Methods of treating anemia and increasing the absorption of non-heme iron by administration of lactobacillus plantarum

Abstract

The present invention relates to the use of at least one strain of Lactobacillus plantarum chosen from the group comprising Lactobacillus plantarum 299, DSM 6595, Lactobacillus plantarum '299v, DSM 9843, Lactobacillus plantarum HEAL 9, DSM 15312, Lactobacillus plantarum HEAL 19, DSM 15313, Lactobacillus plantarum HEAL 99, DSM 15316, and a part thereof, for the preparation of a composition for increasing the absorption of at least one kind of metal/metal ion in a mammal, preferably a human.

Claims

1. A method for the treatment of anemia, the method comprising orally administering to an anemic mammal in need of anemia treatment non-heme iron and a composition comprising at least one strain of viable Lactobacillus plantarum, in an amount of from about 110.sup.6 to about 110.sup.14 CFU, selected from the group consisting of Lactobacillus plantarum 299, DSM 6595, Lactobacillus plantarum 299v, DSM 9843, Lactobacillus plantarum HEAL 9, DSM 15312, Lactobacillus plantarum HEAL 19, DSM 15313, and Lactobacillus plantarum HEAL 99, DSM 15316.

2. The method according to claim 1, wherein said non-heme iron is bound with another element.

3. The method according to claim 2, wherein said composition comprises a carrier material.

4. The method according to claim 1, wherein said composition comprises a carrier material.

5. The method according to claim 4, wherein said carrier material is selected from the group consisting of oat meal gruel, lactic acid fermented foods, resistant starch, dietary fibres, carbohydrates, proteins, glycosylated proteins, and lipids.

6. The method according to claim 4, wherein said carrier material is fermented with one or more of the strains selected from the group consisting of Lactobacillus plantarum 299, DSM 6595, Lactobacillus plantarum 299v, DSM 9843, Lactobacillus plantarum HEAL 9, DSM 15312, Lactobacillus plantarum HEAL 19, DSM 15313, and Lactobacillus plantarum HEAL 99, DSM 15316.

7. The method according to claim 5, wherein said carrier material is fermented with one or more of the strains selected from the group consisting of Lactobacillus plantarum 299, DSM 6595, Lactobacillus plantarum 299v, DSM 9843, Lactobacillus plantarum HEAL 9, DSM 15312, Lactobacillus plantarum HEAL 19, DSM 15313, and Lactobacillus plantarum HEAL 99, DSM 15316.

8. The method according to claim 1, wherein said composition is selected from the group consisting of a food product, a dietary supplement, and a nutritional product.

9. The method according to claim 8, wherein said food product is selected from the group consisting of beverages, yoghurt, juices, ice cream, bread, biscuits, cereals, health bars, and spreads.

10. The method according to claim 1 wherein said at least one viable strain in the composition is present in an amount from about 110.sup.8 to about 110.sup.12 CFU.

11. The method according to claim 1 wherein said at least one viable strain in the composition is present in an amount from about 110.sup.9 to about 110.sup.11 CFU.

12. The method according to claim 1, wherein the mammal is human.

13. The method according to claim 1, wherein said non-heme iron is in food consumed by the mammal.

Description

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

EXPERIMENTAL

Experiment 1

(1) In the present study, the effect of L. plantarum 299v and its fermentation products, lactic acid and acetic acid, on nonheme iron absorption from a low iron bioavailability meal using a crossover design have been studied. Four different oat gruels were included to test the specific effect of L. plantarum 299v and the organic acids: A fermented oat gruel with active L. plantarum 299v, a pasteurized fermented oat gruel with the fermentation products but inactivated bacteria, a pH-adjusted non-fermented oat gruel, and a non-fermented oat gruel with added organic acids.

(2) Subjects and Methods

(3) Subjects

(4) Seventy women volunteered and were screened 2-4 wk before the study, and 24 women were selected for the study on the basis of relatively low iron stores but non-anaemic, i.e. a serum ferritin concentration 40 g/L and a hemoglobin concentration 110 g/L. The 24 volunteers were healthy young women with a mean age (SD) of 254 y, mean weight of 627 kg, and a mean body mass index of 21.31.9 kg/m.sup.2. All subjects were non-smokers and none of them were pregnant or lactating or took any vitamin or mineral supplements for 2 mo before or during the study. Eighteen subjects used oral contraceptives, but none of the subjects were routinely taking any other medication. Blood donation was not allowed for 2 mo before or during the study. Each participant received oral and written information about the study before written consent was obtained. The study was approved by the Municipal Ethical Committee of Copenhagen and Frederiksberg, Denmark (file no. KF 01-219/03) and the National Institute of Radiation Hygiene, Denmark.

(5) Experimental Design

(6) The study was a completely randomized, double-blinded cross-over trial, in which each subject was served 4 test meals: (A) a fermented oat gruel, (B) a pasteurized fermented oat gruel, (C) a non-fermented oat gruel (pH adjusted with lactic acid), and (D) a non-fermented oat gruel with added organic acids (lactic acid and acetic acid).

(7) Iron absorption from the 4 test meals was determined with the dual label extrinsic tag method. Using this method iron absorption from the 4 test meals was measured by measuring iron absorption from 2 test meals simultaneously in each of 2 periods. The two different test meals in each period were extrinsically labelled with .sup.55Fe and .sup.59Fe, respectively, and served twice on 4 consecutive mornings to minimize potential effects of day-to-day variation, e.g. in the order ABBA. All 12 serving orders were used and assigned randomly to subjects, so that all test meals occurred equal times as first meal served in a period. This was important to be able to validate the possible carry-over effect of the fermented oat gruel with the live colonizing bacteria within a period.

(8) The activities of both isotopes were measured in a blood sample 18 days after ingestion, hereafter the second period was carried out with the remaining test meals. Residual isotope activity from the first period was subtracted from the isotope activity levels in the blood sample from the second period.

(9) Composition of Test Meals and Serving Procedure

(10) The oat gruels were made of oatmeal mixed with water, then treated with enzymes and pasteurized (obtained by Probi AB). Oat gruel A was then fermented with L. plantarum 299v (DSM 9843, viable count 1.110.sup.9 cfu/g) [20]. Oat gruel B was a pasteurized oat gruel A (viable count <10 cfu/g), oat gruel C was the non-fermented basic oat gruel acidified with L-lactic acid to an equivalent pH as oat gruel A, B, and D, and oat gruel D was the non-fermented basic oat gruel added the organic acids DL-lactic acid and acetic acid to an equivalent of what was expected to be produced in oat gruel A during the fermentation. For each test meal 100 g oat gruel (A, B, C, or D) was served with a 140 g whole-wheat roll (60.0 g wheat flour, 20.0 g whole-wheat flour, 2.0 g salt, 2.0 g yeast, 16.0 g rapeseed oil, 40.0 g ultra pure water) with 10 g butter and a glass of ultra pure water (200 mL). The oat gruels were prepared from one batch and stored cold (4 C.) until serving. The whole-wheat rolls were prepared in one batch, stored frozen and reheated in an oven at 200 C. for 10 min before serving.

(11) The test meals were served in the morning after 12 h of fasting. Intake of a maximum of 0.5 L water was allowed overnight. Moderate or hard physical activity or the intake of any alcohol or medication was not allowed during the 12 h before intake of the test meals. After consuming the test meals, the subjects were not allowed to eat or drink for 2 h and intake of alcohol was prohibited for 24 h. The subjects filled in a questionnaire in connection with each test meal to ensure that they adhered to all procedures, and they were instructed to eat and drink alternately and to rinse the glass containing the oat gruel thoroughly with the water to ensure complete intake of the isotope dose. A staff member ensured that everything was eaten.

(12) During the experimental period the subjects filled out a detailed questionnaire on their daily eating habits.

(13) Isotopes and Labelling Procedure

(14) All meals were extrinsically labelled by adding 1 mL isotope solution [.sup.55FeCl.sub.3 (NEN Life Science Products, Inc., Boston, Mass.) or .sup.59FeCl.sub.3 (Amersham Biosciences Corp., Piscataway, N.J.) in 0.1 mol/L HCl] directly to the oat gruels 18 h before serving for isotope exchange. In the first period each dose contained 37 kBq .sup.55FeCl.sub.3 or .sup.59FeCl.sub.3 and in the second period 74 kBq .sup.55FeCl.sub.3 or .sup.59FeCl.sub.3.

(15) Dietary Analyses

(16) The 4 oat gruels and the bread were freeze-dried, homogenized, and analyzed in duplicates for total iron, calcium, zinc and phytic acid. The energy content was calculated with the use of a national food-composition database (Danish Tables of Food Composition, DANKOST 2000, version 1.20, Herlev, Denmark). Total iron, calcium, and zinc were determined by atomic absorption spectrophotometry (Spectra-AA 200, Varian, Mulgrave, Australia) after wet-ashing in a MES 1000 Solvent Extraction System (CEM Corp., Matthews, N.C.) with 65% (w/v) suprapure nitric acid (Merck KgaA, Darmstadt, Germany). A typical diet Standard Reference Material 1548a (National Institute of Standards and Technology, Gaithersburg, Md.) was used as the reference for iron (meanSD: 35.33.77 g/g), calcium (1.960.11 mg/g), and zinc (24.61.79 g/g), and the analyzed values were 33.38 g/g, 2.00 mg/g, and 23.25 g/g, respectively. Phytic acid was analyzed as individual inositol tri- to hexaphosphates (IP.sub.3-6).sup.1 by high-performance ion chromatography. The concentration of organic acids in the oat gruels was determined by capillary gas chromatography.

(17) Determination of Iron Status

(18) Restrictions on intake and exercise before the blood samples were as described for the test meals. Blood samples were drawn from the cubital vein after the subjects had rested for 10 min in a supine position. Hemoglobin analysis was carried out on venous blood (3.5 mL) collected in tubes containing dissolved EDTA (Vacutainer system, Becton Dickinson, Franklin Lakes, N.J.) by using a Sysmex KX-21 automated hematology analyzer (Sysmex America Inc., Mundelein, Ill.) and appropriate controls (Eight check-3WP, 22490822, Sysmex America Inc.). Intraassay and interassay variations were 0.5% (n=12) and 0.6% (n=27), respectively. Serum ferritin and .sub.1-antichymotrypsin (ACT) analyses were carried out on venous blood (5.0 mL) collected in plain tubes (Vacutainer system, Becton Dickinson). Serum ferritin was determined on an Immulite 1000 analyzer (Diagnostic Products Corporation, Los Angeles, Calif.) by a chemiluminescent immunometric assay, and appropriate reference sera were also analyzed (3.sup.rd International standard for ferritin (80/578), WHO, NIBSC, South Mimms, United Kingdom). Intraassay and interassay variations were 2.7% (n=15) and 5.0% (n=15), respectively. ACT was determined on a Cobas Mira analyzer (Roche Diagnostic Systems, F. Hoffman-La Roche Ltd., Basel, Switzerland) with use of an immunoturbidimetric technique and appropriate reference sera were also analyzed (European Commission certified reference material 470, no. 11924, IRMM, Geel, Belgium). Intraassay and interassay variations were 1.4% (n=12) and 3.2% (n=14), respectively.

(19) Determination of Nonheme Iron Absorption

(20) Activity of .sup.55Fe and .sup.59Fe was determined from blood samples (60 mL) collected in tubes containing heparin as anticoagulant (Vacutainer system, Becton Dickinson). Simultaneous determination of .sup.55Fe and .sup.59Fe was performed by dry-ashing followed by recrystallization and solubilization before counting in a Tricarb 2100TR Liquid Scintillation Analyzer (Packard Instruments, Meriden, Conn.) with automatic quench correction by the method described previously.

(21) Statistical Analysis

(22) Nonheme iron absorption data were converted to logarithms before statistical analysis, and the results were reconverted to antilogarithms. All data used for statistical analyses were normally distributed, with variance homogeneity tested by plots of residuals. The nonheme iron absorption from the different meals was compared using a linear mixed model with log (nonheme iron absorption) as the dependent variable, meal, alternate meal, and ferritin as independent fixed variables and subject and subjectperiod interaction as random effects:
Log (nonheme iron absorption)=(meal.sub.i)+(alternate meal.sub.i)+bferritin.sub.i+A(subject.sub.i)+B(subject.sub.iperiod.sub.i)+.sub.i

(23) Data are presented as estimates of least-squares means and differences betweens estimates of means with 95% CIs. The statistical analysis was performed with the SAS statistical software package, version 8.2 (SAS Institute Inc., Cary, N.C.), and values were considered significantly different for P<0.05.

(24) Results

(25) Composition of the Test Meals

(26) The composition of the test meals and the contents of organic acids in the oat gruels are given in Table 1 below.

(27) TABLE-US-00001 TABLE 1 Composition of the test meals, including a whole-wheat roll with butter, and pH and concentrations of organic acids in the oat gruels Basic oat Oat gruel Pasteurized gruel with Fermented fermented (pH organic oat gruel oat gruel adjusted) acids A B C D Energy (MJ) 2.5 2.5 2.5 2.5 Nonheme iron 2.8 2.8 2.5 2.8 (mg) Phytate.sup.1 (mg) 403 393 388 344 (mol) 645 635 621 551 Calcium (mg) 39.6 42.2 39.5 41.1 Zinc (mg) 2.2 2.2 2.1 2.2 Lactic acid 110 89 61 43 (mol/g) Acetic acid 4.0 3.7 1.1 3.7 (mol/g) Succinic acid 0.3 0.3 0 0 (mol/g) pH 3.9 4.1 4.2 4.0 .sup.1Represents individual inositol tetra- to hexaphosphates
Iron Status and Nonheme Iron Absorption

(28) The subjects' hemoglobin concentrations were in the range 111-137 g/L and serum ferritin concentrations in the range 12-40 g/L. As the serum ferritin concentration is sensitive to inflammation, the acute phase protein ACT was determined in serum as a marker of an acute phase response. The concentrations were in the region 0.20-0.37 g/L, indicating no acute phase response (ACT<0.6 g/L) and therefore valid measurement of the subjects iron status.

(29) The nonheme iron absorption from the 4 test meals calculated from the mixed linear model analysis is given in Table 2, see below.

(30) TABLE-US-00002 TABLE 2 Nonheme iron absorption from the meals containing the 4 different oat gruels Oat gruel Pasteurized with Basic Fermented fermented organic oat oat gruel oat gruel acids gruel Nonheme iron 1.1 0.6 0.5 0.5 absorbed in blood (0.8, 1.5)* (0.4, 0.7) (0.4, 0.7) (0.4, 0.7) (%).sup.1 Test meal: control 2.2 1.1 1.0 meal.sup.2 (1.7, 2.9)* (0.8, 1.4) (0.8, 1.3) .sup.1Geometric means of least squares estimates from the mixed linear model analysis with 95% CI in parentheses, n = 24 .sup.2Geometric means of estimates of differences from the mixed linear model analysis with 95% CI in parentheses, n = 24 *Values are significantly different from all the other values in each row (P < 0.0001)

(31) The results show a highly significant effect of the test meal with the fermented oat gruel when comparing both absolute nonheme iron absorption values and the ratios relative to the pH-adjusted non-fermented oat gruel meal (P<0.0001), in which the inter-individual variations are taken into account.

(32) As L. plantarum 299v can colonize the human intestinal mucosa for about 2 weeks and as the test meal with the fermented oat gruel increased nonheme iron absorption the specific carry-over effect of this test meal on the following ingested test meal within a period was evaluated. No general carry-over effect of test meals was seen, but looking at the specific effect of the meal with the fermented oat gruel the carry-over effect was close to reach statistical significance (P=0.06).

(33) The absorption ratios from the different test meals showed a highly significant increase in nonheme iron absorption from the test meal with the lactic acid fermented oat gruel, whereas there was no effect of the pasteurized fermented oat gruel meal and the non-fermented oat gruel meals, serving as different controls. As the content of iron and phytate in the 4 test meals was constant, this significant effect can be directed to an effect of the fermentation of L. plantarum 299v. Whether it is an effect of the active L. plantarum 299v or an effect of the organic acids produced during the fermentation should be determined from comparisons of the absorption ratio for the meal with the fermented oat gruel with the absorption ratios for the meals with the inactivated L. plantarum 299v (pasteurized fermented oat gruel) and the non-fermented oat gruel with added organic acids, as lactic acid and acetic acid was added to the latter in concentrations that are normally produced during the fermentation process. The results from the analysis of organic acids show that it wasn't possible to reach similar levels of organic acids in the 3 oat gruels at the time of ingestion (table 2). The meal with the smallest difference in concentrations of organic acids compared to the fermented oat gruel was the pasteurized fermented oat gruel, in which the concentrations of lactic acid and acetic acid was 19% and 8% lower, respectively. When comparing the iron absorption ratios for these 2 meals the ratio was reduced to 50% for the pasteurized fermented oat gruel. As the level of lactic acid in the oat gruel with added organic acids was 52% lower than in the pasteurized fermented oat gruel and the absorption ratio was reduced with only 9%, it is unlikely that the increase in iron absorption in the fermented oat gruel was due mainly to an effect of the organic acids. The results of the present study therefore indicate that the active lactic acid bacterium, L. plantarum 299v (1.110.sup.11 cfu), was able to increase nonheme iron absorption from a low iron bioavailability meal in young women.

(34) Iron absorption is normally described to occur in the duodenum and proximal small intestine. Small organic acids from the food, such as lactic acid and acetic acid from the pasteurized fermented oat gruel and the oat gruel with added organic acids, is very quickly absorbed in the gastrointestinal tract. A possible explanation of the enhanced nonheme iron absorption from the fermented oat gruel could be the colonization of L. plantarum 299v in the mucosa of the most proximal small intestine or possibly colon, where local production of the organic acids by the active bacterium may both decrease the local pH and the lactic acid may form soluble complexes with iron as has been described by Derman et al. This hypothesis may be strengthened by the fact that the carry-over effect of the meal with the fermented oat gruel was close to reach significance (P=0.06), indicating an effect of L. plantarum 299v on nonheme iron absorption from the meals ingested the following days where the bacterium still colonized the intestine.

(35) When comparing the absorption ratios for the test meals with fermented oat gruel, the pasteurized fermented oat gruel, and the oat gruel with added organic acids (as described above) it seems clear that the increase in absorption from the test meal with the fermented oat gruel can not be assigned to an effect of the organic acids alone, as has been hypothesised before, but that there is a specific effect of the active L. plantarum 299v.

Experiment 2

(36) Reagents. All reagents were from GTF (Gteborg, Sweden) unless otherwise indicated. Caco-2 Cell Culture. Caco-2 cells were obtained from the American Type Culture Collection (Rockville, Md.) at passage 17 and used for experiments at passages 20-35. Stock cultures were maintained in Dulbecco's modified essential medium (DMEM) supplemented with 20% (v/v) fetal calf serum (FSC), 100 units/L penicillin G, and 100 mg/L streptomycin at 37 C. in a huminified atmosphere of 95% air-5% CO2. The growth medium was changed every second to third day. Cells were split at 80% confluence using 0.5 g/L trypsin with 0.5 mmol/L EDTA in Dulbecco's phosphate buffered saline (PBS). Prior to the experiments, 100000 cells in 0.5 mL of supplemented DMEM were seeded on 0.4 m microporous polycarbonate membrane inserts (1 cm2 Tranwell inserts; Corning, Acton, Mass.). The basolateral chamber contained 1.5 mL of supplemented DMEM. The medium on both sides of the filter insert was changed every 2-3 days. All iron uptake and transfer experiments were performed 14-17 days postseeding.

(37) Bacterial cultures. Lactobacillus plantarum 299v (DSM9843), (5), Lactobacillus plantarum 299, (1), Lactobacillus plantarum Heal 9 (DSM 15312), (2), Lactobacillus plantarum Heal 19 (DSM15313), (4), Lactobacillus plantarum 299v mutant (AMJ1277), (3) and Lactobacillus reuteri, (6) were cultured in MRS broth at a rotary shaker (37 C., 200 rpm). The bacteria were harvested in the exponential phase (OD.sub.600, max=1.3). The volume of cell culture corresponding to a certain cell-number was calculated from a predetermined standard curve. The cells were spinned down at 5000 rpm (Sorvall heraeus, multifuge) for 2 minutes and later resuspended in a transport solution of HBSS (PAA), HEPES 2.5% (1 M, PAA) and FeCl.sub.3 10 M. The trial was performed with a cell concentration of 6.710.sup.7 cells/ml for all species but Lactobacillus reuteri which was added in a concentration of 3.3510.sup.7 cells/ml. The trial was repeated twice.

(38) Assay for Cellular .sup.55Fe uptake and Transfer across Monolayers. Fresh supplemented DMEM was provided to the cells 1 day prior to the uptake and transfer assays. To study Fe(III) uptake and transepithelial transfer by Caco-2 cells, bacterial suspensions were traced with .sup.55Fe (Perkin Elmer). Suspensions in volumes of 0.5 mL were placed on the apical side of Caco-2 cells, while the basolateral chamber contained 1.5 ml HBSS/HEPES. Cells were incubated in 37 C. in a humidified atmosphere of 95% air-5% CO2. After 2 h incubation, the cells were washed four times with ice-cold wash buffer (150 mmol/L NaCl, 10 mmol/L HEPES, 1 mmol/L EDTA, pH7) and homogenized in 0.5M NaOH. .sup.55Fe transferred to the basolateral chamber or associated with the Caco-2 lysates was measured by liquid scintillation counting. The integrity of the cell monolayers was monitored before and after the assays by measuring TEER.

(39) Transport of FeCl.sub.3 (10 mol/l) in HBSS/HEPES with the addition of different strains of bacteria (see description of the method).

(40) Control: as above but without bacteria.

(41) TABLE-US-00003 Transport % Control 0.06 1. Lactobacillus plantarum 299 0.58 2. Lactobacillus plantarum Heal 9 0.23 3. Lactobacillus plantarum 299v mutant 0.32 4. Lactobacillus plantarum heal 19 0.69 5. Lactobacillus plantarum 299v 0.38 6. Lactobacillus reuteri 0.11
Results: The strains 1 to 5 affect the transport of Fe compared to the neat Fe solution. An increase of between 3 and 9 fold is observed. In the sample containing Lactobacillus reuteri an increase of the transport is seen, but not comparable with the different Lactobacillus plantarum strains.

(42) Thus, the increased transport observed with the different Lactobacillus plantarum strain shows a similar increase in iron absorption, as shown in the earlier human studie.