LACTOCOCCUS LACTIS FOR USE IN PREVENTING OR TREATING MINERAL DEFICIENCY

Abstract

The present invention relates to the use of Lactococcus lactis suitable for preventing or treating mineral deficiency or insufficiency by increasing the bioavailability of phytate bound minerals. The present invention provides novel strains, compositions comprising said strains and methods for the preparation of such compositions.

Claims

1. A method for preventing or treating at least one symptom of mineral deficiency or insufficiency in a subject, comprising administering an effective amount of a composition comprising L. lactis to the subject.

2. The method of claim 1, wherein said mineral is calcium, iron, copper, magnesium, manganese or zinc.

3. The method of claim 1, wherein said subject follows a plant-based diet.

4. The method of claim 1, wherein said L. lactis increase bioavailability of phytate bound minerals.

5. The method of claim 1, wherein said L. lactis are capable of fermenting a dairy milk.

6. The method of claim 1, wherein said L. lactis is CNCM I-5450.

7. L. lactis strain deposited at the CNCM under reference number CNCM I-5450.

8. A composition comprising at least 10.sup.5 CFU/g of the L. lactis strain of claim 7.

9. The composition according to claim 8, wherein said composition is a fermented composition.

10. The composition according to claim 8, wherein said composition comprises vegetal and/or dairy milk.

11. A method for increasing bioavailability of phytate bound minerals of a food product, comprising adding the L. lactis strain of claim 7 to the food product.

12. A process for the reduction of phytate, comprising: culturing a mixture comprising a vegetal base comprising phytate, and a phytate degrading L. lactis, to provide a reduction of phytates.

13. The process of claim 12, wherein said culturing is carried out to provide a reduction in pH to 4.7 or lower.

14. The process of claim 12, wherein the L. lactis is CNCM I-5450.

Description

DESCRIPTION OF THE FIGURES

[0190] FIG. 1 provides the phytate content of cereal flours determined according to Example 2.

[0191] FIG. 2 provides the % dephosporylation activity of bacteria CNCM I-5450 in cereal flours according to according to Example 2.

[0192] FIG. 3 provides the milk acidification kinetics of bacterial strains tested according to Example 3.

[0193] FIG. 4 provides the milk acidification kinetics of CNCM I-5450 tested according to Example 3.

[0194] FIG. 5 provides the population of bacteria CNCM I-5450 determined according to Example 4

[0195] FIG. 6 provides the % dephosporylation activity of bacteria CNCM I-5450 during the gastrointestinal tests according to Example 4.

EXAMPLES

Example 1: Screening of Phytate-Degrading Bacterial Strains

[0196] Approximately 900 individual bacterial strains from the Applicant's Danone Culture Collection were screened for phytates degradation activity of which 66 strains were identified as having phytates-degrading activities.

Materials & Methods:

[0197] Bacterial strains were growth overnight at 37° C. in 96-wells microplate in a defined medium without phosphate source. A neutral MRS medium was modified to reduce its phosphate concentration. This modification was intended to allow the Inventors to measure the phosphate released by phytate degradation without reaching the saturation threshold of the standard range. To achieve this they removed fractions containing high concentrations of phosphate such as yeast extract and potassium phosphate. To compensate for these elements, a mixture of vitamins B and iron sulfate was added. After fermentation, cultures or supernatants were incubated in presence of sodium phytate and the total available phosphorus released from samples was measured. A quantitative method to measure total “available phosphorus” released from samples was used. Phytase activity was measured in terms of inorganic phosphate released from phytic acid by strains using a Megazyme kit assay.

[0198] Only the strains that showed the highest activity in the first screening (66) were chosen for subsequent experiments done in triplicate.

[0199] The inventors used kit from Megazyme (K-PHYT). This method used a quantification of phosphorous with a colorimetric assay. The amount of molybdenum blue formed in this reaction is proportional to the amount of inorganic phosphate (Pi) present in the sample and is measured by the increase in absorbance at 650 nm. Pi is quantified as phosphorus from a calibration curve generated using standards of known phosphorus concentration.

[0200] A reaction mixture containing 100 μl of cell suspension and 50 μl of phytate substrate (3 mM in acetate buffer) was incubated for 1 h at 37° C. Then the release of inorganic phosphate was measured by adding color reagent, prepared daily, and an incubation 1 h at 37° C. before reading the absorbance at 650 nm.

[0201] Results were compared to a standard curve prepared with inorganic phosphate (K2HPO4).

TABLE-US-00001 TABLE 1 Screening Results Total number of Phytate -degrading Genus/Species strains tested strains L. plantarum 246 23 L. rhamnosus 67 2 L. lactis 142 5 L. fermentum 25 1 Leuconostoc 23 8 Bifidobacterium 254 11 Pediococcus 60 1 L. brevis 16 12 L. reuteri 5 0 L. amylovorus 4 0 L. curvatus 24 3 L. sakei 1 0 867 66

Example 2: Preparation of Fermented Vegetal Product

[0202] The 10 most effective phytate degrading strains from Table 1 were tested for their suitability for the degradation of phytate in plant-based food products as determined by phytate dephosphorylation activity in various plant flours.

[0203] Materials & Methods:

[0204] 15 flours were evaluated for the amount of phytate, and it was decided to test the highest phytate containing flours: soy, lupine, chickpea, brown millet, coconut, complete rice, buckwheat and oat (see FIG. 1).

[0205] Flours were sterilized under UV light. For vegetal fermentation, 15 g of flour was suspended in 100 ml of distilled water. Fermentation was started by inoculation with 1% of overnight culture of individual lactic acid bacteria or bifidobacteria. Fermentation was done for 24 h at 37° C. Enzyme activities was measured at 37° C.

[0206] Prior to fermentation with strains, phytate contained in the different flours was determined by suspending 1 g of flour in 20 mL of hydrochloric acid (0.66 M) and stirred overnight at room temperature. The solution was neutralized by adding 0.75 ml of sodium hydroxyde. Inorganic phosphate total and released allowed the calculation of the amount of phytate in the sample. Selected strains were incubated overnight at 37° C. on each flour in 96-wells plate before testing the amount of phosphorous released during fermentation vs control without bacteria. During culture strain growth was observed using pH as an indicator as the tested strains produce acid that decreases the pH of the media. A further test of growth is the determination of the amount of strain after fermentation i.e. the population determined as cfu (colony forming unit) per ml. The bacteria in the fermented flours were enumerated using MRS agar medium, supplemented with cysteine (0.3 g/l). Plates were incubated under anaerobic conditions at 37° C. for 24 h.

[0207] After fermentation, quantification of phosphorous released during fermentation from phytate was measured. The amount of phytate in each selected flour was determined by mixing 1 g of flour with 20 mL of hydrochloric acid (0.66 M) and stirred overnight at room temperature.

[0208] Results:

[0209] Table 2 provides the % dephosphorylation of the best-performing strains in key flours, where no observable growth/fermentation was observed in a flour fields are left blank.

[0210] FIG. 2 provides the phytate dephosphorylation of key tested flours using strain CNCM I-5450.

TABLE-US-00002 TABLE 2 Brown Unrefined Chickpea Oat Buckwheat Lupin Millet rice Coconut Soy Bifidobacteria 52%  2% 62% 1 Leuconostoc 29% 7% 46% 32% 37% 10% Bifidobacteria 11% 12%  40% 56% 64% 2 Bifidobacteria 38% 5% 26% 46% 48% 3 Lactobacillus 9% 39% 17% 41% 51% 14% CNCM I-5450 41% 1% 46% 21% 31% 44%  7%

[0211] Most strains grew well in all flours, surprisingly it was observed that the ability to dephosphorylate phytate was strain dependent.

Example 3: Preparation of Fermented Dairy Milk Product

[0212] The 10 most effective phytate degrading strains from Table 1 were also tested for their suitability for the preparation of dairy fermented milk products.

[0213] Materials & Methods:

[0214] Fermented milk test products were prepared by preparing a milk base (135 g/L powdered milk, 0.2% yeast extract, 5% galactose, 0.03% cysteine) with 1% vol/vol bacterial culture (about 10.sup.6 CFU/ml). Fermentation was carried out at 37° C. and monitored using a CiNAC probe.

[0215] The aim was to identify phytate degrading strains, ideally combining the capacity to reduce phytate in relevant plant foodstuffs and also able to grow in milk. The aim was to identify strains that were effective in acidifying dairy milk, that could be used to prepare fermented dairy milk products (typically pH lower than 5).

[0216] Results:

[0217] Only three of the strains were able to reduce the starting pH of the milk base (above 6) to lower than 4.75 within the target of 24 hours (extended fermentation times increases contamination risk), said strains achieving the target reduction within about 17 hours (see FIG. 3). Milk acidification kinetics of CNCM I-5450 are provided in FIG. 4.

Example 4: In Vitro Model of Strain Survival

[0218] The inventors aimed to identify strains that not only had a phytate-degrading enzymatic activity, but also good survival capability in the digestive tract in order to provide probiotic strains. For that reason, strains survival in the gastric & intestinal systems was determined using in vitro models.

[0219] These models were also used to confirm if the phytate dephosphorylation activity was present in said gastrointestinal models.

[0220] The aim was to evaluate the tolerance of strains toward stomach acidic pH, bile salts by using an in vitro static test.

[0221] Fermented milks were incubated with or without phytate in gastric & intestinal fluid models to verify the cell survival and to estimate the capacity of strains to resist to gastric and intestinal conditions. During incubation, sodium phytate was added to the reaction mixture to assay phytase activity during digestion. A strain numeration was done at time points (see Figures) to measure the survival of the strain. MRS-cys broth was used and incubated at 37° C. for 24 h.

[0222] FIG. 5 provides the population of bacteria prior to (pre-culture & in fermented milk) and during the in vitro gastrointestinal tests (at various timepoints).

[0223] FIG. 6 provides the % dephosporylation activity during the gastrointestinal tests.

[0224] The strain shows a tolerance to gastric and intestinal conditions with a minimal decrease in cfu during intestinal stress and maintains some dephosphorylation capacity.