Strain of Bifidobacterium animalis ssp. animalis

Abstract

The invention relates to a novel strain of Bifidobacterium animals ssp. animalis, which is able, inter alia, to improve the function of the enteric nervous system, and to compositions said novel strain.

Claims

1. A composition comprising the Bifidobacterium animalis subspecies animalis strain deposited on 6 Mar. 2012 under number I-4602 with the CNCM (Collection Nationale de Cultures de Microorganismes) (strain Bifidobacterium animalis subspecies animalis CNCM I-4602), wherein the composition is a fermented dairy food product.

2. The composition according to claim 1, wherein the composition comprises at least 10.sup.5 cfu, per g dry weight, of the strain Bifidobacterium animalis ssp. animalis CNCM I-4602.

3. The composition according to claim 1, wherein the composition comprises at least 10.sup.6 cfu, per g dry weight, of the strain Bifidobacterium animalis ssp. animalis CNCM I-4602.

4. A method of preventing and/or reducing gastro-intestinal discomfort or gastro-intestinal disorders, comprising administering the strain Bifidobacterium animalis subspecies animalis CNCM I-4602 to a subject in need thereof.

5. The method of claim 4, wherein the strain is administered to a subject having a disorder selected from the group consisting of an intestinal motility disorder, an intestinal permeability disorder, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), constipation, and intestinal infections.

6. A method of preventing and/or reducing gastro-intestinal discomfort or gastro-intestinal disorders, comprising administering a composition according to claim 1 to a subject in need thereof.

7. The method of claim 6, wherein the composition is administered to a subject having a disorder selected from the group consisting of an intestinal motility disorder, an intestinal permeability disorder, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), constipation, and intestinal infections.

8. A method of increasing vaso-active intestinal peptide (VIP) levels of the enteric nervous system in a subject, comprising administering the strain Bifidobacterium animalis subspecies animalis CNCM I-4602 to a subject in need thereof.

9. A method of increasing vaso-active intestinal peptide (VIP) levels of the enteric nervous system in a subject, comprising administering a composition according to claim 1 to a subject in need thereof.

10. A method of improving gastro-intestinal motility, improving intestinal peristalsis and/or decreasing intestinal permeability, comprising administering the strain Bifidobacterium animalis subspecies animalis CNCM I-4602 to a subject in need thereof.

11. A method of improving gastro-intestinal motility, improving intestinal peristalsis and/or decreasing intestinal permeability, comprising administering a composition according to claim 1 to a subject in need thereof.

Description

(1) Details or advantages of the present invention can be found in the non limiting example below.

EXAMPLE 1: EFFECT OF LACTIC ACID BACTERIA ON NEURONAL VIP PRODUCTION IN A COCULTURE MODEL OF ENS PRIMARY CULTURES AND T84 CELLS

(2) Differential response of primary enteric neurons on VIP production following interaction of 107 probiotic strains, including lactic acid bacteria and in particular Bifidobacteria, was studied.

(3) Bacterial Strains and Growth Conditions

(4) Lactobacilli were grown in Man, Rogosa and Sharpe (MRS) medium (Biokar Diagnostics, Beauvais, France), at 37 C. in aerobic conditions for 24 hours.

(5) Bifidobacteria were cultured in a defined medium which consisted of a tryptone peptone (Becton Dickinson, USA) basis supplemented with glucose (Sigma, France), yeast extract (Becton Dickinson, USA), and hemin (Calbiochem, France) and grown at 37 C. in anaerobic conditions for 24 hours. B. animalis ssp. lactis strains were cultured in MRS medium (Biokar Diagnostics), supplemented with cysteine (Sigma, France), and grown at 37 C. in anaerobic conditions for 24 hours.

(6) Obtaining a Co-Culture Model of ENS Primary Cultures and T84 Cells

(7) A co-culture model, adapted from a model developed by Moriez et al. (Biochem. Biophys. Res. Commun., 382(3):577-82, 2009), with enteric neuron cells cultured with intestinal epithelial T84 cells was used for the screening of bacteria.

(8) Primary cultures of rat ENS were obtained as previously described (Chevalier et al., J. Physiol., 586(7):1963-75, 2008).

(9) T84 cell line was cultured in DMEM-F12 (1:1, GIBCO) supplemented with 10% heat-inactivated FBS and 50 IU/ml penicillin and 50 g/ml streptomycin. Cells were seeded in 12-well Transwell filters (Corning, N.Y. USA) at a density of 210.sup.5 cells/insert and cultured to obtain confluence. 24 hours before setting up the co-culture, the culture medium of T84 cells was replaced by primary ENS medium.

(10) One day after epithelial cells arrived to confluence, Transwell filters were transferred in the 12-well plates seeded at the bottom with enteric nervous cells. Epithelial and neuronal cells were co-cultured in the medium for epithelial cells.

(11) Bacterial cultures were washed in PBS and re-suspended in a medium Dulbecco's Modified Eagle Medium (DMEM):Nutrient Mixture F-12 (Ham's) (1:1), containing 1% of N-2 supplement (Life Technologies, Cergy-Pontoise, France). Each strain of bacterium was tested at least in triplicate in order to perform analysis of variance. The strains were added in the filter compartment at a MOI of 40 bacteria/epithelial cell. As a control, no bacteria were added. After 8 hours of co-incubation, the filter compartment containing epithelial cells and bacteria was removed and primary ENS culture was maintained for 22 hours in the incubator (95% air, 5% CO.sub.2) at 37 C. In the control wells, neuronal cells were stimulated with 40 mM KCl when VIP measurements were performed.

(12) VIP Measurements

(13) Then, the ENS cells were collected from the 12-well plates and used to prepare protein extracts for quantification of vaso-active intestinal peptide (VIP). For determination of intracellular VIP, the intracellular proteins were extracted using RIPA lysis buffer (Millipore, Billerica, Mass., USA) containing a protease inhibitor cocktail (Roche Diagnostics, Meylan, France). VIP was quantified using a purchased enzyme immunoassay analysis (EIA) kit (Peninsula laboratories, Bachem, SA).

(14) Statistical Method for ENS Model

(15) All strains VIP marker values are normalized by the negative control (T84) according to the following formula: (VIP strain strainVIP.sub.T84)/VIP.sub.T84.

(16) Strains are ranked according to their estimated normalization value coming from a mixed model with strain as fixed effect. The model gave a P-value associated to each strain estimated normalized value. If the P-value was less than 5% (<0.05), the strain effect was significant.

(17) Results

(18) Results are shown in Table 1 below.

(19) TABLE-US-00001 TABLE 1 Strain reference % VIP increase Control or and/or vs negative Bacterial species deposit number control (T84) P value KCl (positive control) 15.7% <0.0005 Bifidobacterium animalis CNCM I-4602 62.5% <0.0005 ssp. animalis Lactobacillus plantarum CNCM I-4318 59.8% <0.0005 Bifidobacterium breve CNCM I-2219 27.2% 0.018 Bifidobacterium animalis CNCM I-2494 23.5% 0.006 ssp. lactis Bifidobacterium longum 22.8% 0.033 Bifidobacterium breve CNCM I-4321 22.5% 0.008 Bifidobacterium bifidum 0.18% 0.038 Lactobacillus casei 17.4% 0.049 Lactobacillus johnsonii 19.4% 0.03 Bifidobacterium breve 20.0% 0.02 Bifidobacterium breve 21.6% 0.014 Bifidobacterium longum 23.4% 0.009 Bifidobacterium longum 24.1% 0.008 Bifidobacterium longum 25.7% 0.004 Bifidobacterium longum 33.1% 0.0003 Bifidobacterium longum W11 (LMG 25.7% 0.004 P-21586) Bifidobacterium longum 35.0% 0.0007

(20) KCl, used as positive control, increased the level of VIP in average by 15.7%.

(21) Nine strains were able to significantly increase VIP level, from 0.18 to 62.5%. Table 1 gives the results obtained for 7 of them. Among the 9 strains, B. animalis ssp. animalis CNCM I-4602 was shown to increase VIP at the highest level. There was a 62.5% increase versus the level in absence of any strain.

(22) On another hand, it was observed that 15 strains significantly (P<0.05) decreased VIP level. The results for 10 of these 15 strains are indicated in Table 1.

(23) The other strains tested had no significant (P>0.05) effect on VIP, including for instance Bifidobacterium longum NCC 2705 (CNCM I-2618), Lactobacillus rhamnosus GG (ATCC 53103), Bifidobacterium infantis UCC 3564, Bifidobacterium longum Bb536, Bifidobacterium animalis ssp. lactic Bi-07 (ATCC SD 5220), Lactobacillus johnsonii La1 (CNCM I-1225), Lactobacillus plantarum 299v (DSM 9843), and Lactobacillus reuteri SD 2112 (ATCC 55730).