In Vitro Bioactivities of Food Grade Extracts from Yarrow (Achillea millefolium L.) and Stinging Nettle (Urtica dioica L.) Leaves.

Yarrow (Achillea millefolium L., AM) and nettle (Urtica dioica L., UD) are bioactive plants used commercially in functional food and supplement applications and traditionally to alleviate gastric disorders. In this work, the effects of food-grade optimized extracts of Finnish early-season AM and UD were tested on bacterial growth including potential beneficial and foodborne pathogens, as well as murine norovirus (MNV). The anti-inflammatory properties of the extracts were also tested in vitro by NF-κB reporter cells. The food-grade extraction was optimized with the response surface modelling in terms of total carotenoid, chlorophyll, and phenolic compounds contents and antioxidant capacities. The optimal food-grade extraction parameters were a 1-h extraction in 70% ethanol at 45 °C for AM, and at 49 °C for UD. There were no significant effects on the beneficial bacteria (Lacticaseibacillus and Bifidobacterium strains), and the extracts were more effective against gram-positive than gram-negative foodborne bacteria and potential pathogens. Listeria innocua was the most susceptible strain in the optimized extracts with a growth rate of 0.059 ± 0.004 for AM and 0.067 ± 0.006 for UD, p < 0.05 compared to control. The optimized extracts showed a logarithmic growth reduction of 0.67 compared to MNV. The hydroethanolic extracts were cytotoxic to both cell lines, whereas aqueous AM and UD extracts induced and reduced TLR4 signalling in a reporter cell line, respectively. The results provide novel food-grade extraction parameters and support the bioactive effects of AM and UD in functional food applications, but more research is needed to elucidate the precise biological activity in vivo for gastric health.

Omic methodologies for assessing metal(-loid)s-host-microbiota interplay: A review.

Omic methodologies have become key analytical tools in a wide number of research topics such as systems biology, environmental analysis, biomedicine or food analysis. They are especially useful when they are combined providing a new perspective and a holistic view of the analytical problem. Methodologies for microbiota analysis have been mostly focused on genome sequencing. However, information provided by these metagenomic studies is limited to the identification of the presence of genes, taxa and their inferred functionality. To achieve a deeper knowledge of microbial functionality in health and disease, especially in dysbiosis conditions related to metal and metalloid exposure, the introduction of additional meta-omic approaches including metabolomics, metallomics, metatranscriptomics and metaproteomics results essential. The possible impact of metals and metalloids on the gut microbiota and their effects on gut-brain axis (GBA) only begin to be figured out. To this end new analytical workflows combining powerful tools are claimed such as high resolution mass spectrometry and heteroatom-tagged proteomics for the absolute quantification of metal-containing biomolecules using the metal as a "tag" in a sensitive and selective detector (e.g. ICP-MS). This review focus on current analytical methodologies related with the analytical techniques and procedures available for metallomics and microbiota analysis with a special attention on their advantages and drawbacks.

Antioxidant Vitamins and Prebiotic FOS and XOS Differentially Shift Microbiota Composition and Function and Improve Intestinal Epithelial Barrier In Vitro.

Human gut microbiota (HGM) play a significant role in health and disease. Dietary components, including fiber, fat, proteins and micronutrients, can modulate HGM. Much research has been performed on conventional prebiotics such as fructooligosaccharides (FOS) and galactooligosaccharides (GOS), however, novel prebiotics or micronutrients still require further validation. We assessed the effect of FOS, xylooligosaccharides (XOS) and a mixture of an antioxidant vitamin blend (AOB) on gut microbiota composition and activity, and intestinal barrier in vitro. We used batch fermentations and tested the short-term effect of different products on microbial activity in six donors. Next, fecal inocula from two donors were used to inoculate the simulator of the human microbial ecosystem (SHIME) and after long-term exposure of FOS, XOS and AOB, microbial activity (short- and branched-chain fatty acids and lactate) and HGM composition were evaluated. Finally, in vitro assessment of intestinal barrier was performed in a Transwell setup of differentiated Caco-2 and HT29-MTX-E12 cells exposed to fermentation supernatants. Despite some donor-dependent differences, all three tested products showed beneficial modulatory effects on microbial activity represented by an increase in lactate and SCFA levels (acetate, butyrate and to a lesser extent also propionate), while decreasing proteolytic markers. Bifidogenic effect of XOS was consistent, while AOB supplementation appears to exert a specific impact on reducing F. nucleatum and increasing butyrate-producing B. wexlerae. Functional and compositional microbial changes were translated to an in vitro host response by increases of the intestinal barrier integrity by all the products and a decrease of the redox potential by AOB supplementation.

Insights into cancer and neurodegenerative diseases through selenoproteins and the connection with gut microbiota - current analytical methodologies.

Introduction: Selenium plays many key roles in health especially in connection with cancer and neurodegenerative diseases. However, it needs to be appreciated that the essentiality/toxicity of selenium depends on both, a narrow range of concentration and the chemical specie involved. In this context, selenoproteins are essential biomolecules against these disorders, mainly due to its antioxidant action. To this end, analytical methodologies may allow identifying and quantifying individual selenospecies in human biofluids and tissues. Areas covered: This review focus on the role of selenoproteins in medicine, with special emphasis in cancer and neurodegenerative diseases, considering the possible link with gut microbiota. In particular, this article reviews the analytical techniques and procedures recently developed for the absolute quantification of selenoproteins and selenometabolites in human biofluids and tissues. Expert commentary: The beneficial role of selenium in human health has been extensively studied and reviewed. However, several challenges remain unsolved as discussed in this article: (i) speciation of selenium (especially selenoproteins) in cancer and neurodegenerative disease patients; (ii) supplementation of selenium in humans using functional foods and nutraceuticals; (iii) the link between selenium and selenoproteins expression and the gut microbiota and (iv) analytical methods and pitfalls for the absolute quantification of selenoproteins and selenometabolites.

Butyrate-producing bacteria supplemented in vitro to Crohn's disease patient microbiota increased butyrate production and enhanced intestinal epithelial barrier integrity.

The management of the dysbiosed gut microbiota in inflammatory bowel diseases (IBD) is gaining more attention as a novel target to control this disease. Probiotic treatment with butyrate-producing bacteria has therapeutic potential since these bacteria are depleted in IBD patients and butyrate has beneficial effects on epithelial barrier function and overall gut health. However, studies assessing the effect of probiotic supplementation on microbe-microbe and host-microbe interactions are rare. In this study, butyrate-producing bacteria (three mono-species and one multispecies mix) were supplemented to the fecal microbial communities of ten Crohn's disease (CD) patients in an in vitro system simulating the mucus- and lumen-associated microbiota. Effects of supplementation in short-chain fatty acid levels, bacterial colonization of mucus environment and intestinal epithelial barrier function were evaluated. Treatment with F. prausnitzii and the mix of six butyrate-producers significantly increased the butyrate production by 5-11 mol%, and colonization capacity in mucus- and lumen-associated CD microbiota. Treatments with B. pullicaecorum 25-3T and the mix of six butyrate-producers improved epithelial barrier integrity in vitro. This study provides proof-of-concept data for the therapeutic potential of butyrate-producing bacteria in CD and supports the future preclinical development of a probiotic product containing butyrate-producing species.

Transformation of arsenic species during in vitro gastrointestinal digestion of vegetables.

Arsenic is an element widely distributed in the environment, and the diet is the main source of arsenic exposure for most people. However, many of the processes related to steps before intestinal absorption are unknown. This study evaluates the effect of in vitro gastrointestinal digestion on pentavalent arsenic forms [As(V), MMA(V), DMA(V)] present in various vegetables (garlic, broccoli, asparagus, spinach) after soaking or boiling in aqueous solutions of these species. The results showed that the gastrointestinal digest contained trivalent or thiolated arsenic forms different from the pentavalent species added initially. Transformation percentages varied, depending on sample, treatment, and arsenic species. Results showed transformation of up to 22% to As(III), 35% to MMA(III)/MMAS, and 26% to DMA(III)/DMAS. These data indicate that more toxic arsenic species are present in the gastrointestinal digest, and they highlight the need to consider this process when evaluating the toxicological risk associated with ingestion of this metalloid.

Active films based on cocoa extract with antioxidant, antimicrobial and biological applications.

Novel films of ethylene-vinyl alcohol copolymer (EVOH) containing flavonoid-rich cocoa were developed. To understand their potential application as active packaging material, antioxidant and antimicrobial properties of the films were determined as well as the antioxidant activity of the release compounds in Caco-2 human epithelial colorectal adenocarcinoma cells. Exposure of the films to aqueous food simulant showed antioxidant capacity. The release of cocoa extract components was dependent on the antioxidant concentration incorporated in the film and on temperature. Cocoa extract and the fraction obtained after in vitro gastrointestinal digestion presented antioxidant activity against oxidative stress induced by hydrogen peroxide in Caco-2 cells. Films with 10%, 15%, and 20% cocoa extract produced bactericidal effect against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella enterica. The application of films to an infant milk formula, previously inoculated with L. monocytogenes, inhibited the growth of bacteria 1.5 log units the first day and showed sustained release, inhibiting 0.52 and 0.76 log units, respectively, by the sixth day, while cocoa powder added directly did not produce any effect.

In vitro study of intestinal transport of inorganic and methylated arsenic species by Caco-2/HT29-MTX cocultures.

This study characterizes intestinal absorption of arsenic species using in vitro system Caco-2/HT29-MTX cocultures in various proportions (100/0 to 30/70). The species assayed were As(V), As(III), monomethylarsonic acid [MMA(V)], monomethylarsonous acid [MMA(III)], dimethylarsinic acid [DMA(V)], and dimethylarsinous acid [DMA(III)]. The results show that the apparent permeability (P(app)) values of pentavalent species increase significantly in the Caco-2/HT29-MTX cocultures in comparison with the Caco-2 monoculture, probably because of enhancement of paracellular transport. For MMA(III) and DMA(III), P(app) decreases in the Caco-2/HT29-MTX cell model, and for As(III), there is no change in P(app) between the two culture models. Transport studies of arsenic solubilized from cooked foods (rice, garlic, and seaweed) after applying an in vitro gastrointestinal digestion showed that arsenic absorption also varies with the model used, increasing with the incorporation of HT29-MTX in the culture. These results show the importance of choosing a suitable in vitro model when evaluating intestinal arsenic absorption processes.

Mercury and selenium in fish and shellfish: occurrence, bioaccessibility and uptake by Caco-2 cells.

This study evaluates Hg and Se concentrations and bioaccessibility (element solubilised after simulated gastrointestinal digestion) in 16 raw seafood species consumed in Spain. The concentrations varied greatly (Hg, 3.8-1621 ng/g wet weight, ww; Se, 84-1817 ng/g ww). Only one sample of swordfish exceeded the Hg limit permitted in Spain (1mg/kg), and for this sample the Hg/Se molar ratio and Se Health Benefit Value food safety criteria also indicated the presence of a risk. Bioaccessibility of Hg (35-106%) and Se (17-125%) was very variable and the Hg/Se molar ratio in the bioaccessible fraction was less than one for all samples. Transport by Caco-2 cells, an intestinal epithelium model, was also evaluated from the swordfish bioaccessible fraction. Hg and Se transport from the food was less than 14%, and cell retention was much greater for Hg (49-69%) than Se (8-12%).

Comparison of a static and a dynamic in vitro model to estimate the bioaccessibility of As, Cd, Pb and Hg from food reference materials Fucus sp. (IAEA-140/TM) and Lobster hepatopancreas (TORT-2).

Bioaccessibility, the fraction of an element solubilized during gastrointestinal digestion and available for absorption, is a factor that should be considered when evaluating the health risk of contaminants from food. Static and dynamic models that mimic human physiological conditions have been used to evaluate bioaccessibility. This preliminary study compares the bioaccessibility of arsenic (As), cadmium (Cd), lead (Pb) and mercury (Hg) in two food certified reference materials (CRMs) (seaweed: Fucus sp., IAEA-140/TM; Lobster hepatopancreas: TORT-2), using two in vitro gastrointestinal digestion methods: a static method (SM) and a dynamic multicompartment method (TIM-1). There are significant differences (p<0.05) between the bioaccessible values of As, Cd, Pb and Hg obtained by SM and TIM-1 in the two CRMs. The specific form in which the elements studied are present in the CRM may help to explain the bioaccessibility values obtained.