Impact of physicochemical parameters on the decomposition of deoxynivalenol during extrusion cooking of wheat grits

Deoxynivalenol (DON) is a toxic secondary metabolite produced by molds of the Fusarium genus and is known to cause a spectrum of diseases in animals such as vomiting and gastroenteritis. It is found in cereals and cereal products as most processing techniques lead only to a partial reduction of deoxynivalenol levels. One technique with a reported relatively high impact on deoxynivaleol decomposition is extrusion cooking. In the current work, systematic studies of a range of physicochemical parameters, such as temperature, moisture, compression, residence time in the extruder, pH value, and protein content, on their impact on deoxynivalenol decomposition during extrusion cooking were performed. The analysis of deoxynivalenol was made by high-performance liquid chromatography--tandem mass spectrometry using a quick, easy, cheap, effective, rugged, and safe-based cleanup with 15-d(1)-deoxynivalenol as an internal standard. It could be shown that the reduction of deoxynivalenol levels is dependent on a set of parameters partially interacting with each other. Especially the moisture content and compression are key factors for the reduction of deoxynivalenol levels. A correlation between residence time of the mycotoxin in the extruder and deoxynivalenol degradation was also observed when screws without a compression factor were used. Generally, the reduction of deoxynivalenol levels was increased by the use of screws with a high compression factor. As known from cooking, deoxynivalenol could also be easily degraded by extrusion under alkaline conditions. Furthermore, an increase of the protein content of the starting material resulted in higher reduction rates of deoxynivalenol.     

Untersuchungen zum endophytischen Befall von Fusarium proliferatum (Matsushima) Nirenberg in geernteten Stangen von Spargel (Asparagus officinalis L.)

Zusammenfassung Erstmalig wurden mit den vorliegenden Untersuchungen Spargelstangen zur Haupterntezeit auf endophytischen Pilzbefall untersucht. Sie zeigen, dass im Ernteprodukt zwar Fusarium proliferatum als potenzieller Mykotoxinbildner zu finden ist. Eine mögliche natürliche Kontamination mit Fumonisinen bestätigte sich nicht. Von den mit F. proliferatum infizierten Stangen wies nur eine Stange mit grau-rosa-orange farbenen Gewebeveränderungen an der Basis sichtbare Symptome auf. Allgemeine Rückschlüsse auf eine mögliche Gefährdung oder Nichtgefährdung des Verbrauchers beim Verzehr von mit F. proliferatum kontaminierten, symptomlosen Stangen können aus der Analyse nicht gezogen werden. Hierzu müssen weitergehende Untersuchungen zur Wirt-Pathogen-Interaktion erfolgen und die phänotypischen und genotypischen Einflussfaktoren in diesem Prozess noch näher untersucht werden.     

Identification and apoptotic potential of T-2 toxin metabolites in human cells

The mycotoxin T-2 toxin, produced by various Fusarium species, is a widespread contaminant of grain and grain products. Knowledge about its toxicity and metabolism in the human body is crucial for any risk assessment as T-2 toxin can be detected in processed and unprocessed food samples. Cell culture studies using cells of human origin represent a potent model system to study the metabolic fate of T-2 toxin as well as the cytotoxicity in vitro. In this study the metabolism of T-2 toxin was analyzed in a cell line derived from human colon carcinoma cells (HT-29) and primary human renal proximal tubule epithelial cells (RPTEC) using high-performance liquid chromatography coupled with Fourier transformation mass spectrometry (HPLC-FTMS). Both cell types metabolized T-2 toxin to a variety of compounds. Furthermore, cell cycle analysis in RPTEC proved the apoptotic effect of T-2 toxin and its metabolites HT-2 toxin and neosolaniol in micromolar concentrations.     

Analysis of Flavan-3-ols and procyanidins in food samples by reversed phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS)

Concentrations of the main dimeric and trimeric procyanidins (PC) and their monomeric constitutive units catechin (CT) and epicatechin (EC) were determined in food samples by using reversed phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS). In a first step, 12 PCs (PC B1, B2, B3, B4, B5, B6, B7, B8, C1, C2, and A2 and cinnamtannin B1), of which most are not commercially available, were isolated from plant materials or synthesized and purified by a combination of column chromatographic separation techniques with different stationary phases. These PCs in combination with CT and EC were used as standard substances for identification and quantification during the following screening of food samples by RP-HPLC-ESI-MS/MS analysis. The main focus of the newly developed RP-HPLC-ESI-MS/MS method is the compensation of matrix effects by using the echo-peak technique simulating internal standard injection. The suitability of this new method was demonstrated by the determination of recovery rates being 90% or higher. Use of this method allowed the determination of patterns and concentrations of PCs in 55 food samples.     

Structural profiling and quantification of sphingomyelin in human breast milk by HPLC-MS/MS

The sphingolipid composition of food as well as of physiological samples has received considerable interest due to their positive biological activities. This study quantified the total amount of sphingomyelin (SM) in 20 human breast milk samples from healthy volunteers and determined the structures of SM by detailed mass spectrometric studies in combination with enzymatic cleavage. The quantification of SM was performed by hydrophilic interaction liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (HILIC-HPLC-ESI-MS/MS) measuring the characteristic fragment ion of the phosphorylcholine group at m/z 184.2 and by using hexanoylsphingomyelin (C6-SM) and heptadecanoylsphingomyelin (C17-SM) as internal standards. The structures of SM species were identified after enzymatic cleavage with alkaline sphingomyelinase (SMase) to the corresponding ceramides. Structure elucidation of the sphingoid base and fatty acid backbone was performed by reversed-phase HPLC-ESI-MS/MS. The method includes the sphingoid bases dihydrosphingosine (d18:0), sphingosine (d18:1(Δ4)), 4,8-sphingadienine (d18:2(Δ4,8)), 4-hydroxysphinganine (phytosphingosine (t18:0)), and 4-hydroxy-8-sphingenine (t18:1(Δ8)) and fatty acids with even-numbered carbon atoms (C12-C26) as well as their (poly)unsaturated and monohydroxylated analogues. The total amount of SM in human breast milk varied from 3.87 to 9.07 mg/100 g fresh weight. Sphingosine (d18:1) was the predominant sphingoid base, with 83.6 ± 3.5% in human breast milk, followed by 4,8-sphingadienine (d18:2) (7.2 ± 1.9%) and 4-hydroxysphinganine (t18:0) (5.7 ± 0.7%). The main SM species contained sphingosine and palmitic acid (14.9 ± 2.2%), stearic acid (12.7 ± 1.5%), docosanoic acid (16.2 ± 3.6%), and tetracosenoic acid (15.0 ± 3.1%). Interestingly, the fatty acid composition of SM species in this study differs from the total fatty acids in human breast milk, and the fatty acids are not consistently distributed among the different sphingoid bases.     

Intestinal metabolism of two A-type procyanidins using the pig cecum model: detailed structure elucidation of unknown catabolites with Fourier transform mass spectrometry (FTMS)

Procyanidins, as important secondary plant metabolites in fruits, berries, and beverages such as cacao and tea, are supposed to have positive health impacts, although their bioavailability is yet not clear. One important aspect for bioavailability is intestinal metabolism. The investigation of the microbial catabolism of A-type procyanidins is of great importance due to their more complex structure in comparison to B-type procyanidins. A-type procyanidins exhibit an additional ether linkage between the flavan-3-ol monomers. In this study two A-type procyanidins, procyanidin A2 and cinnamtannin B1, were incubated in the pig cecum model to mimic the degradation caused by the microbiota. Both A-type procyanidins were degraded by the microbiota. Procyanidin A2 as a dimer was degraded by about 80% and cinnamtannin B1 as a trimer by about 40% within 8 h of incubation. Hydroxylated phenolic compounds were quantified as degradation products. In addition, two yet unknown catabolites were identified, and the structures were elucidated by Fourier transform mass spectrometry.     

Deconjugation and degradation of flavonol glycosides by pig cecal microbiota characterized by Fluorescence in situ hybridization (FISH)

As the bioavailability of flavonoids is influenced by intestinal metabolism, we have investigated the microbial deconjugation and degradation of several flavonols and flavonol glycosides using the pig cecum in vitro model system developed in our group. For this model system the microbiota was directly isolated from the cecal lumen of freshly slaughtered pigs. The characterization of the cecal microbiota by fluorescence in situ hybridization (FISH) with 16S rRNA-based oligonucleotide probes confirmed the suitability of the model system for studying intestinal metabolism by the human microbiota. We have investigated the microbial degradation of quercetin-3-O-beta-d-rutinoside 1, quercetin-3-O-beta-d-glucopyranoside 2, quercetin-4'-O-beta-d-glucopyranoside 3, quercetin-3-O-beta-d-galactopyranoside 4, quercetin-3- O-beta-d-rhamnopyranoside 5, quercetin-3- O-[alpha-l-dirhamnopyranosyl-(1-->2)-(1-->6)-beta-d-glucopyranoside 6, kaempferol-3-O-[alpha-l-dirhamnopyranosyl-(1-->2)-(1-->6)-beta-d-glucopyranoside 7, apigenin 8, apigenin-8- C-glucoside (vitexin) 9, and feruloyl-O-beta-d-glucopyranoside 10 (100 microM), representing flavonoids with different aglycones, sugar moieties, and types of glycosidic bonds. The degradation rate was monitored using HPLC-DAD. The flavonol O-glycosides under study were almost completely metabolized by the intestinal microbiota within 20 min and 4 h depending on the sugar moiety and the type of glycosidic bond. The degradation rates of the quercetin monoglycosides showed a clear dependency on the hydroxyl pattern of the sugar moiety. The degradation of 2 with all hydroxyl groups of the glucose in the equatorial position was the fastest. The intestinal metabolism of di- and trisaccharides was much slower compared to the monoglycosides. The structure of the aglycone has not much influence on the intestinal metabolism; however, the type of glycosidic bond ( C- or O-glycoside) has substantial influence on the degradation rate. The liberated aglycones were completely metabolized within 8 h. Phenolic compounds such as 3,4-dihydroxyphenylacetic acid 12, 4-hydroxyphenylacetic acid 13, and phloroglucinol 18 were detected by GC-MS as main degradation products.     

Analysis of fumonisin B(1) in Fusarium proliferatum-infected asparagus spears and garlic bulbs from Germany by liquid chromatography-electrospray ionization mass spectrometry

Fusarium proliferatum is one of a group of fungal species that produce fumonisins and is considered to be a pathogen of many economically important plants. The occurrence of fumonisin B(1) (FB(1)) in F. proliferatum-infected asparagus spears from Germany was investigated using a liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method with isotopically labeled fumonisin FB(1)-d(6) as internal standard. FB(1) was detected in 9 of the 10 samples in amounts ranging from 36.4 to 4513.7 ng/g (based on dry weight). Furthermore, the capability of producing FB(1) by the fungus in garlic bulbs was investigated. Therefore, garlic was cultured in F. proliferatum-contaminated soil, and the bulbs were screened for infection with F. proliferatum and for the occurrence of fumonisins by LC-MS. F. proliferatum was detectable in the garlic tissue, and all samples contained FB(1) (26.0-94.6 ng/g). This is the first report of the natural occurrence of FB(1) in German asparagus spears, and these findings suggest a potential for natural contamination of garlic bulbs with fumonisins.