USE OF MICROORGANISMS FOR THE PREVENTION AND TREATMENT OF INTESTINAL DISEASES

Abstract

The invention relates to acetylcholine-producing microorganisms for use in the prevention and/or treatment of intestinal diseases, and/or reduction of risks of intestinal diseases, and/or improvement of intestinal health as well as promoting healthy gut flora. The acetylcholine-producing microorganisms may be provided as a pharmaceutical dosage form or as additive to functional food or food supplemental products. Also encompassed is a method for the production of acetylcholine by use of Lactobacilli. Further the invention refers to microbially produced acetylcholine for use in the treatment and/or prevention of intestinal diseases.

Claims

1. A method of treating and/or reducing the risk of intestinal diseases comprising administering a living acetylcholine-producing microorganism to a patient in need thereof, wherein the intestinal disease is a disorder associated with impaired intestinal motility and secretion or an inflammatory bowel disease and wherein said microorganism is selected from the group consisting of Lactobacillus sanfranciscensis, Lactobacillus rossiae and Lactobacillus brevis.

2. The method according to claim 1, wherein said living acetylcholine-producing microorganism is administered in an amount sufficient for maintaining a healthy gut flora, promoting a healthy gut flora, reducing the toxic effects of the digestive process, stimulating the digestive system and/or improving intestinal control.

3. The method according to claim 1, wherein the intestinal disease is a functional bowel disorder.

4. The method according to claim 1, wherein the intestinal disease is a functional intestinal disorder and/or a disorder associated with secretions of the intestinal wall controlled by the enteric nervous system.

5. The method according to claim 1, wherein the intestinal disease is functional constipation, functional diarrhea and/or irritable bowel syndrome (IBS).

6. The method according to claim 1, wherein the intestinal disease is constipation predominant IBS, alternating IBS or diarrhea predominant IBS.

7. The method according to claim 3, wherein the functional bowel disorder is a chronic or semi-chronic gastrointestinal disorder which is associated with bowel pain, disturbed bowel function and/or social disruption.

8. The method according to claim 1, wherein the intestinal disease is ulcerative colitis and/or Crohn's disease.

9. The method according to claim 1, wherein the microorganism produces >30 mg acetylcholine/kg weight of the medium when culturing with MRS-broth and adjusting the acetyl concentration to a 10.sup.6 bacteria count.

10. The method according to claim 1, wherein the microorganism is a DSM 23090 or DSM 23093 strain.

11. The method according to claim 1, wherein the lactobacillus is selected from the group consisting of strains DSM 26024, DSM 23090, DSM 23091, DSM 23200, DSM 23092, DSM 23093, DSM 23201, DSM 23174 and DSM 23121, and a strain cultivated therefrom.

12. The method according to claim 1, wherein said living acetylcholine-producing microorganism is in a pharmaceutical dosage form, a functional food or in a functional beverage.

13. The method according to claim 12, wherein the functional food is sourdough bread.

14. The method according to claim 1, further comprising administering at least one additional bacterium for maintaining and/or restoring a favorable gut flora.

15. The method according to claim 1, further comprising administering at least one additional medicament for the treatment of intestinal diseases.

16. A method for the production of acetylcholine comprising culturing lactobacilli, and isolating acetylcholine, wherein the lactobacilli are selected from the group consisting of Lactobacillus sanfranciscensis and Lactobacillus rossiae strains.

17. A method for the treatment and/or reducing the risk of intestinal diseases in a patient in need of such treatment, comprising orally administering a therapeutically effective amount of a composition comprising microbially produced acetylcholine to said patient, wherein the intestinal disease is a disorder associated with impaired intestinal motility and secretion or an inflammatory bowel disease.

18. A method for maintaining and/or improving intestinal health comprising administering a living acetylcholine-producing microorganism to a patient in need of such treatment wherein said microorganism is selected from the group consisting of Lactobacillus sanfranciscensis, Lactobacillus rossiae and Lactobacillus brevis.

19. The method according to claim 18, wherein said living acetylcholine-producing microorganism is administered in combination with a pharmaceutically acceptable carrier.

20. The method according to claim 1, wherein the inflammatory bowel disease is chronic.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0062] FIGS. 1A-1B: Metabolite profile in water extracts of sourdough, sourdough bread and analog bread. A: Heatmap of absolute metabolite concentrations in water extract from sourdough (SD), sourdough bread (BR) and analog bread (AN) in triplicates. Water extracts constitute 5-10% of total dough or bread dry mass. B: Principal component analysis (PCA) of metabolites reveals significant differences in metabolite concentrations between sourdough (SD), sourdough bread (BR) and analog bread (AN).

[0063] FIGS. 2A-2B: Sourdough extracts effectively stimulate stomach muscle motility by acting directly on muscarinic acetylcholine receptor (mACHR). A: Muscle tone change induced by either acetylcholine (ACH) (2.5 μM) or extracts of sourdough (SD), sourdough bread (BR) or analog bread (AN BR) at 0.02%. Increase in tone was immediately observed upon addition of the stimulants (arrow sign) except for extract of analog bread. B: Median (n>4) change of muscle tone upon differential treatments. The pro-kinetic effect of acetylcholine (ACH) and extracts of sourdough (SD), sourdough bread (BR) or analog bread (AN BR) is completely abolished by mACHR specific antagonist atropine. This indicates that acetylcholine in extracts directly acts on mACHR.

[0064] FIG. 3: Tetrodotoxin (TTX) has no significant effect on the stimulation of muscle contraction by sourdough (SD)-derived acetylcholine (ACH). The figure shows the muscle tone change stimulated by either acetylcholine (2.5 μM) or 0.02% extracts of sourdough (SD), sourdough bread (BR) or analog bread (AN BR), and the effect of TTX pre-treatment. TTX did not exhibit significant effect on the muscle contraction induced by sourdough ACH suggesting stimulation is induced by direct action on muscle mACHR without neuronal mediation.

[0065] FIGS. 4A-4C: Sourdough and sourdough bread extracts stimulate chloride ions secretion when applied from either serosal or mucosal side of intestinal mucosa. A: Ussing chamber set-up with mucosa piece separating the two chambers. The lower set of electrodes measure transepithelial voltage (V.sub.TE) and the lateral set—the short circuit current (I.sub.SC). The secretion is measured estimated by the change in I.sub.SC necessary to maintain V.sub.TE at 0 mV. B: Representative I.sub.SC traces stimulated by acetylcholine (ACH), sourdough (SD), sourdough bread (SD BR) or analog bread (AN BR) extracts. Area under the curve is calculated using an integral (μA*s/cm.sup.2) where blue shows response to extracts and red-striped shows response to electric field stimulation (EFS). Figure C shows median (n>4) value of change in I.sub.SC upon treatment on either mucosal side (MUC) or serosal side (SER) of guinea pig colonic mucosa. Acetylcholine (ACH) and sourdough (SD) as well as sourdough bread (SD BR) extracts clearly stimulate secretion when applied to either side of mucosa while analog brad extract has no effect. Showing that acetylcholine in sourdough and sourdough bread is responsible for the stimulation.

[0066] FIGS. 5A-5B: Atropine completely abrogates chloride ion secretion stimulated with sourdough (SD) and sourdough bread (SD BR) extracts. Shown is the mean (n>4) value of change in short circuit current (I.sub.SC) over time upon acetylcholine (ACH) (10 μM) and extract (0.1%) application on either mucosal side (A) or serosal side (B) of guinea pig colonic mucosa with and without atropine pretreatment (1 μM). Atropine completely abrogated secretion stimulation suggesting the role of mACHR in the secretory effect of sourdough extracts.

[0067] FIG. 6: Atropine completely abrogates secretion stimulation by ACH and extracts of sourdough (SD), sourdough bread (BR) or analog bread (AN BR) when applied serosally but not mucosally. Shown is the mean (n>4) value of change in short circuit current (I.sub.SC) over time subsequent to ACH and extract treatment on the mucosal side with and without atropine pre-treatment (1 μM). Atropine was added on either serosal or mucosal side. Atropine completely abrogated secretion when applied on serosal but not mucosal side.

[0068] FIGS. 7A-7C: Metabolic profile of sourdough lactic acid bacteria. A: Heatmap of absolute metabolite values in MRS broth after 24 hour lactic acid bacteria inoculation (mean of three experiments). B: PCA plot of metabolites indicates sharp distinction between tested sourdough bacteria and L. paracasei (LC). C: Acetylcholine (ACH) plays the strongest role in the separation observed in PCA plot since L. paracasei does not produce any Acetylcholine.

[0069] FIGS. 8A-8B: Acetylcholine concentration in MRS media upon 24 hours of incubation with lactic acid bacteria. A: Acetylcholine concentration in MRS broth upon 24 hour inoculation with 0.25×10.sup.7 of bacteria/mL. B: Acetylcholine (ACH) concentration adjusted to 10.sup.6/mL bacteria count in MRS. Concentration was determined using LC-MS/MS by comparing the peak area to the area of solutions with known concentration of acetylcholine.

[0070] FIG. 9: Concentrated conditioned media of sourdough lactic acid bacteria significantly inhibits the secretion of interferon inducible protein 10 (IP-10) by tumor necrosis factor (TNF)-activated intestinal epithelial cells (IEC). The concentration of IP-10 in the culture media of Mode-k cells as measured by ELISA is shown. The cells were incubated for 24 hours with concentrated conditioned media (cCM) (black bar) and cCM plus 10 ng/mL TNF (grey bar). L. paracasei (L.p.) expresses Lactocepin PrtP that is capable of efficiently degrading IP-10 and as expected has the highest inhibitory activity. The cCM of sourdough lactic acid bacteria significantly inhibit IP-10 secretion but to a lesser extent. L. sanfranciscensis strains DSM 23174 and DSM 23200 are the most efficient in inhibiting IP-10 secretion next to L. paracasei.

[0071] FIG. 10: Formaldehyde-fixed sourdough lactic acid bacteria significantly inhibit the secretion of interferon inducible protein (IP-10) by TNF-activated intestinal epithelial cells. The concentration of IP-10 in the culture media of Mode-k cells as measured by ELISA is shown. The cells where incubated for 24 hours with 20 MOI of fixed lactic acid bacteria (black bar) and 20 MOI of fixed lactic acid bacteria plus 10 ng/mL tumor necrosis factor (TNF) (grey bar). Fixed L. paracasei (L.p.) has, similar to fixed L. sanfranciscensis strains DSM 23090 and DSM 23092, the highest inhibitory activity on the IP-10 secretion by TNF-activated intestinal epithelial cells (grey bar) as compared to TNF-activated control.

[0072] Further, the invention shall be explained in more detail by the following examples.

EXAMPLES

1) Methods and Materials

1.1) Sourdough and Bread

[0073] Sourdough (Vollsauer) was prepared by traditional propagation of type I sourdough rye starter containing the Lactobacilli strains DSM 26024, DSM 23090, DSM 23091, DSM 23200, DSM 23092, DSM 23093, DSM 23201, DSM 23174 and DSM 23121. The composition of sourdough and sourdough bread is: 71% rye flour, 25% wheat flour, 1.8% salt and 2% bread crumbs (pH 4.5, acidity 9-10). The dough was baked at 298° C. for 1.5 hours. Analog bread was identical to sourdough bread with substitution of sourdough starter with 2.5% sodium bicarbonate, 0.13% acetic acid and 1.2% lactic acid.

1.2) Metabolite Analysis

[0074] Water extracts (<10 kDa) of sourdough, sourdough bread and analog bread as well as MRS growth media of lactic acid bacteria were subjected to LC-MS/MS analysis for metabolite quantification. MRS media was filtered with 10 kDa Vivaspin 500 filters (Sartorius Stedim biotech, Goettingen, Germany) before analysis.

[0075] Samples were measured using:

[0076] Dionex Ultra High Performance Liquid Chromatography UltiMate® 3000 (Dionex, Idstein, Germany) [0077] Pump—HPG-34005D [0078] Degasser—SRD-3400 [0079] Autosampler—WPS-3000TSL [0080] Column oven—TCC-30005D

[0081] API 4000 QTRAP, Linear Ion Trap Quadrupole Mass Spectrometer (AB Sciex, Darmstadt, Germany): [0082] Ionization type—electrospray ionization (ESI) [0083] Instrument control—Analyst software (AbSciex, Darmstadt, Germany) [0084] Stationary phase: TSKgel Amide-80 3 μm (150×2 mm, Tosoh Bioscience, Stuttgart, Germany) [0085] Stationary phase temperature: 40° C. [0086] Mobile phase: eluent A: acetonitrile/5 mM/L ammonium acetate in water (95+5) [0087] eluent B: 5 mM/L ammonium acetate in water (95+5)

TABLE-US-00001 gradient:  0 min 90% A  10% B  5 min 90% A  10% B  10 min 80% A  20% B  15 min 50% A  50% B  18 min  0% A 100% B  21 min  0% A 100% B  24 min 90% A  10% B  30 min 90% A  10% B flow: 200 μl/min
The Chromatograms were analysed with Multiquant 2.0 (AB Sciex, Darmstadt, Germany) and concentrations in the samples were calculated according to the spectra of standards.

1.3) Extraction

[0088] Sourdough, sourdough bread and analog bread were freeze-dried and grinded into powder. 100 g of powdered bread or sourdough was solubilized in 500 mL distilled water and extracted for 3 hours at 50° C. with constant stirring. The suspension was centrifuged at 9000 rpm for 20 min. The supernatant was collected and kept at 4° C. The pellet was re-suspended again in 500 mL distilled water and 3 hour extraction repeated. After centrifugation the pellet was again re-suspended in 500 mL distilled water and extracted overnight. Supernatants after three extraction steps (total volume of approx. 1.5 L) were pooled together and step-wise filtered using Vivaflow 200 cassettes of 0.2 μm, 100 kDa and 10 kDa exclusion thresholds (Sartorius, Goettingen, Germany). The filtrates of the 100 kDa and 10 kDa exclusion thresholds were freeze dried and re-suspended in distilled water to 25% for in vitro assays. 10 g of <10 kDa fraction was extracted from 100 g freeze-dried bread and sourdough.

1.4) Endotoxin Measurement and Clean-Up

[0089] Endotoxin concentrations measurement in water extracts from sourdough, sourdough bread and analog bread were determined using Limulus Amebocyte Lysate (LAL) Chromogenic Endpoint Assay (Hycult biotech, Uden, Netherlands). The assay was performed according to the manufacturer's instructions. Endotoxin contamination in water extracts from sourdough, sourdough bread and analog bread was removed using Detoxi-Gel™ Endotoxin Removing Columns (Thermo Scientific, Rockford, USA), containing a resin with immobilized polymyxin B to bind and remove pyrogens from solution. The removal of endotoxin was performed according to the manufacturer's instructions.

1.5) ELISA

[0090] Interferon inducible protein (IP-10) (murine/human) and (murine) concentrations in cell culture supernatants were determined using the appropriate ELISA kits (R&D Europe, Abington, England) according to the manufacturer's instructions. The ELISA was performed using Nunc MaxiSorp® flat-bottom 96 well plates (Greiner Bio-One GmbH, Frickenhausen, Germany). Briefly 96-well plates were coated with the appropriate capture antibody overnight at RT. Plates were washed 3 times with phosphate buffered saline (PBS), blocked with 1% bovine serum albumin in PBS and incubated with cell culture supernatants for 1.5 h at RT. Plates were washed and incubated with the appropriate detection antibody for 1.5 h at RT. Plates were washed and incubated with a detection enzyme. Plates were washed and incubated with a substrate solution. Protein concentration was determined by photometrical analysis of the reaction of substrate and detection enzyme.

1.6) Bacterial culture

[0091] L. sanfranciscensis strains (DSM 23090, DSM 23091, DSM 23092, DSM 23093, DSM 23174, DSM 23200, DSM 23201) and L. rossiae (DSM 26024) isolated from sourdough, L. sanfranciscensis type strain DSM 20451 (DSMZ GmbH, Braunschweig,Germany), L. plantarum FUA 3038 and L. brevis 3113 (provided by Prof. Ganzle from University of Alberta, Canada), L. paracasei VSL #3 (provided by Dr. DeSimone, L'Aquila, Italy) were grown at 30° C. in MRS broth (pH 5.4) containing freshly added 0.15% L-cystein under anaerobic conditions using Anaerogen packages (Anaerogen, Basingstoke, Oxoid, UK). Fixed bacteria (5% formaldehyde, 4 hours, 4° C.) were washed three times with sterile PBS before use. Concentrated conditioned media (CM) were generated by transferring bacteria (5×10.sup.7 cfu/ml) from anovernight culture to DMEM (1% glutamine, 20 mM HEPES) and anerobical cultivation overnight at 30° C. Bacteria and bacterial supernatant (CM) were separated after centrifugation (4500 g, 10 min, RT). CM was adjusted to pH 7.4, filter sterilized (0.22 μm), and concentrated (100×) using Vivacell filter systems with an exclusion size of 100 kDa (Satorius Stedim Biotech, Goettingen, Germany). Concentrated conditioned media was diluted to 1 × in the cell culture stimulation experiments. Agar plates were obtained by adding 1.5% of agar to the above described respective medium.

1.7) Motility

[0092] Motility measurements were performed with corpus circular muscle preparations from Dunkin Hardley guinea pigs (Sulzfeld and Harlan Winkelmann GmbH, Borchen, Germany). Contractile force of the muscle was measured using force transducer in organ bath using LabChart 5 software (ADInstruments, Spechbach, Germany). Briefly, stomach muscle tissue was dissected from mucosa layer in continuously perfused ice-cold preparation Krebs solution (pH 7.4) (MgCl.sub.2×6H.sub.2O 1.2 mM, CaCl.sub.2×2H.sub.2O 2.5 mM, NaH.sub.2PO.sub.4 1.2 mM, NaCl 117 mM, NaHCO.sub.3 25 mM, C.sub.6H.sub.12O.sub.6 11 mM, KCl 4.7 mM). A 1.5 cm.sup.2 piece of corpus circular muscle was cut out and mounted from both ends with polyamide thread between two electrodes into organ bath in 20 mL experimental Krebs solution (identical to preparation Krebs except for NaHCO.sub.3 20 mM) at 37° C. and aerated continuously with Carbogen (95% O.sub.2 and 5% CO.sub.2). After an equilibration period of 45 min muscle preparations were stimulated by electrical field stimulation (EFS) to test vitality. The change in contractile force during EFS as well experimental treatment was measured by the force transducer. The time lapse between any treatments was always 20 min.

1.8) Ussing Chamber

[0093] The ion movement across intestinal epithelia was measured with Ussing chamber technique (Easy mount chambers, Physiologic instruments, San Diego, USA) and LabChart 5 software (ADInstruments, Spechbach, Germany). Briefly segments, of the distal colon of Dunkin Hardley guinea pigs (Sulzfeld and Harlan Winkelmann GmbH, Borchen, Germany) were dissected, the muscle layers removed and mucosa/submucosa preparations were mounted into slider with a recording area 0.5 cm.sup.2. Apical and basolateral sides were bathed separately in 5 mL Krebs solution. During experimental procedures, the bath was maintained at 37° C. and aerated continuously with Carbogen (95% O.sub.2 and 5% CO.sub.2). After an equilibration period of 45 min tissue was electrically stimulated (Parameters: stimulus strength 6V, duration 10 sec, frequency 10 Hz, single pulse duration 0.5 ms) to assess tissue vitality. For assessment of active ion transport spontaneous occurring transepithelial voltage (V.sub.TE) formed by passive ion transport across the tissue was set to 0 mV by applying short circuit current (I.sub.SC). When the active chloride ion secretion is induced an increase in I.sub.SC is observed necessary to keep V.sub.TE at 0 mV. The change in I.sub.SC is equivalent to the current generated by the anions secretion or cation absorption. Transepithelial resistance (TER=V.sub.TE/I.sub.SC×1000/2) of tissue was measured at the beginning and at the end of each experiment to assess the tissue integrity.

1.9) Statistical Analysis

[0094] Data are expressed as mean values ±standard deviation (SD). All statistical computations were performed using Statistical programming platform R comparing treatment vs. corresponding control group were analyzed using unpaired t-tests. Data comparing several treatments vs. corresponding control group were analyzed using One-Way ANOVA followed by an appropriate multiple comparison procedure. If data was not normally distributed or comprised discontinuous data, non-parametrical tests (Mann-Whitney/Rank sum test, ANOVA on ranks) were used. Differences were considered significant if p-values were <0.05 (*) or <0.01 (**). Principal component analysis (PCA) is described in Pearson, K.; On Lines and Planes of Closest Fit to Systems of Points in Space, Philosophical Magazine (1901), 2 (11), 559-572 and Theodoridis, G., Gika, H. G., Wilson, I. D.; LC-MS-based methodology for global metabolite profiling in metabonomics/metabolomics, TrAC Trends in Analytical Chemistry (2008), 27 (3), 251-260.

2.) Results

[0095] 2.1) LC-MS/MS Analysis of Metabolites in Extracts from Sourdough, Sourdough Bread and Analog Bread

[0096] To compare the effect of fermentation on sourdough and sourdough bread water soluble extracts (<10 kDa, triplicates) of raw sourdough, sourdough bread and analog bread prepared from three different batches were subjected to LC-MS/MS analysis. The concentration of metabolites in extracts was determined by comparison to the standard solution with known concentration of metabolites.

[0097] Principal component analysis (PCA) demonstrated significant differences in metabolites isolated from sourdough, sourdough bread and analog bread (FIG. 1). Raw sourdough has significantly higher amounts of free amino acids reflecting proteolitic acitivity of endogenous flour enzymes as well as lactic acid bacteria proteases. Before baking fresh unfermented flour is added to the sourdough explaining why there are no differences in the free amino acid content between analog and sourdough bread. Acetylcholine is a metabolite that is consistently present in sourdough and sourdough bread but not in analog bread (Table 1). Fermentation by sourdough bacteria is the only difference between sourdough bread and analog bread, suggesting acetylcholine is produced by microorganisms present in sourdough.

TABLE-US-00002 TABLE 1 Sourdough and sourdough bread contain high amounts of acetylcholine of with 38.6 mg/kg in dry bread. Concentration was determined using LC-MS/MS by comparing the peak area to the area of solutions with standard concentrations of acetylcholine. ACH concentration in ACH concentration bread or sourdough, Water extracts in water extracts dry mass Sourdough, 10 kDa 1819 ± 717 μM 26.5 ± 10.4 mg/kg Sourdough bread, 10 kDa 2644 ± 273 μM 38.6 ± 4.03 mg/kg Analog bread, 10 kDa 44.5 ± 1.0 μM 0.64 ± 0.03 mg/kg

2.2) Sourdough-Derived Acetylcholine Triggers Muscle Contraction In Vitro

[0098] Acetylcholine (ACH) is a neurotransmitter that is responsible for the activation of motility in the gastro-intestinal tract by stimulating either muscarinic (mACHR) or nicotinic ACH receptors (nACHR) on the muscle cells. To determine if the sourdough-derived acetylcholine mimicks this activity, isolated corpus muscle of guinea pig was stimulated with extracts of sourdough, sourdough bread and analog bread and the contraction stimulation was measured. Both sourdough and sourdough extracts but not analog bread extract induced muscle contractions similar to acetylcholine at equivalent concentration (FIG. 2). Atropine, mACHR-specific antagonist, was used to determine whether extracts stimulate contraction by activating muscarinic or nicotinic ACHR. Pre-treatment of muscle strips with atropine completely abrogated stimulation by ACH as well as by sourdough and sourdough bread extracts indicating that sourdough-derived acetylcholine is acting via mACHR .

[0099] Furthermore to clarify whether sourdough-derived ACH acts through the activation of neurons that consequently stimulate muscle cells, muscle preparations were pre-treated with tetrodotoxin (TTX). TTX blocks action potential generated by neurons that abrogates downstream signalling. TTX pre-treatment had no significant effect on muscle contraction induced by acetylcholine and extracts suggesting that both directly activate mACHR on muscle cells (FIG. 3). Motility is one of the crucial functions of the GI tract. Agonists and antagonists of serotonin (5-hydroxytryptamine) receptors are a common treatment option for modulating motility and consequently bowel movements of IBS patients (Camilleri, M. and V. Andresen, Current and novel therapeutic options for irritable bowel syndrome management. Dig Liver Dis, 2009. 41(12): p. 854-62). These observations show that external application of ACH at local sites could also be utilised to modulate ENS-mediated motility.

2.3) Sourdough-Derived Acetylcholine Stimulates Secretion by the Intestinal Mucosa from the Luminal Side

[0100] Acetylcholine (ACH), released by the enteric neurons to the serosal side of the intestinal wall, stimulates secretion of chloride ions by the mucosa subsequently driving the passive transport of water into the lumen. This effect is transient due to rapid degradation of acetylcholine by acetylcholine esterase. The effect of sourdough-derived ACH on the intestinal secretory function was tested in guinea pig colon. Extracts of sourdough, sourdough bread, analog bread as well as (pure) ACH were applied on either luminal (mucosal) or serosal side of intestinal mucosa/submucosa preparations from guinea pig distal colon. The experiment was performed in Ussing chamber and the change in short-circuit current (I.sub.SC) was measured. In this system, passive flow of ions across a tissue or epithelial cell layer is eliminated by balancing electrical, osmotic, hydrostatic and chemical gradients across the preparation, such that only active ion transport is measured. In the Ussing chamber, electrodes are placed close to each side of the tissue to allow detection of the spontaneous potential difference (PD) across the epithelium, generated as a consequence of active ion transport (Hirota, C. L. and McKay D. M., Cholinergic regulation of epithelial ion transport in the mammalian intestine, Br. J. Pharmacol., 2006, 149(5):p. 463-79). Surprisingly an increase in I.sub.SC could be observed when the preparations were stimulated from both mucosal and serosal side by ACH as well as ACH-containing extracts (analog bread extract had no effect) (FIG. 4), showing that acetylcholine in sourdough and sourdough bread is suitable for the stimulation.

[0101] The tissue was pre-treated with atropine and the effect of extracts on secretion measured again (FIG. 5). Atropine completely abolished the response corroborating the role of mACHR in the pro-secretory effect of sourdough extracts.

[0102] Earlier research provides evidence of the expression of ACHR in intestinal epithelial cells on the basolateral side but not on the apical side of the cell layer (Hirota, C. L. and McKay D. M., Cholinergic regulation of epithelial ion transport in the mammalian intestine, Br. J. Pharmacol., 2006, 149(5):p. 463-79). Therefore it is highly probable that ACH applied from apical side crosses the cell layer and stimulates the receptors on the basolateral side. This hypothesis was verified by the fact that when ACH is applied apically, i.e. on the mucosal or luminal side, the secretory effect was significantly inhibited when atropine was applied to the serosal but not to the mucosal side (FIG. 6). Atropine applied on the serosal side blocked all mACHR on basolateral side abrogating ACH activity. However, when atropine is applied on the mucosal side this results into smaller amounts reaching basolateral side and thus only partially inhibiting ACH activity. The observation that atropine can cross epithelial layer and block basolateral mACHR was confirmed by the fact that when ACH was applied serosally and atropine mucosally secretion was inhibited (data not shown).

[0103] These observations show that external application of ACH at local sites could also be utilised to modulate ENS-mediated fluid secretion. This is especially important, since fluid secretion into the intestine is hypothesized to provide the ideal environment for enzymatic digestion and to facilitate the passage of stool through the intestinal tract. Furthermore recent studies suggest that acute and locally targeted water secretion serves as a protective measure against epithelial damage at points of particular mechanical stress (Barrett, K. E. and S. J. Keely, Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects. Annu Rev Physiol, 2000. 62: p. 535-72 and Sidhu, M. and H. J. Cooke, Role for 5-HT and ACh in submucosal reflexes mediating colonic secretion. Am J Physiol, 1995. 269(3 Pt 1): p. G346-51).

2.4) LC-MS/MS Analysis Revealed Presence of Acetylcholine in Growth Media of Sourdough Lactic Acid Bacteria

[0104] Seven strains of L. sanfranciscensis (DSM 23090-DSM 23201) and L. rossiae (DSM 26024) isolated from sourdough, L. plantarum FUA 3038 and L. brevis 3113 isolated from another sourdoughs, and L. paracasei (VSL#3) were grown for 24 hours in MRS media. The growth media was collected, filtered and analyzed using LC-MS/MS.

[0105] PCA analysis shows significant difference in the metabolites profiles of all sourdough isolated bacteria compared to L. paracasei. The separation is due in major part to the impact of acetylcholine present in sourdough bacteria growth media but not in L. paracasei media (FIG. 7). The highest ACH producer over 24 hours is L. brevis 3113, which additionally has the highest growth rate (FIG. 8A). However when the concentration is adjusted to a bacterial number, e.g. 10.sup.6/ml, in the broth, the most ACH per bacterial cell is produced by L. sanfranciscensis strains DSM 23090 and DSM 23093 (FIG. 8B).

2.5) Sourdough Lactic Acid Bacteria Inhibit Secretion of Chemokine IP-10 by TNF-Activated Intestinal Epithelial Cells

[0106] Lactocepin PrtP, a serine protease expressed in L. paracasei (VSL#3), selectively degrades pro-inflammatory chemokine interferone inducible protein 10 (IP-10). To investigate if PrtP is also present in the Lactobacilli of the present invention, the total bacterial DNA was isolated from eight sourdough strains: L. sanfranciscensis (DSM 23090, DSM 23091, DSM 23092, DSM 23093, DSM 23174, DSM 23200, DSM 23201) and L. rossiae (DSM 26024) as well as L. paracasei as positive control. DNA was amplified using Lactocepin PrtP specific primers and visualized on agarose gel. No detectable amounts of lactocepin PrtP gene were present in sourdough-isolated lactic acid bacteria. The eight sourdough lactic acid bacteria strains and L. paracasei were tested for their effects on the secretion of pro-inflammatory chemokine IP-10 by unstimulated and TNF-activated Mode-K cells. Interestingly, both conditioned media (FIG. 9) and fixed lactic acid bacteria (FIG. 10) demonstrated IP-10 inhibitory activity despite the fact that no Lactocepin PrtP gene was detected. This suggests that there are both secreted and cell surface-bound factors produced by sourdough lactic acid bacteria suitable to inhibit secretion of pro-inflammatory chemokine IP-10. This result provide a basis for potential treatment of IBD and relief of their symptoms, since IP-10 has been implicated in re-enforcing intestinal inflammation in IBD patients.