A Wake-Up Call for the Efficient Use of the Bacterial Resting Cell Process, with Focus on Low Solubility Products.

Micro-organisms are often subjected to stressful conditions. Owing to their capacity to adapt, they try to rapidly cope with the unfavorable conditions by lowering their growth rate, changing their morphology, and developing altered metabolite production and other stress-related metabolism. The stress-related metabolism of the cells which interrupted their growth is often referred to as resting metabolism and can be exploit for specific and high rate production of secondary metabolites. Although the bacterial resting cell process has been described decades ago, we find it worthwhile to bring the process under renewed attention and refer to this type of processes as non-growing metabolically active (NGMA) cell processes. Despite their use may sound counterproductive, NGMA cells can be of interest to increase substrate conversion rates or enable conversion of certain substrates, not accessible to growing cells due to their bacteriostatic nature or requirement of resistance to a multitude of different stress mechanisms. Biomass reuse is an interesting feature to improve the economics of NGMA cell processes. Yet, for lipophilic compounds or compounds with low solubility, biomass separation can be delicate. This review draws the attention on existing examples of NGMA cell processes, summarizing some developmental tools and highlighting drawbacks and opportunities, to answer the research question if NGMA cells can have a distinct added value in industry. Particular elaboration is made on a novel and more broadly applicable strategy to enable biomass reuse for conversions of compounds with low solubility.

Dietary fiber intake and its association with indicators of adiposity and serum biomarkers in European adolescents: the HELENA study.

PURPOSE: To evaluate total, energy-adjusted dietary fiber (DF), water-soluble fiber (WSF), and water-insoluble fiber (WIF) intakes in European adolescents and to investigate their association with indicators of adiposity and serum biomarkers. METHODS: This study, conducted from 2006 to 2007, included 1804 adolescents aged 12.5-17.5 years (47% males) from eight European cities completing two non-consecutive computerized 24-h dietary recalls. GLM multivariate analysis was used to investigate associations. RESULTS: Mean DF intake (20 g/day) of the sample met the European Food Safety Authority recommendation, but was below those of the World Health Organization and of the Institute of Medicine. Total DF, WSF and WIF intakes were higher in males (P < 0.001), but following energy-adjustments significantly higher intakes were observed among females (P < 0.001). Bread and cereals contributed most to total DF, WSF and WIF intakes, followed by potatoes and grains, energy-dense but low-nutritious foods, fruits and vegetables. Moreover, energy-adjusted WSF and WIF were positively associated with body fat percentage (BF%), waist to height ratio and low-density lipoprotein cholesterol, while energy-adjusted WSF was inversely associated with serum fasting glucose (ß = -0. 010, P = 0.020). CONCLUSION: Total DF intakes are rather low in European adolescents. An inverse association with serum fasting glucose might indicate a possible beneficial role of DF in preventing insulin resistance and its concomitant diseases, even though DF intakes were positively associated with adolescents' BF%. Therefore, further longitudinal studies should elaborate on these potential beneficial effects of DF intake in the prevention of obesity and related chronic diseases.

Dietary animal and plant protein intakes and their associations with obesity and cardio-metabolic indicators in European adolescents: the HELENA cross-sectional study.

BACKGROUND: Previous studies suggest that dietary protein might play a beneficial role in combating obesity and its related chronic diseases. Total, animal and plant protein intakes and their associations with anthropometry and serum biomarkers in European adolescents using one standardised methodology across European countries are not well documented. OBJECTIVES: To evaluate total, animal and plant protein intakes in European adolescents stratified by gender and age, and to investigate their associations with cardio-metabolic indicators (anthropometry and biomarkers). METHODS: The current analysis included 1804 randomly selected adolescents participating in the HELENA study (conducted in 2006-2007) aged 12.5-17.5 y (47% males) who completed two non-consecutive computerised 24-h dietary recalls. Associations between animal and plant protein intakes, and anthropometry and serum biomarkers were examined with General linear Model multivariate analysis. RESULTS: Average total protein intake exceeded the recommendations of World Health Organization and European Food Safety Authority. Mean total protein intake was 96 g/d (59% derived from animal protein). Total, animal and plant protein intakes (g/d) were significantly lower in females than in males and total and plant protein intakes were lower in younger participants (12.5-14.9 y). Protein intake was significantly lower in underweight subjects and higher in obese ones; the direction of the relationship was reversed after adjustments for body weight (g/(kg.d)). The inverse association of plant protein intakes was stronger with BMI z-score and body fat percentage (BF%) compared to animal protein intakes. Additionally, BMI and BF% were positively associated with energy percentage of animal protein. CONCLUSIONS: This sample of European adolescents appeared to have adequate total protein intake. Our findings suggest that plant protein intakes may play a role in preventing obesity among European adolescents. Further longitudinal studies are needed to investigate the potential beneficial effects observed in this study in the prevention of obesity and related chronic diseases.

A Nine-Strain Bacterial Consortium Improves Portal Hypertension and Insulin Signaling and Delays NAFLD Progression In Vivo.

The gut microbiome has a recognized role in Non-alcoholic fatty liver disease (NAFLD) and associated comorbidities such as Type-2 diabetes and obesity. Stool transplantation has been shown to improve disease by restoring endothelial function and insulin signaling. However, more patient-friendly treatments are required. The present study aimed to test the effect of a defined bacterial consortium of nine gut commensal strains in two in vivo rodent models of Non-alcoholic steatohepatitis (NASH): a rat model of NASH and portal hypertension (PHT), and the Stelic animal (mouse) model (STAM™). In both studies the consortium was administered orally q.d. after disease induction. In the NASH rats, the consortium was administered for 2 weeks and compared to stool transplant. In the STAM™ study administration was performed for 4 weeks, and the effects compared to vehicle or Telmisartan at the stage of NASH/early fibrosis. A second group of animals was followed for another 3 weeks to assess later-stage fibrosis. In the NASH rats, an improvement in PHT and endothelial function was observed. Gut microbial compositional changes also revealed that the consortium achieved a more defined and richer replacement of the gut microbiome than stool transplantation. Moreover, liver transcriptomics suggested a beneficial modulation of pro-fibrogenic pathways. An improvement in liver fibrosis was then confirmed in the STAM™ study. In this study, the bacterial consortium improved the NAFLD activity score, consistent with a decrease in steatosis and ballooning. Serum cytokeratin-18 levels were also reduced. Therefore, administration of a specific bacterial consortium of defined composition can ameliorate NASH, PHT, and fibrosis, and delay disease progression.

Plant and animal protein intake and its association with overweight and obesity among the Belgian population.

The objective of the present study was to assess animal and plant protein intakes in the Belgian population and to examine their relationship with overweight and obesity (OB). The subjects participated in the Belgian National Food Consumption Survey conducted in 2004. Food consumption was assessed by using two non-consecutive 24 h dietary recalls. About 3083 participants ( ≥ 15 years of age; 1546 males, 1537 females) provided completed dietary information. Animal protein intake (47 g/d) contributed more to total protein intakes of 72 g/d than plant protein intake, which accounted for 25 g/d. Meat and meat products were the main contributors to total animal protein intakes (53 %), whereas cereals and cereal products contributed most to plant protein intake (54 %). Males had higher animal and plant protein intakes than females (P < 0·001). Legume and soya protein intakes were low in the whole population (0·101 and 0·174 g/d, respectively). In males, animal protein intake was positively associated with BMI (ß = 0·013; P = 0·001) and waist circumference (WC; ß = 0·041; P = 0·002). Both in males and females, plant protein intake was inversely associated with BMI (males: ß = - 0·036; P < 0·001; females: ß = - 0·046; P = 0·001) and WC (male: ß = - 0·137; P < 0·001; female: ß = - 0·096; P = 0·024). In conclusion, plant protein intakes were lower than animal protein intakes among a representative sample of the Belgian population and decreased with age. Associations with anthropometric data indicated that plant proteins could offer a protective effect in the prevention of overweight and OB in the Belgian population.

Fibre intake among the Belgian population by sex-age and sex-education groups and its association with BMI and waist circumference.

The objectives of the present study were to assess total dietary fibre intake and the main contributors to fibre intake in the Belgian population by sex-age and sex-education groups and to investigate its relationship with BMI and waist circumference (WC). The participants of the Belgian food consumption survey (2004) were randomly selected. Information about food intake was collected using two repeated, non-consecutive 24 h recall interviews. A total of 3083 individuals ( ≥ 15 years; 1546 men and 1537 women) completed both interviews. The main contributors to total fibre intake (17·8 g/d) were cereals and cereal products (34 %; 5·9 g/d), potatoes and other tubers (18·6 %; 3·3 g/d), fruits (14·7 %; 2·8 g/d) and vegetables (14·4 %; 2·6 g/d). Legume fibre intake was extremely low (0·672 %; 0·139 g/d). In all sex-age and sex-education groups, total fibre intake was below the recommendations of the Belgian Superior Health Council. Men (21 g/d) consumed significantly more fibre than women (17·3 g/d) (P < 0·001). Lower educated men and higher educated women reported the highest fibre intake. A significant inverse association was found between total fibre intake and WC (ß = - 0·118, P < 0·001). Fruit-derived fibre was positively associated with WC (ß = 0·731, P = 0·001). In summary, total fibre intake was inversely associated with WC, whereas fruit-derived fibre intake was positively associated with WC in the Belgian population.

Dietary sources of animal and plant protein intake among Flemish preschool children and the association with socio-economic and lifestyle-related factors.

BACKGROUND: The aims of this study were to assess the intake of animal, plant and food group-specific protein, and to investigate their associations with socio-economic and lifestyle-related factors in Flemish preschoolers. METHODS: Three-day estimated dietary records were collected from 661 preschoolers aged 2.5-6.5 y (338 boys and 323 girls). Multiple linear regression analysis was used to investigate the association between animal, plant, and food group-specific protein intake and socio-economic and lifestyle factors. RESULTS: Animal proteins (mean 38 g/d) were the main source of total protein (mean 56 g/d), while mean plant protein intake amounted to 18 g/d. The group of meat, poultry, fish and eggs was the main contributor (51%) to animal protein intake, followed by milk and milk products (35%). Bread and cereals (41%) contributed most to the plant protein intake, followed by low-nutritious, energy-dense foods (21%). With higher educated fathers and mothers as reference, respectively, preschoolers with lower secondary and secondary paternal education had lower animal, dairy-, and meat-derived protein intakes, and those with lower secondary and secondary maternal education consumed less plant, and bread and cereal-derived proteins. Compared to children with high physical activity levels, preschoolers with low and moderate physical activity had lower animal and plant protein intakes. Significantly higher potatoes and grains-, and fish- derived proteins were reported for children of smoking mothers and fathers, respectively, compared to those of non-smoking mothers and fathers. CONCLUSIONS: The total protein intake of Flemish preschoolers was sufficient according to the recommendations of the Belgian Superior Health Council. Parental level of education and smoking status might play a role in the sources of children's dietary proteins.

Dietary sources of fiber intake and its association with socio-economic factors among Flemish preschool children.

The objectives were to assess total dietary fiber intake, identify the major sources of dietary fiber, and examine its association with socio-economic factors among Flemish preschoolers. Three-day estimated dietary records were collected from a representative sample of preschoolers 2.5-6.5 years old (n = 661; 338 boys, 323 girls). The mean dietary fiber intake (13.4 g/d) was lower than the intake level recommended by the Belgian Superior Health Council (70% boys and 81% girls below the guidelines). The most important contributor was the group of bread and cereals (29.5%), followed by fruits (17.8%), potatoes and grains (16.0%), energy-dense, low-nutritious foods (12.4%), and vegetables (11.8%). Multiple linear regression analyses showed that total fiber intake was associated with maternal education and parents' employment. Overall, fiber intakes from high-nutritious foods (vegetables and fruits) were higher in preschoolers of higher educated mothers and those with one or both parents being employed. In conclusion, the majority of the preschoolers had dietary fiber intakes below the recommended level. Hence, dietary fiber should be promoted among parents of preschoolers and low socio-economic status families should be addressed in particular.

Aggregate size and architecture determine microbial activity balance for one-stage partial nitritation and anammox.

Aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB) cooperate in partial nitritation/anammox systems to remove ammonium from wastewater. In this process, large granular microbial aggregates enhance the performance, but little is known about granulation so far. In this study, three suspended-growth oxygen-limited autotrophic nitrification-denitrification (OLAND) reactors with different inoculation and operation (mixing and aeration) conditions, designated reactors A, B, and C, were used. The test objectives were (i) to quantify the AerAOB and AnAOB abundance and the activity balance for the different aggregate sizes and (ii) to relate aggregate morphology, size distribution, and architecture putatively to the inoculation and operation of the three reactors. A nitrite accumulation rate ratio (NARR) was defined as the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate. The smallest reactor A, B, and C aggregates were nitrite sources (NARR, >1.7). Large reactor A and C aggregates were granules capable of autonomous nitrogen removal (NARR, 0.6 to 1.1) with internal AnAOB zones surrounded by an AerAOB rim. Around 50% of the autotrophic space in these granules consisted of AerAOB- and AnAOB-specific extracellular polymeric substances. Large reactor B aggregates were thin film-like nitrite sinks (NARR, <0.5) in which AnAOB were not shielded by an AerAOB layer. Voids and channels occupied 13 to 17% of the anoxic zone of AnAOB-rich aggregates (reactors B and C). The hypothesized granulation pathways include granule replication by division and budding and are driven by growth and/or decay based on species-specific physiology and by hydrodynamic shear and mixing.

Microbial equol production attenuates colonic methanogenesis and sulphidogenesis in vitro.

Hydrogen gas produced during colonic fermentation is excreted in breath and flatus, or removed by hydrogen-consuming bacteria such as methanogens and sulphate-reducing bacteria. However, recent research has shown that H2 is also consumed by equol-producing bacteria during the reduction of daidzein into equol. In this study, the interactions between methanogens, sulphate-reducing, and equol-producing bacteria were investigated under in vitro simulated intestinal conditions. In the presence of daidzein, the equol-producing bacterial consortium EPC4 gave rise to equol production in cultures of Methanobrevibacter smithii or Desulfovibrio sp. as well as in faecal samples with methanogenic or sulphate-reducing abilities. Moreover, this supplementation significantly (P<0.001) decreased the methanogenesis and sulphidogenesis. The attenuation did not occur in the absence of a daidzein source. Additionally, there was no influence of soy germ powder, daidzein or equol as such, excluding a possible inhibition by these compounds. Finally, a stronger decrease was observed with increasing amounts of EPC4 and a constant equol production, suggesting that the observed effect was only partly caused by the action of daidzein as a hydrogen sink. These findings are of relevance since abdominal discomfort such as bloating and flatulence, are related to colonic gas production, whereas equol has potential health benefits.

Exploration of isoxanthohumol bioconversion from spent hops into 8-prenylnaringenin using resting cells of Eubacterium limosum.

Hops is an almost unique source of the potent phytoestrogen 8-prenylnaringenin (8-PN). As hops contain only low levels of 8-PN, synthesis may be more attractive than extraction. A strain of the Gram-positive Eubacterium limosum was isolated previously for 8-PN production from more abundant precursor isoxanthohumol (IX) from hops. In this study, spent hops, an industrial side stream from the beer industry, was identified as interesting source of IX. Yet, hop-derived compounds are well-known antibacterial agents and the traces of a large variety of different compounds in spent hops interfered with growth and IX conversion. Critical factors to finally enable bacterial 8-PN production from spent hops, using a food and feed grade medium, were evaluated in this research. The use of bacterial resting cells and complex medium at a pH of 7.8-8 best fulfilled the requirements for 8-PN production and generated a solid basis for development of an economic process.

Live Biotherapeutic Products, A Road Map for Safety Assessment.

Recent developments in the understanding of the relationship between the microbiota and its host have provided evidence regarding the therapeutic potential of selected microorganisms to prevent or treat disease. According to Directive 2001/83/EC, in the European Union (EU), any product intended to prevent or treat disease is defined as a medicinal product and requires a marketing authorization by competent authorities prior to commercialization. Even if the pharmaceutical regulatory framework is harmonized at the EU level, obtaining marketing authorisations for medicinal products remains very challenging for Live Biotherapeutic Products (LBPs). Compared to other medicinal products currently on the market, safety assessment of LBPs represents a real challenge because of their specific characteristics and mode of action. Indeed, LBPs are not intended to reach the systemic circulation targeting distant organs, tissues, or receptors, but rather exert their effect through direct interactions with the complex native microbiota and/or the modulation of complex host-microbiota relation, indirectly leading to distant biological effects within the host. Hence, developers must rely on a thorough risk analysis, and pharmaceutical guidelines for other biological products should be taken into account in order to design relevant non-clinical and clinical development programmes. Here we aim at providing a roadmap for a risk analysis that takes into account the specificities of LBPs. We describe the different risks associated with these products and their interactions with the patient. Then, from that risk assessment, we propose solutions to design non-clinical programmes and First in Human (FIH) early clinical trials appropriate to assess LBP safety.

Cosupplementation of isoflavones, prenylflavonoids, and lignans alters human exposure to phytoestrogen-derived 17beta-estradiol equivalents.

The microbial metabolism of dietary phytoestrogens varies considerably among individuals and influences the final exposure to bioactive compounds. In view of the increasing number of food supplements combining several classes of phytoestrogens, the microbial potential to activate various proestrogens within an individual was evaluated in 3 randomized dietary crossovers. Treatment allocation was based on participants' eligibility (>45% in vitro bioactivation of >or=2 separate proestrogens by fecal cultures; n = 40/100). After a run-in of >or=4 d, participants were given soy-, hop-, and/or flax-based food supplements dosed either separately (SOY: 2.83 mg daidzein aglycone equivalents/supplement, HOP: 1.20 mg isoxanthohumol (IX)/supplement, or FLAX: 2.08 mg secoisolariciresinol (SECO) aglycone equivalents/supplement; reference intervention) or simultaneously (MIX; test intervention) 3 times/d for 5 d, followed by a wash-out period (>or=7 d) and the second intervention. Before and after each (co)supplementation, spot urine and serum were collected. In total, 22 equol, 19 8-prenylnaringenin (8-PN), and 21 enterolactone (ENL) producers completed the SOY+MIX, HOP+MIX, and FLAX+MIX trials, respectively. The microbial bioactivation of daidzein, IX, and SECO, generally decreased upon coincubation in vitro (equol: 4.4%, P = 0.164; 8-PN: 20.5%, P < 0.001; ENL: 44.3%, P < 0.001) and cosupplementation in vivo (equol: 28.3%, P = 0.009; 8-PN: 35.4%, P = 0.107; ENL: 35.9%, P = 0.003). Although the bioavailabilities of total isoflavones, prenylflavonoids, and lignans were not significantly affected upon coadministration, participants were exposed to lower phytoestrogen-derived 17beta-estradiol equivalents. In conclusion, the bioavailability of phytoestrogens, especially when given in mixtures, is subject to high interindividual variation. These findings support the importance of personalized screening when assessing the efficacy of such products and mixtures.

Exploratory analysis of the human breast DNA methylation profile upon soymilk exposure.

Upon soy consumption, isoflavone metabolites attain bioactive concentrations in breast tissue possibly affecting health. Though in vitro epigenetic activity of soy metabolites has been described, the in vivo impact on the epigenome is largely unknown. Therefore, in this case-control study, the breast glandular tissue DNA methylome was explored in women undergoing an aesthetic breast reduction. After a run-in phase, 10 generally healthy Belgian or Dutch women received soymilk for 5 days. MethylCap-seq methylation profiles were compared with those of 10 matched controls. Isoflavones and their microbial metabolites were quantified in urine, serum, and glandular breast tissue (liquid chromatography-mass spectrometry) and 17ß-estradiol in glandular breast tissue (immunoassay). Global DNA methylation levels were obtained for 6 cases and 5 controls using liquid chromatography-mass spectrometry. Although lower MethylCap-seq coverages were observed, mass spectrometry results and computational LINE-1 methylation analysis did not provide evidence supporting global methylation alterations upon treatment. At a false discovery rate of 0.05, no differentially methylated loci were identified. Moreover, a set of previously identified loci was specifically tested, but earlier reported results could not be validated. In conclusion, after a 5-day soymilk treatment, no major general epigenetic reprogramming in breast tissue could be found in this exploratory study.

Metabolism of isoflavones, lignans and prenylflavonoids by intestinal bacteria: producer phenotyping and relation with intestinal community.

Many studies have investigated the importance of the intestinal bacterial activation of individual phytoestrogens. However, human nutrition contains different phytoestrogens and the final exposure depends on the microbial potential to activate all different groups within each individual. In this work, interindividual variations in the bacterial activation of the different phytoestrogens were assessed. Incubation of feces from 100 individuals using SoyLife EXTRA, LinumLife EXTRA and isoxanthohumol suggested that individuals could be separated into high, moderate and low O-desmethylangolensin (O-DMA), equol, enterodiol (END), enterolactone (ENL) or 8-prenylnaringenin producers, but that the metabolism of isoflavones, lignans and prenylflavonoids follows separate, independent pathways. However, O-DMA and equol production correlated negatively, whereas a positive correlation was found between END and ENL production. In addition, END production correlated negatively with Clostridium coccoides-Eubacterium rectale counts. Furthermore, O-DMA production was correlated with the abundance of methanogens, whereas equol production correlated with sulfate-reducing bacteria, indicating that the metabolic fate of daidzein may be related to intestinal H(2) metabolism.

Equine-Induced Pluripotent Stem Cells Retain Lineage Commitment Toward Myogenic and Chondrogenic Fates.

Induced pluripotent stem cells (iPSCs) hold great potential not only for human but also for veterinary purposes. The equine industry must often deal with health issues concerning muscle and cartilage, where comprehensive regenerative strategies are still missing. In this regard, a still open question is whether equine iPSCs differentiate toward muscle and cartilage, and whether donor cell type influences their differentiation potential. We addressed these questions through an isogenic system of equine iPSCs obtained from myogenic mesoangioblasts (MAB-iPSCs) and chondrogenic mesenchymal stem cells (MSC-iPSCs). Despite similar levels of pluripotency characteristics, the myogenic differentiation appeared enhanced in MAB-iPSCs. Conversely, the chondrogenic differentiation was augmented in MSC-iPSCs through both teratoma and in vitro differentiation assays. Thus, our data suggest that equine iPSCs can differentiate toward the myogenic and chondrogenic lineages, and can present a skewed differentiation potential in favor of the source cell lineage.

Characterization of the bactericidal effect of dietary sphingosine and its activity under intestinal conditions.

Sphingosine is known as a natural antimicrobial agent, protecting the human skin from bacterial colonization and possibly affecting the intestinal microbial community after ingestion. In this study we further investigated the antibacterial spectrum of dietary d-eythro-sphingosine in saline towards three intestinal pathogens and to the health promoting lactobacilli and bifidobacteria. The degree of bactericidal effect was studied using plate counts and Live/Dead analysis combined with flow cytometry. To assess activity under complex intestinal conditions, sphingosine was dosed to the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) for a period of 11 days. Finally, we tried to elucidate the factors influencing the activity and the mode of action of sphingosine. In all performed experiments, high correlation occurred between plate counts and Live/Dead analysis. In saline a strong antibacterial effect was seen to all tested species, Gram-negative and Gram-positive, and sphingosine not selectively acted against pathogens, as health promoting bacteria were also affected. Under simulated intestinal conditions however, no shifts in bacterial concentrations were detected. Experiments with individual medium components thought that the effect of sphingosine is very easily neutralized by BSA, stearic acid and surfactants. Based on our results, d-erythro-sphingosine would only be active when protonated and its mode of action would imply electrostatic attraction to the bacteria and disruption of membrane integrity. In conclusion, the application of sphingosine is limited to specific environments, as activity was very sensitive to inhibition. Yet, because of its broad spectrum membrane disrupting activity, it could be very useful under controlled conditions.

Gut metabotypes govern health effects of dietary polyphenols.

Polyphenols are thought to be responsible for some of the health effects conferred by a diet rich in fruit and vegetables. Both the formation of bioactive polyphenol-derived metabolites and the modulation of colonic microbiota contribute to these health benefits. Therefore, one cannot infer biological responses from dietary intake records without considering polyphenol-microbiota interactions. However, the latter are complex and subject to large interindividual variability, leading to different polyphenol-metabolizing phenotypes or 'metabotypes'. Based on accurate measurements of intake, exposure and effect on carefully selected samples, the physiological relevance of dietary polyphenols can be evaluated for each metabotype. Ultimately, this will lead to predictive modeling and the development of (personalized) functional foods and other nutraceuticals with maximized health benefits.

The intestinal microbiome: a separate organ inside the body with the metabolic potential to influence the bioactivity of botanicals.

For many years, it was believed that the main function of the large intestine was the resorption of water and salt and the facilitated disposal of waste materials. However, this task definition was far from complete, as it did not consider the activity of the microbial content of the large intestine. Nowadays it is clear that the complex microbial ecosystem in our intestines should be considered as a separate organ within the body, with a metabolic capacity which exceeds the liver with a factor 100. The intestinal microbiome is therefore closely involved in the first-pass metabolism of dietary compounds. This is especially true for botanical supplements, which are now marketed for various health applications. Being of natural origin, their structural building blocks, such as polyphenols, are often highly recognized by the human and especially the intestinal microbial metabolism machinery. Intensive metabolism results in often low circulating levels of the original products, with the consequence that final health effects of botanicals are often related to specific active metabolites which are produced in the body rather than being related to the product's original composition. Understanding how such metabolic processes contribute to the in situ exposure is therefore crucial for the proper interpretation of biological responses. A multidisciplinary approach, characterizing the food and phytochemical intake as well as the metabolic potency of the gut microbiota, while measuring biomarkers of both exposure and response in target tissues, is therefore of critical importance. With polyphenol metabolism as example, this review describes how the incorporation of microbial metabolism as an important variable in the evaluation of the final bioactivity of botanicals strongly increases the relevance and predictive value of the outcome. Moreover, knowledge about intestinal processes may offer innovative strategies for targeted product development.

Production of monoclonal antibodies against hop-derived (Humulus lupulus L.) prenylflavonoids and the development of immunoassays.

Monoclonal antibodies against the hop-derived prenylated chalcone xanthohumol (X) and the prenylated flavonoids isoxanthohumol (IX) and 8-prenylnaringenin (8-PN) were developed. Carboxylic acid haptens of X, IX and 8-PN were synthesized by linking a spacer to their C4'-OH group followed by subsequent coupling to bovine serum albumin (BSA) to form conjugates that were employed as immunogens in BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the established hybridoma cell lines proved, in cross-reactivity studies, to possess highly specific binding capacities in an optimized competitive indirect ELISA. The immunoassays make use of immunogen-coated microtiterplates and a peroxidase-labeled anti-mouse IgG(1) secondary antibody with ABTS as a chromogenic substrate. For X the IC(50) value derived from the standard curve was 62.91 ng mL(-1), and for both IX and 8-PN 37.15 ng mL(-1). The assay was validated for the quantitative analysis of X, IX and 8-PN in urine and serum. A simple sample pretreatment procedure using a diethyl ether extraction was optimized and the recoveries and matrix effects were assessed. The validity of the established assay was tested and mean inter- and intra-assay variations in urine were 2.32% and 1.91%, respectively for X, 6.24% and 2.39%, respectively for IX and 7.18% and 0.74%, respectively for 8-PN. In serum, the mean inter- and intra-assay variations were 8.90% and 1.37%, respectively for X, 6.13% and 1.57%, respectively for IX and 6.13% and 2.43%, respectively for 8-PN. Furthermore, the method demonstrated excellent accuracy and significant correlation with measurements by an established and validated HPLC-MS method.