Probiotic composition comprising <i>Lactobacillus rhamnosus </i>and <i>Lactobacillus paracasei </i>and methods thereof

Abstract

The present invention relates to a composition comprising a given Lactobacillus rhamnosus strain and a given Lactobacillus paracasei strain wherein the ratio of the given Lactobacillus rhamnosus strain to the given Lactobacillus paracasei strain (LRα:LPα) is higher or equal to 8:1 and to methods thereof.

Claims

1. A composition comprising: a Lactobacillus rhamnosus CNCM I-3690 strain and a Lactobacillus paracasei CNCM I-3689 strain wherein the ratio of the Lactobacillus rhamnosus CNCM I-3690 strain to the Lactobacillus paracasei CNCM I-3689 strain (LR.sub.α:LP.sub.α) is between 9:1 and 15:1.

2. The composition according to claim 1 wherein the composition comprises at least 10.sup.6 Colony Forming Unit per ml (CFU/ml) of said given Lactobacillus rhamnosus strain.

3. The composition according to claim 1, wherein the composition comprises at least one additional Lactobacillus paracasei strain.

4. The composition according to claim 3, wherein the additional Lactobacillus paracasei strain is Lactobacillus paracasei CNCM I-1518.

5. The composition according to claim 1, wherein the composition is a food composition.

6. The composition according to claim 1, wherein the composition is a dairy product.

7. A method for improving the intestinal epithelial barrier integrity comprising administering to a subject the composition according to claim 1.

8. A method for treating a gastrointestinal disorder, type 1 diabetes or obesity comprising administering to a subject the composition according to claim 1.

9. The method according to claim 8, wherein the gastrointestinal disorder is an inflammatory bowel disease (IBD) and/or irritable bowel syndrome (IBS).

10. A process for preparing a composition comprising: adding to a medium a Lactobacillus rhamnosus CNCM I-3690 strain and a Lactobacillus paracasei CNCM I-3689 strain to obtain a composition LR.sub.α:LP.sub.α ratio between 9:1 and 15:1 or inoculating a fermentation medium with the Lactobacillus rhamnosus CNCM I-3690 strain and the Lactobacillus paracasei CNCM I-3689 strain and fermenting the inoculated medium by incubating it at a temperature suitable for growth of said strains, until the LR.sub.α:LP.sub.α ratio is between 9:1 and 15:1.

Description

FIGURES

(1) FIG. 1 shows the effect of the administration of the following compositions on the gut barrier integrity protection in the DNBS-induced low-grade inflammation model of mice. The legend is as follow: vehicule+PBS: mice not challenged with DNBS and not treated with probiotic strains DNBS+PBS: mice challenged with DNBS and not treated with strains DNBS+CNCM-13690: mice challenged with DNBS and treated with L. rhamnosus (10.sup.9 CFU/ml) DNBS+Mix 1:1: mice challenged with DNBS and treated with the same concentration (10.sup.9 CFU/ml) of L. rhamnosus and L. paracasei DNBS+Mix 1:10: mice challenged with DNBS and treated with the ratio of L. rhamnosus:L. paracasei 10:1 (10.sup.9 CFU/ml: 10.sup.8 CFU/ml) DNBS+CNCM-13689: mice challenged with DNBS and treated with L. paracasei (10.sup.9 CFU/ml)

EXAMPLES

(2) Material and Methods:

(3) Bacterial Growth Conditions and Animals

(4) Lactobacillus rhamnosus CNCM I-3690 and Lactobacillus paracasei CNCM I-3689 were grown in MRS medium (Difco, USA) under anaerobic conditions at 37° C.

(5) Male C57BL/six mice (6-8 weeks old; Janvier, Le Genest Saint Isle, France) were maintained at the animal care facilities of the National Institute of Agricultural Research (IERP, INRA, Jouy-en-Josas, France) under specific pathogen-free (SPF) conditions. Mice were housed under standard conditions for a minimum of 1 week before experimentation. All experiments were performed in accordance with European Community rules for animal care and were approved by the relevant local committee (Comethea) (Protocol number 02550.0).

(6) Experimental Design

(7) Inflammation was induced as previously described (Laval et al., 2015). Briefly, mice where challenged under anaesthesia with a first intra-rectal dose of 100 mg/kg of dinitro-benzenesulfonicacid (DNBS) solution (ICN, Biomedical Inc.) in 30% ethanol (EtOH). Control mice (without colitis) received only 30% EtOH.

(8) Thirteen days after the first DNBS injection, 10.sup.9 CFU of viable Lactobacillus rhamnosus CNCM I-3690 alone, 10.sup.9 CFU of viable Lactobacillus rhamnosus CNCM I-3690 and 10.sup.9 CFU of viable Lactobacillus paracasei CNCM I-3689 or 10.sup.9 CFU of viable Lactobacillus rhamnosus CNCM I-3690 and 10.sup.8 CFU of viable Lactobacillus paracasei CNCM I-3689 in 200 μl of PBS or PBS alone were administered intra-gastrically, daily for 10 days (gavage period).

(9) Finally, 21 days after the first challenge, the mice were challenged again with a second administration of 50 mg/kg of DNBS or EtOH.

(10) Weight loss was monitored during 3 days following the second DNBS injection to assess possible clinical signs of distress. To confirm the absence of over inflammation, colonic macroscopic and histological scores as well as colonic myeloperoxidase (MPO) activity (a marker of the degree of infiltration by polymorpho-nuclear neutrophils) and serum lipocalin-2 levels (an early inflammation marker) were determined as previously described (Shashidharamurthy et al., 2013; Martin et al., 2014; Laval et al., 2015).

(11) Intestinal Permeability In Vivo

(12) Permeability in vivo was assessed using fluorescein iso-thiocyanate-conjugated dextran (FITC-dextran 3000-5000 Da, Sigma-Aldrich) tracer as previously described (Tambuwala et al., 2010). Briefly, at the endpoint 0.6 mg/g body weight of FITC-dextran dissolved in PBS was administered to mice by oral gavage. To measure the presence of FITC-dextran in blood, 3.5 h after the gavage blood samples were recovered from the retro-orbital venous plexus and kept in dark at 4.0 until analysis. Mice were housed under standard conditions during this period with un-limited access to water and food. Serum was separated by centrifugation and plasma FITC levels were determined using a fluorescence micro-plate reader (excitation 485 nm and emission 530 nm; Tecan, Lyon, France).

(13) Statistical Analysis

(14) GraphPad software (GraphPad Sofware, LaJolla, Calif., USA) was used for statistical analysis. Results are presented as bar graphs or dot plots with means±SEM. Comparisons involved the non-parametric Kruskal-Wallistest followed by a Dunn's Multiple Comparison test. A p value of less than 0.05 was considered significant.

(15) Results:

(16) L. rhamnosus and L. paracasei strains were both tested in a chronic murine model of low-grade inflammation induced by two intrarectal administrations of dinitrobenzene sulfonic acid (DNBS) separated by a recovery period. This model is characterized by an increase of colon barrier permeability.

(17) As shown in FIG. 1, L. rhamnosus CNCM-I-3690, but not L. paracasei CNCM-I-3689 is able to restore the integrity of the intestinal barrier. When the L. rhamnosus CNCM-1-3690 and the L. paracasei CNCM-I-3689 strains were mixed in the ratio 1:1, L. rhamnosus CNCM-I-3690 lost its capacity to protect the barrier but when in the mixture is at a ratio 10:1 where L. paracasei CNCM-I-3689 was present 10 times less than L. rhamnosus CNCM-I-3690, the L. rhamnosus CNCM-I-3690 beneficial effect is still present.