Dose-Dependent Alterations to In Vitro Human Microbiota Composition and Butyrate Inhibition by a Supercritical Carbon Dioxide Hops Extract.

Hop cones (Humulus lupulus L.) have been used throughout history as an additive in beer brewing and as herbal supplements with medicinal and culinary properties. The objective of this study was to ascertain the effect of a range of concentrations of a supercritical CO2 extract of hops on the composition and metabolism of human gut bacterial communities using in vitro batch culture systems. Fermentations were conducted over 24 h using a mixed human fecal inoculum. Microbial metabolism was assessed by measuring organic acid production and microbial community alterations were determined by 16S rRNA gene sequencing. Butyrate, an important short chain fatty acid in maintaining colonic well-being, decreased at elevated concentrations of hops, which may partly be accounted for by the concomitant reduction of Eubacterium and Coprococcus, known butyrate-producing genera, and also the inhibition of Bifidobacterium, a beneficial organism that has a butyrogenic effect through metabolic cross-feeding with intestinal commensals. The hops compounds also caused dose-dependent increases in the potentially pathogenic Enterobacteriaceae and potentially beneficial Akkermansia. Thus, hops compounds had a significant impact on the structure of the bacterial consortium, which warrants further study including human clinical trials.

Dose Effects of Orally Administered Spirulina Suspension on Colonic Microbiota in Healthy Mice.

Oral supplemented nutraceuticals derived from food sources are surmised to improve the human health through interaction with the gastrointestinal bacteria. However, the lack of fundamental quality control and authoritative consensus (e.g., formulation, route of administration, dose, and dosage regimen) of these non-medical yet bioactive compounds are one of the main practical issues resulting in inconsistent individual responsiveness and confounded clinical outcomes of consuming nutraceuticals. Herein, we studied the dose effects of widely used food supplement, microalgae spirulina (Arthrospira platensis), on the colonic microbiota and physiological responses in healthy male Balb/c mice. Based on the analysis of 16s rDNA sequencing, compared to the saline-treated group, oral administration of spirulina once daily for 24 consecutive days altered the diversity, structure, and composition of colonic microbial community at the genus level. More importantly, the abundance of microbial taxa was markedly differentiated at the low (1.5 g/kg) and high (3.0 g/kg) dose of spirulina, among which the relative abundance of Clostridium XIVa, Desulfovibrio, Eubacterium, Barnesiella, Bacteroides, and Flavonifractor were modulated at various degrees. Evaluation of serum biomarkers in mice at the end of spirulina intervention showed reduced the oxidative stress and the blood lipid levels and increased the level of appetite controlling hormone leptin in a dose-response manner, which exhibited the significant correlation with differentially abundant microbiota taxa in the cecum. These findings provide direct evidences of dose-related modulation of gut microbiota and physiological states by spirulina, engendering its future mechanistic investigation of spirulina as potential sources of prebiotics for beneficial health effects via the interaction with gut microbiota.

Novel carbohydrate binding modules in the surface anchored α-amylase of Eubacterium rectale provide a molecular rationale for the range of starches used by this organism in the human gut.

Gut bacteria recognize accessible glycan substrates within a complex environment. Carbohydrate binding modules (CBMs) of cell surface glycoside hydrolases often drive binding to the target substrate. Eubacterium rectale, an important butyrate-producing organism in the gut, consumes a limited range of substrates, including starch. Host consumption of resistant starch increases the abundance of E. rectale in the intestine, likely because it successfully captures the products of resistant starch degradation by other bacteria. Here, we demonstrate that the cell wall anchored starch-degrading α-amylase, Amy13K of E. rectale harbors five CBMs that all target starch with differing specificities. Intriguingly these CBMs efficiently bind to both regular and high amylose corn starch (a type of resistant starch), but have almost no affinity for potato starch (another type of resistant starch). Removal of these CBMs from Amy13K reduces the activity level of the enzyme toward corn starches by ∼40-fold, down to the level of activity toward potato starch, suggesting that the CBMs facilitate activity on corn starch and allow its utilization in vivo. The specificity of the Amy13K CBMs provides a molecular rationale for why E. rectale is able to only use certain starch types without the aid of other organisms.

Effect of Dietary Chestnut or Quebracho Tannin Supplementation on Microbial Community and Fatty Acid Profile in the Rumen of Dairy Ewes.

Ruminants derived products have a prominent role in diets and economy worldwide; therefore, the capability to control the rumen microbial ecosystem, for ameliorating their quality, is of fundamental importance in the livestock sector. The aim of this study was to evaluate the effect of dietary supplementation with chestnut and quebracho tannins on microbial community and fatty acid profile, in the rumen fluid of dairy ewes. Multivariate analysis of PCR-DGGE profiles of rumen microbial communities showed a correlation among the presence of chestnut or quebracho in the diet, the specific Butyrivibrio group DGGE profiles, the increase in 18:3 cis9, cis12, and cis15; 18:2 cis9 and cis12; 18:2 cis9 and trans11; 18:2 trans11 and cis15; and 18:1 trans11 content, and the decrease in 18:0 concentration. Phylogenetic analysis of DGGE band sequences revealed the presence of bacteria representatives related to the genera Hungatella, Ruminococcus, and Eubacterium and unclassified Lachnospiraceae family members, suggesting that these taxa could be affected by tannins presence in the diets. The results of this study showed that tannins from chestnut and quebracho can reduce the biohydrogenation of unsaturated fatty acids through changes in rumen microbial communities.

Biodegradation of asphalt by Garciaella petrolearia TERIG02 for viscosity reduction of heavy oil.

Petroleum hydrocarbon is an important energy resource, but it is difficult to exploit due to the presence of dominated heavy constituents such as asphaltenes. In this study, viscosity reduction of Jodhpur heavy oil (2,637 cP at 50°C) has been carried out by the biodegradation of asphalt using a bacterial strain TERIG02. TERIG02 was isolated from sea buried oil pipeline known as Mumbai Uran trunk line (MUT) located on western coast of India and identified as Garciaella petrolearia by 16S rRNA full gene sequencing. TERIG02 showed 42% viscosity reduction when asphalt along with molasses was used as a sole carbon source compared to only asphalt (37%). The viscosity reduction by asphaltene degradation has been structurally characterized by Fourier transform infrared spectroscopy (FTIR). This strain also shows an additional preference to degrade toxic asphalt and aromatics compounds first unlike the other known strains. All these characteristics makes TERIG02 a potential candidate for enhanced oil recovery and a solution to degrading toxic aromatic compounds.

Diversity and similarity of microbial communities in petroleum crude oils produced in Asia.

To understand microbial communities in petroleum crude oils, we precipitated DNA using high concentrations of 2,2,4-trimethylpentane (isooctane) and purified. Samples of DNA from five crude oils, (Middle East, 3; China, 1; and Japan, 1) were characterized based upon their 16S rRNA gene sequences after PCR amplification and the construction of clone libraries. We detected 48 eubacterial species, one cyanobacterium, and one archaeon in total. The microbial constituents were diverse in the DNA samples. Most of the bacteria affiliated with the sequences of the three oils from the Middle East comprised similar mesophilic species. Acinetobacter, Propionibacterium, Sphingobium and a Bacillales were common. In contrast, the bacterial communities in Japanese and Chinese samples were unique. Thermophilic Petrotoga-like bacteria (11%) and several anaerobic-thermophilic Clostridia- and Synergistetes-like bacteria (20%) were detected in the Chinese sample. Different thermophiles (12%) and Clostridia (2%) were detected in the Japanese sample.

Eubacterium limosum activates isoxanthohumol from hops (Humulus lupulus L.) into the potent phytoestrogen 8-prenylnaringenin in vitro and in rat intestine.

Recently, it was shown that the exposure to the potent hop phytoestrogen 8-prenylnaringenin (8-PN) depends on intestinal bacterial activation of isoxanthohumol (IX), but this occurs in only one-third of tested individuals. As the butyrate-producing Eubacterium limosum can produce 8-PN from IX, a probiotic strategy was applied to investigate whether 8-PN production could be increased in low 8-PN producers, thus balancing phytoestrogen exposure. Using fecal samples from high (Hop +) and low (Hop -) 8-PN-producing individuals, a Hop + and Hop - dynamic intestinal model was developed. In parallel, Hop + and Hop - human microbiota-associated rats were developed, germ-free (GF) rats acting as negative controls. IX and then IX + E. limosum were administered in the intestinal model and to the rats, and changes in 8-PN production and exposure were assessed. After dosing IX, 80% was converted into 8-PN in the Hop + model and highest 8-PN production, plasma concentrations, and urinary and fecal excretion occurred in the Hop + rats. Administration of the bacterium triggered 8-PN production in the GF rats and increased 8-PN production in the Hop - model and Hop - rats. 8-PN excretion was similar in the feces (294.1 +/- 132.2 nmol/d) and urine (8.5 +/- 1.1 nmol/d ) of all rats (n = 18). In addition, butyrate production increased in all rats. In conclusion, intestinal microbiota determined 8-PN production and exposure after IX intake. Moreover, E. limosum administration increased 8-PN production in low producers, resulting in similar 8-PN production in all rats.

Molecular and biochemical characterization of two tungsten- and selenium-containing formate dehydrogenases from Eubacterium acidaminophilum that are associated with components of an iron-only hydrogenase.

Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit ( fdhA-I, fdhA-II) and one for an electron-transferring subunit ( fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbor five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level and contain five putative iron-sulfur clusters. Four genes thought to encode the subunits of an iron-only hydrogenase are located upstream of the FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron-sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, and HymC the putative catalytic subunit containing motifs for four iron-sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, and HymA and HymB, identified after determination of their N-terminal sequences. Thus, this complex might represent the most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor, and zinc, but no molybdenum. FDH-II had a two-fold higher K(m) for formate (0.37 mM) than FDH-I and also catalyzed CO(2) reduction to formate. Reverse transcription (RT)-PCR pointed to increased expression of FDH-II in serine-grown cells, supporting the isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in-frame UGA codon for selenocysteine incorporation was read in the heterologous system only as stop codon, although its potential SECIS element exhibited a quite high similarity to that of E. coli FDH.

Interaction of selenoprotein PA and the thioredoxin system, components of the NADPH-dependent reduction of glycine in Eubacterium acidaminophilum and Clostridium litorale [corrected].

Purification of protein PA of the glycine reductase complex from Eubacterium acidaminophilum and Clostridium litorale [corrected] was monitored by a new spectrophotometric assay. The procedure depended on a specific two- to threefold stimulation of a dihydrolipoamide dehydrogenase activity that is elicited by the interaction of a thioredoxin reductase-like flavoprotein and thioredoxin from both organisms. Protein PA isolated from E. acidaminophilum by 75Se labeling and monitoring of the dithioerythritol-dependent glycine reductase activity was identical in its biochemical, structural, and immunological properties to the protein isolated by using the stimulation assay. Proteins PA from both organisms were glycoproteins of Mr about 18,500 and exhibited very similar N-terminal amino acid sequences. Depletion of thioredoxin from crude extracts of E. acidaminophilum totally diminished the NADPH-dependent but not the dithioerythritol-dependent glycine reduction. The former activity could be fully restored by adding thioredoxin. Antibodies raised against the thioredoxin reductase-like flavoprotein or thioredoxin inhibited to a high extent NADPH-dependent but not dithioerythritol-dependent glycine reductase activity. These results indicate the involvement of the thioredoxin system in the electron flow from reduced pyridine nucleotides to glycine reductase.