Entête

Chapitre III: Effets des fractions peptidiques et protéiques du cytoplasme de bifidobactéries sur la prolifération des lymphocytes de souris et la production de cytokines. Effects of bifidobacterial cytoplasm peptide and protein fractions on mouse lymphocyte proliferation and cytokine production.

Table des matières

T. Amrouche a,b,c, Y. Boutin b,d, and I. Fliss a,b*

a Dairy Research Center STELA, Pavillon Paul-Comtois, Université Laval, Québec (Qc) G1K 7P4

b Institute of Nutraceutical and Functional Foods INAF, Université Laval, Québec (Qc) G1K 7P4

c Department of Food Technology, Faculty of Agronomy and Biological Sciences, University of Tizi-Ouzou, Algeria

d TransBiotech, CEGEP Lévis Lauzon, Lévis (Qc) G6V 9V6

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Dans le premier chapitre, nous avions mentionné que le contenu cytoplasmique des bifidobactéries, notamment celui de la B. lactis Bb12 , exerce un effet stimulant sur la prolifération des lymphocytes et la production de cytokines. A cet effet, nous avions postulé que cet effet immunostimulant a été induit par les peptides et les protéines retrouvés dans le cytoplasme des cellules de bifidobactéries. Afin de vérifier cette hypothèse, nous avons réalisé les travaux rapportés dans ce chapitre. Nous avons fractionné et caractérisé les peptides et les protéines cytoplasmiques de B. lactis Bb12. Les fractions peptidiques et protéiques ont été ensuite étudiées pour leurs propriétés immunomodulatrices.

Les bifidobactéries sont des bactéries très utilisées dans les produits fermentées probiotiques. L’une des propriétés intéressantes des bifidobactéries est leur capacité à rétablir ou améliorer la fonction immunitaire chez l’homme et l’animal. Dans une étude antérieure, nous avions démontré que le contenu cytoplasmique de certaines souches de bifidobactéries stimule la prolifération des splénocytes et la sécrétion de cytokines. Cependant, les composants responsables de l’effet immunostumulant observé ne sont pas encore identifiés. La présente étude a pour but de caractériser et d’étudier l’effet des peptides et des protéines du cytoplasme de Bifidobacterium lacis Bb12 sur la prolifération des splénocytes de souris et la production de cytokines. L'analyse par SDS-PAGE de l’extrait cytoplasmique de B. lacis Bb12 cultivée sur le milieu MRS en anaérobiose a révélé la présence d’un groupe hétérogène de peptide et de protéines. Les peptides et les protéines ont été fractionnés par électrofocalisation (pH isoélectrique ou pI) en fraction acide (pI 2-5) et fraction basique (pI 5-12). Analysées par RP-HPLC, chacune des deux fractions montre la prédominance de peptides et de protéines relativement moins hydrophobes. Le poids moléculaire des peptides et des protéines des pics majeurs a été estimé par spectrométrie de masse à environ 510.16 à 10048.03 Da et plus de 13142.83 Da respectivement. Quatre pics majeurs ont été sélectionnés dans chaque fraction puis testés pour leur activité mitogénique sur des splénocytes de souris. Le cytoplasme brut (avant fractionnement) de la souche B. lactis Bb 12 s’est montré immunostimulant (indice de stimulation (IS) = 2,96 ± 0,35) comparativement à la PHA (IS=1,66 ± 0,20) en confirmant les effets immunostimulants observés dans notre étude antérieure. La fraction acide (100 µg/ml) stimule la prolifération des splénocytes (SI=1,61 ± 0,08) plus que la fraction basique (SI=1,33± 0,02). Les deux cytokines IFN-γ et IL-4 ont été détectées dans le milieu de culture à différentes concentrations indiquant la stimulation des cellules T helper. Une forte sécrétion de IFN-γ (1199,57±127,60 pg/ml) a été induite par l’extrait cytoplasmique brut (40 µg/ml) indiquant probablement une forte stimulation des cellules Th1. Une augmentation notable de la production de IFN-γ (578,55±195,16 pg/ml) a été également générée par la fraction basique (40 µg/ml). Une stimulation maximale de la sécrétion de IL-4 (9,14±4,04 pg/ml) induite par le contenu cytoplasmique à une concentration de 40 µg/ml indiquerait la stimulation des cellules Th2. Enfin, les effets immunostimulants ainsi démontrés laissent suggérer une possibilité d’utilisation de l’extrait cytoplasmique de bifidobactéries comme nutraceutique (agent immunomodulateur).

Key words: Bifidobactéries, prolifération cellulaire, cytokines, cytoplasme, peptides, protéines.

Bifidobacteria are probiotic bacteria shown to possess immunonodulatory properties in human and animal. Cellular components such as cytoplasmic content from bifidobacteria were demonstrated to elicit a mitogenic activity when tested in vitro with immune cells. Based on previously reported immunostimulating effects of bifidobacterial cytoplasm, we tested the potentiating effects of representative peptides and proteins from. B. lactis Bb12 cytoplasm on immune response measuring splenocyte proliferation and secreted cytokines. SDS-PAGE analysis of cytoplasmic extract resulted in a profile indicating an heterogeneous protein and peptide group in B. lactis Bb12. Cytoplasmic peptides and proteins were fractionated into acidic and basic fractions by liquid-phase electrofocalisation based on amino acid isoelectric pH. Both acidic and basic fractions were further fractionated by RP-HPLC into different peptides and protein fraction based on their hydrophobicity. The most abundant components in both fractions were eluted early indicating their low hydrophobicity. Four majors peaks selected in each fraction were analysed by mass spectrometry and showed individually an abundant peptide or protein with contaminants. The molecular weight of the components ranged from 510.16 to 10048.03 Da (peptides) or higher than 13142.83 Da (proteins). Each fraction was tested for its mitogenic activity on mouse splenocytes. The acidic fraction (100 µg/ml) was found to stimulate cell proliferation more (stimulation index (SI)= 1,61 ± 0,08) than the basic fraction (SI=1,33± 0,02), but less than the cytoplasmic content (2,96 ± 0,35). Secreted IFN-γ and IL-4 were detected in culture media at different level indicating the stimulation of helper T cells. The basic fraction was shown to induce more IFN-γ (578,55±195,16) than the acidic fraction (191,08±7,50 pg/ml). The results demonstrate that several peptides and proteins from B. lactis Bb12 exhibit an immunostimulating activity suggesting the possibility of the use of cytoplasmic content as nutraceutical products to enhance immune responses in children or elderly persons with reduced immune function.

Key words: Bifidobacteria, cell proliferation, cytokines, cytoplasm, peptides, proteins.

Probiotics such as bifidobacteria have been recently reported to enhance the human and animal immune system. Immunomodulatory effects of probiotic bacteria were shown to be associated not only with live microorganisms but also with nonviable organisms (Tejada-Simon and Pestka, 1999; Christensen et al., 2002; Kankaanpa et al., 2003; McCarthy et al., 2003; Chukeatirote, 2003; Mastrandrea et al., 2004). In our previous work, we demonstrated that cell wall and cytoplasm from bifidobacteria stimulated in vitro the lymphocyte proliferation and cytokines production. In addition, Lee et al. (2004) mentioned that cytoplasmic preparations of lactic acid bacteria were found to have antitumor effects in vivo with the modulation of cellular immunity.

According to Reid et al. (2002), several novel peptides from Lactobacillus fermentum RC-14 may prove to be significant with respect to its probiotic activity. Recently, Nouaille et al.(2003) mentioned that a new application for lactic acid bacteria is their use as live delivery vectors for antigenic or therapeutic protein or peptides in mucosal surfaces enhancing both mucosal and systemic immune responses. Chadwick et al. (1988) reported that peptides produced by intestinal bacteria could promote an inflammatory response and should cross the 'mucosal barrier'. They mentioned that these peptides activate in vitro and in vivo human neutrophils inducing chemotaxis and lysosomal enzyme. Furthermore, a bacterial formylpeptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP), has been reported by Arbour et al. (1996) to induce and modulate various cellular responses and secretion of pro-inflammatory cytokines by peripheral blood mononuclear cells.

In addition, some prokaryotic proteins such as heat shock proteins were shown to be a potential source of microbial peptides generated by digested bacteria and could enhance presentation of peptides to CD8 + T cells (Castellino et al., 2000; Aaron et al., 2004, Tobian et al., 2004). However, cytoplasm-derived peptides inducing the immunomodulatory effects are few investigated in bifidobacteria. In this study, we aimed to characterize the peptides or proteins fractions from bifidobacteria (Bifidobacterium lactis Bb12) cytoplasm responsible for immunomodulation.

A proliferation assay was carried out to evaluate the mononuclear cell response to cytoplasm fractions from B. lactis Bb12. Spleens were removed aseptically from euthanized Balb/C mice and single cell suspensions were prepared by mechanically disrupting the tissues through a cell strainer into RPMI 1640 medium (Gibco BRL Inc., Paisley, Scotland). Erythrocytes were removed from cell suspension by osmotic shock with 2 mL of 0.87 % NH4Cl solution for 2 min at 37oC. Spleen cells were then washed three times at 4°C, suspended in RPMI 1640 complete medium (with 10% fetal calf serum (FSC), 0.1 mL mL-1 50 mM mercaptoethanol , 100 U mL-1 penicillin, and 100 µg mL-1 streptomycin, all from Gibco). The crude cytoplasmic extract and acidic and basic fractions were diluted in supplemented RPMI to 0.8, 8, 80 and 200 μg/mL; whereas the fractionated components (peaks) were diluted in supplemented RPMI at 0.8, 8 and 80 μg/mL. All the samples were filtered (0.22 μm). Cells were plated at a final concentration of 5x105 cells mL-1. A mitogenic agent, phytohemagglutinin (PHA) (10 µg mL-1) was used alone as control for cell proliferation. Medium alone, PHA or above-mentioned concentrations of bifidobacterial components were added to wells in duplicate. Finally, AlamarBlue indicator (BioSource International, Inc., USA) was added to wells (20 µL/well) to measure cell proliferation. AlamarBlue system incorporates an oxidation-reduction indicator that both fluoresces and changes color in response to chemical reduction of culture media resulting from cell growth (Lancaster, 1996). Cell cultures were incubated for 48 h in a humidified 5 % CO2 atmosphere at 37 °C. At the end of incubation, the microplates were centrifuged and supernatants were transferred to a black opaque 96-well microplate (Fisher Scientific, Pittsburgh, USA) to determine fluorescence values. Fluorescence measurements were made by FLUOstar spectrofluorometric microtiter well plate reader (BMG Labtechnologies, Offenburg, Germany) (excitation: 544 nm; emission: 610 nm; gain 026). Proliferation responses were expressed as a stimulation index calculated as the ratio of mean fluorescence value obtained for stimulated culture to the mean fluorescence value obtained for nonstimulated control cell cultures.

To compare the cytoplasmic peptide and protein profile of three isolates of bifidobacteria and B. lactis Bb12 cytoplasm extracts were analysed by SDS-PAGE. The results reported in Figure 3.1 showed difference and similarities in protein profile between the strains tested.

The band corresponding to the molecular weight (MW) of 75 kDa was shown only by B. lactis Bb12. While, a common band with MW of 34 kDa was found to be characteristic to the isolates RBL81, RBL82, and RBL64. Bands with high MW (>45 kDa) and weak MW (<31 kDa) allowing discrimination between B. lactis Bb12 and isolates tested were also observed. More proteins and peptides appear to be biosynthesized by B. lactis Bb12 compared to other bifidobacterial strains. However, as can be seen in the Figure 3.1, peptides seem to be produced in quantities non detectable by blue coomassie staining compared to proteins. Marasco et al. (1984) reported that bacteria elaborate in very low concentrations numerous peptides of similar composition, which may differ markedly in their specific biological activities. The differences observed in protein and peptides profile of bifidobacteria should be attributed to the variability and diversity of strain proteome. Champomier-Verges et al. (2002) mentioned that lactic acid bacteria strains grown in the same rich synthetic MRS medium show some common (a dozen) proteins which represent almost 20% of the total protein detected on the acidic proteomic map. However, lactic acid bacteria indicating metabolic adaptation to media conditions show some differences in protein content. Indeed, different stress treatments (e.g., heat, low pH, osmotic shock, etc.) have been reported to induce transiently general and specific proteins (e.g., heat shock proteins) by physiological changes enhancing bacterial ability to survive in adverse environmental conditions (Ang et al., 1991; Prasad et al., 2003, De Angelis et al., 2004).

The band corresponding to the molecular weight (MW) of 75 kDa was shown only by B. lactis Bb12. While, a common band with MW of 34 kDa was found to be characteristic to the isolates RBL81, RBL82, and RBL64. Bands with high MW (>45 kDa) and weak MW (<31 kDa) allowing discrimination between B. lactis Bb12 and isolates tested were also observed. More proteins and peptides appear to be biosynthesized by B. lactis Bb12 compared to other bifidobacterial strains. However, as can be seen in the Figure 3.1, peptides seem to be produced in quantities non detectable by blue coomassie staining compared to proteins. Marasco et al. (1984) reported that bacteria elaborate in very low concentrations numerous peptides of similar composition, which may differ markedly in their specific biological activities. The differences observed in protein and peptides profile of bifidobacteria should be attributed to the variability and diversity of strain proteome. Champomier-Verges et al. (2002) mentioned that lactic acid bacteria strains grown in the same rich synthetic MRS medium show some common (a dozen) proteins which represent almost 20% of the total protein detected on the acidic proteomic map. However, lactic acid bacteria indicating metabolic adaptation to media conditions show some differences in protein content. Indeed, different stress treatments (e.g., heat, low pH, osmotic shock, etc.) have been reported to induce transiently general and specific proteins (e.g., heat shock proteins) by physiological changes enhancing bacterial ability to survive in adverse environmental conditions (Ang et al., 1991; Prasad et al., 2003, De Angelis et al., 2004).

In order to separate several peptides and proteins with subtle differences in their amino acid composition, the acidic and basic fractions were analysed individually by RP-HPLC. The chromatogram of each fraction is shown in Figure 3.2 and Figure 3.3. Chromatography system resulted in marked differences in retention time for several peptides and proteins when applied to the column as a mixture. In both fractions, we observed numerous peaks throughout the elution gradient indicating the presence of heterogeneous group of peptides and proteins. However, most of materials that are eluted earlier (5 to 10 mn) gave high absorbance (A210 > 1.0).

The early peaks in the HPLC fractionation probably would correspond to the peptides or proteins containing hydrophilic amino acids such as aspartic acid, serine, glutamic acid, alanine, and glycine (Schiffmann et al., 1975). Furthermore, some peptides or proteins could not easily be separated from others. Several minor peaks appear later (12 mn - 30 mn) in chromatogram indicating that more hydrophobic peptides and proteins would be present in cytoplasm in minute concentration. These peptides could be formyl methionyl peptides cleaved post-translationally from the NHs-terminal regions of newly synthesized proteins. The formyl methionine character of these peptides has been suspected since a large number of bacteria were shown to elaborate similar substances (Ward et al., 1968). It has been reported that both the biological activity of peptides and their affinity for the peptide receptor increase as the hydrophobic character of the constituent amino acids increases (Freer et al., 1982). However, complete characterization of these peptides and proteins has been hampered by their weak concentrations.

Four peaks with high absorbance (major peaks) were selected in each fraction and were collected individually by preparative HPLC to be analyzed by mass spectrometry and tested for their bioactivity. Mass spectrometry analysis of labelled peaks in chromatograms resulted in variable m/z corresponding to several components in each peak. Indeed, the mass spectra (not shown) demonstrated more than one component when individual fraction (labelled peak) was infused in spectrometry system. These results indicated partially purified peptide or proteins. The extraneous peaks present in the total mass spectra of each peptide or protein fraction (or labelled peak) are contaminants from other peaks due presumably to the interaction of peptides and proteins with column or other factors. Because of small quantities of HPLC-fractionated peptides and proteins selected, we were not able to enhance purity of the materials by subsequent HPLC runs. Then, the data analysis obtained for each labelled peak did not allow identification of peptide or protein fractionated.

Moreover, the spectrometry data allow distinction between labelled peaks based on the molecular weight of peptides or proteins previously fractionated. The average molecular weight of the most abundant peptides or proteins fractionated are presented in Table 3.1. The results indicated that peptides with molecular weigh ranging from 510.16 to 10048.03 Da are biosynthesized in B. lactis Bb12. These peptides were detected in both acidic and basic fractions. Also, proteins with molecular weight higher than 13142.83 Da were shown by mass spectra.

In bacteria, most secreted proteins are synthesized as precursors containing the mature protein and an N-terminal signal peptide that is an essential signature for protein secretion. The signal peptide primary sequences are poorly conserved and show a common tripartite structure including a positively charged N-terminus, a hydrophobic core and a neutral or negatively charged C-terminus containing the signal peptide cleavage site (von Heijne, 1990). Bacterial protein synthesis starts with a formylated methionine residue, and this residue is sequentially cleaved away by a unique peptide deformylase and a methionine aminopeptidase to generate mature proteins (Fu et al., 2003). Several bioactive formyl oligopeptides were shown to be produced by different species of enteric bacteria (Hobson et al., 1990).

The immunoactivity of semi-purified peptides and proteins from acidic and basic fractions were assessed measuring splenocyte proliferation and hallmark Th1 and Th2 cytokines IFN-γ and IL-4 production in the presence of these peptides or proteins at different concentrations in culture media. The cell proliferation was carried out with non-stimulated cells (in PHA-free media). Wells with PHA were used as positive control. The stimulation index (SI) obtained by both acidic and basic are presented in Figure 3.4.

The results indicate that a cell proliferation stimulation was induced by cytoplasmic peptides and proteins depending on fraction and concentration used. The acidic fraction is shown to significantly stimulate (1,616 ± 0,08) splenocyte proliferation compared to the nonstimulated cells (cells alone) when used at high concentration (100 µg/ml). The cell proliferation induced by the acidic fraction was comparable with that obtained by PHA (1,66 ± 0,20); the SI given by basic fraction was estimated at 1,3349 (± 0,02). High stimulation of cell proliferation was obtained by cytoplasmic extract which showed a SI increasing from 1,41 (± 0,15) to 2,96 ( ± 0,35) when the extract concentration in culture media varied from 4 to 100 µg/ml respectively. Bacterial peptides and proteins (enzymes) were reported to be immunomodulatory and to influence the specific and nonspecific immune response (Houba et al., 1992; Dobrotina et al., 1992, Panaro and Mitolo, 1999).

These results are in accordance with those obtained in our previous study. Increased cell proliferation induced by crude cytoplasmic extract could be explained by the presence of synergistic components in cytoplasm of B. lactis Bb12. Indeed, Iliev et al. (2005) demonstrated that chromosomal DNA motifs from Lactobacillus rhamnosus GG are active in both murine and human immune cells. Nevermore, these results agree with those recently reported by Lee et al. (2004) who mentioned that administration of cytoplasmic fraction of Lactobacillus casei and Bifidobacterium longum to mice for 4 weeks enhanced the number of total T cells, NK cells and MHC class II+ cells, and CD4 -CD8 + T cells.

In order to compare the immunostimulatory effects of peptides and proteins in the acidic fraction, we tested separately four majors peaks (labelled peaks) at concentrations of 4 and 40 µg/ml. Significant increased cell proliferation was induced by semi purified peptides corresponding to peak 1 (1,50± 0,03) and peak 2 (1,68 ± 0,22) when used at 40 µg/ml. It has been reported that several peptides from bacteria are target of both T and B cell responses and could be exploited for vaccine purposes (Chua-Intra et al., 1998; Hussain et al., 2004). Peptides receptor such as formyl-peptide receptor (FPR) and its variant FPRL1 (FPR-like 1) have been found to be expressed at high levels on human leucocytes involved in inflammatory processes induced by bacteria (Le et al., 2002; Ernst et al., 2004). The nature of amino acids was reported to influence the immunoactive properties of the peptide or protein which contains them. For example, a D-methionine-containing peptide, Trp-Lys-Tyr-Met-Val-D-Met-NH2, has been found to be a stronger activator of neutrophils than f-Met-Leu-Phe. D-isomers amino acid are used in protein synthesis only by prokaryotes and then generate danger signals to the innate immune system (Svensson et al., 2002).

The overall results suggest that the cytoplasmic peptides (and proteins) are immunoactive components that contribute to immune stimulation induced by cytoplasm of B. lactis B12. The differences in cell proliferation could be attributed to the difference in nature or amino acid composition between peptide and protein from cytoplasm. The low hydrophobicity of peptides and proteins tested does not appear to be a factor influencing greatly the immune response. Vinderola et al. (2004) have investigated the relationship between interaction sites in the gut, hydrophobicity, mucosal immunomodulating capacities and cell wall protein profiles in lactobacilli, bifidobacteria and enterococci. They mentioned that the highest interaction capacity with Peyer’s patches and nodules was observed for enterococci, which presented the smallest hydrophobicity values. While, bifidobacteria and actobacilli, which showed a lower interaction capacity with lymphoid follicles, presented higher values of hydrophobicity.

To determine the effect of cytoplasmic peptides and proteins from B. lactis B12 on cytokines secreted by activated lymphocytes, we analyzed the production of IFN-γ and interleukin IL-4. It is well established now that Helper T cells (Th) are heterogeneous with regard to cytokine secretion and their functions. Th1 cells produce mainly IL-2 and IFN-γ, while Th2 cells predominantly produce IL-4 and IL-6. These two cytokines are shown to have antagonistic functions ( Yazdanbakhsh et al.,1999; Valentini et al., 2001).

The production of IFN-γ and IL-4 by splenocytes cultured with various concentration of cytoplasmic extract, and the acidic and basic fractions is illustrated by Figure 3.5 and Figure 3.6.

According to these results, both crude cytoplasmic extract and basic fraction induce a bell-shaped dose-response curve. As shown in Figure 3.5, splenocytes stimulated by cyplasmic extract from B. lactis Bb12 secreted approximately twofold IFN-γ (1199,57 ±127,60) higher than when stimulated by the basic fraction (578,55±195,16) and fourfold when cultured with the acidic fraction (191,08±7,50 pg/ml). While, IFN-γ production obtained with PHA was estimated at 127,10±101,50 pg/ml. Th1 cells appear to be more stimulated by basic fraction compared to acidic fraction.

The results reported in Figure 3.6 demonstrate low IL-4 production compared to those of IFN-γ obtained by cytoplasmic content, and both acidic and basic fractions. Hence, Th2 cells seem to be weakly stimulated. The most significant production of IL-4 was obtained by crude cytoplasmic extract (9,14±4,04) and basic fraction (4,14±2,02). While, the production of IL-4 increased slightly upon treatment with acidic fraction (< 3,86 pg/ml). Th1 lymphocytes stimulate cell-mediated immunity, which is characterized by intense phagocytic activity. Conversely, Th2 cells stimulate humoral immunity, which is characterized by high antibody production. Evidence suggested that coinduction of T2 immunity maintains immune homeostasis during T1-mediated defence reactions. Th1 cells also stimulate moderate levels of antibody production, whereas Th2 cells actively suppress phagocytosis (Spellberg and Edwards, 2001; Bot et al., 2004).

It has been demonstrated that synthetic peptides stimulate T cell chemotaxis showing unique signalling and provide a helpful tool to understand the T cell activation mechanism (Zachariae et al., 1992; Kim et al., 2001). The synthetic peptides were shown to stimulate the formation of inositol phosphates in lymphocyte cells (Baek et al., 1996). Wang et al. (2004) reported that a fungal immunomodulatory protein from Flammulina velutipes (FIP-fve) exhibited potent mitogenic effects on human peripheral blood lymphocytes, inducing G1/G0 to S phase proliferation. The activation of T cells resulted in significant production and secretion of IFN-γ associated with intercellular adhesion molecule 1 expression but low detectable levels of interleukin-4 in vitro or in vivo .

These results obtained confirmed previous observations that cytoplasm from B. lactis Bb12 stimulate the cell proliferation and cytokines production in mouse splenocytes. In this study, we have demonstrated that B. lactis Bb12 cytoplasm contains an heterogeneous group of peptides and proteins characterized by a wide range of isoelectic pH and molecular weight. The cytoplasmic peptides and proteins were found to have low hydrophobicity, and to be heat-stable and immunostimulatory. The acidic fraction (100 µg/ml) stimulated cell proliferation (SI= 1,616 ± 0,085), whereas the basic fraction induced more IFN-γ secretion (578,55±195,16 pg/ml). The most significant immunostimulation (SI= 2,96 ± 0,35) was obtained by cytoplasmic content suggesting that the cytoplasm contains a components acting synergistically with immunostimulatory peptides or proteins. However, IL-4 secreted by lymphocytes stimulated by cytoplasmic content, and both acidic and basic fractions, was slightly increased. Further studies could be performed to improve separation of peptides and/or protein using ionic exchange chromatography, and to identify these components by sequencing method. Furthermore, nonlabelled peaks showing high hydrophobicity could be explored for their mitogenicity producing high quantities of material in optimized conditions.

Evidence demonstrates that peptides and proteins derived from B. lactis Bb12 cytoplasm could be delivered in intestinal environment and activate the immunocompetent cells of the mucosal immune system. The potent immunostimulating effect of cytoplasm could suggest the possibility of use of bifidobacaterial extract as nutraceutical products.

ACKNOWLEDGMENTS

This work was supported by grants from FCAR, NOVALAIT, MAPAQ and NSERC. T. Amrouche was recipient of scholarships from the World Bank. We thank ADVITEC team mainly S. Gauthier and N. Attouri for their contribution.

© Tahar Amrouche, 2005