High Bile Salt Concentrations Research Articles

Optimization, kinetics and radioprotective potential of a heteropolysaccharide from L. rhamnosus RVPI.

The probiotic potential of Lactobacillus rhamnosus RVP1 isolated from Sardinella longiceps was investigated in vitro. The bacterium exhibited highest tolerance at low pH, high bile salt concentration and demonstrated good antioxidant activity, hydrophobicity and inhibited both gram-negative and gram-positive indicator bacteria. To aid in process design and to unravel the fermentation kinetics, response surface methodology was devised to optimize the EPS production from L. rhamnosus and mechanistic models were developed to describe the fermentation kinetics. The optimum pH, dextrose and peptone concentrations for EPS production were 7.07, 19.995 g/L and 23.4 g/L, respectively, with a predicted yield of 724 mg/L. The actual yield under these conditions was 708±29 mg/L which was within the 95% confidence interval. The simulated mechanistic model fit the experimental values with a high degree of correlation with R2 = 0.99, 0.96 and 0.97 for the logistic growth, substrate consumption and EPS production and degradation curves respectively. The kinetic constants μ_max = 0.29 hr-1 , Xmax = 3.44 g/L, kf = 348 mg of EPS/ g of dry biomass and kd = 0.53 hr-1 were derived from the model. The EPS administration improved the survival of irradiated mice by 50% proving it radioprotective potential and showed positive effects on structural integrity of intestinal tissue. This article is protected by copyright. All rights reserved.

Evaluation of the hypoglycemic effect of probiotics via directly consuming glucose in intestines of STZ-induced diabetic mice and glucose water-induced diabetic mice

Abstract Diabetes has steadily increased in the past few decades and causes huge influences on human health care and socioeconomic development, and drugs with minimal or no side effects for diabetes are imminent. In the present study, probiotic combination containing five selected probiotics were used to treat diabetes, the high glucose consumption capability and sound probiotic characteristics in vitro showed that the probiotics could consume all glucose within 24 h, and could tolerate low potential of hydrogen (pH) (pH = 2) and high bile salt concentration (0.3%), and they also had sound oxidation resistance [2,2-diphenyl-1-picrylhydrazyl (DPPH),∙O2−, OH, Fe2+ chelation, and the reducing powers] and antibacterial activity, highly adhere to HT-29 cells and markedly reduced the chance for pathogens to adhere to HT-29 cell (p

Probiotic Properties of Lactobacillus paracasei subsp. paracasei L1 and Its Growth Performance-Promotion in Chicken by Improving the Intestinal Microflora.

Lactobacillus paracasei subsp. paracasei L1 was previously isolated from sweet potato sour liquid. This bacterial species specifically binds onto starch granular surfaces, triggering the enzymatic hydrolysis of raw starch. We investigated the functional and safety properties of strain L1 in vitro to establish its probiotic potential, and analyzed its effect on growth performance and intestinal microflora of chicken in feeding experiments. The optimal growth conditions of strain L1 included low pH and high concentrations of bile salts and NaCl. Its 1-, 2-, and 24-h autoaggregation values were 15.8 ± 1.2%, 20.4 ± 2.3%, and 47.2 ± 0.8%, respectively, with the surface hydrophobicity value at 560 nm of 38.1 ± 2.7%. Further, its adhesion rate to Caco-2 cells was 22.37 ± 1.44%. Strain L1 was resistant to erythromycin and azithromycin, but sensitive to other antibiotics tested. For the feeding experiments, 240 chickens with similar weights were randomly divided into a control (C) group and strain L1 (L) group and fed for 8 weeks. Strain L1 promoted the weight gain of chickens in L group. A significant increase in the population size of the two phyla and 23 genera in the small intestine was observed in the presence of strain L1 (P < 0.05), with 0 phyla and 4 genera showing significant increase in the cecum (P < 0.05). In the small intestine, the abundance of six functional genes at Kyoto Encyclopedia of Genes and Genomes (KEGG) level 2 and 49 genes at KEGG level 3 was significantly increased in group L (P < 0.05), with lesser changes noted in the cecum. An increase in the metabolic pathway functions, including enzyme families and the digestive system, was observed in the intestinal microbiota in the L group compared to the C group. However, the other metabolic pathway functions, including metabolism of fatty acid biosynthesis, as well as metabolism of glycerolipids and propanoate, increased in the cecal microbiota of the L group relative to the C group. These changes are most likely related to the changes in the gut microbiota composition. Collectively, strain L1 supplementation may promote growth performance and improve the intestinal microflora in chicken although further studies are needed to confirm this.

Viability of 4 Probiotic Bacteria Microencapsulated with Arrowroot Starch in the Simulated Gastrointestinal Tract (GIT) and Yoghurt.

Probiotic bacteria are usually encapsulated to increase their survival through passage of the simulated gastrointestinal tract (GIT). Four Lactobacilli were freeze-dried and encapsulated with maltodextrin (maltodextrin 1.25 g, whey 0.25 g, bacteria 0.5 g, and water 2 mL) and arrowroot starch (arrowroot 1.25 g, whey 0.25 g, bacteria 0.5 g, and water 2 mL). The effects of different coatings were evaluated for their viability in the GIT and yogurt. The findings indicated no significant differences at p > 0.05 in the survival of the encapsulated cells with increased concentrations of arrowroot and maltodextrin. The viability of the encapsulated bacteria was increased in the simulated GIT with high counts of 109 cfu/mL after 30 min stiffening in 1 µm size beads. However, the bead fermented yogurt exhibited insignificant difference on the survivability of the organisms in a simulated GIT after 15 days. Lactobacillus plantarum, Weissela paramesenteroides, Enterococcus faecalis, and Lactobacillus paraplantarum showed a significant increase of viable cells at p > 0.05 after freeze-drying in comparison with free cells at high bile salt concentrations and low acidity. This study confirmed that arrowroot starch and maltodextrin combinations in encapsulation might be an effective method that could allow viable probiotic bacteria to reach the large intestine.

Streptococcus macedonicus strains isolated from traditional fermented milks: resistance to gastrointestinal environment and adhesion ability.

In this study, Streptococcus macedonicus (S. macedonicus) strains were identified from Algerian traditional fermented milks (Lben and Rayeb). Important prerequisites of probiotic interest such as acidity, bile salts tolerance, and adhesion ability to epithelial cells were investigated. A combination of phenotypic (ability to grow on Bile Esculin Azide medium, BEA; on high salt content medium NaCl 6.5%; on alkaline medium pH9.6) and genotypic approaches (16S rRNA, ITS genes sequencing and MLST technique) allowed to identify four genetically distinct strains of S. macedonicus. These four strains and two references, Streptococcus thermophilus LMD-9 and Lactobacillus rhamnosus GG (LGG), were tested for their capacity to survive at low pH values, and at different concentrations of an equimolar bile salts mixture (BSM). Two different cell lines, Caco-2 TC7 and HT29-MTX, were used for the adhesion study. The results show that S. macedonicus strains selected constitute a distinct genetic entity from the Greek strain S. macedonicus ACA-DC-198. They were able to survive up to pH3 and could tolerate high concentrations of bile salts (10mM), unlike LMD-9 and LGG strains. Our strains also display in vitro adhesion similar to the LGG strain on Caco-2 TC7 and higher adhesion than the LMD-9 strain to Caco-2 TC7 and HT29-MTX cell models. This first characterization allows considering S. macedonicus as a potential candidate for possible probiotic effects that need to be investigated.

&amp;lt;i&amp;gt;In Vitro&amp;lt;/i&amp;gt; Antimicrobial Characterization of &amp;lt;i&amp;gt;Lactobacillus&amp;lt;/i&amp;gt; Isolates Towards Their Use as Probiotic Alternatives to Antibiotic Growth Promoters

In the present study, the probiotic potential of Lactobacillus isolates selected from fecal samples of farmyard chickens and ducks was scientifically validated for their use as alternatives to antibiotics in poultry. A total of 129 Lactobacillus isolates were characterized of which four produced inhibitory substances with antimicrobial activities. They were further identified on the basis of their carbohydrate fermentation profile and High-Resolution Melting analysis as Lactobacillus paracasei MW-37CGZ, Lactobacillus paracasei MW-38CGZ, Lactobacillus plantarum MW-48CGZ and Lactobacillus plantarum MW-18CGZ. The obtained results revealed that L. plantarum MW-18CGZ and L. paracasei MW-37CGZ showed strong antagonistic activities against human (nine) and zoonotic pathogens (eleven). The antimicrobial substance produced by L. plantarum MW-18CGZ was found to be proteinaceous, thus indicating that this substance may belong to a group of potent antimicrobial peptides produced by some microorganisms including lactic acid bacteria (LAB). Both viable and non-viable cells of the four isolates demonstrated good hydrophobicity in xylene with L. plantarum MW-48CGZ exhibiting higher hydrophobicity than other isolates (77.64±5.18%). They were susceptible to chloramphenicol, clindamycin, ampicilin and erythromycin with Minimum Inhibitory Concentration (MIC) below cut-off values established by the European Food Safety Authority (EFSA). Among the four Lactobacillus, L. plantarum MW-18CGZ and L. paracasei MW-37CGZ displayed high autoaggregation and coaggregation towards pathogens and all isolates survived in low-pH, high bile salt concentrations and none exhibited virulent factors. According to the obtained results, L. plantarum MW-18CGZ and L. paracasei MW-37CGZ could be considered as future biotherapeutic substitutes for antibiotics to reduce antibiotic residues in food derived from poultry as well as the generation and spread of antibiotic resistance.

E. coli Nissle microencapsulation in alginate-chitosan nanoparticles and its effect on Campylobacter jejuni in vitro

Microencapsulation enhances the oral delivery of probiotic bacteria. In this study, the probiotic Escherichia coli Nissle 1917 (EcN) was microencapsulated using alginate and chitosan nanoparticles. The result showed 90% encapsulation yield of EcN, and the encapsulated EcN displayed significantly (P < 0.05) increased survival in low pH (1.5), high bile salt concentration (4%), and high temperature (70°C). The most effective cryopreservatives of EcN during freezing and thawing was skim milk and sucrose. Exposure to microencapsulated EcN significantly (P < 0.05) reduced the Campylobacter jejuni growth by 2 log CFU. The rate of EcN release from microcapsule was 9.2 × 105cellmin-1, and the appropriate model to describe its release kinetics was zero order. Importantly, the entrapment of EcN inside the microcapsule did not eliminate the exterior diffusion of EcN produced antioxidant compounds. In addition, the EcN microcapsule efficiently adhered to intestinal HT-29 cells and the pre-treatment of HT-29 cells with EcN-microcapsule for 4h significantly (P < 0.05) reduced the invasion (1.9 log) of C. jejuni; whereas, completely abolished the intracellular survival. Furthermore, HT-29 cells pre-treated with encapsulated EcN in PCR array showed decreased expression (> 1.5-fold) of genes encoding chemokines, toll-like receptors, interleukins, and tumor necrosis factors. In conclusion, the alginate-chitosan microcapsule can provide effectual platform to deliver probiotic EcN and thereby can reduce the Campylobacter infection in chickens and humans.

Investigation of Lactic Acid Bacteria Isolated from Giant Panda Feces for Potential Probiotics In Vitro.

The present study aimed to isolate an optimal lactic acid bacterial strain from the feces of healthy giant pandas. The strain exhibited good stability at low pH and high bile salt concentrations, activity against pathogens relevant to pandas, and antibiotic susceptibility. In the current study, 25 isolates were obtained from de Man, Rogosa, and Sharpe agar. Two (E21 and G83) and eight (E1, E2, E16, E18, E21, E69, E70, and G83) isolates demonstrated good performance at pH2.0 and bile 2% (w/v), respectively. Three isolates (G83, G88, and G90) possessed better antimicrobial effect on enterotoxigenic Escherichia coli CVCC196 (ETEC) than the rest. One isolate (G83) strongly affected Salmonella, whereas three (G83, G87, and G88) exhibited inhibitory activity against Staphylococcus aureus. All isolates were multi-drug resistant. These isolates were identified as Lactobacillus (5 isolates) and Enterococcus (20 isolates) by 16S rRNA sequencing. Virulence genes were detected in Enterococcus isolates. Isolate G83 was identified as Lactobacillus plantarum and was considered as the best probiotic candidate among all of the experimental isolates. This study provided necessary and important theoretical guidance for further experiments on G83 in vivo.

Functional Characterization of Probiotic Potential of Novel Pigmented Bacterial Strains for Aquaculture Applications.

The bioprospecting proficient of novel pigmented probiotic strains with respect to aquaculture industry was unexplored hitherto. In this study, we investigated the probiotic potential of novel pigmented bacterial strains isolated from the indigenous soil sediments in their vicinal habitats, which were screened for their antimicrobial activity against aquatic pathogens using agar well diffusion assay. The strains namely Exiguobacterium acetylicum (S01), Aeromonas veronii (V03), and Chryseobacterium joostei (V04) were phenotypically identified and confirmed by 16S rRNA gene sequence analysis. Further characterization revealed that strains S01 and V03 survive relatively in lower pH and higher bile salt concentrations and possess good adherence ability and broad-spectrum antibiotic susceptibility. The isolate S01 exhibited the higher adhesion ability to hydrocarbons (82%) and mannose-specific adhesion (msa) gene expression. Additionally, the probiotic effects were evaluated in Artemia nauplii fed with algae supplemented with S01, V03, and V04 strains (2.7 × 107cfu/mL) for 3days under axenic environment. We observed a significant increase (p < 0.05) in the survival rate of Artemia nauplii treated with S01 (83 ± 5%) and V03 (55 ± 5%), whereas the survival rate was only 30 ± 0% in the untreated group. Moreover, the individual length (IL) was increased in treated group S01 (156.7 ± 2.2μm), V03 (146.1 ± 3.4μm), and V04 (134.4 ± 2.5μm) compared with untreated group (116.0 ± 4.8μm). Our results revealed that E. acetylicum S01 exhibits desirable functional probiotic attributes compared to A. veronii and C. joostei and it would be a promising probiotic strain, which can be efficiently used in the aquaculture applications.

Novel Aggregation Promoting Factor AggE Contributes to the Probiotic Properties of Enterococcus faecium BGGO9-28.

The understanding of mechanisms of interactions between various bacterial cell surface proteins and host receptors has become imperative for the study of the health promoting features of probiotic enterococci. This study, for the first time, describes a novel enterococcal aggregation protein, AggE, from Enterococcus faecium BGGO9-28, selected from a laboratory collection of enterococcal isolates with auto-aggregation phenotypes. Among them, En. faecium BGGO9-28 showed the strongest auto-aggregation, adhesion to components of ECM and biofilm formation. Novel aggregation promoting factor AggE, a protein of 178.1 kDa, belongs to the collagen-binding superfamily of proteins and shares similar architecture with previously discovered aggregation factors from lactic acid bacteria (LAB). Its expression in heterologous enterococcal and lactococcal hosts demonstrates that the aggE gene is sufficient for cell aggregation. The derivatives carrying aggE exhibited the ten times higher adhesion ability to collagen and fibronectin, possess about two times higher adhesion to mucin and contribute to the increase of biofilm formation, comparing to the control strains. Analysis for the presence of virulence factors (cytolysin and gelatinase production), antibiotic resistance (antibiotic susceptibility) and genes (cylA, agg, gelE, esp, hylN, ace, efaAfs, and efaAfm) showed that BGGO9-28 was sensitive to all tested antibiotics, without hemolytic or gelatinase activity. This strain does not carry any of the tested genes encoding for known virulence factors. Results showed that BGGO9-28 was resistant to low pH and high concentrations of bile salts. Also, it adhered strongly to the Caco-2 human epithelial cell line. In conclusion, the results of this study indicate that the presence of AggE protein on the cell surface in enterococci is a desirable probiotic feature.

In vitro probiotic potential of Enterococcus species isolated from tungrymbai, a fermented soybean product of Meghalaya, India

Lactic acid bacteria were isolated from tungrymbai, a traditional fermented soybean product of Meghalaya, India and assessed for their probiotic properties. Survival under simulated gastric conditions was tested and five acid tolerant strains were selected. By using 16S rDNA sequencing, these isolates were confirmed to be Enterococcus species. The strains exhibited good survivability in high concentrations of bile salts. The strains were susceptible to antibiotics and they were also found to inhibit growth of Staphylococcus aureus and Escherichia coli. The strains were gelatinase negative and non-haemolytic. The strains also assimilated cholesterol up to a certain level. The study confirmed that all identified five strains have in vitro probiotic properties.

IN VITRO CHARACTERIZATION OF LACTIC ACID BACTERIA ISOLATED FROM LASODA BARI- A RARE FERMENTED FOOD OF HIMACHAL PRADESH-INDIA FOR POTENTIAL PROBIOTIC ATTRIBUTES

Probiotic lactic acid bacteria (LAB) are health promoting microorganisms which are recently been used as food additives and therapeutic supplements. Recently, there has been an increase of interest regarding the commercial utilization of probiotic LAB strains isolated from traditional and naturally fermented food products. Therefore, the present study was aimed to isolate and screen lactic acid bacteria from Lasoda bari – a rare fermented food of Himachal Pradesh, for the first time for their probiotic potential. Total eight isolates were obtained out of which one isolate was selected based on its broadest antagonistic spectrum. This strain was identified using 16S rRNA technique as Pediococcus pentosaceus LB-CC. The analysis of acid resistance, bile tolerance, antibiotic sensitivity, auto-aggregation and microbial adhesion to organic solvents were established. The results revealed normal growth of P. pentosaceus LB-CC in the presence of low pH, high bile salt concentration and ability to produce antimicrobial compounds. No gelatinase and DNase enzyme activity was detected. Natural susceptibility to the tested antibiotics was observed. Thus, according to these results, this probiotic strain could be proposed as safe potential probiotic culture and can be exploited for the production of nutraceutical agents.

Isolation, Identification and In vitro Characterisation of Probiotic Yeast Strains

In the present study, ten yeasts were isolated from fruit juices and subjected to in vitro tests for screening probiotic potentiality. Three isolates exhibited higher tolerance for pH and heat stress. Based on preliminary screening, isolates were subjected to molecular identification and identified as Wickerhamomyces anomalusVIT-ASN01, Saccharomyces cerevisiae VIT- ASN03 and Yarrowia lipolytica VIT- ASN04. The screened yeast isolates survived and grew well at human gastrointestinal conditions in pH 2.5 and 0.3% (w/v) oxgall at 37°C. The antagonistic activity of yeast isolates were tested against clinical pathogens, viz.S. typhii, B. subtilis and E. coli. W. anomalusVIT-ASN01 showed maximum tolerance to high bile salt concentration and acidity, could remove cholesterol 51.28% during 48 h and produced 586.55 mg/L of exopolysaccharide. It can be concluded that the screened novel yeast isolates can serve as promising probiotics in various fields of food industry.

Adsorption of Lactobacillus acidophilus on attapulgite: Kinetics and thermodynamics and survival in simulated gastrointestinal conditions

In the present study, we investigated the optimum adsorption conditions, adsorption characteristics and surviva of Lactobacillus acidophilus adsorbed on attapulgite (ATT). Results of single factor experiments showed that the optimum adsorption parameters were ATT concentration of 10%, initial pH of 6.5 and temperature of 35 °C. From the static experiments of adsorption characteristics, we found that the experimental data were fitted best to the pseudo-secondorder kinetics and Langmuir isotherm models, and the adsorption was a spontaneous and endothermic process. Bacteria survival test showed that the microencapsulated L. acidophilus stored at −18 °C for 30 days achieved the highest survival rate (83.2% ± 5.8) and obtained better protection at simulated conditions of gastric pH and at high bile salt concentrations when compared with free bacteria. The study demonstrated that microencapsulation of L. acidophilus in ATT was an effective technique of protection against low-temperature injury and under simulated gastrointestinal environment.

Displacement of itraconazole from cyclodextrin complexes in biorelevant media: In vitro evaluation of supersaturation and precipitation behavior

Intestinal fluids contain several constituents with affinity for cyclodextrins that have the potential of displacing drugs from the cyclodextrin cavity by competition. In this study, the solubilizing capacity of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) for itraconazole was studied in presence of selected bile salts and phosphatidylcholine. Despite the fact that these competing agents significantly lowered the solubility of itraconazole in presence of cyclodextrins, the addition of concentrated solutions of these bile constituents to a solution containing itraconazole solubilized by HP-β-CD did not result in precipitation, even at bile salt and phospholipid concentrations where itraconazole precipitation would be anticipated based on solubility studies. This phenomenon was further investigated in more dynamic conditions via in vitro transfer studies, mimicking the gastrointestinal transfer of HP-β-CD solutions saturated with itraconazole. Intestinal supersaturation upon transfer was observed for all conditions tested and a concentration dependent impact of bile salts and phospholipids on the precipitation behavior of itraconazole was demonstrated: high concentrations of bile salts and phospholipids generated the highest degrees of supersaturation shortly after the transfer step but also resulted in stronger itraconazole precipitation at later time points. These findings demonstrate the possible impact of the variable intestinal fluid composition on the behavior of cyclodextrin containing formulations.

Proteomic Analysis of the Rat Canalicular Membrane Reveals Expression of a Complex System of P4-ATPases in Liver.

Transport processes in the canalicular membrane are key elements in bile formation and are the driving force of the enterohepatic circulation of bile salts. The canalicular membrane is constantly exposed to the detergent action of bile salts. One potential element protecting the canalicular membrane from the high canalicular bile salt concentrations may be bile salt resistant microdomains, however additional factors are likely to play a role. To obtain more insights into the molecular composition of the canalicular membrane, the proteome of highly purified rat canalicular membrane vesicles was determined. Isolated rat canalicular membrane vesicles were stripped from adhering proteins, deglycosylated and protease digested before subjecting the samples to shot gun proteomic analysis. The expression of individual candidates was studied by PCR, Western blotting and immunohistochemistry. A total of 2449 proteins were identified, of which 1282 were predicted to be membrane proteins. About 50% of the proteins identified here were absent from previously published liver proteomes. In addition to ATP8B1, four more P4-ATPases were identified. ATP8A1 and ATP9A showed expression specific to the canalicular membrane, ATP11C at the bLPM and ATP11A in an intracellular vesicular compartment partially colocalizing with RAB7A and EEA1 as markers of the endosomal compartment. This study helped to identify additional P4-ATPases from rat liver particularly in the canalicular membrane, previously not known to be expressed in liver. These P4-ATPases might be contributing for maintaining transmembrane lipid homeostasis in hepatocytes.

English

The aim of this work was to investigate the effect of different microencapsulation materials on the stability of probiotic bacterium (Lactobacillus plantarum DSM 20174). Microencapsulation methods with alginates were carried out using sodium chloride, canola oil, olive oil, and chitosan. The recorded data showed that the encapsulated probiotic bacterium was more stable compared with free cells. Olive oil capsules recorded the highest stability at pH 2 after incubation period of 24 h with stability up to 0.00004%. Olive oil and chitosan capsules showed stability with high concentration of bile salts (0.5%) with stability percent of 82 and 65% respectively, after 2 h of incubation. Sodium chloride and chitosan capsules gave the best stability percent of 0.026 and 0.00005%, respectively, at heat treatment up to 65°C for 30 min. Storage treatment at 4°C for 17 days reduced the stability of all capsule types, whereas sodium chloride and chitosan capsule showed stability percent up to 59 and 56%, respectively. Key words: Microencapsulation, Lactobacillus plantarum, olive oil and alginate.

Novel probiotic evidence of lactobacilli on immunomodulation and regulation of satiety hormones release in intestinal cells

Lactobacilli strains with probiotic traits were tested for their ability to survive to the digestion process modelled during this study. These strains managed to sustain the harsh conditions of the gastric and duodenal phases and showed good adhesion capacities to human Caco-2 cell line. These probiotic microorganisms have survived during these steps, exposed to low pH, high concentration of bile salts and enzymes occurring in the digestion and virtually reached the duodenal compartment in sufficient amount with limited population loss. These lactobacilli strains appeared to be non-cytotoxic after contact with Caco-2 cells for 24 h. Importantly, some of these strains showed immunomodulatory effect, lowering the pro-inflammatory cytokine IL-8 and promoting secretion of the anti-inflammatory IL-10. Besides, Lactobacillus gasseri CMUL34 and Lactobacillus acidophilus CMUL67 strains were able to modulate secretion and expression of two intestinal hormones: the Glucagon-Like Peptide 1 (GLP-1) and the cholecystokinin (CCK) in STC-1 cells.

Differential proteomic analysis of outer membrane enriched extracts of Bacteroides fragilis grown under bile salts stress

Bacteroides fragilis is the most commonly isolated anaerobic bacteria from infectious processes. Several virulence traits contribute to the pathogenic nature of this bacterium, including the ability to tolerate the high concentrations of bile found in the gastrointestinal tract (GIT). The activity of bile salts is similar to detergents and may lead to membrane permeabilization and cell death. Modulation of outer membrane proteins (OMPs) is considered a crucial event to bile salts resistance. The primary objective of the current work was to identify B. fragilis proteins associated with the stress induced by high concentration of bile salts. The outer membrane of B. fragilis strain 638R was isolated after growth either in the presence of 2% conjugated bile salts or without bile salts. The membrane fractions were separated on SDS-PAGE and analyzed by ESI-Q/TOF tandem mass spectrometry. A total of 37 proteins were identified; among them nine were found to be expressed exclusively in the absence of bile salts whereas eight proteins were expressed only in the presence of bile salts. These proteins are related to cellular functions such as transport through membrane, nutrient uptake, and protein-protein interactions. This study demonstrates the alteration of OMPs composition in B. fragilis during bile salts stress resistance and adaptation to environmental changes. Proteomics of OMPs was also shown to be a useful approach in the identification of new targets for functional analyses.

Heterologous overexpression and biochemical characterization of the (galactophospho)lipase from Fusarium solani in Pichia pastoris that is expressed in planta

High-level extracellular production of Fusarium solani (galactophospho)lipase, named FSL, was achieved using a Pichia pastoris X33 expression system. The (galactophospho) lipase encoding gene was cloned into pGAPZαA with the Saccharomyces cerevisiae α-factor signal sequence by two different ways. The two constructs consist of an additional sequence of a (His)6-tag of the vector fused to the N-terminus of this enzyme (tFSL) while the other expression vector was constructed without any additional sequence (rFSL). Compared to the native enzyme (nFSL) (18.75mg/L), a high level secretion of rFSL (310mg/L) and tFSL (240mg/L) was achieved providing an important improvement in enzyme production.Biochemical characterization showed that pure recombinant proteins (rFSL and tFSL) presented similar behaviour towards triglycerides, phospholipid and galactolipid. Like the nFSL, rFSL and tFSL are active at high concentration of bile salts (4mM) and calcium ions enhanced lipase activity.During plant infection, transcripts of this fungal lipase gene were detected 3, 7 and 10 days post infection.