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Although the use of probiotics based on Bacillus strains to fight off intestinal pathogens and antibiotic-associated diarrhea is widespread, the mechanisms involved in producing their beneficial effects remain unclear. Here, we studied the ability of compounds secreted by the probiotic Bacillus clausii strain O/C to counteract the cytotoxic effects induced by toxins of two pathogens, Clostridium difficile and Bacillus cereus , by evaluating eukaryotic cell viability and expression of selected genes. Coincubation of C. difficile and B. cereus toxic culture supernatants with the B. clausii supernatant completely prevented the damage induced by toxins in Vero and Caco-2 cells. The hemolytic effect of B. cereus was also avoided by the probiotic supernatant. Moreover, in these cells, the expression of rhoB , encoding a Rho GTPase target for C. difficile toxins, was normalized when C. difficile supernatant was pretreated using the B. clausii supernatant. All of the beneficial effects observed with the probiotic were abolished by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Suspecting the involvement of a secreted protease in this protective effect, a protease was purified from the B. clausii supernatant and identified as a serine protease (M-protease; GenBank accession number Q99405 ). Experiments on Vero cells demonstrated the antitoxic activity of the purified protease against pathogen supernatants. This is the first report showing the capacity of a protease secreted by probiotic bacteria to inhibit the cytotoxic effects of toxinogenic C. difficile and B. cereus strains. This extracellular compound could be responsible, at least in part, for the protective effects observed for this human probiotic in antibiotic-associated diarrhea.

Opuntia ficus indica has been identified for its bioflocculant properties in water treatment; however, its underlying mechanism and active compounds have not been clearly identified. Flocculent molecules of cactus solid material (CSM) under alkaline conditions were extracted at pH 10 and then precipitated under neutral conditions (pH 7). The precipitate was fractionated by ultrafiltration systems and analyzed using inverted phase chromatography and enzymatic treatments. This approach revealed that quercetin and starch constitute the active agents found in the fractionated parts at ≤3,000 and ≥10,000 Da, respectively. The use of quercetin or (potato) starch alone at 18 mg/L yielded 72% ± 2% and 54% ± 3% of turbidity removal, respectively. With a combination of both these components, a higher flocculation activity (84% ± 2%) could be obtained. From these experimental results, a flocculation model based on identified active constituents is being proposed in order to improve process knowledge.

Industrial processing of raspberries into juice and jam results in the production of with high content of lipophilic and hydrophilic phytochemicals. Usually considered as waste, raspberry pomace is occasionally cold-pressed to recover specialty oil. However, the resulting pomace press-cake (PPC) still contains 30% to 35% of lipophilic compounds, such as essential fatty acids, tocols, phytosterols, and ellagitannins initially present in pomace. In a perspective of sustainable development, we investigate an eco-friendly process using an aqueous enzyme-assisted extraction (AEAE) to simultaneously and effectively recover lipophilic compounds and polyphenols from the PPC. The performance of different combinations of carbohydrases and proteases was compared. After selecting the best enzymatic system, a definitive screening design involving six factors was then implemented to optimize the process. Under optimized conditions, 1.2 units of thermostable alkaline protease/100 g PPC, pH 9, 60 degrees C, and 2 hr hydrolysis, more than 38% of total PPC lipophilic content were recovered in the aqueous medium. The recovery of polyphenols and antioxidant activity was, respectively, 48% and 25% higher than obtained by extraction with methanol/acetone/water mixture. Such an AEAE extract might prove useful in food and nutraceutical applications. Practical Application Raspberry pomace, a food industrial by-product, is often considered as waste. However, it is a rich source of phytochemicals, such as tocols, polyphenols, and polyunsaturated fatty acids. To overcome the drawbacks of organic solvent use, an enzyme-assisted extraction process was developed as an eco-friendly alternative to recover these bioactive compounds. Definitive screening design experimental design was used to enhance polyphenols and lipophilics extraction yields while reducing process costs. This extract is an oil-in-water emulsion, with high content in antioxidant phytochemicals, which might have potential for use in nutraceutical applications. Therefore, this green process developed for the valorization of raspberry pomace is considered as a perspective of sustainable development.

We report the preparation of a cellulose fabric bearing derivative protoporphyrin IX units covalently attached to the cellulose backbone of a fabric. Ce-(IV) redox system radical polymerization was used to polymerize methacrylic acid (MAA) onto a cotton material and to obtain cotton-g-polyMAA. Attachment of the photosensitizer, a protoporphyrin IX (PpIX) amino derivative, on cotton-g-polyMAA was realized successfully by a classical peptidic covalent link. The modified surfaces were characterized by ATR-FTIR, DRUV, TGA, and SEM methods. Under visible light irradiation, protoporphyrinic cotton showed antibacterial activity against Staphyloccoccus aureus. This concept is very promising in the field of bacterial decontamination (sterile area, hospital equipment, etc.).

Prostaglandin E2 (PGE2 ) plays a critical role in intestinal mucosal tolerance and barrier integrity. Cyclooxygenase-2 (COX-2)-dependent PGE2 production involves mobilisation of arachidonic acid. Lactobacillus rhamnosus GG (LbGG) is one of the most widely used probiotics reported to colonise the colonic mucosa. LbGG contributes to the protection of the small intestine against radiation injury through the repositioning of mucosal COX-2 expressing cells. However, it is unknown if LbGG modulates PGE2 production in the colonic mucosa under homeostasis and the major cellular elements involved in these processes. Colonic epithelial and CD90+ mesenchymal stromal cells, also known as (myo) fibroblasts (CMFs), are abundant innate immune cells in normal colonic mucosa able to produce PGE2 . Herein, we tested the hypothesis that under colonic mucosal homeostasis, LbGG modulates the eicosanoid pathway resulting in increased PGE2 production in both epithelial and stromal cells. Among the five tested human colonic epithelial cell lines, only exposure of Caco-2 to LbGG for 24 hr led to the mobilisation of arachidonic acid with concomitant increase in the components within the leukotriene and COX-2-dependent PGE2 pathways. By contrast, CMFs isolated from the normal human colonic mucosa responded to LbGG with increased expression of COX-2 and PGE2 in the prostaglandin pathway, but not 5-LO in the leukotriene pathway. Oral gavage of C57BL/6 mice for 5 days with LbGG (5 × 108 Colony-Forming Unit (CFU)/dose) increased COX-2 expression in the colonic mucosa. The majority of cells upregulating COX-2 protein expression were located in the colonic lamina propria and colocalised with α-SMA+ cells corresponding to the CMF phenotype. This process was myeloid differentiation factor-88-dependent, because silencing of myeloid differentiation factor-88 expression in CMFs abrogated LbGG-induced upregulation of COX-2 in culture and in vivo. Taken together, our data suggest that LbGG increases release of COX-2-mediated PGE2 , contributing to the maintenance of mucosal homeostasis in the colon and CMFs are among the major contributors to this process.

International audience; Probiotics represent a potential strategy to influence the host's immune system thereby modulating immune response. Lipoteichoic Acid (LTA) is a major immune-stimulating component of Gram-positive cell envelopes. This amphiphilic polymer, anchored in the cytoplasmic membrane by means of its glycolipid component, typically consists of a poly (glycerol-phosphate) chain with d-alanine and/or glycosyl substitutions. LTA is known to stimulate macrophages in vitro, leading to secretion of inflammatory mediators such as Nitric Oxide (NO). This study investigates the structure-activity relationship of purified LTA from three probiotic Bacillus strains (Bacillus cereus CH, Bacillus subtilis CU1 and Bacillus clausii O/C). LTAs were extracted from bacterial cultures and purified. Chemical modification by means of hydrolysis at pH 8.5 was performed to remove d-alanine. The molecular structure of native and modified LTAs was determined by 1H NMR and GC-MS, and their inflammatory potential investigated by measuring NO production by RAW 264.7 macrophages. Structural analysis revealed several differences between the newly characterized LTAs, mainly relating to their d-alanylation rates and poly (glycerol-phosphate) chain length. We observed induction of NO production by LTAs from B. subtilis and B. clausii, whereas weaker NO production was observed with B. cereus. LTA dealanylation abrogated NO production independently of the glycolipid component, suggesting that immunomodulatory potential depends on d-alanine substitutions. d-alanine may control the spatial configuration of LTAs and their recognition by cell receptors. Knowledge of molecular mechanisms behind the immunomodulatory abilities of probiotics is essential to optimize their use.

Resume La recherche de produits de construction non energivores, resistants et moins couteux est de nos jour une preoccupation mondiale du fait des ressources (energetiques, financieres,…) limitees. Dans ce travail, nous avons associe la decoction de gousses de Parkiabiglobosa (Nere) riche en tanins, notamment en acide gallique, epicatechine et epigallocatechine gallate, a un melange argile–sable afin de repondre a cette problematique. L’argile utilisee est referencee KORO et contient de la montmorillonite, de l’illite, du quartz, de l’albite, de l’orthose, de la kaolinite et de la goethite. L’etude des proprietes mecaniques des geomateriaux formules a partir du melange montre qu’ils presentent des valeurs de resistances mecaniques plus elevees et que leur comportement devient viscoplastique. Cela est du a la formation de complexes chimiques entre les atomes de fer des oxy-hydroxydes dans l’espace interfoliaire des mineraux argileux et les radicaux carboxyliques des macromolecules de tanins. Ce processus est accentue par la sorption des tanins a la surface des mineraux de l’argile.

Among the lytic enzymes secreted by the phytopathogen fungus Sclerotinia sclerotiorum, a starch-degrading activity has been isolated and characterized. Two extracellular α-amylases were produced in culture medium in presence of oats flour as carbons sources. An endoamylase named ScAmy1 was purified to homogeneity by ammonium sulfate precipitation, phosphocellulose and cation exchange high performance liquid chromatographies. Molecular mass of purified ScAmy1 was estimated as 54 kDa. Amylase exhibits maximal activity at pH 5 to 6 and at temperature 60C. ScAmy1 was stable in a pH range of (5–11) and at 50C. Initial activity was still conserved 40%, after heating at 60C during 30 min. In addition, Ca2+activate and stabilize the enzyme. Starch end products were determined as low molecular oligoglucanes, the liquefying power of ScAmy1 was also tested with the Amylograph Brabender, results suggest a suitable application of ScAmy1 in several industrial process. PRACTICAL APPLICATIONS α-Amylase ScAmy1 was highly produced from Sclerotinia sclerotiorum on oats flour – a cheaper by-agro-substrate product. The enzyme was purified and biochemical characterized. ScAmy1 was applied in starch liquefaction treatments assay. The enzyme allows a decrease in peak viscosity after gelatinization and therefore has an important liquefying power. ScAmy1 has a nearly liquefaction effect on flour compared to the commercial enzyme Novamyl, from Novozymes, donated by Novo Nordisk Co. (Denmark). Enzyme end products were analyzed and identified as oligoglucanes and dextrins. Those are widely applied in food, paper, textile and pharmacological industries. Oligosaccharides are useful as prebiotics as dietary fiber or slowly digestible starch derivatives, and they can be used in form of supplement to certain foodstuffs.

International audience; The group of exported proteins of a bacterium are those proteins that are sorted from the cytoplasm to the bacterial surface or to the surroundings of the microorganism. In probiotic bacteria, these proteins are of special relevance because they might determine important traits such as adhesion to intestinal surfaces and molecular cross-talking with the host. Current knowledge about the presence and biological relevance of exported proteins produced by the main genera of probiotic bacteria in the gastrointestinal environment is reviewed in this minireview. As will be seen, some of these proteins are involved in host adhesion or are able to modify certain signalization pathways within host cells, whereas others are important for the physiology of probiotic bacteria in the gastrointestinal tract.

International audience; AIMS: To study the exopolysaccharides (EPSs) produced by three novel moderately halophilic species belonging to the family Alteromonadaceae to optimize EPS yields, characterize their physical and chemical properties and evaluate possible biotechnological applications for these polymers. METHODS AND RESULTS: EPSs synthesized by Idiomarina fontislapidosi F32(T), Idiomarina ramblicola R22(T) and Alteromonas hispanica F23(T) were collected and analysed under optimum conditions: MY medium supplemented with 7.5% (w/v) salts; 32 degrees C; and 1% (w/v) glucose. Polymers were synthesized mainly during the early stationary growth phase with yields ranging from 1 to 1.5 g l(-1). The Idiomarina species each produced an anionic EPS composed mainly of glucose, mannose and galactose. A. hispanica synthesized an anionic EPS composed mainly of glucose, mannose and xylose. Solutions of all the polymers were low in viscosity and pseudoplastic in their behaviour. They showed emulsifying activity and the capacity to bind some metals. CONCLUSIONS: The Alteromonadaceae species studied in this work produced EPSs with physical and chemical properties different from those produced by other halophilic and nonhalophilic bacteria, suggesting that the wide diversity of micro-organisms being encountered nowadays in hypersaline environments offers enormous potential resources for biotechnological applications. SIGNIFICANCE AND IMPACT OF THE STUDY: We have optimized the EPS production and analysed new biopolymers produced by some recently described, moderately halophilic bacteria. These biopolymers are chemically and physically different from others already in use in biotechnology and offer hopes for new applications, especially in the case of A. hispanica, which may prove to be a viable source of xylo-oligosaccharides.

A Lactobacillus plantarum strain producing exopolysaccharides (EPSs) was isolated from corn silage. When this strain, named L. plantarum EP56, was grown on a chemically defined medium, two EPS fractions were isolated. The cell-bound EPS fraction (EPS-b) was composed of a single high-molecular-mass polymer of 8.5x10(5) Da containing glucose, galactose and N-acetylgalactosamine in a molar ratio of approximately 3:1:1 and traces of glycerol and phosphoglycerol. The released EPS fraction (EPS-r) was composed of the high-molecular-mass bound polysaccharide and a second polymer of 4x10(4) Da containing glucose, galactose and rhamnose in a molar ratio of 3:1:1 and traces of glycerol and phosphoglycerol. EPS-b and EPS-r contained phosphate which contributes to their negative net charge. Studies on polysaccharide production and location showed that both polymers were synthesized during the exponential growth phase and that the EPS-b polymer was progressively released into the culture medium during the stationary growth phase. Carbon source and temperature influenced EPS synthesis when L. plantarum EP56 was grown in a chemically defined medium. Lactose was the most efficient carbon source among the five tested (glucose, galactose, fructose, lactose and sucrose). EPS production was also increased when the incubation temperature is lowered.

Streptomyces strain K 1-02 , which was identified as a strain of Streptomyces albidoflavus , secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70°C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH 2 -terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K.

International audience; Probiotics and prebiotics play an important role in human nutrition. In recent years there has been a significant increase in research on the characterization and verification potential health benefits associated with the use of probiotic and prebiotic. The main effects attributed to selected probiotics/prebiotic products have been proved by clinical trials, while others have been acquired on the basis of in vitro tests which require in vivo transposition in order to be validated. The main clinical reports in the literature for the application of probiotic have been done for the treatment of infectious diseases including viral, bacterial or antibiotic associated diarrhoea, relief of chronic bowel inflammatory diseases, immuno-modulation, lowering of serum cholesterol, decreased risk of colon cancer, improve lactose digestion, reduce allergies, and effect on intestinal microbiota. Although the large investigation for the health benefits, information on probiotic species, a specific strain-therapeutic application, and sufficient dosages, is not sufficiently studied to allow practical and rational consumption. Moreover, prebiotic oligosaccharides although provided curative and nutritional values, they are poorly understood in regard to their origin, the processes employed to generate them, their fermentation profiles, and dosages required for health effects. The present review summarizes guidelines reported on the literature in regard to clinician or therapeutic trials of probiotic and prebiotic.

International audience; In the present work, we report on the synthesis of cellulose cotton fibers bearing different types of photosensitizers with the aim to prepare new efficient polymeric materials for antimicrobial applications. Anionic, neutral, and cationic amino porphyrins have been covalently grafted on cotton fabric, without previous chemical modification of the cellulosic support, using a 1,3,5-triazine derivative as the linker. The obtained porphyrin-grafted cotton fabrics were characterized by infrared (ATR-FTIR), diffuse reflectance UV-vis (DRUV) spectroscopies, and thermogravimetric analysis (TGA) to confirm the triazine linkage. Antimicrobial activity of porphyrin-cellulose materials was tested under visible light irradiation against Staphylococcus aureus and Escherichia coli . The results showed excellent activity on the Gram-positive bacterium, showing structure-activity relationship, although no photodamage of the Gram-negative microorganism was recorded. A mechanism of bacterial inactivation by photosensitive surfaces is proposed.

International audience; We describe a new approach to the formation of silver nanoparticles (Ag NPs) using cellulose nanocrystals. The process involves periodate oxidation of cellulose nanocrystals, generating aldehyde functions which, in turn, are used to reduce Ag+ into Ag0 in mild alkaline conditions. The nanoparticles were characterized using transmission electron microscopy (TEM) and ultraviolet-visible absorption spectroscopy. From the microscope studies (TEM) we observed that Ag NPs have spherical shape with a size distribution comprise between 20 and 45 nm. The antibacterial activity was assessed using the minimum inhibitory concentration. The antibacterial assays compare favourably with most of other experiments conducted with the same species.

Anionic, neutral and cationic amino porphyrins were synthesized as precursors of photodynamic antimicrobial agents with an aim to functionalize cotton surface through 1,3,5-triazine link. Structures of porphyrin-triazine derivatives were characterized by 1H NMR, MS and UV-vis confirming the feasibility of this novel concept. Porphyrinic cotton fabrics have been developed from these derivatives, and tested in vitro against Staphylococcus aureus. These novel photodynamic surfaces showed strong and varied antimicrobial activity.

International audience; This paper examines the probiotic bacterium Lactobacillus rhamnosus GG, and how it reacts to the presence of mucin in its extracellular milieu. Parameters studied included cell clustering, adhesion to mucin, extracellular protein production, and formation of final metabolites. L. rhamnosus GG was found to grow efficiently in the presence of glucose, N-acetylglucosamine, or mucin (partially purified or purified) as sole carbon sources. However, it was unable to grow using other mucin constituents, such as fucose or glucuronic acid. Mucin induced noticeable changes in all the parameters studied when compared with growth using glucose, including in the formation of cell clusters, which were easily disorganized with trypsin. Mucin increased adhesion of the bacterium, and modulated the production of extracellular proteins. SDS-PAGE revealed that mucin was not degraded during L. rhamnosus GG growth, suggesting that this bacterium is able to partially use the glucidic moiety of glycoprotein. This study goes some way towards developing an understanding of the metabolic and physiological changes that L. rhamnosus GG undergoes within the human gastrointestinal tract.

SeveralBacillusstrains isolated from commercial probiotic preparations were identified at the species level, and their adhesion capabilities to three different model intestinal surfaces (mucin, Matrigel and Caco-2 cells) were assessed. In general, adhesion of spores was higher than that of vegetative cells to the three matrices, and overall strainBacillus cereusCHdisplayed the best adhesion. Different biochemical treatments revealed that surface proteins ofB. cereusCHwere involved in the adhesion properties of the strain. Surface-associated proteins from vegetative cells and spores ofB. cereusCHwere extracted and identified, and some proteins such as S-layer components, flagellin and cell-bound proteases were found to bind to mucin or fibronectin. These facts suggest that those proteins might play important roles in the interaction of this probioticBacillusstrain within the human gastrointestinal tract.

International audience; The aim of this study was to provide new insight into the mechanism whereby the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locates to cell walls of Lactobacillus plantarum 299v. After purification, cytosolic and cell wall GAPDH (cw-GAPDH) forms were characterized and shown to be identical homotetrameric active enzymes. GAPDH concentration on cell walls was growth-time dependent. Free GAPDH was not observed on the culture supernatant at any time during growth, and provoked cell lysis was not concomitant with any reassociation of GAPDH onto the cell surface. Hence, with the possibility of cw-GAPDH resulting from autolysis being unlikely, entrapment of intracellular GAPDH on the cell wall after a passive efflux through altered plasma membrane was investigated. Flow cytometry was used to assess L. plantarum 299v membrane permeabilization after labeling with propidium iodide (PI). By combining PI uptake and cw-GAPDH activity measurements, we demonstrate here that the increase in cw-GAPDH concentration from the early exponential phase to the late stationary phase is closely related to an increase in plasma membrane permeability during growth. Moreover, we observed that increases in both plasma membrane permeability and cw-GAPDH activity were delayed when glucose was added during L. plantarum 299v growth. Using a double labeling of L. plantarum 299v cells with anti-GAPDH antibodies and propidium iodide, we established unambiguously that cells with impaired membrane manifest five times more cw-GAPDH than unaltered cells. Our results show that plasma membrane permeability appears to be closely related to the efflux of GAPDH on the bacterial cell surface, offering new insight into the understanding of the cell wall location of this enzyme.

A novel alpha-amylase (alpha-1,4-alpha-D-glucan glucanohydrolase, E.C. 3.2.1.1), ScAmy43, was found in the culture medium of the phytopathogenic fungus Sclerotinia sclerotiorum grown on oats flour. Purified to homogeneity, ScAmy43 appeared as a 43 kDa monomeric enzyme, as estimated by SDS-PAGE and Superdex 75 gel filtration. The MALDI peptide mass fingerprint of ScAmy43 tryptic digest as well as internal sequence analyses indicate that the enzyme has an original primary structure when compared with other fungal alpha- amylases. However, the sequence of the 12 N-terminal residues is homologous with those of Aspergillus awamori and Aspergillus kawachii amylases, suggesting that the new enzyme belongs to the same GH13 glycosyl hydrolase family. Assayed with soluble starch as substrate, this enzyme displayed optimal activity at pH 4 and 55oC with an apparent Km value of 1.66 mg/ml and Vmax of 0.1 micromol glucose x min-1 x ml-1. ScAmy43 activity was strongly inhibited by Cu2+, Mn2+, and Ba2+, moderately by Fe2+, and was only weakly affected by Ca2+ addition. However, since EDTA and EGTA did not inhibit ScAmy43 activity, this enzyme is probably not a metalloprotein. DTT and beta-mercaptoethanol strongly increased the enzyme activity. Starting with soluble starch as substrate, the end products were mainly maltotriose, suggesting for this enzyme an endo action.

International audience; AIMS: The ability of 31 Lactobacillus plantarum strains to adhere to biological matrixes was evaluated, and the molecules involved in adherence were studied. METHODS AND RESULTS: Mucin, basement membrane proteins and Caco-2 cells were used in adhesion tests. These in vitro assays, together with a yeast agglutination test, were found to be discriminative for screening Lact. plantarum strains for adhesion. Some strains, such as 299v, CBE, BMCM12, Col4S and T25, were shown to possess interesting adhesion properties in at least two models. The adhesion of these strains was strongly inhibited when the bacterial cells were pretreated with trypsin. Lithium chloride and methyl-alpha-D-mannoside also inhibited adhesion to a lower extent. CONCLUSIONS: The adhesion of Lact. plantarum depends on both the model and the strain used. The chemical and enzymatic pretreatments applied to the bacterial cells suggested that lectin-like adhesins and other proteinaceous cell-surface structures are involved in adhesion of these strains. SIGNIFICANCE AND IMPACT OF THE STUDY: We found a great diversity in the adhesion properties between Lact. plantarum strains. Based upon the adhesive property of these strains interesting candidates were identified, that will undergo further study as potential probiotics.

International audience; We studied exopolysaccharides (EPSs) produced by Halomonas ventosae and Halomonas anticariensis, two novel species of halophilic bacteria. Under optimum environmental and nutritional conditions, H. ventosae strains Al12(T) and Al16 excreted 28.35 mg and 28.95 mg of EPS per 100 ml of culture medium (34.55 and 38.6 mg of EPS per gram of dry cell weight) respectively. The molecular masses of the polymers were about 50 kDa and their main components were glucose, mannose and galactose. They had high protein fractions and showed emulsifying activity on several hydrophobic substrates. Under optimum environmental and nutritional conditions, H. anticariensis strains FP35(T) and FP36 excreted about 29.65 and 49.95 mg of EPS per 100 ml of culture medium (43.6 and 50.95 mg of EPS per gram of dry cell weight) respectively. The molecular masses of the polymers were about 20 and 46 kDa respectively and were composed mainly of glucose, mannose and galacturonic acid. All EPSs produced solutions of low viscosity and pseudoplastic behaviour. They also had a high capacity for binding cations and incorporated considerable quantities of sulphates, which is highly unusual in bacterial polysaccharides. All strains assayed formed biofilms both in polystyrene wells and borosilicate test tubes.

One of the most interesting groups of phenolic compounds is comprised of the low molecular weight phenylpropanol derivative substances named isocoumarins, which possess important biological activities. In this study, the isocoumarin production and genetic diversity of 51 Bacillus strains isolated from different geographical and ecological niches were studied. Using molecular identification techniques, 47 strains were identified as B. subtilis, three as B. licheniformis and one as B. pumilus. When these strains were screened for isocumarin production, 11 belonging to the species B. subtilis produced amicoumacins, antibiotics of the isocoumarin group. RAPD analysis demonstrated that these strains fell into two groups which contained only these amicoumacin producers. No association was detected between RAPD profiles and the geographic origin or habitat of the strains tested. In conclusion, production of amicoumacin antibiotics by B. subtilis is a common characteristic of individual strains that presented genetic and physiological homogeneity.

A limited number of antibiotics can be used against Helicobacter pylori infection, and resistance jeopardizes the success of treatment. Therefore, a search for new agents is warranted. The use of probiotics to enhance gastrointestinal health has been proposed for many years, but the scientific basis of the prophylactic and therapeutic actions of probiotics has not yet been clearly delineated. Probiotic strain Bacillus subtilis 3, whose safety has previously been demonstrated, is known to have antagonistic properties against species of the family Enterobacteriaceae . In the present study, it was also found to inhibit H. pylori . The anti- H. pylori activity present in the cell-free supernatant was not related to pH or organic acid concentration. It was heat stable and protease insensitive. At least two antibiotics, detected by thin-layer chromatography ( R f values, 0.47 and 0.85, respectively) and confirmed by high-performance liquid chromatographic analysis, were found to be responsible for this anti- H. pylori activity. All H. pylori strains tested were sensitive to both compounds. One of these compounds was identified as amicoumacin A, an antibiotic with anti-inflammatory properties. MICs for H. pylori determined in solid and liquid media ranged between 1.7 and 6.8 μg/ml and 0.75 and 2.5 μg/ml, respectively. The underestimation of MICs determined in solid medium may be due to physicochemical instability of the antibiotic under these test conditions. An additive effect between amicoumacin A and the nonamicoumacin antibiotic against H. pylori was demonstrated.

A plasmid-linked antimicrobial peptide, named coagulin, produced by Bacillus coagulans I 4 has recently been reported (B. Hyronimus, C. Le Marrec and M. C. Urdaci, J. Appl. Microbiol. 85:42–50, 1998). In the present study, the complete, unambiguous primary amino acid sequence of the peptide was obtained by a combination of both N-terminal sequencing of purified peptide and the complete sequence deduced from the structural gene harbored by plasmid I 4 . Data revealed that this peptide of 44 residues has an amino acid sequence similar to that described for pediocins AcH and PA-1, produced by different Pediococcus acidilactici strains and 100% identical. Coagulin and pediocin differed only by a single amino acid at their C terminus. Analysis of the genetic determinants revealed the presence, on the pI 4 DNA, of the entire 3.5-kb operon of four genes described for pediocin AcH and PA-1 production. No extended homology was observed between pSMB74 from P. acidilactici and pI 4 when analyzing the regions upstream and downstream of the operon. An oppositely oriented gene immediately dowstream of the bacteriocin operon specifies a 474-amino-acid protein which shows homology to Mob-Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from gram-positive bacteria. This is the first report of a pediocin-like peptide appearing naturally in a non-lactic acid bacterium genus.

The clinical benefits observed with probiotic use are mainly attributed to the antimicrobial substances produced by probiotic strains and to their immunomodulatory effects. Currently, the best-documented probiotic bacteria used in human therapy are lactic acid bacteria. In contrast, studies aiming to characterize the mechanisms responsible for the probiotic beneficial effects of Bacillus are rare. The current work seeks to contribute to such characterization by evaluating the antimicrobial and immunomodulatory activities of probiotic B. clausii strains. B. clausii strains release antimicrobial substances in the medium. Moreover, the release of these antimicrobial substances was observed during stationary growth phase and coincided with sporulation. These substances were active against Gram-positive bacteria, in particular against Staphylococcus aureus, Enterococcus faecium, and Clostridium difficile. The antimicrobial activity was resistant to subtilisin, proteinase K, and chymotrypsin treatment, whereas it was sensitive to pronase treatment. The evaluation of the immunomodulatory properties of probiotic B. clausii strains was performed in vitro on Swiss and C57 Bl/6j murine cells. The authors demonstrate that these strains, in their vegetative forms, are able to induce NOS II synthetase activity, IFN-gamma production, and CD4 T-cell proliferation.

Probiotics are live microorganisms, which when administered in adequate amounts confer a health benefit on the host. Bacillus probiotic strains are not able to colonize the gut, and are considered as transient residents of the microbiota. Prebiotic are non-digestible food ingredients that could stimulate growth of bacteria in the gut. This work investigates the in vitro effect of a prebiotic isomaltooligosaccharide (IMOS) on the growth of a probiotic strain Bacillus subtilis. This study highlights the ability of the strain to use IMOS as unique carbon source. A comparative proteomic analysis investigates the main enzymes implicated in the prebiotic metabolism, and biomarkers possibly involved in probiotic effects. This preliminary work, which studies the synbiotic association between a probiotic and a prebiotic, will lead to in vivo assays. Beneficial effects of probiotic Bacilli, mainly modulation of intestinal immune system, result from interaction between bacterial cell-wall molecules and intestinal cells. The molecular origin of immunomodulatory mechanisms are poorly understood, while understanding is needed to optimize the use of probiotics. A second part of this work consists in comparing the structure of a molecular cell-wall component named lipoteichoic acids (LTA) from three Bacillus probiotic, a molecular cell-wall component of Gram positive bacteria, and their immunological activity. A structural study, using biochemical determinations and NMR spectroscopic analysis, highlights the structural diversity between LTAs from different Bacillus species. The key role of D-alanine substituents in the biological activity of these bacterial antigens has been demonstrated.; Les probiotiques sont des microorganismes vivants qui, lorsqu’ils sont ingérés en quantité adéquate, peuvent exercer des propriétés bénéfiques sur la santé de l’hôte. Les souches de Bacillus utilisées en tant que probiotiques ne sont pas colonisatrices du tractus intestinal, mais sont des résidents transitoires du microbiote. Ce travail fait l’investigation in vitro de l’association, qualifiée de synbiotique, entre une souche probiotique de Bacillus subtilis et une source carbonée prébiotique, composé alimentaire peu digéré par les enzymes intestinales mais utilisable par les bactéries dans l’intestin. L’étude de cette association met en évidence la capacité de la souche à utiliser les isomaltooligosaccharides (IMOS) prébiotiques comme unique substrat carboné. L’effet positif de ce substrat sur la tolérance à la bile de la souche a été démontré in vitro. Les résultats d’une analyse protéomique faisant l’étude des enzymes clés impliquées dans le métabolisme des IMOS, ainsi que d’autres biomarqueurs d’intérêt probiotique, sont en cours d’exploitation. Ce travail préliminaire d’investigation de l’association synbiotique entre les IMOS prébiotiques et la souche probiotique B. subtilis, aboutira à des essais in vivo. Les effets bénéfiques des probiotiques du genre Bacillus, notamment au niveau de la modulation du système immunitaire, résultent de l’interaction entre les molécules de la surface bactérienne et les cellules de l’intestin. Les mécanismes moléculaires à l’origine de l’immunomodulation sont mal connus, alors que leur compréhension est nécessaire à l’optimisation de l’utilisation du probiotique. Un deuxième volet de ce travail concerne la comparaison des structures d’entités moléculaires de surface de trois Bacilli probiotiques, les acides lipotéichoïques (LTAs), et leurs activités immunologiques respectives. Une étude structurale des LTAs par des méthodes biochimiques et par RMN a permis de mettre en évidence la diversité structurale au sein du même genre Bacillus. Le rôle clé de la D-alanine dans l’activité biologique de ces antigènes bactériens a été démontré.