Bifidobacterium longum and functional GI disorders

Abstract

The present invention generally relates to the field of probiotic bacteria. In particular it relates to methods for treating functional GI disorders comprising administering Bifidobacterium longum, such as Bifidobacterium longum ATCC BAA-999.

Claims

1. A method for the treatment of irritable bowel syndrome and anxiety, the method comprising: administering to a patient suffering from the same a composition comprising Bifidobacterium longum ATCC BAA-999, wherein the composition is selected from the group consisting of a dietary supplement, a nutraceutical, a drink, and a medical composition.

2. The method of claim 1, wherein the composition comprises at least one other type of food grade bacteria.

3. The method of claim 1, wherein the composition comprises at least one prebiotic.

4. The method of claim 3, wherein the prebiotic is selected from the group consisting of oligosaccharides, dietary fibers, and mixtures thereof.

5. The method of claim 1, wherein at least 5% of Bifidobacterium longum ATCC BAA-999 cells are viable in the composition.

6. The method of claim 1, wherein at least 80% of Bifidobacterium longum ATCC BAA-999 cells are non-replicating in the composition.

7. The method of claim 1, wherein the composition comprises between 10.sup.4 and 10.sup.10 cells of Bifidobacterium longum ATCC BAA-999 per g dry weight of the composition.

8. The method of claim 1, wherein the composition comprises between 10.sup.2 and 10.sup.8 cells of Bifidobacterium longum ATCC BAA-999 per g dry weight of the composition.

9. The method of claim 1, wherein the composition comprises a protein source comprising hydrolyzed whey protein.

10. The method of claim 1, wherein the composition is in powder form having a water activity of less than 0.2.

11. The method of claim 2, wherein the at least one other type of food grade bacteria is selected from the group consisting of lactic acid bacteria, propionibacteria and mixtures thereof.

Description

(1) Further features and advantages of the present invention result from the following Examples and Figures:

(2) FIG. 1 shows the result of a dark box/bright box test: total time spent in bright box, latency to re-enter bright box and latency to step down in mice infected with Trichuris muris (Tm). Tm-medium and Tm-B. longum are Tm infected mice treated with fresh medium (negative control) and Bifidobacterium longum ATCC BAA-999, respectively; a Tm group treated with L. rhamnosus (L. rh) strain is shown for comparison.

(3) FIG. 2 shows the results of the in situ hybridization in the brain hippocampal region of mice infected with a Trichuris muris (Tm). Tm-B. longum is Tm infected mice treated with Bifidobacterium longum ATCC BAA-999; a Tm group of mice treated with L. rhamnosus strain is shown for comparison. Quantification of 35S signals was performed by autoradiography and image analysis (right upper panel).

(4) FIG. 3 shows the results obtained on colonic inflammation measured by myeloperoxidase activity assay (left panel) and mononuclear cell infiltration (right panel) in mice infected with Trichuris muris (Tm). Tm-medium and Tm-B. longum are Tm infected mice treated with fresh medium (negative control) and Bifidobacterium longum ATCC BAA-999; a Tm group of mice treated with L. rhamnosus strain is shown for comparison.

EXAMPLES

(5) Material and Methods

(6) Bacterial Culture Conditions:

(7) Probiotics (Bifidobacterium longum ATCC BAA-999 and L. rhamnosus NCC4007 for comparison) were grown under anaerobic conditions in Man-Rogosa-Sharpe (MRS, BioMerieux) broth (bifidobacteria with 0.5% cysteine). After 24 h at 37? C., the number of bacteria was estimated by measuring the optical density at 600 nm (1 OD600=10.sup.8 bacteria/mL). Bacterial cells were pelleted by centrifugation at 5000?g for 15 min at 4? C. and further resuspended at a concentration of 10.sup.10/mL in their spent culture medium. Aliquots of 1 mL were kept frozen until use.

(8) Animals:

(9) Male BALB/c or AKR mice (Harlan, Canada) were purchases at the age of 6-8 wks and housed in a conventional specific pathogen free Unit at McMaster University Central Animal Facility. All experiments were conducted with approval from the McMaster University Animal Care Committee.

(10) Design:

(11) Chronic T. muris Infection:

(12) Male AKR mice were gavaged with T. muris (300 eggs/mouse) (n=26) or with placebo (n=9). Infected mice were then gavaged daily with L. rhamnosus, B. longum or fresh MRS from day 30 for 10 days. Uninfected mice were gavaged with fresh MRS on a daily basis from day 30 to day 40. At the end of probiotic or placebo administration, the mice underwent dark box/bright box and step-down tests. The mice were sacrificed thereafter and tissue samples were obtained. Colon samples were fixed in formalin for histological analysis or were snap frozen for MPO determination. Brains were snap frozen in liquid nitrogen and stored for in situ hybridization.

(13) Behavior Testing:

(14) Dark Box/Bright Box:

(15) Anxiety behavior was assessed individually in mice using dark box/bright box as described in the literature. Briefly, each mouse was placed in the center of illuminated bright box (30?30 cm) connected by an opening (10?3 cm) to smaller dark box (30?15 cm). The locomotor behavior of each mouse in the bright box was recorded for 10 min by a digital video camera and stored in a computer for an off-line analysis. Several parameters were assessed by a blinded observer including total time spent in bright box, latency to re-enter bright box (time spent in dark box after first entry), and number of crossovers (number of crossing from dark box to bright box).

(16) Step-Down Test:

(17) Anxiety behavior was assessed using step down test as described in the literature. Briefly, each mouse was placed in the center of an elevated platform (7.5 cm in diameter, 3 cm high) positioned in the middle of a black floor. Latency to step down from the pedestal was measured by a stop-watch; maximum duration of the test was 5 min.

(18) Histology:

(19) Colon samples were fixed in 10% formalin and then stained with hematoxylin-eosin. The slides were examined under light microscopy to grade for acute and chronic inflammatory infiltrate.

(20) Myeloperoxidase Activity Assay:

(21) In order to assess acute intestinal inflammation, myeloperoxidase activity (MPO) assay was performed on frozen tissues as described previously. MPO activity is expressed in units per mg of tissue, where one unit of MPO is defined as a quantity of the enzyme able to convert 1 ?mol of hydrogen peroxide to water in 1 minute at room temperature.

(22) In Situ Hybridization in the CNS:

(23) Levels of BDNF in hippocampus and CRH in hypothalamus (paraventricular nucleus) were assessed by in situ hybridizations using 35S-labeled RNA probes on frozen brain sections as described previously (Whitfield et al., 1990; Foster et al., 2002). Briefly, brains were removed and rapidly frozen by immersion in 2-methylbutane at ?60? C., and stored at ?70? C. Cryostat-cut 12-pin-thick coronal sections were thaw-mounted onto gelatine-coated slides, dried, and stored at ?35? C. Tissue sections were fixed with 4% formaldehyde, acetylated with 0.25% acetic anhydride in 0.1M triethanolamine-HCl, pH 8.0, dehydrated, and delipidated with chloroform. Antisense BDNF ribonucleotide probe (gift of Dr. J. Lauterborn and Dr. C. Gall, Univeristy of California Irvine) and anti-sense CRH ribonucleotide probe (gift of Dr. James Herman, University of Cincinnati) was transcribed from linearized plasmid using the Riboprobe System (Promega Biotech, Burlington, ON) with ?-35S-UTP (specific activity>1000 Ci/mmol; Perkin-Elmer, Boston, Mass.) and T3 and T7 polymerases respectively. Radiolabelled probes were diluted in a hybridization buffer (0.6M NaCl, 10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 10% Dextran sulfate, 0.01% sheared salmon sperm DNA, 0.05% total yeast RNA, type XI, 0.01% yeast tRNA, 1?Denhardt's solution) and applied to brain sections (approximately 500,000 CPM/section). Slides were incubated overnight at 55? C. in a humidified chamber. To reduce nonspecific binding of the probe, slides were washed in 20 ?g/ml RNase solution for 30 min at room temperature, followed by 1 h each in 2?SSC at 50? C., 0.2?SSC at 55? and 60? C. Slides were dehydrated and air-dried for autoradiography. Slides and 14 C plastic standards were placed in x ray cassettes, apposed to film (BioMax MR; Eastman Kodak, Rochester, N.Y.) for 5 days and developed (Kodak Medical X-Ray Processor). Autoradiographic film images of brain sections and standards were digitized with a solid-state camera with a 60 mm camera lens manufactured by Nikon and using QCapture software (Qicam; Quorum Technologies Inc., Guelph, ON) and a Macintosh computer-based image analysis system with Image software. Light transmittance through the film was measured by outlining the structure on the monitor. For BDNF mRNA, transmittance was converted to radioactivity levels using the Rodbard curve applied to the standards. For CRH mRNA, the density slice feature was utilized to measure both the light transmittance and the area of mRNA signal. The calculated DPM were then multiplied by area to produce a measurement of integrated density. Illustrations were made directly from the captured images.

(24) Statistical Analysis:

(25) Data are presented as mean?standard deviation or medians with interquartile ranges as appropriate. Data was analysed using either two-way ANOVA, test or non-paired t-test as appropriate. A p value of <0.05 was considered as statistically significant.

(26) Results:

(27) Mice chronically infected with the parasite Trichuris muris showed an increase in anxiety-like behaviour in two behavioural tests: 1) In the dark box/bright box test, infected animals showed a decrease in the time spent in the bright box and an increase of the latency to re-enter the bright box; 2) In the step down test, the infection increased the latency to step down from the pedestal (FIG. 1). Treatment with Bifidobacterium longum ATCC BAA-999 but not with L. rhamnosus NCC4007 induced a reduction of anxiety-like behaviour towards normality. The effect on behaviour was correlated with a normalization of Trichuris muris-mediated decrease in BDNF levels in the hippocampus by B. longum only (FIG. 2). In contrast, treatment with B. longum as well as with L. rhamnosus resulted in a reduction of myeloperoxidase activity and mononuclear infiltration previously induced by Trichuris muris infection (FIG. 3) indicating that the normalization of behaviour was independent of the anti-inflammatory effect of the bacteria.