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.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. application Ser. No. 14/116,996, filed on Dec. 18, 2013 incorporated herein by reference, which, in turn, is the US national stage of International Patent Application PCT/IB2012/000895 filed on May 9, 2012 which, in turn, claims priority to Italian Patent Application MI2011A000791 filed on May 9, 2011.

The present invention relates to a selection of lactic bacterial strains and bifidobacteria of human intestinal origin capable of metabolizing oxalic acid and/or the salts thereof (oxalates). Moreover, the present invention relates to a food composition or supplement product or medical device or pharmaceutical composition containing said bacterial strains.

Oxalate (salt of oxalic acid) is an ubiquitous compound in the plant kingdom, widely present in all human diets. The daily intake ranges from 70 to 920 mg (average 495 mg˜5.6 mM), but these values are easily exceeded in the diets of vegetarians. Oxalic acid (dicarboxylic acid) is one of the most highly oxidized organic compounds and thus acts as a powerful chelating agent for cations, in particular the ion Ca2+. Because of this property, the salts of oxalic acid (oxalates) are of very little use in catabolic processes and energy production. Moreover, oxalic acid is toxic for the majority of living beings and particularly for mammals.

For this reason, the accumulation of oxalic acid and oxalates in man can trigger and exacerbate a series of pathological conditions, among which we shall mention hyperoxaluria, urolithiasis, kidney failure, cardiomyopathies and other cardiac disorders. In particular, oxalic acid combines with calcium to form the corresponding calcium oxalate, an insoluble salt which is responsible for over 70% of the kidney stones diagnosed. Moreover, oxalic acid is a powerful inflammatory agent affecting the intestinal mucosa. Therefore, an excess presence of this acid in the lumen can compromise the natural barrier function of the epithelium by altering its permeability and consequently provoking an increased absorption of oxalate. In particular, the colon is the main site of absorption of oxalate, with an intake of 3-5% in normal physiological conditions. Reducing the oxalate in the intestinal lumen could therefore contribute to reducing absorption. This would then lead to a decrease in the concentration of oxalates in plasma and in urine, thus reducing the hazardousness thereof.

Moreover, high levels of oxalates in the blood can lead to diverticulosis or diverticulitis. Diverticulosis, also known as “diverticular disease”, is a medical condition characterized by diverticula in the colon; these are eversions of the mucosa and submucosa of the colon through areas of relative weakness of the muscular layer in the wall of the colon. Diverticula are decidedly more common in the sigmoid colon, which is a portion of the intestine characterized by greater pressure, a factor facilitating the formation of diverticula. Diverticulitis is a pathology of the digestive tract, characterized by the inflammation of one or more diverticula. The majority of cases of diverticulitis are localized in the colon (in particular the descending and sigmoid colon).

Therefore, it is important to be able to reduce the amount of oxalates in the intestinal lumen, plasma and urine in such a way as to avoid the complications connected with high values of oxalates, such as, for example, hyperoxaluria, urolithiasis, kidney failure, cardiomyopathies and other cardiac disorders, kidney stones, diverticulosis and diverticulitis.

In particular, it is desirable to be able to reduce the levels of oxalate in the urine of two types of subjects:

• • Hyperoxaluric subjects who do not tend toward a diet with a high oxalate content.
  • Normooxaluric subjects who tend toward to a diet with a high oxalate content.

The Applicant has provided an answer to the above-mentioned needs following an intense activity of research, at the end of which it identified, from a highly vast set of strains, a selection of bacterial strains belonging to the genera Lactobacillus and Bifidobacterium; said strains exhibit a marked ability to quantitatively degrade oxalates. The selected strains show the ability to use oxalate as a source of energy, removing it from the environment in which said oxalate was originally to be found. Therefore, the selected strains are capable of degrading oxalates.

The subject matter of the present invention relates to a bacterial strain belonging to the genera Lactobacillus and Bifidobacterium and having the ability to degrade oxalates, as disclosed in the appended independent claim.

The subject matter of the present invention also relates to a food composition or supplement product or medical device or pharmaceutical composition containing said bacterial strains, as disclosed in the appended independent claim.

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

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

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

FIG. 3 shows a chromatogram for the bacterial strain B. breve BR03 DSM 16604.

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

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.

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.

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

(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.
(ii) L. gasseri LGS 01, deposited by the company Probiotical SpA of Novara (Italy) on 24 May 2006, with deposit number DSM 18299.
(iii) L. gasseri LGS 02, deposited by the company Probiotical SpA of Novara (Italy) on 24 May 2006, with deposit number DSM 18300.
(iv) L. acidophilus LA 07, deposited by the company Probiotical SpA of Novara (Italy) on 23 Nov. 2010, with deposit number DSM 24303.
(v) L. acidophilus LA 02, deposited by the company Probiotical SpA of Novara (Italy) on 6 Aug. 2008, with deposit number DSM 21717.
(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,
(vii) L. reuteri LRE 03, deposited by the company Probiotical SpA of Novara (Italy) on 5 Aug. 2010, with deposit number DSM 23879.
(viii) L. reuteri LRE 02, deposited by the company Probiotical SpA of Novara (Italy) on 5 Aug. 2010, with deposit number DSM 23878.
(ix) B. breve BR 03, deposited by the company Probiotical SpA of Novara (Italy) on 16 Jul. 2004, with deposit number DSM 16604.
(x) B. longum BL 03, deposited by the company Probiotical SpA of Novara (Italy) on 20 Jul. 2004, with deposit number DSM 16603.
(xi) L. rhamnosus GG, ATCC 53103, available in the ATCC public collection.
(xii) L. reuteri LRE 04, deposited by the company Probiotical SpA of Novara (Italy) on 5 Aug. 2010, with deposit number DSM 23880.
(xiii) L. rhamnosus LR 06, deposited by the company Probiotical SpA of Novara (Italy) on 14 Nov. 2008, with deposit number DSM 21981.
(xiv) B. lactis BA 05, deposited by the company Probiotical SpA of Novara (Italy) on 15 Jun. 2006, with deposit number DSM 18352.
(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.

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).

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.

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%. Advantageously, said ability is greater than 60%. Advantageously, said ability is greater than 70%.

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

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.

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. 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.

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

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).

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).

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.

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.

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 gasseriis 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 gasseriis selected from the group consisting of the strain L. gasseri LGS 01 DSM 18299 and the strain L. gasseri LGS 02 DSM 18300.

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.

EXPERIMENTAL PART

1. Bacterial Strains Analyzed

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.

	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 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

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 of 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.

2. Culture Conditions Adopted

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.

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

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. 1 A-B. The protocol used was the following

Type of SPE column: Phenomenex Strata-XA
Activation: 1 ml of methanol
Conditioning: 2 ml of sodium formiate 20 mM
Sample loading: 1 ml sample
Washing of impurities: 1 ml ammonium acetate 25 mM+1 ml methanol

Elution: 2×500 μl HCl 1 M+2×500 μl HCl 3 M

4. HPLC Analysis

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:

Type of column: Phenomenex Hydro-RP 250×4.6 mm
Type of detector: UV-Vis with reading at 220 nm
Elution flow rate: 0.7 ml/min
Injection volume: 20 μl
Column temperature: 30° C.
Type of elution: isocratic
Mobile phase: 20 mM Potassium phosphate pH 2.0

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).

A. Determination of the Acidification Curves for the Strain L. paracasei Spp. Paracasei LPC 09 DSM 24243.

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-GI, —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′).

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

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

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.

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.

	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

Claims

1.-5. (canceled)

6. A food composition or supplement product or medical device or pharmaceutical composition comprising at least one bacterial strain L. paracasei spp. paracasei LPC 09 DSM 24243 and a fibre selected from the group consisting of fructo-oligosaccharides (FOS), inulin, xylo-oligosaccharides (XOS), and galacto-oligosaccharides with galactose residual (GOS-Gal in an effective amount for preventive and/or curative treatment of at least one of hyperoxaluria, urolithiasis, kidney failure, cardiopathies, kidney stones, diverticulosis and diverticulitis.

7. (canceled)

8. The food composition or supplement product or medical device or pharmaceutical composition according to claim 6, wherein said bacterial composition further comprises the bacterial strain L. gasseri LGS 01 DSM 18299 and the bacterial strain L. gasseri LGS 02 DSM 18300.

9. The food composition or supplement product or medical device or pharmaceutical composition according to claim 6, wherein said bacterial composition further comprises the bacterial strain L. acidophilus LA02 DSM 21717 and the bacterial strain L. acidophilus LA 07 DSM 24303.

10. The food composition or supplement product or medical device or pharmaceutical composition according to claim 6, 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.

11. The food composition or supplement product or medical device or pharmaceutical composition according to claim 6, wherein said fibre comprises fructo-oligosaccharides and inulin.

12. The food composition or supplement product or medical device or pharmaceutical composition according to claim 6, wherein the fructo-oligosaccharides and inulin are comprised in an amount from 3 to 15%, by weight.

13. The food composition or supplement product or medical device or pharmaceutical composition according to claim 6, wherein the fructo-oligosaccharides and inulin are comprised in an amount from 5 to 10% by weight.