Colonic metabolism of dietary polyphenols: influence of structure on microbial fermentation products

The metabolism of chlorogenic acid., naringin, and rutin, representative members of three common families of dietary polyphenols, the hydroxycinnamates, the flavanones, and the flavonols, respectively, was studied in an in vitro mixed culture model of the human colonic microflora. Time- and concentration-dependent degradation of all three compounds was observed, which was associated with the following metabolic events after cleavage of the ester or glycosidic bond: reduction of the aliphatic double bond of the resulting hydroxycinnamate caffeic acid residue; dehydroxylation and ring fission of the heterocyclic C-ring of the resulting deglycosylated flavanone, naringenin, and of the deglycosylated flavonol, quercetin (which differed depending on the substitution). The metabolic events, their sequences, and major phenolic end products, as identified by GC-MS or LC-MS/MS, were elucidated from the structural characteristics of the investigated compounds. The major phenolic end products identified were 3-D-hydroxyphenyl)propionic acid for chlorogenic acid, 3-(4-hydroxyphenyl)-propionic acid and 3-phenylpropionic acid for naringin, and 3-hydroxyphenylacetic acid and 3-(3-hydroxyphenyl)-propionic acid for rutin. The degree of degradation of the compounds studied was significantly influenced by the substrate concentration as well as individual variations in the composition of the fecal flora. The results support extensive metabolism of dietary polyphenols in the colon, depending on substrate concentration and residence time, with resultant formation of simple phenolics, which can be considered biomarkers of colonic metabolism if subsequently absorbed. It is also apparent that a relatively small number of phenolic degradation products are formed in the colon from the diverse group of natural polyphenols. (C) 2003 Elsevier Inc. All rights reserved.

Opportunities and challenges in high pressure processing of foods

Consumers increasingly demand convenience foods of the highest quality in terms of natural flavor and taste, and which are freedom additives and preservatives. This demand has triggered the need for the development of a number of nonthermal approaches to food processing, of which high-pressure technology has proven to be very valuable. A number of recent publications have demonstrated novel and diverse uses of this technology. Its novel features, which include destruction of microorganisms at room temperature or lower, have made the technology commerically attractive. Enzymes forming bacteria can be by the application of pressure-thermal combinations. This review aims to identify the opportunities and challenges associated with this technology. In addition to discussing the effects of high pressure on food components, this review covers the combined effects of high pressure processing with: gamma irradiation, alternating current, ultrasound, and carbon dioxide or anti-microbial treatment. Further, the applications of this technology in various sectors-fruits and vegetables, dairy and meat processing-have been dealt with extensively. The integration of high-pressure with other matured processing operations such as blanching, dehydration, osmotic dehydration, rehyrdration, frying, freezing/thawing and solid-liquid extraction has been shown to open up new processing options. The key challenges identified include: heat transfer problems and resulting non-uniformity in processing, obtaining reliable and reproducible data, for process validation, lack of detailed knowledge about the interaction between high pressure, and a number of food constituents, packaging and statutory issues.

Modulation of the microbial ecology of the human colon by probiotics, prebiotics and synbiotics to enhance human health: an overview of enabling science and potential applications

The application of probiotics and prebiotics to the manipulation of the microbial ecology of the human colon has recently seen many scientific advances. The sequencing of probiotic genomes is providing a wealth of new information on the biology of these microorganisms. In addition, we are learning more about the interactions of probiotics with human cells and with pathogenic bacteria. An alternative means of modulating the colonic microbial community is by the use of prebiotic oligosaccharides. Increasing knowledge of the metabolism of prebiotics by probiotics is allowing us to consider specifically targeting such dietary intervention tools at specific populatiori groups and specific disease states. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Predicting the thermal inactivation of bacteria in a solid matrix: simulation studies on the relative effects of microbial thermal resistance parameters and process conditions

A combined mathematical model for predicting heat penetration and microbial inactivation in a solid body heated by conduction was tested experimentally by inoculating agar cylinders with Salmonella typhimurium or Enterococcus faecium and heating in a water bath. Regions of growth where bacteria had survived after heating were measured by image analysis and compared with model predictions. Visualisation of the regions of growth was improved by incorporating chromogenic metabolic indicators into the agar. Preliminary tests established that the model performed satisfactorily with both test organisms and with cylinders of different diameter. The model was then used in simulation studies in which the parameters D, z, inoculum size, cylinder diameter and heating temperature were systematically varied. These simulations showed that the biological variables D, z and inoculum size had a relatively small effect on the time needed to eliminate bacteria at the cylinder axis in comparison with the physical variables heating temperature and cylinder diameter, which had a much greater relative effect. (c) 2005 Elsevier B.V All rights reserved.

New mathematical modeling approach for predicting microbial inactivation by high hydrostatic pressure

A new primary model based on a thermodynamically consistent first-order kinetic approach was constructed to describe non-log-linear inactivation kinetics of pressure-treated bacteria. The model assumes a first-order process in which the specific inactivation rate changes inversely with the square root of time. The model gave reasonable fits to experimental data over six to seven orders of magnitude. It was also tested on 138 published data sets and provided good fits in about 70% of cases in which the shape of the curve followed the typical convex upward form. In the remainder of published examples, curves contained additional shoulder regions or extended tail regions. Curves with shoulders could be accommodated by including an additional time delay parameter and curves with tails shoulders could be accommodated by omitting points in the tail beyond the point at which survival levels remained more or less constant. The model parameters varied regularly with pressure, which may reflect a genuine mechanistic basis for the model. This property also allowed the calculation of (a) parameters analogous to the decimal reduction time D and z, the temperature increase needed to change the D value by a factor of 10, in thermal processing, and hence the processing conditions needed to attain a desired level of inactivation; and (b) the apparent thermodynamic volumes of activation associated with the lethal events. The hypothesis that inactivation rates changed as a function of the square root of time would be consistent with a diffusion-limited process.

Modulation of detoxification enzymes by watercress: in vitro and in vivo investigations in human peripheral blood cells

Epidemiological studies indicate that consumption of cruciferous vegetables (CV) can reduce the risk of cancer. Supposed mechanisms are partly the inhibition of phase I and the induction of phase II enzymes. The aim of this study was to investigate in vitro and in vivo effects of watercress (WC), a member of the CV family, on chemopreventive parameters using human peripheral blood mononuclear cells (PBMC) as surrogate cells. We investigated the hypothesis that WC reduces cancer risk by inducing detoxification enzymes in a genotype-dependent manner. In vitro gene expression and enzyme activity experiments used PBMC incubated with a crude extract from fresh watercress (WCE, 0.1-10 mu L/mL with 8.2 g WC per 1 mL extract) or with one main key compound phenethyl isothiocyanate (PEITC, 1-10 mu M). From an in vivo perspective, gene expression and glutathione S-transferase (GST) polymorphisms were determined in PBMC obtained from a human intervention study in which subjects consumed 85 g WC per day for 8 weeks. The influence of WC consumption on gene expression was determined for detoxification enzymes such as superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPX1), whilst the SOD and GPX activities in red blood cells were also analysed with respect to GST genotypes. In vitro exposure of PBMC to WCE or PEITC (24 h) increased gene expression for both detoxification enzymes GPX1 (5.5-fold, 1 mu L/mL WCE, 3.7-fold 1 mu M PEITC) and SOD2 (12.1-fold, 10 mu L/mL WCE, 7.3-fold, 10 mu M PEITC), and increased SOD2 activity (1.9-fold, 10 mu L/mL WCE). The WC intervention had no significant effect on in vivo PBMC gene expression, as high individual variations were observed. However, a small but significant increase in GPX (p = 0.025) and SOD enzyme activity (p = 0.054) in red blood cells was observed in GSTM1*0, but not in GSTM1*1 individuals, whilst the GSTT1 genotype had no impact. The results indicate that WC is able to modulate the enzymes SOD and GPX in blood cells in vitro and in vivo, and suggest that the capacity of moderate intake of CV to induce detoxification is dependent in part on the GSTM1 genotype.

Synthesis of isomaltooligosaccharides and oligodextrans by the combined use of dextransucrase and dextranase

Batch syntheses of isomaltooligosaccharides (IMO) from sucrose, using the enzymes dextransucrase and dextranase were performed with the aim of understanding the reaction mechanism and the parameters which affect product characteristics and molecular size. Both activities described for dextransucrase (dextran formation and acceptor reaction) achieved synthesis whilst the hydrolytic activity of dextranase regulated the product molecular size and acceptor availability. Depending on the reaction conditions, the product oligosaccharide mixtures contained mainly sugars (up to 36%) with degrees of polymerization (DP) varying between 10 and 60 together with lower concentrations of both lower and higher molecular weight sugars. Alterations in substrate and dextranase concentrations (50-400 mg ml(-1) and 2.5-46 U ml(-1), respectively) affected the molecular weight of IMO, the reaction rate and the formation of leucrose. This permitted manipulation of the product characteristics. It was found that higher substrate and dextranase concentrations gave rise to products with lower molecular sizes and a dextransucrase:dextranase ratio of 1: 1 or 1:2 appeared to produce a polymer with a molecular weight which is desirable for prebiotic use. (C) 2004 Elsevier Inc. All rights reserved.

Dietary modulation of the human colonic microbiota: updating the concept of prebiotics

Prebiotics are non-digestible (by the host) food ingredients that have a beneficial effect through their selective metabolism in the intestinal tract. Key to this is the specificity of microbial changes. The present paper reviews the concept in terms of three criteria: (a) resistance to gastric acidity, hydrolysis by mammalian enzymes and gastrointestinal absorption; (b) fermentation by intestinal microflora; (c) selective stimulation of the growth and/or activity of intestinal bacteria associated with health and wellbeing. The conclusion is that prebiotics that currently fulfil these three criteria are fructo-oligosaccharides, galacto-oligosaccharides and lactulose, although promise does exist with several other dietary carbohydrates. Given the range of food vehicles that may be fortified by prebiotics, their ability to confer positive microflora changes and the health aspects that may accrue, it is important that robust technologies to assay functionality are used. This would include a molecular-based approach to determine flora changes. The future use of prebiotics may allow species-level changes in the microbiota, an extrapolation into genera other than the bifidobacteria and lactobacilli, and allow preferential use in disease-prone areas of the body.

Assessing the impact of nano- and micro-scale zerovalent iron particles on soil microbial activities: Particle reactivity interferes with assay conditions and interpretation of genuine microbial effects

The effects of nano-scale and micro-scale zerovalent iron (nZVI and mZVI) particles on general (dehydrogenase and hydrolase) and specific (ammonia oxidation potential, AOP) activities mediated by the microbial community in an uncontaminated soil were examined. nZVI (diameter 12.5 nm; 10 mg gÿ1 soil)apparently inhibited AOP and nZVI and mZVI apparently stimulated dehydrogenase activity but had minimal influence on hydrolase activity. Sterile experiments revealed that the apparent inhibition of AOP could not be interpreted as such due to the confounding action of the particles, whereas, the nZVIenhanced dehydrogenase activity could represent the genuine response of a stimulated microbial population or an artifact of ZVI reactivity. Overall, there was no evidence for negative effects of nZVI or mZVI on the processes studied. When examining the impact of redox active particles such as ZVI on microbial oxidation–reduction reactions, potential confounding effects of the test particles on assay conditions should be considered.

Twists and turns: a tale of two shikimate-pathway enzymes

We are studying two enzymes from the shikimate pathway, dehydroquinate synthase (DHQS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Both enzymes have been the subject of numerous studies to elucidate their reaction mechanisms. Crystal structures of DHQS and EPSPS in the presence and absence of substrates, cofactors and/or inhibitors are now available. These structures reveal movements of domains, rearrangements of loops and changes in side-chain positions necessary for the formation of a catalytically competent active site. The potential for using complementary small-angle X-ray scattering (SAXS) studies to confirm the presence of these structural differences in solution has also been explored. Comparative analysis of crystal structures, in the presence and absence of ligands, has revealed structural features critical for substrate-binding and catalysis. We have also analysed these structures by generating GRID energy maps to detect favourable binding sites. The combination of X-ray crystallography, SAXS and computational techniques provides an enhanced analysis of structural features important for the function of these complex enzymes.

Predicting microbial pollution concentrations in UK rivers in response to land use change

The Water Framework Directive has caused a paradigm shift towards the integrated management of recreational water quality through the development of drainage basin-wide programmes of measures. This has increased the need for a cost-effective diagnostic tool capable of accurately predicting riverine faecal indicator organism (FIO) concentrations. This paper outlines the application of models developed to fulfil this need, which represent the first transferrable generic FIO models to be developed for the UK to incorporate direct measures of key FIO sources (namely human and livestock population data) as predictor variables. We apply a recently developed transfer methodology, which enables the quantification of geometric mean presumptive faecal coliforms and presumptive intestinal enterococci concentrations for base- and high-flow during the summer bathing season in unmonitored UK watercourses, to predict FIO concentrations in the Humber river basin district. Because the FIO models incorporate explanatory variables which allow the effects of policy measures which influence livestock stocking rates to be assessed, we carry out empirical analysis of the differential effects of seven land use management and policy instruments (fiscal constraint, production constraint, cost intervention, area intervention, demand-side constraint, input constraint, and micro-level land use management) all of which can be used to reduce riverine FIO concentrations. This research provides insights into FIO source apportionment, explores a selection of pollution remediation strategies and the spatial differentiation of land use policies which could be implemented to deliver river quality improvements. All of the policy tools we model reduce FIO concentrations in rivers but our research suggests that the installation of streamside fencing in intensive milk producing areas may be the single most effective land management strategy to reduce riverine microbial pollution.

Platelets release novel thiol isomerase enzymes which are recruited to the cell surface following activation

The thiol isomerase enzymes protein disulphide isomerase (PDI) and endoplasmic reticulum protein 5 (ERp5) are released by resting and activated platelets. These re-associate with the cell surface where they modulate a range of platelet responses including adhesion, secretion and aggregation. Recent studies suggest the existence of yet uncharacterised platelet thiol isomerase proteins. This study aimed to identify which other thiol isomerase enzymes are present in human platelets. Through the use of immunoblotting, flow cytometry, cell-surface biotinylation and gene array analysis, we report the presence of five additional thiol isomerases in human and mouse platelets and megakaryocytes, namely; ERp57, ERp72, ERp44, ERp29 and TMX3. ERp72, ERp57, ERp44 and ERp29 are released by platelets and relocate to the cell surface following platelet activation. The transmembrane thiol isomerase TMX3 was also detected on the platelet surface but does not increase following activation. Extracellular PDI is also implicated in the regulation of coagulation by the modulation of tissue factor activity. ERp57 was identified within platelet-derived microparticle fractions, suggesting that ERp57 may also be involved in the regulation of coagulation as well as platelet function. These data collectively implicate the expanding family of platelet-surface thiol isomerases in the regulation of haemostasis.

Sulforaphane protects cortical neurons against 5-S-cysteinyl-dopamine-induced toxicity through the activation of ERK1/2, Nrf-2 and the upregulation of detoxification enzymes

The degeneration of dopaminergic neurons in the substantia nigra has been linked to the formation of the endogenous neurotoxin 5-S-cysteinyl-dopamine. Sulforaphane (SFN), an isothiocyanate derived from the corresponding precursor glucosinolate found in cruciferous vegetables has been observed to exert a range of biological activities in various cell populations. In this study, we show that SFN protects primary cortical neurons against 5-S-cysteinyl-dopamine induced neuronal injury. Pre-treatment of cortical neurons with SFN (0.01-1 microM) resulted in protection against 5-S-cysteinyl-dopamine-induced neurotoxicity, which peaked at 100 nM. This protection was observed to be mediated by the ability of SFN to modulate the extracellular signal-regulated kinase 1 and 2 and the activation of Kelch-like ECH-associated protein 1/NF-E2-related factor-2 leading to the increased expression and activity of glutathione-S-transferase (M1, M3 and M5), glutathione reductase, thioredoxin reductase and NAD(P)H oxidoreductase 1. These data suggest that SFN stimulates the NF-E2-related factor-2 pathway of antioxidant gene expression in neurons and may protect against neuronal injury relevant to the aetiology of Parkinson's disease.