Compositions and methods for increasing or maintaining <i>Faecalibacterium prausnitzii </i>populations

Abstract

The present invention relates to the use of at least one lactic acid bacterium, or a composition comprising thereof or conditioned thereby, for increasing or maintaining a Faecalibacterium prausnitzii population.

Claims

1. A fermented food product comprising milk and a Streptococcus thermophilus strain deposited at the CNCM under reference number I-3862, wherein the milk is heat treated prior to preparing the fermented food product.

2. The fermented food product of claim 1, wherein the food product is a dairy product.

3. The fermented food product of claim 2, wherein the dairy product is yogurt, set yogurt, stirred yogurt, pourable yogurt, a yogurt drink, frozen yogurt, kefir, buttermilk, quark, sour cream, or cheese.

4. The fermented food product of claim 1, wherein the food product is a drinkable composition.

5. The fermented food product of claim 4, wherein the food product is a yogurt drink or kefir.

6. The fermented food product of claim 1, wherein the food product is spoonable.

7. The fermented food product of claim 6, wherein the food product is set or stirred yogurt.

8. The fermented food product of claim 1, wherein the milk is animal milk.

9. The fermented food product of claim 1, wherein the milk is vegetal milk.

10. The fermented food product of claim 1, wherein the milk is pasteurized prior to preparing the fermented food product.

11. A method of preparing a fermented food product of claim 1, comprising culturing heat treated milk and a Streptococcus thermophilus strain deposited at the CNCM under reference number I-3862.

12. The method of claim 11, wherein the culturing is performed from about 36 C. to about 44 C.

13. The method of claim 12, wherein the culturing is performed from about 37 C. to about 40 C.

14. The method of claim 11, wherein the culturing reduces the pH of the product to a pH of equal to or lower than 5.

15. The method of claim 14, wherein the culturing reduces the pH of the product to between about 3 and 4.5.

16. The method of claim 11, wherein the food product is a dairy product.

17. The method of claim 16, further comprising culturing Lactobacillus delbruckei subsp. bulgaricus.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 represents the optical density (OD) at 600 nm (vertical axis) of a culture of Faecalibacterium prausnitzii without (black bar, control) or with the addition of a culture supernatant from Lactococcus lactis subsp. lactis (dotted bar) or from Streptococcus thermophilus (hatched bar), after a culture time of 40 hours and 65 hours (horizontal axis).

(2) FIG. 2 represents the concentration of butyrate (in mmol/L, vertical axis) of a culture of Faecalibacterium prausnitzii without (black bar, control) or with the addition of a culture supernatant from Lactococcus lactis subsp. lactis (dotted bar) or from Streptococcus thermophilus (hatched bar), after a culture time of 40 hours (horizontal axis).

EXAMPLE

(3) Material and Methods

(4) Bacterial Strains, Media and Supernatants Production

(5) The bacterial strains used in this study are listed in Table 1. Purity of all strains was regularly assessed by microscopy observation after cell staining. Faecalibacterium prausnitzii was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (DSM 17677). They were all grown in Brain-heart infusion medium (Becton Dickinson) supplemented with 0.5% yeast extract (Becton Dickinson) and 5 mg/L hemin chloride (Calbiochem), supplemented with cellobiose (1 g/L; Sigma-Aldrich), maltose (1 g/L; Sigma-Aldrich), and cysteine (0.5 g/L; Sigma-Aldrich) in an anaerobic chamber, with the following gas mixture, 80% N.sub.2 10% CO.sub.2 10% H.sub.2. When the culture was grown, glycerol solution was added (16 g/L; Sigma-Aldrich). 2 mL aliquots stock were prepared and kept at 20 C. For each experiment, a new aliquot was used. For probioticsFaecalibacterium prausnitzii interaction tests, all strains were grown in YCFA medium (pH 6) that was optimized in order to allow growth of all bacterial strains (Table 2). This medium consisted of glucose (20 g/L, Sigma-Aldrich), tryptone (10 g/L, Becton Dickinson), yeast extract (5 g/L, Becton Dickinson), sodium acetate (5 g/L, Sigma-Aldrich), monohydrate lactose (5 g/L, Sigma-Aldrich), sodium bicarbonate (4 g/L, Sigma-Aldrich), cellobiose (2 g/L, Sigma-Aldrich), sodium chloride (0.9 g/L, Sigma-Aldrich), ammonium sulfate (0.9 g/L, Sigma-Aldrich), cysteine (0.5 g/L, Sigma-Aldrich), dibasic potassium phosphate (0.45 g/L, Sigma-Aldrich), magnesium sulfate (0.09 g/L, Sigma-Aldrich), calcium chloride (0.09 g/L, Sigma-Aldrich), hemin chloride (0.01 g/L Sigma-Aldrich), resazurine sodium salt (0.001 g/L, Alfa Aesar). This medium was reduced in anaerobic chamber and autoclaved before use.

(6) TABLE-US-00001 TABLE 1 List of strains used in this study Name Usual medium Lactobacillus lactis subsp lactis CNCM I-1631 M17 with lactose Streptococcus thermophilus CNCM I-3862 M17 with lactose F. prausnitzii DSM 17 677 BHI

(7) TABLE-US-00002 TABLE 2 Composition of YCFA optimized for this study Component g/L (1 L) Yeast Extract 5 Celllobiose 2 Tryptone 10 Dibasic Potassium phosphate 0.45 (450 mg) Sodium chloride 0.9 (900 mg) Ammonium sulfate 0.9 (900 mg) Magnesium sulfate 0.09 (90 mg) Calcium chloride 0.09 (90 mg) Resazurine sodium salt 0.01% 10 mL Sodium bicarbonate 4% 100 mL Sodium acetate 5% 100 mL Hemin chloride 1% + 0.4 mL NaOH 5M 1 mL Monohydrate lactose 5% 100 mL Glucose10% 200 mL Cysteine 5% 10 mL
Bacterial Supernatants Preparation

(8) Bacterial supernatants were produced as follows: Strains were grown in 2 mL of usual growth media and atmosphere (Table 1) at 37 C. After 24 h-growth, the cells were inoculated at 1% in 10 mL of usual growth media and atmosphere (Table 1) at 37 C. Lastly, these cultures were inoculated at 1% in 50 mL of YCFA, previously reduced as described above, at 37 C. for 24 h. Cultures were pelleted at 7500 g for 10 min, supernatants were filtered at 0.2 m and directly frozen at 20 degrees.

(9) Tests of Stimulation of Faecalibacterium prausnitzii DSM 17677 by Lactic Acid Bacteria

(10) Before use, the bacterial supernatants were allowed to reduce in the anaerobic chamber for 2 hours. Then, 4 ml of reduced supernatants from the bacterial strains were added to 50 mL of YCFA. As a control, 4 ml of sterile YCFA were added to 50 ml of YCFA. Subsequently, a 48 hours culture of F. praunistzii was inoculated at 1% into the YCFA medium. Growth was monitored over 72 hours. OD and pH were measured at regular interval during the growth (hours). Samples were collected and stored for DNA extraction (see below). Additional samples were collected for further analysis (DNA, SCFA analysis, metabolomics and transcriptomics).

(11) Metabolites Analysis

(12) 2 mL of fresh culture were collected and centrifuged at 10 000 g for 15 minutes. Supernatants were filtered (0.2 m) and stored at 20 C. until analysis.

(13) Samples for short-chain fatty acid (SCFA) and branched chain fatty acids (BCFA) analysis were collected after 16, 22 and 40 h. They were measured quantitatively. Short-chain fatty acid (SCFA) Acetate, propionate, butyrate, valerate, caproate and branched chain fatty acids (BCFA) isobutyrate, isovalerate and isocaproate as well as lactate were measured at Prodigest as follow: SCFA were extracted from the samples with diethyl ether, after the addition of 2-methyl hexanoic acid as an internal standard. Extracts were analysed using a GC-2014 gas chromatograph (Shimadzu, S-Hertogenbosch, the Netherlands), equipped with a capillary fatty acid-free EC-1000 Econo-Cap column (dimensions: 25 mm 0.53 mm, film thickness 1.2 mM; Alltech, Laarne, Belgium), a flame ionization detector and a split injector. The injection volume was 1 mL and the temperature profile was set from 110 to 160 C., with a temperature increase of 6 C./min. The carrier gas was nitrogen and the temperature of the injector and detector were 100 and 220 C., respectively. The production of unbranched and branched SCFA was calculated by summing the molar concentrations of acetate, propionate, butyrate, valerate and caproate, and summing isobutyrate, isovalerate and isocaproate molar concentrations, respectively. The total SCFA production was defined as the sum of unbranched and branched SCFA. Lactate was measured using a D-lactate/L-lactate kit (R-Biopharm, Mannheim, Germany), according to the manufacturer's protocols.

(14) Results

(15) The results of the stimulation of F. prausnitzii growth by the culture supernatants of S. thermophilus and Lactococcus lactis subsp. lactis are shown in FIG. 1. It can be seen that both supernatants yield an increase of the growth of F. prausnitzii after 40 hours and 65 hours of culture.

(16) The results of the stimulation of butyrate production by F. prausnitzii growth by the culture supernatants of S. thermophilus and Lactococcus lactis subsp. lactis are shown in FIG. 2. After 40 hours of incubation, there was a 15% increase of butyrate production in presence of S. thermophilus and L. lactis lactis supernatant compared to the single culture (19.2 mmol/L3.6 mmol/L, 19.0 mmol/L0.8 mmol/L and 16.7 mmol/L1 mmol/L respectively).