Bacterial strains capable of metabolizing oxalates

Abstract

A selection of strains of lactic bacteria and bifidobacteria of human intestinal origin capable of metabolizing oxalates is described. Moreover, a food composition or supplement product or pharmaceutical composition containing said bacterial strains is also described.

Claims

1. A method for preventive and/or curative treatment in a subject of hyperoxaluria, urolithiasis, kidney failure, cardiopathies, kidney stones, diverticulosis and diverticulitis, the method comprising administering to the subject a food composition or supplement product or medical device or pharmaceutical composition comprising a bacterial composition, said bacterial composition comprising the strain L. paracasei spp. paracasei LPC 09 DSM 24243.

2. The method according to claim 1, wherein the composition further comprises at least one bacterial strain selected from the group consisting of the bacterial strain L. gasseri LGS 01 DSM 18299 and the bacterial strain L. gasseri LGS 02 DSM 18300.

3. The method according to claim 1, wherein the composition further comprises bacterial strain L. acidophilus LA02 DSM 21717 and the bacterial strain L. acidophilus LA 07 DSM 24303.

4. The method according to claim 1, wherein the composition further comprises fructo-oligosaccharides and inulin.

5. The method according to claim 1, wherein said bacterial composition consists of L. paracasei spp. paracasei LPC 09 DSM 24243, L. acidophilus LA02 DSM 21717 and L. acidophilus LA 07 DSM 24303.

6. The method according to claim 2, wherein the composition further comprises fructo-oligosaccharides and inulin.

Description

(1) Preferred embodiments of the present invention will be illustrated in the detailed description that follows.

(2) FIG. 1 shows a comparison between the chromatogram for a culture medium containing 5 mM of oxalate before (A) and after (B) SPE purification.

(3) FIG. 2 shows a chromatogram for a culture medium containing 5 mM of oxalate (positive reference).

(4) FIG. 3 shows a chromatogram for the bacterial strain B. breve BRO3 DSM 16604.

(5) FIG. 4 shows a chromatogram for the bacterial strain L. paracasei spp. paracasei LPC09 DSM 24243.

(6) FIG. 5 shows acidification curves (pH value) obtained as a function of time (T=0, 3, 6, 8 and 10 hours) when the strain L. paracasei spp. paracasei LPC 09 DSM 24243 was made to grow in a sugar-free MRS culture medium (carbon source), to which other carbon sources (fibres) were respectively added.

(7) The Applicant has developed a method capable of identifying and quantifying the oxalate degrading ability of cultures of strains belonging to the genera Lactobacillus and Bifidobacterium.

(8) The Applicant has found that the following bacterial strains have a demonstrated ability to use oxalates as an energy source:

(9) (i) L. paracasei spp. paracasei LPC 09, deposited by the company Probiotical SpA of Novara (Italy) on 23 Nov. 2010, with deposit number DSM 24243.

(10) (ii) L. gasseri LGS 01, deposited by the company Probiotical SpA of Novara (Italy) on 24 May 2006, with deposit number DSM 18299.

(11) (iii) L. crasseri LGS 02, deposited by the company Probiotical SpA of Novara (Italy) on 24 May 2006, with deposit number DSM 18300.

(12) (iv) L. acidophilus LA 07, deposited by the company Probiotical SpA of Novara (Italy) on 23 Nov. 2010, with deposit number DSM 24303.

(13) (v) L. acidophilus LA 02, deposited by the company Probiotical SpA of Novara (Italy) on 6 Aug. 2008, with deposit number DSM 21717.

(14) (vi) L. plantarum LP 01, deposited by the company Mofin Srl of Novara (Italy) with the Depositary Institution BCCM-LMG (Belgium) on 16 Oct. 2001, with deposit number LMG-P-21021,

(15) (vii) L. reuteri LRE 03, deposited by the company Probiotical SpA of Novara (Italy) on 5 Aug. 2010, with deposit number DSM 23879.

(16) (viii) L. reuteri LRE 02, deposited by the company Probiotical SpA of Novara (Italy) on 5 Aug. 2010, with deposit number DSM 23878.

(17) (ix) B. breve BR 03, deposited by the company Probiotical SpA of Novara (Italy) on 16 Jul. 2004, with deposit number DSM 16604.

(18) (x) B. longum BL 03, deposited by the company Probiotical SpA of Novara (Italy) on 20 Jul. 2004, with deposit number DSM 16603.

(19) (xi) L. rhamnosus GG, ATCC 53103, available in the ATCC public collection.

(20) (xii) L. reuteri LRE 04, deposited by the company Probiotical SpA of Novara (Italy) on 5 Aug. 2010, with deposit number DSM 23880.

(21) (xiii) L. rhamnosus LR 06, deposited by the company Probiotical SpA of Novara (Italy) on 14 Nov. 2008, with deposit number DSM 21981.

(22) (xiv) B. lactis BA 05, deposited by the company Probiotical SpA of Novara (Italy) on 15 Jun. 2006, with deposit number DSM 18352.

(23) (xv) L. casei spp. rhamnosus LR 04, deposited by the company Probiotical SpA of Novara (Italy) on 20 Jul. 2004, with deposit number DSM 16605.

(24) In a preferred embodiment, the composition comprises or, alternatively, consists of at least one strain selected from among those indicated above by (i) to (xv); preferably the strains are selected from among those indicated above by (i) to (viii).

(25) In the context of the present invention, “bacterial strain” means the live and/or dead cells and/or parts, components/derivatives and/or enzymes thereof.

(26) The selected bacterial strains belong to the genus Lactobacillus and have an ability to degrade and use oxalate as a source of energy in an amount greater than 50%.

(27) Advantageously, said ability is greater than 60%. Advantageously, said ability is greater than 70%.

(28) The selected bacterial strains belong to the species Lactobacillus paracasei. A preferred strain is L. paracasei spp. paracasei LPC 09 DSM 24243.

(29) The selected bacterial strains belong to the species Lactobacillus gasseri. Several preferred strains are selected from the group comprising or, alternatively, consisting of L. gasseri LGS 01 DSM 18299 and L. gasseri LGS 02 DSM 18300.

(30) The selected bacterial strains belong to the species Lactobacillus acidophilus. Several preferred strains are selected from the group comprising or, alternatively, consisting of L. acidophilus LA02 DSM 21717 and L. acidophilus LA 07 DSM 24303.

(31) A composition in accordance with the present invention comprises at least one bacterial strain, for use in the treatment of hyperoxaluria, urolithiasis, kidney failure, cardiopathies, kidney stones, diverticulosis and diverticulitis.

(32) The composition can be a food composition or supplement product or medical device or pharmaceutical composition.

(33) The composition for use in the treatment of hyperoxaluria, urolithiasis, kidney failure, cardiopathies, kidney stones, diverticulosis and diverticulitis comprises or, alternatively, consists of at least two strains selected from among those indicated above by (i) to (xv), preferably the strains are selected from among those indicated above by (i) to (viii).

(34) The composition for use in the treatment of hyperoxaluria, urolithiasis, kidney failure, cardiopathies, kidney stones, diverticulosis and diverticulitis comprises or, alternatively, consists of at least two strains selected from among those indicated above by (i) to (v).

(35) The composition for use in the treatment of hyperoxaluria, urolithiasis, kidney failure, cardiopathies, kidney stones, diverticulosis and diverticulitis comprises or, alternatively, consists of: (a) L. paracasei spp. paracasei LPC 09 -DSM 24243; or (b) L. paracasei spp. paracasei LPC 09 -DSM 24243 and L. gasseri LGS 01 -DSM 18299; or (c) L. paracasei spp. paracasei LPC 09 -DSM 24243 and L. gasseri LGS 02 -DSM 18300; or (d) L. paracasei spp. paracasei LPC 09 -DSM 24243, L. gasseri LGS 01 -DSM 18299 and L. gasseri LGS 02 -DSM 18300; or (e) L. paracasei spp. paracasei LPC 09 -DSM 24243, L. gasseri LGS 01 -DSM 18299, L. gasseri LGS 02 -DSM 18300 and L. acidophilus LA 07 -DSM 24303; or (f) L. paracasei spp. paracasei LPC 09 -DSM 24243, L. gasseri LGS 01 -DSM 18299, L. gasseri LGS 02 -DSM 18300 and L. acidophilus LA 02 -DSM 21717; or (g) L. paracasei spp. paracasei LPC 09 -DSM 24243, L. gasseri LGS 01 -DSM 18299, L. gasseri LGS 02 -DSM 18300, L. acidophilus LA 07 -DSM 24303 and L. acidophilus LA 02 -DSM 21717.

(36) All of the above-described compositions, and particularly the compositions (a) to (g) listed above, can further comprise fructo-oligosaccharides (FOS) and/or inulin. Fructo-oligosaccharides (FOS) and/or inulin are included in an amount comprised from 1 to 30% by weight, relative to the weight of the composition, preferably from 3 to 15%, even more preferably from 5 to 10% by weight.

(37) The subject matter of the present invention relates to a bacterial strain belonging to the species Lactobacillus paracasei or Lactobacillus gasseri and which is capable of degrading oxalic acid and/or the salts thereof in an amount greater than 60%. Said strain is capable of degrading oxalic acid and/or the salts thereof in an amount greater than 70%. Said strain belonging to the species Lactobacillus paracasei is L. paracasei spp. paracasei LPC 09 DSM 24243. Said strain belonging to the species Lactobacillus gasseri is selected from the group comprising the strain L. gasseri LGS 01 DSM 18299 and the strain L. gasseri LGS 02 DSM 18300. Said strain belonging to the species Lactobacillus gasseri is selected from the group consisting of the strain L. gasseri LGS 01 DSM 18299 and the strain L. gasseri LGS 02 DSM 18300.

(38) The subject matter of the present invention relates to a food composition or supplement product or medical device or pharmaceutical composition comprising a bacterial composition; said bacterial composition comprises at least one bacterial strain as described above, for use in the preventive and curative treatment of hyperoxaluria, urolithiasis, kidney failure, cardiopathies, kidney stones, diverticulosis and diverticulitis. Said bacterial composition comprises the strain L. paracasei spp. paracasei LPC 09 DSM 24243. Said bacterial composition comprises the strain L. gasseri LGS 01 DSM 18299 and the strain L. gasseri LGS 02 DSM 18300. Said bacterial composition further comprises the strain L. acidophilus LA02 DSM 21717 and the strain L. acidophilus LA 07 DSM 24303. Said bacterial composition consists of L. paracasei spp. paracasei LPC 09 DSM 24243, L. acidophilus LA02 DSM 21717 and/or L. acidophilus LA 07 DSM 24303. Said composition further comprises fructo-oligosaccharides and/or inulin.

(39) Experimental Part

(40) 1. Bacterial Strains Analyzed

(41) About 70 strains belonging to the genera Bifidobacterium and Lactobacillus were studied; they came from the internal strain collection of the company Probiotical SpA of Novara and international collections such as, for example, the DSMZ-Germany, or were found in the literature. The selected strains are shown in Table 1, which shows the percentage of oxalate degradation by the bacterial strains tested. The experiment was conducted using a culture medium containing 5 mM ammonium oxalate.

(42) TABLE-US-00001 TABLE 1 % Deposit No. Species/strain Degradation DSM 24243 L. paracasei LPC 09 73.50 DSM 18299 L. gasseri LGS 01 73.40 DSM 18300 L. gasseri LGS 02 71.20 DSM 24303 L. acidophilus LA 07 59.25 DSM21717 L. acidophilus LA 02 56.35 LMG P-21021 L. plantarum L LP 01 40.31 DSM 23879 L. reuteri LRE 03 33.86 DSM 23878 L. reuteri LRE 02 31.42 DSM 16604 B. breve BR 03 28.16 DSM 16603 B. longum BL 03 25.29 ATCC 53103 L. rhamnosus GG 23.59 DSM 23880 L. reuteri LRE 04 16.79 DSM 21981 L. rhamnosus LR 06 15.70 DSM 18352 B. lactis BA 05 15.45 DSM 16605 L. rhamnosus LR 04 12.89

(43) The bacterial strains (i) to (v), (vii) to (x) and (xii) to (xv) listed in Table 1 were deposited by the company Probiotical SpA of Novara (Italy). Strain (vi) was deposited by the company Mofin Srl cf Novara (Italy). Strain (xi) is available from the ATCC collection. All strains are available and accessible to the public under the conditions established by the Budapest Treaty.

(44) 2.Culture Conditions Adopted

(45) The preparation of the strains to be submitted to analysis consisted in a series of three sequential subcultures in MRS broth (+1% cys-HCl, anaerobiosis, for the bifids) incubated at 37° C. until adequate growth was reached. This culture strategy enables complete activation of the strain. The strains were subsequently inoculated at the same percentage of inoculum (2%) in an experimental medium, specifically conceived to ensure maximum growth of lactobacilli and bifidobacteria, supplemented with 5 mM ammonium oxalate (amount equal to the average daily intake of oxalic acid). The cultures thus prepared were incubated for 24 hours at 37° C.

(46) 3. SPE (Solid Phase Extraction) Purification of the Samples

(47) At the end of the incubation period, the broth cultures were centrifuged and the supernatant was filtered through a 0.22 μm filter. HPLC injection of the samples brought to light an unclear chromatographic profile. In particular, the chromatographic peak of oxalic acid appeared to overlap the glucose peak present in the samples. In order to remedy the aforesaid problem, the samples were purified using SPE (solid phase extraction) columns specific for organic acids. The protocol for purification by SPE columns required an optimization step in order to obtain the best final yield. In particular, different reagents were used in relation to the column conditioning step and final elution of the analyte. This solid-phase purification made it possible to obtain a distinctly cleaner chromatographic peak of oxalic acid, as can be seen in FIG. 1A-B. The protocol used was the following

(48) Type of SPE column: Phenomenex Strata-XA

(49) Activation: 1 ml of methanol

(50) Conditioning: 2 ml of sodium formiate 20 mM

(51) Sample loading: 1 ml sample

(52) Washing of impurities: 1 ml ammonium acetate 25 mM+1 ml methanol

(53) Elution: 2×500 μl HCl M+2×500 μl HCl 3 M

(54) 4. HPLC Analysis

(55) The amount of oxalate degraded by each individual strain was analyzed by HPLC, calculating the difference between the concentration of oxalate present in the culture medium (5 mM) at T0 (before fermentation) and the residual concentration after growth of the microorganism. The results of the individual strains are expressed as percentages, considering the concentration of oxalate at T0 to be 100. For example, the result of the strain L. paracasei spp. paracasei LPC 09 DSM 24243, equal to around 70%, indicates that the latter is able to use an amount of oxalate equal to around 3.5 mM of oxalate (70% of 5 mM). The HPLC protocol used was the following:

(56) Type of column: Phenomenex Hydro-RP 250×4.6 mm

(57) Type of detector: UV-Vis with reading at 220 nm

(58) Elution flow rate: 0.7 ml/min

(59) Injection volume: 20 μl

(60) Column temperature: 30° C.

(61) Type of elution: isocratic

(62) Mobile phase: 20 mM Potassium phosphate pH 2.0

(63) The bacterial strains belonging to the genus Lactobacillus which showed a high degradation activity towards oxalic acid are the ones indicated above by (i) to (v).

(64) A. Determination of the Acidification Curves for the Strain L. paracasei spp. paracasei LPC 09 DSM 24243.

(65) The strain LPC09 was reactivated prior to the experiment by subculture in MRS and incubated under aerobiosis at 37° C. The reactivation steps were repeated three times prior to the experiment with overnight incubation. At the end of the third reactivation step, the cells were pelleted, washed with sterile water and resuspended before being inoculated into the culture media supplemented with fibre. The media used were based on sugar-free MRS (carbon sources), supplemented respectively with: Glucose (solution sterilized by heat treatment, 121° C. 15′), control medium. Fructo-oligosaccharides—FOS (solution sterilized by filtration, 0.20 μl filter). GOS-Gal,—Galacto-oligosaccharides with glucose residual (solution sterilized by filtration, 0.20 μl filter). GOS-Gal,—Galacto-oligosaccharides with galactose residual (solution sterilized by filtration, 0.20 μl filter). XOS,—xylo-oligosaccharides (solution sterilized by filtration, 0.20 μl filter). Larex,—larch fibre (solution sterilized by heat treatment, 121° C. 15′). Inulin (solution sterilized by heat treatment, 121° C. 15′).

(66) The final concentration of carbon sources for all media was 20 g/l.

(67) The media thus prepared were then inoculated with the strain LPC09, at a percentage of 4%, and incubated at 37° C. under aerobiosis.

(68) At time 0 and at 3, 6, 8 and 10 hours, pH measurements were made in order to construct the acidification curves shown in the graph of FIG. 5.

(69) Table 2 shows the acidification curves (pH value) obtained as a function of time (T=0, 3, 6, 8 and 10 hours) when the strain the L. paracasei spp. paracasei LPC 09 DSM 24243 was made to grow in a culture medium as described above.

(70) TABLE-US-00002 TABLE 2 0 3 6 8 10 LPC09 Glu 6.46 5.84 4.65 4.24 4.03 Fos 6.48 6.02 5.57 4.62 4.13 Xos 6.47 5.98 5.83 5.76 5.73 Gos-gal 6.48 6.03 6.02 6.09 6.14 Gos-glu 6.49 5.9 5.5 5.14 5.09 Inu 6.5 5.98 4.96 4.3 4.03 Lar 6.43 5.93 5.95 5.99 6.03