Identification of Marker Peptides for the Whey Protein Quantification in Edam-Type Cheese.

Several technologies are available for incorporating whey proteins into a cheese matrix. However, there is no valid analytical method available to determine the whey protein content in matured cheese, to date. Consequently, the aim of the present study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of individual whey proteins based on specific marker peptides ('bottom-up' proteomic approach). Therefore, the whey protein-enriched model of the Edam-type cheese was produced in a pilot plant and on an industrial scale. Tryptic hydrolysis experiments were performed to evaluate the suitability of identified potential marker peptides (PMPs) for α-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG). Based on the findings, α-LA and ß-LG appeared to be resistant to proteolytic degradation during six weeks of ripening and no influence on the PMP was observed. Good levels of linearity (R2 > 0.9714), repeatability (CVs < 5%), and recovery rate (80% to 120%) were determined for most PMPs. However, absolute quantification with external peptide and protein standards revealed differences in model cheese depending on the PMP, e.g., 0.50% ± 0.02% to 5.31% ± 0.25% for ß-LG. As protein spiking prior to hydrolysis revealed differing digestion behavior of whey proteins, further studies are required to enable valid quantification in various cheese types.

Detection and quantification of zearalenone and its modified forms in enzymatically treated oat and wheat flour.

An analytical method for the analysis of the mycotoxin zearalenone (ZEN) and its modified forms was developed. Sample preparation was performed based on a modified QuEChERS method combined with liquid chromatography coupled to a triple quadrupole mass spectrometry detection. The method was tested for linearity, precision, limits of detection and quantification and recoveries. The evaluation of the above-mentioned parameters was performed on oat flour. The method was applied to oat and wheat flours that were submitted to an amylolytic treatment (α-amylase and amyloglucosidase), similar to the one used in the cereal-based baby food production process. A decrease in ß-zearalenol (ß-ZEL) and ß-ZEL-14-sulfate of approximately 40% after 90 min incubation was observed, the other analytes did not show any significant changes. To our knowledge, this is the first method that approaches the identification and assessment of ZEN-sulfate derivates in a cereal matrix. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05683-6.

Transfer of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and polychlorinated biphenyls (PCBs) from oral exposure into cow's milk - Part I: state of knowledge and uncertainties.

Polychlorinated dibenzo-para-dioxins (PCDDs) and dibenzofurans (PCDFs) (collectively and colloquially referred to as 'dioxins') as well as polychlorinated biphenyls (PCBs) are persistent and ubiquitous environmental contaminants that may unintentionally enter and accumulate along the food chain. Owing to their chronic toxic effects in humans and bioaccumulative properties, their presence in feed and food requires particular attention. One important exposure pathway for consumers is consumption of milk and dairy products. Their transfer from feed to milk has been studied for the past 50 years to quantify the uptake and elimination kinetics. We extracted transfer parameters (transfer rate, transfer factor, biotransfer factor and elimination half-lives) in a machine-readable format from seventy-six primary and twenty-nine secondary literature items. Kinetic data for some toxicologically relevant dioxin congeners and the elimination half-lives of dioxin-like PCBs are still not available. A well-defined selection of transfer parameters from literature was statistically analysed and shown to display high variability. To understand this variability, we discuss the data with an emphasis on influencing factors, such as experimental conditions, cow performance parameters and metabolic state. While no universal interpretation could be derived, a tendency for increased transfer into milk is apparently connected to an increase in milk yield and milk fat yield as well as during times of body fat mobilisation, for example during the negative energy balance after calving. Over the past decades, milk yield has increased to over 40 kg/d during high lactation, so more research is needed on how this impacts feed to food transfer for PCDD/Fs and PCBs.

Transfer of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and polychlorinated biphenyls (PCBs) from oral exposure into cow's milk - part II: toxicokinetic predictive models for risk assessment.

Understanding the transfer of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) as well as polychlorinated biphenyls (PCBs) from oral exposure into cow's milk is not purely an experimental endeavour, as it has produced a large corpus of theoretical work. This work consists of a variety of predictive toxicokinetic models in the realms of health and environmental risk assessment and risk management. Their purpose is to provide mathematical predictive tools to organise and integrate knowledge on the absorption, distribution, metabolism and excretion processes. Toxicokinetic models are based on more than 50 years of transfer studies summarised in part I of this review series. Here in part II, several of these models are described and systematically classified with a focus on their applicability to risk analysis as well as their limitations. This part of the review highlights the opportunities and challenges along the way towards accurate, congener-specific predictive models applicable to changing animal breeds and husbandry conditions.

Enzyme immunoassays for the detection of mycotoxins in plant-based milk alternatives: pitfalls and limitations.

Plant-based milk alternatives (PBMAs) are a potential source of mycotoxin uptake. To ensure food safety, simple and rapid testing methods of PBMAs for mycotoxins are therefore required. This study investigated the applicability of enzyme immunoassay (EIA) methods for direct testing of PBMAs without sample extraction. Mycotoxin analyses included aflatoxin B1 (AFB1), sterigmatocystin (STC), ochratoxin A (OTA), deoxynivalenol (DON), and T-2/HT-2-toxin (T-2/HT-2). It was found that the PBMA matrix negatively affected the EIA to varying degrees, thus affecting the reliability of the results. A dilution of PBMAs of at least 1:8 was necessary to overcome matrix interference. This resulted in calculated detection limits of 0.4 µg/L (AFB1), 2 µg/L (STC), 0.08 µg/L (OTA), 16 µg/L (DON), and 0.4 µg/L (T-2/HT-2). After analysis of 54 PBMA products from German retail stores, positive results in at least one test system were obtained for 23 samples. However, most positive results were near the calculated detection limit. Control analyses of selected samples by LC-MS/MS for AFB1, STC, and OTA qualitatively confirmed the presence of trace amounts of STC in some samples, but quantitative agreement was poor. It was concluded that the high diversity of ingredients used in PBMAs led to a highly variable degree of sample matrix interference even in a 1:8 dilution. Since the use of higher dilutions conflicts with the need to achieve low detection limits, the application of EIA for routine mycotoxin analysis in PBMA for mycotoxins requires further study on the development of a feasible sample preparation method.

Growth and toxin production of phomopsin A and ochratoxin A forming fungi under different storage conditions in a pea (Pisum sativum) model system.

Phomopsins are mycotoxins mainly infesting lupines, with phomopsin A (PHOA) being the main mycotoxin. PHOA is produced by Diaporthe toxica, formerly assigned as toxigenic Phomopsis leptostromiformis, causing infections in lupine plants and harvested seeds. However, Diaporthe species may also grow on other grain legumes, similar to Aspergillus westerdijkiae as an especially potent ochratoxin A (OTA) producer. Formation of PHOA and OTA was investigated on whole field peas as model system to assess fungal growth and toxin production at adverse storage conditions. Field pea samples were inoculated with the two fungal strains at two water activity (aw) values of 0.94 and 0.98 and three different levels of 30, 50, and 80% relative air humidity.After 14 days at an aw value of 0.98, the fungi produced 4.49 to 34.3 mg/kg PHOA and 1.44 to 3.35 g/kg OTA, respectively. Strains of D. toxica also tested showed higher PHOA concentrations of 28.3 to 32.4 mg/kg.D. toxica strains did not grow or produce PHOA at an aw values of 0.94, while A. westerdijkiae still showed growth and OTA production.Elevated water activity has a major impact both on OTA and, even more pronouncedly, on PHOA formation and thus, proper drying and storage of lupins as well as other grain legumes is crucial for product safety.

Metabolic fate and toxicity reduction of aflatoxin B1 after uptake by edible Tenebrio molitor larvae.

The use of insects as food and feed is gaining more attention for ecological and ethical reasons. Despite the high tolerance of edible yellow mealworm (Tenebrio molitor) larvae to aflatoxin B1 (AFB1), the metabolic fate of the toxin along with its toxic potential in the insect is uncertain. The present study aimed at investigating the AFB1 mass balance and the metabolite formation in a feeding trial with AFB1-contaminated grain flour. T. molitor larvae tolerated the AFB1 level of 10,700 µg/kg in the feed, however, weight gain was decreased by 15% over a 4-weeks feeding period. The investigation of the phase I metabolite pattern revealed the formation of AFM1 and a novel presumably monohydroxylated compound in larvae extracts that was not formed by reference incubation with rat, bovine or porcine liver microsomes. Mass balance quantification of ingested AFB1 revealed that 87% of the initial toxin remain undetected in larval body or residue. Analysis of histone H2Ax phosphorylation in human liver cells as a surrogate for genotoxicity showed that extracts from exposed larvae did not exhibit an elevated toxic potential. Although toxicological uncertainties remain due to the undetected transformation products, the resulting mutagenicity of the edible larvae appears to be low.

Distribution of T-2 toxin and HT-2 toxin during experimental feeding of yellow mealworm (Tenebrio molitor).

Within the European Union (EU), edible insects need to be approved as "Novel Food" according to Regulation (EU) 2015/2283 and must comply with the requirements of European food law with regard to microbiological and chemical food safety. Substrates used for feeding insects are susceptible to the growth of Fusarium spp. and consequently to contamination with trichothecene mycotoxins. Therefore, the current study aimed to investigate the influence of T-2 and HT-2 toxins on the larval life cycle of yellow mealworm (Tenebrio molitor (L.)) and to study the transfer of T-2, HT-2, T-2 triol and T-2 tetraol in the larvae. In a 4-week feeding study, T. molitor larvae were kept either on naturally (oat flakes moulded with Fusarium sporotrichioides) or artificially contaminated oat flakes, each at two levels (approximately 100 and 250 µg/kg total T-2 and HT-2). Weight gain and survival rates were monitored, and mycotoxins in the feeding substrates, larvae and residues were determined using LC-MS/MS. Larval development varied between the diets and was 44% higher for larvae fed artificially contaminated diets. However, the artificially contaminated diets had a 16% lower survival rate. No trichothecenes were detected in the surviving larvae after harvest, but T-2 and HT-2 were found both in the dead larvae and in the residues of naturally and artificially contaminated diets.

Impact of experimental thermal processing of artificially contaminated pea products on ochratoxin A and phomopsin A.

Fungi of Aspergillus and Penicillium genus can infect peas (Pisum sativum), leading to a contamination with the nephrotoxic and carcinogenic ochratoxin A (OTA). Under unfavourable conditions, a fungus primarily found on lupines, Diapothe toxica, may also grow on peas and produce the hepatotoxic phomopsin A (PHOA). To study the effect of processing on OTA and PHOA content, two model products-wheat/rye-mixed bread with pea flour addition and pea pasta-were manufactured at small-business scale from artificially contaminated pea flour. The decrease of OTA and PHOA contents were monitored along the production process as indicators for toxin transformation. Pea bread dough was subjected to proofing for 30-40 min at 32 °C and baked at 250 °C to 230 °C for 40 min. OTA content (LODs < 0.1 µg/kg) showed a reduction in the bread crust (initially 17.0 µg/kg) to 88% and no reduction in the crumb (110%). For PHOA (LODs < 3.6 µg/kg), a decrease to approximately 21% occurred in the bread crust (initially 12.5 µg/kg), whilst for crumb, a less intense decrease to 91% was found. Pea pasta prepared with two toxin levels was extruded at room temperature, dried and cooked for 8 min in boiling water. In pea pasta, OTA was reduced from 29.8 to 13.9 µg/kg by 22% each after cooking, whilst 15% and 10% of the initial toxin amounts were found in the cooking water, respectively. For PHOA, 60% and 78% of initially 14.3 µg/kg and 7.21 µg/kg remained in the cooked pasta. As only the decrease of the initial content was measured and no specific degradation products could be detected, further research is needed to characterise potential transformation products. Heat treatment reduces the initial PHOA content stronger than the OTA content during pasta cooking and bread making. However, significant amounts of both toxins would remain in the final products.

A loop-mediated isothermal amplification (LAMP) based assay for the rapid and sensitive group-specific detection of fumonisin producing Fusarium spp.

Fumonisins are mycotoxins that contaminate maize and maize-based food products, and feed. They have been associated with nerve system disorders in horses, pulmonary edema in swine as well as neural tube defects and esophageal cancer in humans. The fum1 gene codes for a polyketide synthase involved in the biosynthesis of fumonisins. It is present in the genomes of all fumonisin producing Fusarium spp. Reliable detection of fum1 can provide an estimate of the toxicological potential of cultures and food sources. Therefore, a fum1 specific LAMP assay was developed and tested with purified DNA of 48 different species from the Fusarium fujikuroi species complex (FFSC). The fum1 gene was detected in 22 species among which F. fujikuroi, F. globosum, F. nygamai, F. proliferatum, F. subglutinans and F. verticillioides were the most prominent fumonisin producers. None out of 92 tested non-Fusarium species showed cross reactions with the new assay. The lowest limit of detection (LOD) was 5 pg of genomic DNA per reaction for F. fujikuroi, F. nygamai and F. verticillioides. Higher LODs were found for other LAMP positive species. Apart from pure genomic DNA, the LAMP assay detected fumonisin-producers when 103 conidia/reaction were used as template after mechanical lysis. LAMP-results were well correlated with FB1 production. This is the first report on fumonisin production in strains of F. annanatum, F. coicis, F. mundagurra, F. newnesense, F. pininemorale, F. sororula, F. tjataeba, F. udum and F. werrikimbe. Usefulness of the LAMP assay was demonstrated by analyzing fumonisin contaminated maize grains. The new LAMP assay is rapid, sensitive and reliable for the diagnosis of typical fumonisin producers and can be a versatile tool in HACCP concepts that target the reduction of fumonisins in the food and feed chain.

Formation of Zearalenone Metabolites in Tempeh Fermentation.

Tempeh is a common food in Indonesia, produced by fungal fermentation of soybeans using Rhizopus sp., as well as Aspergillus oryzae, for inoculation. Analogously, for economic reasons, mixtures of maize and soybeans are used for the production of so-called tempeh-like products. For maize, a contamination with the mycoestrogen zearalenone (ZEN) has been frequently reported. ZEN is a mycotoxin which is known to be metabolized by Rhizopus and Aspergillus species. Consequently, this study focused on the ZEN transformation during tempeh fermentation. Five fungal strains of the genera Rhizopus and Aspergillus, isolated from fresh Indonesian tempeh and authentic Indonesian inocula, were utilized for tempeh manufacturing from a maize/soybean mixture (30:70) at laboratory-scale. Furthermore, comparable tempeh-like products obtained from Indonesian markets were analyzed. Results from the HPLC-MS/MS analyses show that ZEN is intensely transformed into its metabolites α-zearalenol (α-ZEL), ZEN-14-sulfate, α-ZEL-sulfate, ZEN-14-glucoside, and ZEN-16-glucoside in tempeh production. α-ZEL, being significantly more toxic than ZEN, was the main metabolite in most of the Rhizopus incubations, while in Aspergillus oryzae fermentations ZEN-14-sulfate was predominantly formed. Additionally, two of the 14 authentic samples were contaminated with ZEN, α-ZEL and ZEN-14-sulfate, and in two further samples, ZEN and α-ZEL, were determined. Consequently, tempeh fermentation of ZEN-contaminated maize/soybean mixture may lead to toxification of the food item by formation of the reductive ZEN metabolite, α-ZEL, under model as well as authentic conditions.

Feeding study for the mycotoxin zearalenone in yellow mealworm (Tenebrio molitor) larvae-investigation of biological impact and metabolic conversion.

Edible insects as additional food and/or feed source may represent one important component to solve the problem of food security for a growing human population. Especially for covering the rising demand for protein of animal origin, seven insect species currently allowed as feed constituents in the European Union are gaining more interest. However, before considering insects such as yellow mealworm larvae (Tenebrio molitor) as suitable for, e.g. human consumption, the possible presence and accumulation of contaminants must be elucidated. The present work investigates the effects of the mycotoxin zearalenone (ZEN) and its metabolites on insect larvae. Seven different diets were prepared: toxin-free control, spiked and artificially contaminated (both containing approx.500 µg/kg and approx. 2000 µg/kg of ZEN) as well as two naturally contaminated diets (600 µg/kg and 900 µg/kg ZEN). The diets were used in a multiple-week feeding trial using T. molitor larvae as model insects. The amount of ZEN and its metabolites in the feed, larvae and the residue were measured by HPLC-MS/MS. A significantly enhanced individual larval weight was found for the insects fed on the naturally contaminated diets compared to the other feeding groups after 8 weeks of exposure. No ZEN or ZEN metabolites were detected in the T. molitor larvae after harvest. However, ZEN, α- and ß-stereoisomers of zearalenol were found in the residue samples indicating an intense metabolism of ZEN in the larvae. No further ZEN metabolites could be detected in any sample. Thus, ZEN is not retained to any significant amount in T. molitor larvae.

Natural diversity of hydroxycinnamic acid derivatives, flavonoid glycosides, carotenoids and chlorophylls in leaves of six different amaranth species.

Amaranth species are globally grown food crops. However, knowledge about the composition of their secondary metabolites is insufficient. Here, selected hydroxycinnamic acid derivatives, flavonoid glycosides, carotenoids and chlorophylls in the leaves of 14 genotypes from six different amaranth species were identified and quantified. For the first time, caffeic acid esters of isocitric and several aldaric acids were isolated and quantified in a leafy food matrix. High concentrations of hydroxycinnamic acid derivatives and chlorophylls, and moderate amounts of flavonoids and carotenoids were detected. A hierarchical clustering method of the metabolic profiles followed by Random Amplification of Polymorphic DNA (RAPD)-PCR fingerprinting was used to group the genotypes. Using this combined approach, three main groups of amaranth species were assigned. The information provided in this study increases the attractiveness of the amaranth genus as a food crop due to its strong diversity of plant secondary metabolites that are associated with numerous health-promoting benefits.

Biosynthesis and Characterization of Zearalenone-14-Sulfate, Zearalenone-14-Glucoside and Zearalenone-16-Glucoside Using Common Fungal Strains.

Zearalenone (ZEN) and its phase II sulfate and glucoside metabolites have been detected in food and feed commodities. After consumption, the conjugates can be hydrolyzed by the human intestinal microbiota leading to liberation of ZEN that implies an underestimation of the true ZEN exposure. To include ZEN conjugates in routine analysis, reliable standards are needed, which are currently not available. Thus, the aim of the present study was to develop a facilitated biosynthesis of ZEN-14-sulfate, ZEN-14-glucoside and ZEN-16-glucoside. A metabolite screening was conducted by adding ZEN to liquid fungi cultures of known ZEN conjugating Aspergillus and Rhizopus strains. Cultivation conditions and ZEN incubation time were varied. All media samples were analyzed for metabolite formation by HPLC-MS/MS. In addition, a consecutive biosynthesis was developed by using Fusarium graminearum for ZEN biosynthesis with subsequent conjugation of the toxin by utilizing Aspergillus and Rhizopus species. ZEN-14-sulfate (yield: 49%) is exclusively formed by Aspergillus oryzae. ZEN-14-glucoside (yield: 67%) and ZEN-16-glucoside (yield: 39%) are formed by Rhizopus oryzae and Rhizopusoligosporus, respectively. Purities of ≥73% ZEN-14-sulfate, ≥82% ZEN-14-glucoside and ≥50% ZEN-16-glucoside were obtained by ¹H-NMR. In total, under optimized cultivation conditions, fungi can be easily utilized for a targeted and regioselective synthesis of ZEN conjugates.

Mutual Interaction of Phenolic Compounds and Microbiota: Metabolism of Complex Phenolic Apigenin-C- and Kaempferol-O-Derivatives by Human Fecal Samples.

Human colonic bacteria have an important impact on the biotransformation of flavonoid glycosides and their conversion can result in the formation of bioactive compounds. However, information about the microbial conversion of complex glycosylated flavonoids and the impact on the gut microbiota are still limited. In this study, in vitro fermentations with selected flavonoid O- and C-glycosides and three different fecal samples were performed. As a result, all flavonoid glycosides were metabolized via their aglycones yielding smaller substances. Main metabolites were 3-(4-hydroxyphenyl)propionic acid, 3-phenylpropionic acid, and phenylacetic acid. Differences in the metabolite formation due to different time courses between the donors were determined. Therefore, from all fermentations, the ones with a specific donor were always slower resulting in a lower number of metabolites compared to the others. For example, tiliroside was totally degraded from 0 h (105 ± 13.2 µM) within the first 24 h, while in the fermentations with fecal samples from other donors, tiliroside (107 ± 52.7 µM at 0 h) was not detected after 7 h anymore. In general, fermentation rates of C-glycosides were slower compared to the fermentation rates of O-glycosides. The O-glycoside tiliroside was degraded within 4 h while the gut microbiota converted the C-glycoside vitexin within 13 h. However, significant changes (p < 0.05) in the microbiota composition and short chain fatty acid levels as products of carbohydrate fermentation were not detected between incubations with different phenolic compounds. Therefore, microbiota diversity was not affected and a significant prebiotic effect of phenolic compounds cannot be assigned to flavonoid glycosides in food-relevant concentrations.

Chlorogenic acid versus amaranth's caffeoylisocitric acid - Gut microbial degradation of caffeic acid derivatives.

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.

Detection of a Toxic Methylated Derivative of Phomopsin A Produced by the Legume-Infesting Fungus Diaporthe toxica.

Phomopsin A (PHO-A), produced by the fungus Diaporthe toxica, is a mycotoxin known to be responsible for fatal liver disease of lupin-fed sheep. The full spectrum of the toxic secondary metabolites produced by D. toxica is still unknown. PHO-A and the naturally occurring derivatives B-E have been subject to several studies to reveal their structures as well as chemical and toxicological properties. In this work, a methylated derivative (1) of PHO-A isolated from lupin seeds inoculated with D. toxica is described. It was characterized by high-resolution mass and NMR data and shown to be the N-methylated derivative of PHO-A. 1 is cytotoxic against HepG2 cells.

Detection of freshwater cyanotoxins and measurement of masked microcystins in tilapia from Southeast Asian aquaculture farms.

Recently, there has been a rise in freshwater harmful algal blooms (HABs) globally, as well as increasing aquaculture practices. HABs can produce cyanotoxins, many of which are hepatotoxins. An ultra-performance liquid chromatography tandem mass spectrometry method was developed and validated for nine cyanotoxins across three classes including six microcystins, nodularin, cylindrospermopsin and anatoxin-a. The method was used to analyse free cyanotoxin(s) in muscle (n = 34), liver (n = 17) and egg (n = 9) tissue samples of 34 fish sourced from aquaculture farms in Southeast Asia. Conjugated microcystin was analysed by Lemieux oxidation to ascertain the total amount of microcystin present in muscle. Some tilapia accumulated free microcystin-LR in the muscle tissue at a mean of 15.45 µg/kg dry weight (dw), with total microcystin levels detected at a mean level of 110.1 µg/kg dw, indicating that the amount of conjugated or masked microcystin present in the fish muscle accounted for 85% of the total. Higher levels of cyanotoxin were detected in the livers, with approximately 60% of those tested being positive for microcystin-LR and microcystin-LF, along with cylindrospermopsin. Two fish from one of the aquaculture farms contained cylindrospermopsin in the eggs; the first time this has been reported. The estimated daily intake for free and total microcystins in fish muscle tissue was 2 and 14 times higher, respectively, than the tolerable daily intake value. This survey presents the requirement for further monitoring of cyanotoxins, including masked microcystins, in aquaculture farming in these regions and beyond, along with the implementation of guidelines to safeguard human health. Graphical abstract ᅟ.

Evaluation of an Aqueous Extract from Horseradish Root (Armoracia rusticana Radix) against Lipopolysaccharide-Induced Cellular Inflammation Reaction.

Horseradish (Armoracia rusticana) is a perennial crop and its root is used in condiments. Traditionally, horseradish root is used to treat bacterial infections of the respiratory tract and urinary bladder. The antiphlogistic activity, determined in activated primary human peripheral blood mononuclear cells (PBMC), was evaluated for an aqueous extract and its subfractions, separated by HPLC. Compound analysis was done by UHPLC-QToF/MS and GC-MS. The aqueous extract concentration-dependently inhibited the anti-inflammatory response to lipopolysaccharide (LPS) in terms of TNF-α release at ≥37 µg/mL. Further, the cyclooxygenase as well as lipoxygenase pathway was blocked by the extract as demonstrated by inhibition of COX-2 protein expression and PGE2 synthesis at ≥4 µg/mL and leukotriene LTB4 release. Mechanistic studies revealed that inhibition of ERK1/2 and c-Jun activation preceded COX-2 suppression upon plant extract treatment in the presence of LPS. Chemical analysis identified target compounds with a medium polarity as relevant for the observed bioactivity. Importantly, allyl isothiocyanate, which is quite well known for its anti-inflammatory capacity and as the principal pungent constituent in horseradish roots, was not relevant for the observations. The results suggest that horseradish root exerts an antiphlogistic activity in human immune cells by regulation of the COX and LOX pathway via MAPK signalling.

Alamethicin for using in bioavailability studies? - Re-evaluation of its effect.

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.