Isolation, identification and characterisation of three novel probiotic strains (Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036) from the faeces of exclusively breast-fed infants.

The aim of the present study was to isolate, identify and characterise novel strains of lactic acid bacteria and bifidobacteria with probiotic properties from the faeces of exclusively breast-fed infants. Of the 4680 isolated colonies, 758 exhibited resistance to low pH and tolerance to high concentrations of bile salts; of these, only forty-two exhibited a strong ability to adhere to enterocytes in vitro. The identities of the isolates were confirmed by 16S ribosomal RNA (rRNA) sequencing, which permitted the grouping of the forty-two bacteria into three different strains that showed more than 99 % sequence identity with Lactobacillus paracasei, Lactobacillus rhamnosus and Bifidobacterium breve, respectively. The strain identification was confirmed by sequencing the 16S-23S rRNA intergenic spacer regions. Strains were assayed for enzymatic activity and carbohydrate utilisation, and they were deposited in the Collection Nationale de Cultures de Microorganismes (CNCM) of the Institute Pasteur and named L. paracasei CNCM I-4034, B. breve CNCM I-4035 and L. rhamnosus CNCM I-4036. The strains were susceptible to antibiotics and did not produce undesirable metabolites, and their safety was assessed by acute ingestion in immunocompetent and immunosuppressed BALB/c mouse models. The three novel strains inhibited in vitro the meningitis aetiological agent Listeria monocytogenes and human rotavirus infections. B. breve CNCM I-4035 led to a higher IgA concentration in faeces and plasma of mice. Overall, these results suggest that L. paracasei CNCM I-4034, B. breve CNCM I-4035 and L. rhamnosus CNCM I-4036 should be considered as probiotic strains, and their human health benefits should be further evaluated.

The antioxidant effect of ß-caryophyllene protects rat liver from carbon tetrachloride-induced fibrosis by inhibiting hepatic stellate cell activation.

Plant-based whole foods provide thousands of bioactive metabolites to the human diet that reduce the risk of developing chronic diseases. ß-Caryophyllene (CAR) is a common constituent of the essential oil of numerous plants, vegetables, fruits and medicinal herbs, and has been used as a flavouring agent since the 1930 s. Here, we report the antioxidant activity of CAR, its protective effect on liver fibrosis and its inhibitory capacity on hepatic stellate cell (HSC) activation. CAR was tested for the inhibition of lipid peroxidation and as a free radical scavenger. CAR had higher inhibitory capacity on lipid peroxidation than probucol, α-humulene and α-tocopherol. Also, CAR showed high scavenging activities against hydroxyl radical and superoxide anion. The activity of 5-lipoxygenase, an enzyme that actively participates in fibrogenesis, was significantly inhibited by CAR. Carbon tetrachloride-treated rats received CAR at 2, 20 and 200 mg/kg. CAR significantly improved liver structure, and reduced fibrosis and the expression of Col1a1, Tgfb1 and Timp1 genes. Oxidative stress was used to establish a model of HSC activation with overproduction of extracellular matrix proteins. CAR (1 and 10 µm) increased cell viability and significantly reduced the expression of fibrotic marker genes. CAR, a sesquiterpene present in numerous plants and foods, is as a natural antioxidant that reduces carbon tetrachloride-mediated liver fibrosis and inhibits hepatic cell activation.

Metaproteogenomic insights beyond bacterial response to naphthalene exposure and bio-stimulation.

Microbial metabolism in aromatic-contaminated environments has important ecological implications, and obtaining a complete understanding of this process remains a relevant goal. To understand the roles of biodiversity and aromatic-mediated genetic and metabolic rearrangements, we conducted 'OMIC' investigations in an anthropogenically influenced and polyaromatic hydrocarbon (PAH)-contaminated soil with (Nbs) or without (N) bio-stimulation with calcium ammonia nitrate, NH(4)NO(3) and KH(2)PO(4) and the commercial surfactant Iveysol, plus two naphthalene-enriched communities derived from both soils (CN2 and CN1, respectively). Using a metagenomic approach, a total of 52, 53, 14 and 12 distinct species (according to operational phylogenetic units (OPU) in our work equivalent to taxonomic species) were identified in the N, Nbs, CN1 and CN2 communities, respectively. Approximately 10 out of 95 distinct species and 238 out of 3293 clusters of orthologous groups (COGs) protein families identified were clearly stimulated under the assayed conditions, whereas only two species and 1465 COGs conformed to the common set in all of the mesocosms. Results indicated distinct biodegradation capabilities for the utilisation of potential growth-supporting aromatics, which results in bio-stimulated communities being extremely fit to naphthalene utilisation and non-stimulated communities exhibiting a greater metabolic window than previously predicted. On the basis of comparing protein expression profiles and metagenome data sets, inter-alia interactions among members were hypothesised. The utilisation of curated databases is discussed and used for first time to reconstruct 'presumptive' degradation networks for complex microbial communities.

Functional metagenomics unveils a multifunctional glycosyl hydrolase from the family 43 catalysing the breakdown of plant polymers in the calf rumen.

Microbial communities from cow rumen are known for their ability to degrade diverse plant polymers at high rates. In this work, we identified 15 hydrolases through an activity-centred metagenome analysis of a fibre-adherent microbial community from dairy cow rumen. Among them, 7 glycosyl hydrolases (GHs) and 1 feruloyl esterase were successfully cloned, expressed, purified and characterised. The most striking result was a protein of GH family 43 (GHF43), hereinafter designated as R_09-02, which had characteristics very distinct from the other proteins in this family with mono-functional ß-xylosidase, α-xylanase, α-L-arabinase and α-L-arabinofuranosidase activities. R_09-02 is the first multifunctional enzyme to exhibit ß-1,4 xylosidase, α-1,5 arabinofur(pyr)anosidase, ß-1,4 lactase, α-1,6 raffinase, α-1,6 stachyase, ß-galactosidase and α-1,4 glucosidase activities. The R_09-02 protein appears to originate from the chromosome of a member of Clostridia, a class of phylum Firmicutes, members of which are highly abundant in ruminal environment. The evolution of R_09-02 is suggested to be driven from the xylose- and arabinose-specific activities, typical for GHF43 members, toward a broader specificity to the glucose- and galactose-containing components of lignocellulose. The apparent capability of enzymes from the GHF43 family to utilise xylose-, arabinose-, glucose- and galactose-containing oligosaccharides has thus far been neglected by, or could not be predicted from, genome and metagenome sequencing data analyses. Taking into account the abundance of GHF43-encoding gene sequences in the rumen (up to 7% of all GH-genes) and the multifunctional phenotype herein described, our findings suggest that the ecological role of this GH family in the digestion of ligno-cellulosic matter should be significantly reconsidered.

Inter-conversion of catalytic abilities in a bifunctional carboxyl/feruloyl-esterase from earthworm gut metagenome.

Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(k(cat)/K(m))](CE)/[(k(cat)/K(m))](FAE) factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys(281), Asp(282), Asn(316) and Lys(317)) situated close to the catalytic core (Ser(143)/Asp(273)/His(305)) and a deletion of a 34-AA-long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in k(cat)/K(m) values) and enzymes with inverted specificity ((k(cat)/K(m))(CE)/(k(cat)/K(m))(FAE) ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to -5.6 J mol(-1)), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to -13.7 J mol(-1)) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of 'hot spot' mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.

Interplay of metagenomics and in vitro compartmentalization.

In recent years, the application of approaches for harvesting DNA from the environment, the so-called, 'metagenomic approaches' has proven to be highly successful for the identification, isolation and generation of novel enzymes. Functional screening for the desired catalytic activity is one of the key steps in mining metagenomic libraries, as it does not rely on sequence homology. In this mini-review, we survey high-throughput screening tools, originally developed for directed evolution experiments, which can be readily adapted for the screening of large libraries. In particular, we focus on the use of in vitro compartmentalization (IVC) approaches to address potential advantages and problems the merger of culture-independent and IVC techniques might bring on the mining of enzyme activities in microbial communities.

Active hexose correlated compound acts as a prebiotic and is antiinflammatory in rats with hapten-induced colitis.

Active hexose correlated compound (AHCC) is a product prepared from the mycelium of edible Basidiomycete fungi that contains oligosaccharides. Here we have studied the antiinflammatory effect of AHCC in the trinitrobenzenesulfonic acid (TNBS) model of colitis in rats. Rats received AHCC (100 or 500 mg/kg) daily starting 2 d before (pretreatment) colitis induction and were killed 6 d after the TNBS challenge. The status of the rats was assessed by morphological and biochemical methods. The effect of AHCC on the colonic microflora was also assessed by studying the bacteria profile in feces by standard culture techniques. AHCC administration attenuated colonic inflammation, improving rat weight, food intake, damage score, extension of necrosis, colonic weight, colonic weight-to-length ratio, myeloperoxidase and alkaline phosphatase activities, glutathione concentration, and the expression of proinflammatory cytokines and chemokines (IL-1beta, IL-1 receptor antagonist, TNF, and monocyte chemoattractant protein-1) and of mucins 2-4 and trefoil factor 3. The magnitude of the antiinflammatory effect of AHCC was similar to that of sulfasalazine (200 mg/kg). The study of colonic microflora indicated that rats treated with AHCC had higher aerobic and lactic acid bacteria counts as well as higher bifidobacteria counts, whereas clostridia were reduced when compared with the TNBS group. Therefore, our results indicate that AHCC is antiinflammatory and could be useful as a prebiotic to design functional foods for inflammatory bowel disease patients.

Characterization of human cd200 glycoprotein receptor gene located on chromosome 3q12-13.

An immunomodulatory membrane protein, CD200R displays an expression pattern restricted to myeloid cells in mice. It is the receptor for a ligand, CD200, expressed by a broad range of cell types. In this study, we describe the cloning and characterization of the human homologue of the CD200R gene. This gene maps closely to the CD200 gene on human chromosome 3q12-13. The human CD200R gene spans a region of 52 kb, consists of nine exons, and encodes a 348-amino-acid cell-surface protein consisting of two IgFF domains in a typical V/C2 arrangement. The 59-amino-acid cytoplasmic domain has two tyrosine residues, one of which is contained within a NPXY motif. In common with other IgSF genes, the CD200R gene can generate different protein isoforms through alternative splicing. An alternative spliceout form, which has not yet been described in mice, encodes a 188-amino-acid truncated soluble polypeptide containing only the V immunoglobulin domain. In contrast to murine CD200R protein, the human membrane-bound and soluble CD200R proteins have an insertion of 23 amino acids at position 23, encoded by exon 2, which generates a putative dihydroxyacid dehydratase domain. The splicing of exon 2 generates two new isoforms, encoding the membrane and soluble proteins but lacking the dyhydroxyacid dehydratase domain. Northern-blot analysis shows that both membrane-bound and soluble isoforms are expressed in the thymus, liver, spleen and placenta. By RT-PCR, we have analyzed the expression of the four transcript variants in human placenta, spleen, liver, brain and kidney.