Biotyping using matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectroscopy (MS) has revolutionized microbiology by allowing clinicians and scientists to rapidly identify microbes at genus and species levels. The present study extensively assesses the suitability and reliability of MALDI-ToF biotyping of 14 different aerobic and anaerobic bacterial species as pure and mixed cultures. Reliable identification at species level was possible from biomaterial of older colonies and even frozen biomaterial, although this was species dependent. Using standard instrument settings and direct application of biomaterial onto the MALDI-ToF target plates, it was determined that the cell densities necessary for completely reliable identification of pure cultures varied between 2.40 × 108 and 1.10 × 1010 viable cell counts (VCCs) per mL, depending on the species. Evaluation of the mixed culture algorithm of the Bruker Biotyper® software showed that the performance of the algorithm depends greatly on the targeted species, on their phylogenetic distance, and on their ratio of VCC per mL in the mixed culture. Hence, the use of MALDI-ToF-MS with incorporation of the mixed culture algorithm of the software is a useful pre-screening tool for early identification of contaminants, but due to the great variability in performance between different species and the usually unknown percentage of the possible contaminant in the mixture, it is advisable to combine this method with other microbiology methods.Electronic supplementary materialThe online version of this article (10.1007/s00216-019-02080-x) contains supplementary material, which is available to authorized users.
Understanding individual responses to nutrition and medicine is of growing interest and importance. There is evidence that differences in bitter taste receptor (TAS2R) genes which give rise to two frequent haplotypes, TAS2R38-PAV (functional) and TAS2R38-AVI (non-functional), may impact inter-individual differences in health status. We here analyzed the relevance of the TAS2R38 receptor in the regulation of the human immune response using the TAS2R38 agonist allyl isothiocyanate (AITC) from Brassica plants. A differential response in calcium mobilization upon AITC treatment in leucocytes from healthy humans confirmed a relevance of TAS2R38 functionality, independent from cation channel TRPV1 or TRPA1 activation. We further identified a TAS2R38-dependence of MAPK and AKT signaling activity, bactericidal (toxicity against E. coli) and anti-inflammatory activity (TNF-alpha inhibition upon cell stimulation). These in vitro results were derived at relevant human plasma levels in the low micro molar range as shown here in a human intervention trial with AITC-containing food.
Vegetables of the plant order Brassicales are believed to have health-promoting properties, as they provide high contents of glucosinolates (GLS) and deriving from these, enzymatically and heat-induced breakdown products, such as isothiocyanates (ITC). Besides their positive physiological effects, ITC are electrophilic and can undergo reactions with food components such as proteins. Following the trend of improving traditional food products with GLS-rich ingredients, interactions of ITC with proteins can diminish the properties of both components—protein’s value and functionality as well as ITC’s bioactivity. In vegetable-enriched bread, where cresses (Lepidium sativum L. or Tropaeolum majus L.) are added to the initial dough, together with benzyl cyanide, benzyl isothiocyanate (BITC) is formed during the baking process. The aim of the present study was to investigate the possible migration behavior of the GLS breakdown products and the formation of ITC-wheat protein conjugates. After the baking process, the breads’ proteins were enzymatically hydrolyzed, and the ITC-amino acid conjugates analyzed using a LC-ESI-MS/MS methodology. In all samples, BITC-protein conjugates were detected as thiourea derivatives, while formation of dithiocarbamates could not be detected. The study showed that GLS and their breakdown products such as ITC migrate into the surrounding food matrix and undergo reactions with proteins, which could in turn lead to modified protein properties and reduce the bioavailability of ITC and lysine.
ScopeAs prostaglandin E2 (PGE2) has important roles in physiological and inflammatory functions, a double-blind randomized controlled crossover study to investigate the potential of nasturtium (Tropaeolum majus) for modulating PGE2 was conducted, aiming at clarifying the role of benzyl isothiocyanate (BITC). As secondary parameters leukotriene 4 (LTB4), and cytokine release (tumor necrosis factor alpha, TNF-α; interleukins IL-1β, IL-10, and IL-12) were quantified.Methods and resultsThirty-four healthy female participants consumed 1.5 g nasturtium containing BITC, (verum) or no BITC (control) twice a day for 2 weeks each. Nasturtium intervention resulted in an increase in mean PGE2 levels in serum samples (verum: 1.76-fold, p ≤ 0.05; control: 1.78-fold, p ≤ 0.01), and ex vivo stimulated peripheral blood mononuclear cells (PBMC) (verum: 1.71-fold, p ≤ 0.01; control: 1.43-fold). Using a pre-to-post responder analysis approach, 18 of 34 subjects showed a > 25% PGE2 increase in serum, while it was >25% decreased for 9 subjects (stimulated PBMC: 14 and 8 of 28, respectively). Under the selected conditions, the BITC content of nasturtium did not affect the observed changes in PGE2. Verum intervention also increased mean LTB4 serum level (1.24-fold, p ≤ 0.01), but not in LPS stimulated PBMC, and significantly increased TNF-α release in stimulated PBMC after 3 h (verum: 1.65-fold, p = 0.0032; control: 1.22-fold, p = 0.7818). No change was seen in the anti-inflammatory cytokine IL-10, or the pro-inflammatory cytokines IL-1β, and IL-12.ConclusionIn contrast to the previously reported in vitro results, on average, LPS activated PBMC and serum from both groups showed increased PGE2 levels. Further analyses suggest that PGE2 release after intervention could possibly depend on the baseline PGE2 level. Identification of phenotypes that respond differently to the nasturtium intervention could be useful to establish personalized approaches for dosing phytopharmaceuticals medicines.
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