New Strain Of Lactobacillus Casei With Ability To Degrade The Immunotoxic Peptide From Gluten

Abstract

The invention relates to a bacterial strain, Lactobacillus casei IPLA12038 with deposit number CECT 8590, and its use for the degradation of gluten, gliadin or derivative peptides. The strain has been isolated from an acidic dough used to make bread. Its advantages include the ability to use gliadin as nitrogen source, possess peptidase activities involved in the degradation of it and be able to completely degrade the immunotoxic (33) amino acid peptide involved in triggering of celiac disease in a short period of time. The fact of considering it a GRAS and QPS microorganism, isolated from a fermented food, sensitive to antimicrobial gastrointestinal agents and resistant to gastrointestinal conditions tested in vitro, guarantees its use as a probiotic or nutraceutical supplement.

Claims

1. A composition comprising an effective amount of the isolated strain of Lactobacillus casei deposited in the Spanish Type Culture Collection (CECT) under the accession number CECT 8590.

2. (canceled)

3. The composition according to claim 1, wherein the isolated strain has been fermented in an artificial medium and submitted to a post-treatment after the fermentation, to obtain bacterial cells, and wherein the resulting bacterial cells are in a liquid medium or in a solid form.

4. The composition according to claim 3, wherein the post-treatment is selected from the group consisting of: drying, freezing, freeze-drying, fluid bed-drying, spray-drying and refrigerating in liquid medium.

5. The composition according to claim 1, wherein the strain has been genetically modified.

6-8. (canceled)

9. The composition according to claim 1 further comprising appropriate amounts of at least one acceptable excipient or food ingredient for use as a pharmaceutical product, a veterinary product, a medical food, a food product, a food supplement or a sourdough, a probiotic food composition, a dietary supplement, a symbiotic composition, a biotherapeutic composition, or combinations thereof.

10. A method to obtain a mutant of the strain of Lactobacillus casei deposited in the Spanish Type Culture Collection (CECT) under the accession number CECT 8590, wherein the method comprises using the deposited strain as starting material and applying mutagenesis, and wherein the obtained mutant further retains or enhances at least the ability of the deposited strain to degrade gliadin and the immunotoxic 33-mer peptide having SEQ ID NO: 1.

11. A method for the prevention and/or treatment of disorders associated with the intake of gluten, comprising administering to a subject in need thereof an effective amount of the composition as defined in claim 1.

12. The method of claim 11, wherein the disorders associated with the intake of gluten include celiac disease and non-celiac gluten sensitivity (NCGS).

Description

DESCRIPTION OF THE FIGURES

[0097] FIG. 1. PepX, PepI, PepQ and PepN enzymatic activities of L. casei CECT 8590 strain.

[0098] FIG. 2. Chromatogram obtained when the 33-mer peptide was incubated with L. casei CECT 8590 for 0 h (top line) or 12 h (middle line). Note that the peptide disappeared completely after 12 h of incubation with the strain. A reaction containing the strain alone was used to check the appearance of peptides derived from its normal metabolism (bottom line). The chromatogram section that contains the peak corresponding to the 33-mer peptide is shown enlarged in the inset.

[0099] FIG. 3. Survival to in vitro simulated gastrointestinal conditions of L. casei CECT 8590 strain. CS means cell survival. L. casei strain CECT 8590 was resuspended in milk and subjected to in vitro gastrointestinal conditions to determine cell survival. To simulate the gastric stress (G) was incubated with pepsin and lysozyme decreasing pH values. To simulate the gastrointestinal stress (GI) samples from the pH 5, 4.1 and 3 were incubated with bile salts and pancreatic enzymes for 20 (Gla) and 120 (Glb) minutes.

EXAMPLES

[0100] The following examples are used to illustrate the invention and they should not be considered as limitative of the scope of the invention.

Example 1

Isolation and Selection of the Strain Object of the Present Invention

[0101] The strain of the invention, Lactobacillus casei CECT 8590, has been isolated from a sourdough used to make bread, from Poland. Ten grams of sample were collected and homogenized for 5 min in 90 ml Ringer saline solution (Merck, Darmstadt, Germany) using a Lab-Blender 400 stomacher (Seward Ltd., London, UK). Once homogenized, serial dilutions in Ringer saline solution were performed and 50 l were plated in a chemically defined medium containing gliadin as sole nitrogen source (AHG-M), and incubated in (PARALLEL in) three different conditions: aerobically at 30 C., aerobically at 37 and in anaerobiosis at 37 C., (IN A MAC 500 ANAEROBIC WORKSTATION (DON WHITLEY SCIENTIFIC, WEST YORKSHIRE, UK)). The culture medium was supplemented with cycloheximide (50 g ml.sup.1) to inhibit the growth of fungi and yeasts. Only 27 strains were able to grow on this medium. Of these, only 8 were able to form a halo around the colony indicating degradation of gliadin in the AHG-M medium, and according to its characteristics and to the profile determined by PFGE one was the strain object of the present invention, Lactobacillus casei CECT 8590.

Example 2

Peptidase Activities with Synthetic Substrates

[0102] The ability of the strain to hydrolyze proteins and peptides derived from gluten was carried out by quantifying the activity of intracellular peptidases of broad spectrum and with specificity to hydrolyze peptidic sequences containing proline, present in the peptides responsible for immune and toxic responses of gluten. For the preparation of cell extracts, 20 ml of bacterial culture grown 20-22 hours in MRS was used. They were collected by centrifugation (8000 g for 10 min), washed twice and resuspended in 2 ml of the buffer corresponding to the enzymatic activity to be tested. Then, the samples were broken using a French Press to 2.3 kba (Constant Cell Disruption Systems [Low March, Daventry, Northents, UK]). Cell debris were removed by centrifugation at 10,000 g at 4 for 30 min and the cell-free extracts were assayed immediately.

[0103] Peptidase activities were characterized using specific chromogenic substrates (BACHEM, BUBENDORF, SWITZERLAND). PepN, PepI and PepX and were determined using Leu-p-nitroanilide (Leu-p-NA), Pro-p-NA and Gly-Pro-p-NA respectively. The reaction mixture contained 825 l of 10 mM phosphate buffer (pH 7.5), 75 l of THE ADEQUATE substrate (1.2 mM) and 150 l of the cell extract. After 1 hour of incubation at 37 C., the reaction was stopped by adding 500 l of 20% trichloroacetic acid. The released p-NA was measured spectrophotometrically at 410 nm (U-2800 DIGILAB, HITACHI HIGH-TECHNOLOGIES CORPORATION, TOKYO, JAPAN). PepQ was determined FOLLOWING THE NINHYDRIN METHOD (YARON AND MLYNAR. 1968) using Val-Pro AS A SUBSTRATE. The reaction mixture contained 100 l of the cell extract in 162.5 l of citrate buffer (sodium citrate [20 mM] and zinc sulfate [2.5 mM]) pH 6.5, and 37.5 l of substrate (1.2 mM). It was incubated for 30 min at 40 C. To stop the reaction 350 l of glacial acetic acid was added and to form the color 350 l of the reaction mixture [ninhydrin (3% w/v), phosphoric acid (60% v/v) and glacial acetic acid (40% v/v)] were added. It was incubated for 10 min at 90 C. The color is measured spectrophotometrically at 515 nm. Enzyme assays were performed in triplicate and expressed as milliunits per mg protein, defining one unit (U) as the amount of enzyme required to release 1 mol of p-NA, or 1 pmol of amino acid in the case of PepQ, per minute under the conditions tested. Total protein in each sample was determined using the Pierce BCA Protein Assay kit (PIERCE, ROCKFORD, USA). The results are presented in FIG. 1.

Example 3

Degradation of the Immunotoxic Peptide of 33 Amino Acids

[0104] The ability of the strain to degrade the immunotoxic peptide of 33 amino acids (33-mer) responsible for the triggering of celiac disease (Shan et al. 2002 Structural basis for gluten intolerance in celiac sprue Science 297:2275-2279) was quantified by REVERSE-PHASE HIGH PERFORMANCE LIQUID chromatographic analysis (RP-HPLC). The 33-mer peptide was chemically synthesized with a purity of 95% (Immunosteps S. L., Salamanca, Spain) and dissolved in PBS BUFFER, pH 7.4 (10 mM). Cell culture was grown in MRS BROTH at 37 C. to stationary phase. Cells were collected by centrifugation (8000 g for 10 min), they were washed twice with PBS (pH 7.4) and were resuspended in 1 ml of the same buffer. The cell suspension (OD.sub.600=35) was incubated with the peptide (50 M=195 ppm) at 37 C. and samples (200 l) were taken to inject on HPLC (50 l) at 0, 8 and 12 hours. The samples were heat inactivated and STORED at 20 C. until analysis. Prior to injection the samples were filtered using 0.2 M filter membrane HT Tuffryn. The substrate concentration as well as intermediate products were analyzed by RP-HPLC (ACCORDING TO SHAN ET AL 2002). The HPLC system consists of an Alliance 2795 separation module, PDA with a photodiode detector 2996, and software data acquisition Empower (Waters, Milford, Mass.). One XTerra MS C18 5 m column, 4.6150 mm column with pre column Nova-Pak C18 4 m, 3.920 mm (Waters), with a thermostat at 30 C. was used. Elution phases consisted of (A) water with 0.1% (v/v) TFA (trifluoroacetic acid) and (B) acetonitrile with 0.1% (v/v) TFA. The gradient program started with 100% of solvent A and 0% of solvent B, and changed linearly to reach 62.6% of solvent A and 37.4% of solvent B in 34 minutes The column was washed with 100% solvent B for 5 minutes and equilibrated to initial conditions for 10 min. UV absorbance at 215 nm and 280 nm was collected. The results obtained are shown in FIG. 2. L. casei CECT 8590 degraded the peptide by up to 82% within 8 h, and completely degraded it within 12 h (FIG. 2). Interestingly, no intermediate hydrolysis products were observed when this latter chromatogram was compared to that of a parallel incubation containing solely the strain (i.e., with no substrate).

Example 4

Evaluation of the Resistance to Conditions of Gastrointestinal Stress

[0105] The resistance of the strain object of the invention to the acidic conditions of the gastric juices, which constitute the first barrier limiting the viability of the microorganisms following ingestion, and the action of bile salts and pancreatic enzymes was evaluated. Cell suspensions of CECT 8590 strain (108 cfu ml.sup.1) in milk were prepared. Cell cultures were grown in 30 ml MRS. The cells were recovered by centrifugation (15 min, 3000 g), washed twice with 0.85% NaCl (w/v) and resuspended in a volume of skimmed milk powder 10% (Oxoid). Then these cell suspensions were subjected to the in vitro simulated gastrointestinal tract conditions (GIT). To start with 0.1% (w/v) lysozyme mimicking human saliva was added. Gastric stress (G) was simulated by adding pepsin (Sigma) 0.3% (w/v) and decreasing the pH every 20 minutes (pH 5, pH 4.1, pH 3, pH 2.1 and pH 1.8). The gastrointestinal stress (GI) was tested after stress G in the samples from the pH 5, 4.1 and 3. To simulate small bowel digestion, bile salts (0.3% w/v), a cocktail of pancreatic enzymes (1 mg ml.sup.1 of amylase, 1 mg ml.sup.1 trypsin and 1 mg ml.sup.1 of chymotrypsin) to a final concentration of 5 mg ml.sup.1 were added and the pH was raised to 6.5. Samples were taken after 20 and 120 minutes of incubation in the GI conditions. A sample was collected at each condition and serial dilutions in saline solution of 0.85% NaCl (w/v) were performed. Samples were plated on MRS with 2% agar and viable count was performed after incubation for 48 h at 37 C. The results obtained are presented in FIG. 3.

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Tratado de Farmacia Galenica de C. Faulf i Trillo, 10 Edition, 1993, Luzn 5, S.A. Editions,

[0107] Shan et al. 2002 Structural basis for gluten intolerance in celiac sprue Science 297:2275-2279.