Franck Grattepanche

In Vitro Continuous Fermentation Model (PolyFermS) of the Swine Proximal Colon for Simultaneous Testing on the Same Gut Microbiota

In vitro gut modeling provides a useful platform for a fast and reproducible assessment of treatment-related changes. Currently, pig intestinal fermentation models are mainly batch models with important inherent limitations. In this study we developed a novel in vitro continuous fermentation model, mimicking the porcine proximal colon, which we validated during 54 days of fermentation. This model, based on our recent PolyFermS design, allows comparing different treatment effects on the same microbiota. It is composed of a first-stage inoculum reactor seeded with immobilized fecal swine microbiota and used to constantly inoculate (10% v/v) five second-stage reactors, with all reactors fed with fresh nutritive chyme medium and set to mimic the swine proximal colon. Reactor effluents were analyzed for metabolite concentrations and bacterial composition by HPLC and quantitative PCR, and microbial diversity was assessed by 454 pyrosequencing. The novel PolyFermS featured stable microbial composition, diversity and metabolite production, consistent with bacterial activity reported for swine proximal colon in vivo. The constant inoculation provided by the inoculum reactor generated reproducible microbial ecosystems in all second-stage reactors, allowing the simultaneous investigation and direct comparison of different treatments on the same porcine gut microbiota. Our data demonstrate the unique features of this novel PolyFermS design for the swine proximal colon. The model provides a tool for efficient, reproducible and cost-effective screening of environmental factors, such as dietary additives, on pig colonic fermentation.

Development of a real-time RT-PCR method for enumeration of viable Bifidobacterium longum cells in different morphologies.

Viability of probiotic bacteria is traditionally assessed by plate counting which has several limitations, including underestimation of cells in aggregates or chains morphology. We describe a quantitative PCR (qPCR)-based method for an accurate enumeration of viable cells of Bifidobacterium longum NCC2705 exhibiting different morphologies by measuring the mRNA levels of cysB and purB, two constitutively expressed housekeeping genes. Three primer-sets targeting short fragments of 57-bp of cysS and purB and one 400-bp fragment of purB were used. Cell quantification of serially diluted samples showed a good correlation coefficient of R(2) 0.984 +/- 0.003 between plate counts and qRT-PCR for all tested primer sets. Loss of viable cells exposed to a lethal heat stress (56 degrees C, 10, 20 and 30 min) was estimated by qRT-PCR and plate counts. No significant difference was observed using qRT-PCR targeting the 400-bp fragment of purB compared to plate counts indicating that this fragment is a suitable marker of cell viability. In contrast, the use of the 57-bp fragments led to a significant overestimation of viable cell counts (18 +/- 3 and 7 +/- 2 fold for cysB and purB, respectively). Decay of the mRNA fragments was studied by treatment of growing cells with rifampicin prior qRT-PCR. The 400-bp fragment of purB was faster degraded than the 57-bp fragments of cysB and purB. The 400-bp fragment of purB was further used to enumerate viable cells in aggregate state. Cell counts were more than 2 log(10) higher using the qRT-PCR method compared to plate counts. Growing interest in probiotic characteristics of aggregating bacteria cells make this technique a valuable tool to accurately quantify viable probiotic bacteria exhibiting heterogeneous morphology.

Novel Polyfermentor intestinal model (PolyFermS) for controlled ecological studies: validation and effect of pH.

In vitro gut fermentation modeling offers a useful platform for ecological studies of the intestinal microbiota. In this study we describe a novel Polyfermentor Intestinal Model (PolyFermS) designed to compare the effects of different treatments on the same complex gut microbiota. The model operated in conditions of the proximal colon is composed of a first reactor containing fecal microbiota immobilized in gel beads, and used to continuously inoculate a set of parallel second-stage reactors. The PolyFermS model was validated with three independent intestinal fermentations conducted for 38 days with immobilized human fecal microbiota obtained from three child donors. The microbial diversity of reactor effluents was compared to donor feces using the HITChip, a high-density phylogenetic microarray targeting small subunit rRNA sequences of over 1100 phylotypes of the human gastrointestinal tract. Furthermore, the metabolic response to a decrease of pH from 5.7 to 5.5, applied to balance the high fermentative activity in inoculum reactors, was studied. We observed a reproducible development of stable intestinal communities representing major taxonomic bacterial groups at ratios similar to these in feces of healthy donors, a high similarity of microbiota composition produced in second-stage reactors within a model, and a high time stability of microbiota composition and metabolic activity over 38 day culture. For all tested models, the pH-drop of 0.2 units in inoculum reactors enhanced butyrate production at the expense of acetate, but was accompanied by a donor-specific reorganization of the reactor community, suggesting a concerted metabolic adaptation and trigger of community-specific lactate or acetate cross-feeding pathways in response to varying pH. Our data showed that the PolyFermS model allows the stable cultivation of complex intestinal microbiota akin to the fecal donor and can be developed for the direct comparison of different experimental conditions in parallel reactors continuously inoculated with the exact same microbiota.

Improved tolerance to bile salts of aggregated Bifidobacterium longum produced during continuous culture with immobilized cells.

The effect of cell immobilization and continuous culture was studied on selected physiological and technological characteristics of Bifidobacterium longum NCC2705 cultivated for 20 days in a two stage continuous fermentation system. Continuous immobilized cell (IC) cultures with and without glucose limitation exhibited formation of macroscopic cell aggregates after 12 and 9 days, respectively. Auto-aggregation resulted in underestimation of viable cell counts by plate counts by more than 2 log units CFU/ml compared with qPCR method. Modifications of cell membrane composition might partially explain aggregate formation in IC cultures. Decreases in the ratio of unsaturated to saturated fatty acid content from 1.74 to 0.58 might also contribute to the enhanced tolerance of IC cells to porcine bile salts and aminoglycosidic antibiotics compared with free cells from batch cultures. The enhanced resistance against bile salts in combination with auto-aggregation may confer an advantage to probiotic bacteria produced by IC technology.

New method for selection of hydrogen peroxide adapted bifidobacteria cells using continuous culture and immobilized cell technology

BackgroundOxidative stress can severely compromise viability of bifidobacteria. Exposure of Bifidobacterium cells to oxygen causes accumulation of reactive oxygen species, mainly hydrogen peroxide, leading to cell death. In this study, we tested the suitability of continuous culture under increasing selective pressure combined with immobilized cell technology for the selection of hydrogen peroxide adapted Bifidobacterium cells. Cells of B. longum NCC2705 were immobilized in gellan-xanthan gum gel beads and used to continuously ferment MRS medium containing increasing concentration of H2O2 from 0 to 130 ppm.ResultsAt the beginning of the culture, high cell density of 1013 CFU per litre of reactor was tested. The continuous culture gradually adapted to increasing H2O2 concentrations. However, after increasing the H2O2 concentration to 130 ppm the OD of the culture decreased to 0. Full wash out was prevented by the immobilization of the cells in gel matrix. Hence after stopping the stress, it was possible to re-grow the cells that survived the highest lethal dose of H2O2 and to select two adapted colonies (HPR1 and HPR2) after plating of the culture effluent. In contrast to HPR1, HPR2 showed stable characteristics over at least 70 generations and exhibited also higher tolerance to O2 than non adapted wild type cells. Preliminary characterization of HPR2 was carried out by global genome expression profile analysis. Two genes coding for a protein with unknown function and possessing trans-membrane domains and an ABC-type transporter protein were overexpressed in HPR2 cells compared to wild type cells.ConclusionsOur study showed that continuous culture with cell immobilization is a valid approach for selecting cells adapted to hydrogen peroxide. Elucidation of H2O2 adaptation mechanisms in HPR2 could be helpful to develop oxygen resistant bifidobacteria.

Fast screening of Bifidobacterium longum sublethal stress conditions in a novel two-stage continuous culture strategy

A central issue in the application of probiotics as food additives is their fastidious production and their sensitivity to many environmental stresses. The importance of inducible cell-protective mechanisms triggered by application of sublethal stresses for survival under stress conditions has been demonstrated. Continuous cultures could be a suitable and more efficient method to test stress factors on one culture instead of several repeated batch cultures. In this study, the application of a two-stage continuous culture of Bifidobacterium longum NCC2705 was investigated. The first reactor was operated under fixed conditions at 37 °C and pH 6.0 and used to produce cells with controlled physiology, mimicking cells in the late exponential growth phase. Stress pretreatment combinations of pH (6.0, 5.0 and 4.0), temperature (37, 45 and 47 °C) and NaCl (0, 5 and 10%) were tested in the second reactor. Of all tested combinations, only those of pH 4.0 significantly decreased cell viability in the second reactor compared to control conditions (37 °C, pH 6.0, 0% NaCl) and, therefore, could not be considered as sublethal stresses. Pretreatments with 5 or 10% NaCl had a negative effect on cell viability after gastric lethal stress. A significant improvement in cell resistance to heat lethal stress (56 °C, 5 min) was observed for cells pretreated at 47 °C. In contrast, heat pretreatment negatively affected cell viability after freeze drying and osmotic lethal stresses. The two-stage continuous culture allowed for efficient screening of several stress pretreatments during the same experiment with up to four different conditions tested per day. Optimal sublethal stress conditions can also be applied for producing cells with traditional batch cultures.

Transcriptome analysis and physiology of Bifidobacterium longum NCC2705 cells under continuous culture conditions

A central issue in the use of probiotics in food and food supplements is their sensitivity to many environmental stress factors. The resistance of probiotic cells to lethal stress can be improved by application of homologous or heterologous sub-lethal stress during culture. This screening procedure is generally performed using batch cultures. Continuous cultures could be a suitable and more efficient method to test different stress factors on one culture instead of repeating several batch cultures. However, before testing stresses using continuous cultures, the physiological stability of continuously produced cells over a considered time period must be first evaluated. A continuous culture of Bifidobacterium longum NCC2705 was maintained for 211 h at a dilution rate of 0.1 per h, mimicking a deceleration growth phase culture. Stable viable cell counts were measured over the culture period, decreasing only moderately from 8.8 to 8.6 log10 cfu/ml. A slight shift in metabolite production, characterized by increased lactate and decreased acetate, formate and ethanol concentrations was observed. Susceptibilities to antibiotics and stress conditions were stable (cefotaxim, ampicillin, ceftazidime) or moderately affected (simulated gastric juices, heat, bile salts, tetracycline, chloramphenicol, penicillin, vancomycin and neomycin) over culturing time. Comparison of gene transcription profiles between samples collected after 31 h of continuous culture and samples collected after 134 and 211 h revealed only limited changes in expression of 1.0 and 3.8% of total genes, respectively. Based on these results, we propose that continuous culture can be used to produce bacterial cells with stable physiological properties suitable for fast and efficient screening of sub-lethal stress conditions.

Exopolysaccharides from co-cultures of Weissella confusa 11GU-1 and Propionibacterium freudenreichii JS15 act synergistically on wheat dough and bread texture.

The storage of bread is limited by both physical (staling) and microbial (mainly fungal) spoilage. Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) and organic acids from propionibacteria (PAB) have been used to enhance texture and extend shelf-life of bakery products. In this study the functionality of EPS of Weissella confusa A3/2-1 (dextran), W. confusa F3/2-2 (dextran and levan), W. confusa 11GU-1 (dextran and ropy capsular polysaccharide) was evaluated in wheat bread. Two strains of Propionibacterium freudenreichii (Pf), shown to produce a heteropolysaccharide (Pf JS15) or a β-glucan (Pf DF30), were tested in single and mixed cultures with W. confusa (Wc). The EPS fermentates were prepared by batch fermentation of cereal- or malt-based medium using sucrose (Wc) or lactic acid (Pf) as carbon source. Incorporation of EPS from single culture fermentates and 1:1 Weissella-Propionibacterium fermentate mixtures revealed strong positive effects of dextran and ropy capsular polysaccharide produced by Wc 11GU-1 on bread staling retardation, with synergistic effects of EPS mixture from Wc 11GU-1 and Pf JS15. A co-fermentation of Wc 11GU-1 and Pf JS15 was developed to produce EPS together with antifungal organic acid mixture (acetate and propionate) in a single step process. The addition of 15% (w/w flour base) co-culture, yielding EPS, acetate and propionate concentrations of 1.5, 0.5 and 1g/kg dough, respectively, resulted in improved bread texture, increased loaf volume and decreased crumb firming during storage for 3days compared with control breads and breads supplemented with equivalent levels of chemical organic acids. Our data showed that EPS could compensate for the negative effects of chemical acetate and propionate in a concentration range exerting antifungal effects. The natural bioingredient produced by Wc 11GU-1 and Pf JS15 has potential for applications as antifungal, texture-building and anti-staling agent in breads, consistent with consumer demands for clean label products.

Development of a rapid screening protocol for selection of strains resistant to spray drying and storage in dry powder.

An efficient screening method for selection of Bifidobacterium longum strains resistant to spray drying and storage was developed based on randomly amplified polymorphic DNA (RAPD) for identification of the best survivors in mixed strains bacterial preparations. Three different primers were used to generate RAPD profiles of 22 B. longum strains. All strains were distinguished according to their RAPD profiles except for the strain NCC2705 and its H(2)O(2) resistant derivative variant. The 22 strains were grouped in 3 batches of 7, 7 and 8 strains and subjected to spray drying and storage at 30 and 37 °C under anaerobic conditions. Batch survival rates after spray drying reached 17.1±4.4%. Strains showing the highest prevalence and/or resistance to storage at 37 °C were selected from individual batches for subsequent spray drying and storage testing. After 67 days of storage, NCC572 was identified as the dominant strain in powder. The stability of strain NCC572 was confirmed by performing single spray drying and storage tests. Out of 22 B. longum strains, a robust strain was identified by combining RAPD with a simultaneous screening test for survival under spray drying and storage. The method allowed a fast screening of B. longum strains in mixture for resistance to spray drying and storage compared to traditional screening procedures carried out with individual strains, in the same conditions. This approach could be applied to other stress conditions.

Production of viable probiotic cells

Abstract: When considering the probiotic definition of FAO/WHO, products claiming probiotic effects should contain a sufficient number of viable cells to confer efficacy. Probiotic bacteria, being of intestinal origin, are sensitive to many stresses outside their natural habitat and therefore are difficult to propagate, store and deliver in a viable form. This chapter addresses different aspects of processes that aim to reach maximal cell yields with robust physiology and high resistance to conditions, from production to delivery to the gut. Novel culture and stabilization technologies aiming at the improvement of intrinsic tolerance of probiotic cells or conferring a physical protection against stresses are also discussed.