching in 1-3% of the European population upon contact with the skin. The detection of these chemical changes forms an integral part of quality control and can be prevented as far as possible by suitable production, transport, and storage strategies. Due to their volatility, essential oils are mainly analyzed by gas chromatography. However, due to their instability, the detection of hydroperoxides places considerable demands on common analytical methods. For this reason, a novel spectrophotometric method for the detection of peroxides and hydroperoxides in terpenes and essential oils was developed (paper 1). The oxidation of N-N-dimethyl-p-phenylenediamine by peroxides yielding an intensely red-colored cation (Wursters red) allowed colorimetric detection and quantitation of even smallest amounts (LOD: 0.5 ppm). The minimal sample amount of only a few milligrams, as well as simple and fast performance predestine this method for daily laboratory routine (paper 1). Among plant terpenoids, the monoterpene R-(+)-limonene is very widespread. Thus, it is not only found in citrus oils but also of in caraway oil, where its proportion amounts to almost 50%. To investigate the storage stability, R-(+)-limonene, S-(+)-carvone, different caraway oils, and the corresponding caraway seeds were stored in desiccators at 25 °C and 40 °C for eighteen months (paper 2). The samples were analyzed monthly by GC/MS and GC/FID, as well as HPLC/DAD-MS/MS. This showed that the comparison of seed, isolated essential oil, and pure substance, whichhad not been considered in storage studies so far, was of extraordinary importance. Here, both the plant matrix and the essential oil had a protective effect on individual terpenes and delayed their degradation (paper 2). Further, a clear difference between photo-oxidation and autoxidation was observed. Light-induced oxidation of terpenes primarily resulted in the formation of hydroperoxides, whereas autoxidation led to a variety of compounds such as alcohols, ketones, and epoxides. Thus, the secondary products can serve as specific markers for conclusions about the preload and quality of essential oils. In the study presented in paper 3, further photo-oxidation experiments were conducted with beta-pinene, R-(+)-limonene, and gamma-terpinene, with added furocoumarins. Furocoumarins can absorb UV-A light in the range of 320 – 380 nm and enter an energetically excited state. This energy difference between the ground state and excited state can be dissipated again by the emission of fluorescent and phosphorescent light. In this process, short-wave energy-rich UV light is converted into lower-energy visible light (bathochromic shift). For this reason, the UV light-induced degradation of the terpenes beta-pinene, R-(+)-limonene, and gamma-terpinene could be significantly reduced by adding 5% each of xanthotoxin, bergapten, bergaptol, and bergamottin. The effect of adding bergaptol was most pronounced in the photooxidation of gamma-terpinene (paper 3). Consequently, in citrus essential oils from which the natural furocoumarins had been previously removed, irradiation with UV light resulted in a strong degradation of the terpenes. This process could be markedly reduced by the re-addition of 5% of the previously removed plant-specific furocoumarins (paper 4). In summary, it can be concluded that not only the plant matrix and the essential oil as a multicomponent mixture but also potential interactions with other substances forming part of the essential oil such as furocoumarins may significantly slow down the oxidation of terpenoids.
Einfluss moderner Pflanzenschutzmittel auf die Mobilität von POP-belasteten Agrarflächen am Beispiel von DDT : ein Feldversuch
(2023) Neitsch, Julia Simone; Vetter, Walter
Due to their recalcitrance, the chloropesticide DDT and its structurally related compounds (DDX) are difficult to degrade. Consequently, farmers are still frequently confronted with DDX contamination in their fields that was left over from the 1960s. This problem is particularly prevalent in contaminated soils that are intended to cultivate plants of the Cucurbitaceae family. These plants release so-called root exudates, which function as natural surfactants that mobilize the DDX present in the soils. Furthermore, surfactants are a common constituent of modern plant protection product (PPP) formulations, which can likewise cause DDX mobilization. The higher mobility of DDX caused by these surfactants can result in the absorption and accumulation of chlorinated pesticides in plants. The side effects of such surfactant-containing PPP formulations have historically been overlooked in the context of standard spraying protocols. The potential mobilization of DDX in soils and its accumulation in Cucurbita pepo due to the surfactants present in standard PPPs formulations was investigated using two field trials. One field was treated with a conventional PPP, while the other was treated with a biological PPP; a control field was left untreated, within which pumpkins were cultivated. Soil samples were taken before and after the application of PPP. The DDX content was subsequently determined in extracts from the soil phase samples and soil water fractions. The background DDX contamination of the soils was comparable in all three test fields. The comparative evaluation showed that the field treated with the biological PPP formulation exhibited a considerable increase in DDX mobility compared to the untreated and conventionally cultivated areas (Paper 1). An analysis of its respective water fraction revealed that it was more contaminated with DDX than the control treatments. This increase suggests a higher bioavailability that can be traced back to the presence of surfactants and oils in the PPP formulations (Paper 1). This higher bioavailability may have been accompanied by an increase in the DDX uptake of the cultivated plants. Furthermore, it was found that treatment with specific formulations of emulsifiable concentrates (EC) promoted DDX mobilization. This mobilizing effect was most likely due to the differing composition of the surfactant and proportions of oils in the PPPs. The second field test focused on differential DDX accumulation in Cucurbita pepo cv. Howden by different PPPs. Fields were treated with PPP in accordance with the official spraying plans and regulations set out by the Federal Ministry of Food and Agriculture (BMEL). Samples from the pumpkin plants roots, shoots, as well as the pumpkins themselves were taken during the cultivation period. The DDX content in the roots from the control fields and the fields with conventional PPP treatments remained virtually unchanged; however, the DDX content increased in the biologically treated area (Paper 2). The pumpkin shoots did not exhibit any increases in DDX concentration during the growing phase regardless of the field sampled. However, an increased DDX content was detected in the shoots of the plants in all test fields shortly before harvesting. At the end of the growing phase, fruits from the biologically treated area showed a higher DDX content than those from the control and conventionally treated areas. In addition, the most critical DDT metabolite, DDE, was found to have been transported to distant parts of the plant, while DDD was detectable in the roots and shoots but not in the fruits of the pumpkins (Paper 2). An assessment of the results of both experiments confirmed a direct correlation between DDX mobilization in the soil and plant uptake. In addition, the bioaccumulation factors of the biologically treated areas were markedly higher than those seen in the conventionally treated and control areas. The results of the field trials show that the mobilization of DDT, as well as the likely mobilization of other lipophilic contaminants, can become problematic for farmers using surfactant-containing EC formulations. However, this observation also provides opportunities for improved phytoremediation by applying EC formulations with high mobilization potentials. The field trials indicate that the mobilizing effects of DDT prompted by EC mixtures depend on the surfactant content in the PPP formulations as well as environmental conditions such as soil conditions, soil water content, and precipitation. Unravelling the optimal range of surfactant-rich formulations and environmental conditions could lead to a promising strategy for soil phytoremediation.
Development of strategies for the prioritization of organic trace substances in water by effect-directed analysis
(2020) Stütz, Lena; Schwack, Wolfgang
The protection of the aquatic environment and the supply of clean drinking water to people all over the world are central challenges of our time. Monitoring of the aquatic environment and the input of anthropogenic trace substances into it is therefore very important. However, since aquatic environmental samples often consist of complex substance mixtures, their characterization and evaluation is very demanding. By using generic target analysis methods, selected known anthropogenic trace substances can be detected and quantified very sensitively. For the detection of previously unknown substances, non-target analysis methods have been increasingly used in recent years. However, these methods do not provide information on the relevance of the anthropogenic trace substances occurring in water. In this context, especially all those trace substances are regarded as relevant from which a harmful effect on humans or water organisms is to be expected. For the detection of such effective substances, effect-directed analysis (EDA) can be used. In EDA, a bioassay is combined with a fractionation method and subsequent chemical analysis, the aim being to identify the bioactive substance. The separation method used in this work is high-performance thin-layer chromatography (HPTLC). After chromatography, the bioassay is performed directly on the HPTLC plate. If an effective zone appears in the bioassay, a prioritization strategy is used to clarify the identity of the substance. Due to the complex aquatic samples, a large number of different substances in a zone must still be expected despite the applied HPTLC separation, which makes it difficult to identify the effective substance. Therefore, a strategy to simplify the identification of effective substances should be developed. The aim was to reduce the complexity by multidimensional separation in such a way that chemical analysis can be used to prioritize to a few candidates in the effective fraction. In the first part of the work, a selective two-dimensional HPTLC separation was developed to reduce the number of substances in a bioactive zone. After the first separation dimension (1D) the acetylcholinesterase inhibition assay (AChE assay) was performed and afterwards only the effective zones were extracted from the HPTLC plate. The selected effective zones were separated in a second separation dimension (2D) and the bioassay was performed again. With this 2D separation, the peak capacity could be increased by a factor of 7 compared to a 1D HPTLC gradient development. If real water samples are examined for their effects, an additional structural elucidation must be carried out to clearly identify the unknown bioactive substances. In this work, the developed 2D EDA was therefore connected to a high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (HRMS) and a non-target screening (NTS) was performed. Using three water samples(drinking water, surface water and purified sewage water) spiked with six effective substances, it was shown that the developed strategy is suitable for the identification of effective substances and that these can be recovered despite repeated extraction. When applying the developed methodology to real samples, it was also possible to assign and quantify the detected effect in several waters to the substance lumichrome and to linear alkylbenzene sulfonates. Genotoxicity is a crucial endpoint for the effect assessment of water samples. However, this endpoint with metabolic activation cannot yet be performed directly on the HPTLC plate. Since many of the genotoxic substances have an indirect genotoxic effect, i.e. they only acquire their activity after metabolic activation; this endpoint was investigated in the present work with the umu assay in the microtiter plate. However, separation with HPTLC, subsequent extraction of effective zones and non-target analysis of the extracts, should also be performed for this assay. Therefore the umu assay in the microtiter plate was integrated into the existing EDA-with-HPTLC concept. In laboratory experiments, sodium hypochlorite was added to the drug metformin in order to simulate the behavior of the substance during water treatment (chlorination). The metformin sample treated with hypochlorite was examined with the umu assay and a genotoxic effect was detected. After HPTLC separation of the chlorinated sample, zones were extracted over the entire retardation range. When the extracted zones were examined with the umu assay, the genotoxic effect could be clearly assigned to one fraction. Using high-resolution mass spectrometry, the genotoxic effect could be assigned to an already known transformation product of metformin. The HPTLC separation and extraction of the zones from the plate led to a reduction of the possible effective candidate masses by a factor of 10 and thus to a clear prioritization in HRMS analysis.
Identification of markers for dietary intake and health status by GC-MS based metabolite profiling approaches
(2020) Mack, Carina I.; Kulling, Sabine E.
Markers are compounds that can be used as indicators of an exposure, a metabolic state, or any other effect. Metabolomics and metabolite profiling approaches for marker discovery will increasingly gain significance. In the context of food, diet, and health, these approaches allow among others the identification of dietary intake markers, which can complement and verify traditional dietary assessment methods in epidemiologic studies. Consequently, the investigation of associations between diet and health status in general, and also in particular diet-related diseases will be improved. Compared to classical biomarker studies, metabolomics enables a more comprehensive investigation of clinical markers for diagnosis, prognosis and monitoring of diseases, such as type 2 diabetes mellitus. Especially, early diagnosis in pre-disease states, where symptoms are not yet evident, are of particular interest. The aim of this thesis was to evaluate the application of GC-MS based metabolite profiling approaches for the identification of markers for dietary intake and health status. In this respect, volatile organic compounds and sugar compounds were analyzed to discover marker candidates in urine and plasma samples from a cross-sectional study with 300 participants, as well as from a human intervention study with diabetic, prediabetic and healthy participants. In the past, the search for markers of dietary intake mostly focused on non-volatile metabolites. To explore the potential of the volatilome, urine samples of a cross-sectional study were analyzed aiming to exemplary identify markers of coffee consumption using an untargeted HS-SPME-GC×GC-MS method. Six marker candidates were identified from a profile of 138 volatile organic compounds with the most robust represented by 3,4-dimethyl-2,5-furandione. Moreover, the correlation with the general dietary intake data highlighted the volatilome as a particularly interesting source for the detection of new dietary markers. The chromatographic separation of sugar compounds is often insufficient due to the high structural similarities. Therefore, in most studies common and well-known sugar compounds are analyzed in human body fluids. Within the scope of this thesis, a semitargeted GC-MS sugar profiling method was developed, which revealed a more complex sugar profile, both in urine and plasma, than described so far or expected. Rare sugar compounds such as psicose and trehalose were detected. However, the knowledge about their origin and presence in urine or plasma is limited to date. Moreover, the maltose concentration in urine was shown to be dependent on sex and menopause status (pre- and post-menopausal) – a relationship with the vaginal microbiota is suggested here. In addition, the association of the urinary sugar profile with dietary intake data enabled the identification and confirmation of several new and also known marker candidates as for example, for consumption of avocado, dairy products and alcohol. The plasma sugar profiles of healthy, prediabetic and diabetic volunteers after an oral glucose tolerance test could be clearly distinguished, independent of glucose. Remarkably, a variety of sugar compounds showed marked postprandial differences dependent on health status. For example, trehalose showed a profile similar to the insulin-dependent profile of glucose. However, the origin and underlying biological mechanism for those sugar compounds remain to be elucidated. During the application of the one-dimensional GC-MS sugar profiling method to urine and plasma samples, it became evident that even more sugar compounds might be present, although in low concentrations, but were not detected due to limitations of the analytical method. Therefore, the one-dimensional method was transferred into a two-dimensional GC×GC-MS method. Improved sensitivity and separation finally enabled the detection of 84 instead of 55 sugar compounds in urine. The two-dimensional method was applied in an intervention study with apples, and revealed marker candidates for apple consumption for future validation. Overall, the results illustrate the benefit of a comprehensive analysis of sugar compounds in urine and plasma, including minor and rare sugar derivatives. The GC-MS based metabolite profiling approaches addressing the volatilome and the sugar profile, respectively, were demonstrated to be promising approaches for the identification of markers for dietary intake and health status. Future work should address the identification of unknown compounds, the adaptation of the GC×GC-MS sugar profiling method for quantitative purposes, and especially the validation of the identified marker candidates with respect to their suitability to more accurately assess dietary intake or diabetic state. High priority should also be given to the biochemical mechanisms and the origin of the compounds as well as their physiological or pathophysiological function in human metabolism.
Determination of potentially hazardous oxidation products in cosmetics containing lanolin or 1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)ethanone (OTNE)
(2019) Schrack-Belschner, Sonja Miriam Irmgard; Schwack, Wolfgang
Cosmetic products are important consumer goods in the "non-food" sector, which should not have negative effects of the human health. Critical compounds, however, can be formed by the oxidation of an unsaturated organic compound.Thereby formed oxidation products with potentially adverse properties are well known from the food sector. As the oxidation of cosmetic ingredients, however, has less been studied, the oxidation of selected cosmetic ingredients with respect to the formation of potentially critical compounds was investigated within the framework of this thesis.The oxidation of cholesterol to various cholesterol oxidation products (COPs) was investigated in a first step.COPs are known from the food sector and are suspected of causing certain diseases such as arteriosclerosis.Cosmetic products have not yet been tested for COPs, although a versatile ingredient used only in cosmetic products, lanolin, contains above-average levels of the cholesterol, which is the precursor.Total COPs contents in cosmetics containing lanolin, namely lip care products, fat creams and ointments for nursing women were in the low percent range (up to 3 %) and were thus several orders of magnitudes higher than the contents found in food.The oxidation of fragrances was studied in the second part of this work.The subject is not new as the oxidation of terpenes to contact allergens has been studied in earlier studies. The oxidation of other fragrances was hardly investigated. In order to extend our knowledge in this field, the oxidation of a synthetic fragrance frequently used in perfumes, octahydro tetramethyl naphthalenyl ethanone (OTNE) was studied. Obtained results indicated that peroxides of OTNE were formed during oxidation.It was found out that the OTNE oxidation even occurs, when perfumes are stored indoors under normal temperature and light conditions. An in-vivo test showed that OTNE oxidation can be expected on the skin after application of a perfume.
Development of a planar yeast estrogen screen as screening tool for estrogen active compounds
(2018) Schick, Dinah; Schwack, Wolfgang
Substances that disrupt or impair the hormone system (endocrine system) or that show an irreversible influence on it are referred to as endocrine disruptors or xenohormones. Concerning this, also estrogen active compounds (EAC) are endocrine disruptors, that are under suspicion of being involved in the formation of tumors or to induce disruption during development and reproduction, and are, for example, blamed for being responsible for the feminization of fish. At this, EAC can be natural (human, phytoestrogens) but also synthetic substances, which are discharged to the environment by humans (e.g. pharmaceuticals, pesticides, additives). Regarding the ubiquitous presence of EAC, suitable methods for the analysis of EAC are required. An in vitro method for the determination of EAC is the YES assay (yeast estrogen screen) that is executed in liquid solutions in microtiter plates and that works with genetically modified yeasts, which contain the human estrogen receptor (hER) and a reporter gene encoding for the enzyme beta-galactosidase. In presence of EAC, the enzyme is produced and subsequently cleaves a substrate that is used to measure the receptor activity and thus the estrogenic activity. The transfer of the YES assay to high-performance thin-layer chromatography (HPTLC) was successfully demonstrated and advanced, thus resulting in the combination of a chromatographic separation of analytes and the detection of EAC using genetically modified yeast cells directly on the HPTLC plate (HPTLC planar yeast estrogen screen, HPTLC-pYES). Usually, the substrate 4-methylumbelliferyl-beta-D-galactopyranoside is used for pYES, releasing blue fluorescing 4-methylumbelliferone (MU) after enzymatic cleavage. Various matrices, however, often contain a plenty of different components, partly showing native fluorescences (blue, red), why the detection of the blue fluorescing MU can be interfered. By applying the substrate resorufin-beta-D-galactopyranoside (RGP) and by using automated devices, the RGP-pYES as fast screening tool for EAC was developed and successfully applied to waste water samples and extracts of hops pellet samples. A screening method using HPTLC simultaneously represents a planar clean-up, why samples do not have to undergo complex steps of sample preparation or purification. The chromatographic separation in combination with the detection of estrogenic activity using genetically modified yeasts directly on the plate allowed the detection, the determination and the identification of single EAC. Using RGP, which releases orange fluorescing resorufin after enzymatic cleavage as positive signal of estrogenic activity, enabled a clear differentiation between fluorescences due to estrogenicity and the native fluorescence of sample components. Application of the RGP-pYES to spiked water samples and sewage samples showed high recovery rates and a good precision, and thus the applicability of the method as screening tool for environmental samples. By means of suitable evaluation methods, additionally the generation of dose-response curves of known and unknown EAC and thus the generation of so-called logit-log plots was possible. This enabled the determination of estradiol equivalent factors of known EAC as well as the determination of estradiol equivalent concentrations and amounts, respectively, of known and unknown EAC in liquid and solid samples. Thus, the possibility to estimate the estrogenic potential of a sample or single sample components was given. The coupling of pYES to mass spectrometry additionally allowed the identification of unknown EAC, demonstrated exemplarily by investigation of extracts of hops pellet samples, in which the only detected EAC in the hops extracts was identified as prenylnaringenin. Since the method uses a planar system, the pYES advantageously reveals all chromatographically separated sample components at one look and, as bioassay, additionally detects a possible estrogenic activity (activity at the hER) of single substances, while a differentiation between native occurring fluorescences of sample contaminants and the fluorescence as positive signal for estrogenicity of a substance is granted.
Investigations on (photo) reactions of cosmetic UV filters towards skin proteins
(2014) Stiefel, Constanze; Schwack, Wolfgang
Although UV filters are important, widespread used cosmetic ingredients, their reaction potential towards skin proteins has hardly been studied so far. Therefore, the aim of the present thesis was to investigate the reactivity of widespread UV filter substances towards skin proteins using increasingly complex protein and skin model systems and different analytical techniques. At first, the development of a rapid high-performance thin-layer chromatographic (HPTLC) screening method on an amino phase as protein model provided an easy and rapid way to estimate the reactivity of the common UV filters benzophenone-3 (BP-3), hydroxymethoxybenzoyl sulfonic acid (HMBS), butyl methoxydibenzoylmethane (BM-DBM), 3-benzylidene camphor (3 BC), 4 methylbenzylidene camphor (4 BMC), octocrylene (OCR), ethylhexyl methoxycinnamate (EHMC), ethylhexyl salicylate (EHS), diethylhexyl butamido triazone (DEBT), ethylhexyl triazone (EHT), and octyldimethyl p-aminobenzoic acid (OD-PABA) towards amino groups under thermal and irradiation conditions. A direct comparison of the results of the screening with (photo) patch test data of the dermatological practice showed that especially those UV filters, which are known to be common triggers for (photo) allergic reactions, showed the highest tendency to bind to the amino phase. This indicates that the screening may be well suited to identify possible skin sensitizers as part of a multistage testing strategy. The observation that the reactivity of the different UV filters was influenced by both heat and UV irradiation was verified during the subsequent studies with butylamine and ethanolamine. The UV filters showed individual, time- and temperature-dependent reactivities towards the amines. Benzophenone imines, enamines, and amides were identified as typical reaction products by means of electrospray ionization mass spectrometry (ESI-MS), Fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR) spectroscopy. BP-3, HMBS, the dibenzoylmethanes, OCR, and EHS showed by far the highest reactivity what was in good correlation with the previous screening, indicating a different contact-allergic potential of the UV filters. In contrast, the esters EHMC and EHT showed a significantly lower reactivity, and for the UV filters 3 BC, 4-MBC, and OD-PABA no conversion was observable at all. The formation of the reaction products had partly big influence on the respective UV filter spectra. In the case of BP-3, HMBS, and EHS, the conversions led to a strong bathochromic shift and hence to approved UVA protection. In contrary, in the case of DBM and BM-DBM and especially OCR, a breakage of the original molecule structures was observed, resulting in a significant decrease of the respective absorption strength. The same reaction tendencies could also be observed, when using Boc-protected lysine, the tetrapeptide Boc-Gly-Phe-Gly-Lys-OH (Boc-GFGK), and bovine serum albumin (BSA) as increasingly complex protein or skin models. OCR and BM-DBM confirmed to be most reactive towards the lysine side chains of the mentioned model systems, followed by DBM > BP 3 > EHS > EHMC > EHT in decreasing order. To determine the covalent binding of the UV filters to the protein BSA, beside the extraction of the unbound UV filters, the increase of the molar mass of the formed BSA-adducts was additionally exemplarily determined for EHMC and DBM by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Both methods gave comparable results. Binding to BSA did not affect the UV absorption properties of BM-DBM, EHMC, and EHT, but led to a bathochromic shift in the cases of BP-3 and EHS. For OCR, a strong hypsochromic shift and a nearly complete loss of UVA+B protection was observable. To better reflect the usual application conditions, a thin gelatine layer was chosen as further skin model. The UV filter amounts applied were adapted according to the existing ISO norm for the determination of the SPF. Afterwards, UV irradiation was performed. The binding amounts were determined both by extraction of the unbound UV filters and by isotope-ratio mass spectrometry (IRMS), where two synthetized, stable-isotope labelled UV filter analogues (EHC-d5 and DBM-d5) were used. In contrast to the esters EHMC and EHT, which showed comparatively small binding amounts, for the UV filters OCR, BP-3, EHS, and BM-DBM significant reaction tendencies towards gelatin were observed. Finally, various commercial sunscreens and personal care products with UV protection were applied on either prepared porcine skin or glass plates, followed by UV irradiation. Significant differences were observed for the amounts of UV filters extracted from glass and skin. The lower recoveries in the case of the skin indicated an additional reaction of the UV filters towards the skin samples. BP-3 showed the highest discrepancy between the recoveries from glass and skin after irradiation, followed by EHS > BM DBM > OCR > EHMC > EHT in decreasing order. The present dissertation showed that cosmetic UV filters were able to react with amino structures of different proteins under thermal and irradiation conditions. As the formation of protein adducts is seen as key event in the development of (photo) allergic reactions, the results indicate a specific skin sensitization potential of the UV filters. This is confirmed by the experience of dermatological practice. Since such reactions have partly strong influence on the respective UV filter spectra, the existing in vitro methods using PMMA or quartz glass as substrates have to be questioned, since those methods cannot capture such skin-typical reactions.

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