<i>Lactobacillus fermentum </i>for treating fructose-related diseases

Abstract

The present invention is directed to a composition of Lactobacillus fermentum for use in the treatment of a fructose-related disease and a related method of treatment.

Claims

1. A method for the therapeutic or prophylactic treatment of hereditary fructose intolerance and/or fructose malabsorption, comprising the steps of: providing a composition comprising Lactobacillus fermentum; and administering the composition to a subject having hereditary fructose intolerance and/or fructose malabsorption in a pharmaceutically effective amount, wherein the method effectuates the therapeutic or prophylactic treatment of hereditary fructose intolerance and/or fructose malabsorption, wherein the composition comprises a Lactobacillus fermentum selected from the group consisting of LF2 (DSM 32733), LF3 (DSM 32734), LF4 (DSM 32735), LF5 (DSM 32736), LF6 (DSM 32737), and LF7 (DSM 32738).

2. The method according to claim 1, wherein the composition comprises a Lactobacillus fermentum selected from the group consisting of LF4 (DSM 32735), LF6 (DSM 32737), and LF7 (DSM 32738).

3. The method according to claim 1, wherein the composition is a human food composition or an animal feed composition.

4. The method according to claim 1, wherein the composition is administered in an amount of about 10.sup.3-10.sup.14 CFU L. fermentum per day.

5. The method according to claim 1, wherein the composition is administered in an amount of about 10.sup.6-10.sup.13 CFU L. fermentum per day.

6. The method according to claim 1, wherein the composition is administered in an amount of about 10.sup.8-10.sup.12 CFU L. fermentum per day.

7. The method according to claim 1, wherein the composition is administered in an amount of about 10.sup.9-10.sup.11 CFU L. fermentum per day.

8. The method according to claim 2, wherein the composition is a human food composition or an animal feed composition.

9. The method according to claim 1, wherein the composition is administered orally.

10. The method according to claim 2, wherein the composition is administered in an amount of about 10.sup.3-10.sup.14 CFU L. fermentum per day.

11. The method according to claim 2, wherein the composition is administered in an amount of about 10.sup.6-10.sup.13 CFU L. fermentum per day.

12. The method according to claim 2, wherein the composition is administered in an amount of about 10.sup.8-10.sup.12 CFU L. fermentum per day.

13. The method according to claim 2, wherein the composition is administered in an amount of about 10.sup.9-10.sup.11 CFU L. fermentum per day.

14. The method according to claim 2, wherein the composition is administered orally.

Description

FIGURES

(1) FIG. 1 shows the calculated effects of one daily dose of Fructozym (FIG. 1A, three capsules of 30 mg) and Sweetzyme IT (FIG. 1B, 300 mg granulate). The graphs show the expected percentage of fructose degradation in the intestine with a load of 180 g, 90 g, 45 g, 23 g, 12 g and 6 (top to bottom in each FIGS. 1A and 1B) of fructose over a period of 24 h. In any case, less than 12% of the fructose load was degraded by the isomerase enzyme.

(2) FIGS. 2A-E depicts a comparison of the sugar metabolism of 5 different organisms. On each figure, the y-axis indicates the sugar concentration in mM after 20 h incubation with the organisms at 37° C. The x-axis denotes different media compositions and different organism concentrations (100 mg/L, 10 mg/L and 1 mg/L organism concentration). Each set of three bars depicts the measured amount of either glucose or fructose and each individual bar represents a medium comprising either glucose (dotted bar), glucose and fructose (solid black with white stripes) or only fructose (while with black stripes) from left to right. FIG. 2A shows the results for Bifidobacterium breve, FIG. 2B for Bifidobacterium infantis, FIG. 2C for Bifidobacterium lactis, FIG. 2D for Lactobacillus delbrueckii ssp. bulgaricus and FIG. 2E for Lactobacillus fermentum. Clearly, only L. fermentum was able to metabolize all fructose in the presence of glucose.

(3) FIGS. 3A-F show growth rates (optical density) of the preferred L. fermentum strains for use according to the present invention in different media comprising either glucose, fructose or different mixtures of fructose and glucose. The y-axis notes the optical density of the solution, the x-axis shows different media compositions and the z-axis denotes the time of growth. All strains grow equally well and at equal rates in media comprising fructose, glucose or mixtures thereof. This finding demonstrates that the strains metabolize fructose and glucose at equally high rates and are therefore suitable for the use according to the present invention. FIG. 3A shows the result for LF2 (DSM 32733), FIG. 3B for LF3 (DSM 32734), FIG. 3C for LF4 (DSM 32735), FIG. 3D for LF5 (DSM 32736), FIG. 3E for LF6 (DSM 32737) and FIG. 3F for LF7 (DSM 32738).

(4) FIG. 4 summarizes the growth (optical density) of the strains for use according to the invention in media comprising glucose, fructose and mixtures thereof. All strains are suited for metabolizing fructose in the presence of glucose.

(5) FIGS. 5A-B show how much ethanol is produced by the strains for use according to the present invention when cultured in media comprising glucose, fructose or mixtures thereof. Importantly, all strains produce minimal amounts of ethanol when cultured with an excess of fructose (LB2-7 correspond to LF2-7).

(6) FIG. 6 characterizes the pH sensitivity of the strains for use according to the present invention (see Example 5). All strains were able to grow under pH conditions of between 6.6 and 7.4 and most of them up to a pH of 8 (the term “LB” as depicted in FIG. 6 refers to the same strains named “LF”).

(7) FIG. 7 depicts Fructose utilization of Lactobacillus fermentum strains LF4, LF6 and LF7 in defined sugar media.

EXAMPLES

Example 1: Xylose Isomerase Activity on Fructose Conversion

(8) Glucose concentrations were measured using a standard hexokinase/glucose-6-phosphate-dehydrogenase assay and all samples for measurement were diluted to concentrations of less than 300 μM [fructose+glucose]. Samples of 100 mg Shandong-XI (Shandong Dianmei International Trade Co., Ltd., Shandong, China) and Sweetzyme IT (Novozymes A/S, Denmark) each, 30 mg capsules of Fructozym (Biogena Naturprodukte GmbH & Co KG, Austria) and Xylosolv (SCIOTEC Diagnostic Technologies GmbH, Austria) were suspended in 10 mL PBS and shaken for 1 h at 37° C. The samples were centrifuged. The pellets were suspended in PBS, centrifuged and after discarding the supernatant, the pellets were incubated in 10 mL of a fresh Tris-HCl buffer, pH 8, 30 mM magnesium chloride and 1.25 M fructose at 37° C. 1 mL of the supernatant obtained after the first centrifugation was mixed with 9 mL of the same buffer and incubated at 37° C. Xylosolv and Fructozym showed activities in of 188 mU/mg and 156 mU/mg. Sweetzyme IT showed little activity of the pellet (68 mU/mg after 1 h decreasing to 54 mU/mg after 16 h). Shandong-XI did not have any xylose isomerase activity. For time-correlated measurements, Shandong-XI and Sweetzyme IT (50 mg each) and one 30 mg capsule each of Fructozym and Xylosolv was suspended in 10 mL Tris-HCl buffer, pH 8, 30 mM magnesium chloride and 1.25 M fructose at 37° C. for 1 h and the glucose concentration was monitored for 4 h. Again, Shandong-XI did not show any activity and Sweetzyme IT showed a slightly higher activity (124 mU/mg). Fructozym showed a lower activity than before (31 mU/mg) and Xylosolv showed a higher activity than before (139 mU/mg).

(9) For a simulation of fructose reduction in the intestine (FIG. 1A-B), it was assumed that 6 to 180 g of fructose are consumed with food which are then present in 3 L of intestinal fluid. Using this concentration, it can be calculated that 0.7/0.5 g glucose are converted from a load of 6 g of fructose (12.8/10.2 g for 180 g) by Fructozym (three capsules of 30 mg isomerase)/Sweetzyme IT (300 mg granulate), respectively. This amounts to a reduction of 5 to 12% of the initial fructose load and is not suitable for significantly reducing fructose concentrations or treating fructose intolerance or malabsorption.

Example 2: Comparison of Glucose and Fructose Metabolism of Bifidobacteria and Lactobacilli

(10) For the measurement of fructose metabolism in the comparison of bifidobacteria (see FIGS. 2A-C), a medium of the following composition was prepared anaerobically, then autoclaved and used for cultivation at 37° C.: trypsin digestion of milk protein (casein) 10 g/L, yeast extract 5 g/L, meat extract 5 g/L, tryptic digestion of soy protein 5 g/L, di-potassium hydrogen phosphate 2 g/L, calcium chloride 10 mg/L, manganese chloride 50 mg/L, Tween 80 1 g/L, NaCl 5 g/L, sodium carbonate 400 mg/L, cysteine 500 mg/L, resazurin (25 mg/100 mL) 4 mL/L. 10 g of sugar (glucose, fructose or a 1:1 mixture of both (5 g+5 g)) was added to the medium.

(11) For the measurement of fructose metabolism in the comparison of lactobacilli (see FIGS. 2D-E), a medium of the following composition was prepared anaerobically, then autoclaved and used for cultivation at 37° C.: trypsin digestion of milk protein (casein) 10 g/L, yeast extract 5 g/L, meat extract 10 g/L, Tween 80 1 g/L, di-potassium hydrogen phosphate 2 g/L, sodium acetate 5 g/L, ammonium citrate 2 g/L, magnesium sulfate 200 mg/L, manganese chloride 50 mg/L, cysteine 500 mg/L, resazurin (25 mg/100 mL) 4 mL/L. To this medium, either 10 g/L glucose, 10 g glucose and 10 g fructose, or 20 g fructose was added.

(12) The colony count was carried out in the liquid medium. For evaluating the sugar metabolism, 100 mg/L, 10 mg/L and 1 mg/L of the samples and the remaining sugar content was determined after 20 h at 37° C. by centrifuging the cells off, diluting the medium 1:10 and determining the glucose concentration by the following optical-enzymatic test. Glucose was converted to glucose-6-phosphate with hexokinase and ATP and glucose-6-phosphate was oxidized with glucose-6-phosphate dehydrogenase and NADP.sup.+ to obtain 6-phosphogluconolactone. The NADPH that is formed during this reaction can be quantified at 365 nm and this result was used to determine the glucose concentration (ε=3.4 mM.sup.−1 cm.sup.−1). By adding glucose-6-phosphate isomerase, the fructose concentration could also be determined.

(13) The CFU/g of all samples was then determined:

(14) Bifidobacterium breve: 1.0*10.sup.11 CFU/g in glucose medium, 3.5*10.sup.11 CFU/g in mixed medium (Glc and Fru) and 1.2*10.sup.12 CFU/g in fructose medium; average: 5.6*10.sup.11 CFU/g.

(15) Bifidobacterium infantis: independent of the sugar: 1.0*10.sup.11 CFU/g.

(16) Bifidobacterium lactis: 1.0*10.sup.11 CFU/g in media with glucose or fructose only, 3.0*10.sup.11 CFU/g in mixed medium (Glc and Fru); average: 1.7*10.sup.11 CFU/g.

(17) Lactobacillus delbrueckii ssp. bulgaricus: 1.0*10.sup.11 CFU/g in glucose medium, 3.0*10.sup.11 CFU/g in mixed medium (Glc and Fru) and 3.0*10.sup.11 CFU/g in fructose medium; average: 2.3*10.sup.11 CFU/g.

(18) Lactobacillus fermentum: 3.0*10.sup.11 CFU/g in mixed medium (Glc and Fru) average: 1.7*10.sup.11 CFU/g.

(19) As described above, different amounts of microorganisms (100 mg/L, 10 mg/L and 1 mg/L) were used to determine the residual sugar in the media after 20 h at 37° C. The results are depicted in FIGS. 2A-E. As noted above, Lactobacillus fermentum was by far the most effective organism for the conversion of fructose and selectively converted all fructose available in the medium.

Example 3: Comparison of Growth Rates (Optical Density) of the Preferred L. fermentum Strains for Use According to the Present Invention in Different Media Comprising Either Glucose, Fructose or Different Mixtures of Fructose and Glucose

(20) Lactobacillus minimal media without monosaccharide and low amount of complex sugar was used. Glucose and fructose were added in defined concentrations for sugar metabolism tests separately. Hydrochloric acid (1 M HCl) was added to adjust the pH of the medium to pH 6 at 22° C. MRS media contained 20 g/L dextrose, 10 g/L of pancreatic digest of casein, 10 g/L meat extract, 5 g/L yeast extract, 5 g/L sodium acetate, 2 g/L dipotassium hydrogen phosphate, 2 g/L ammonium citrate, 1 g/L Tween 80, 0.2 g/L magnesium sulfate heptahydrate and 0.05 g/L manganese sulfate heptahydrate. Hydrochloric acid (1 M HCl) was added to adjust the pH of the medium to pH 6 or 8 at 22° C.

(21) L. fermentum preparation: Isolation of individual colonies (LB2-7=LF2-7) from pure L. fermentum cryogenic cultures on MRS agar plates.

(22) MRS media was inoculated with one L. fermentum colony from MRS agar plates. Optical density was measured at 600 nm in a spectrophotometer. Preculture was grown to OD 0.6 and harvested by centrifugation at 3150 rcf for 20 min. Cells were resuspended in 50% glycerol and frozen at −80° C. until use.

(23) Growth of bacteria: Glucose and fructose were filtered sterile and added to minimal media in defined concentrations. L. fermentum cells from cryogenic cultures were washed by diluting 50 times in minimal medium, centrifugation at 3150 rcf for 20 min and removing of supernatant. Defined sugar media were inoculated with 1.26E+11 previously washed cells to an initial optical density of 0.06. The different sugar compositions are denoted on the x-axis of FIGS. 3A-F and 4 in g/L. In comparison pure minimal media without sugar was inoculated and used as a blank value to monitor background growth. Cultures were incubated under anaerobic conditions at 37° C. for 24 h. The increase of L. fermentum growth was monitored for every hour by spectrometric measurement of optical density (OD) at 600 nm with Tecan infinite M1000. Each sugar culture was determined in quadruplicates for each strain and standard deviation was determined. CFU was calculated by multiplication OD with conversion factor 2.09E+12.

(24) The results demonstrate that all strains grow equally well (OD measurement) and at equal rates in media comprising fructose, glucose or mixtures thereof. The data also show that the strains metabolize fructose and glucose at equally high rates and are therefore suitable for the use according to the present invention. Surprisingly, it was found that strains LF6 (DSM 32737) and LF7 (DSM 32738) preferably grow in fructose-containing media and even show better growth with fructose than with glucose alone (see, e.g., FIG. 4).

Example 4: Comparison of Ethanol Fermentation of the L. fermentum Strains for Use According to the Present Invention in Different Media Comprising Either Glucose, Fructose or Different Mixtures of Fructose and Glucose

(25) The procedure of sugar utilization was carried out as described in Example 3. Growth of the cultures (LB2-7=LF2-7) in different sugar media (60 g/L glucose, mixture of 30 g/L glucose and 30 g/L fructose, and 60 g/L fructose) was stopped after 5, 7 and 9 hours with centrifugation at 3150 rcf at 4° C. The cell free supernatant was used for the ethanol assay. Ethanol determination (g/L) was carried out according to protocol from the K-ETOH assay kit from Megazyme (Megzyme c.u., Ireland). For the first pretest (FIG. 5A, a first ethanol test was carried out with all strains to decide for best candidates for subsequent analysis. Ethanol production was determined at 60 g/L sugar concentration. The assay was executed with only one sample dilution.) the samples where diluted 1:10 before the assay. For the ethanol assay (FIG. 5B) the samples where diluted 1:5 and 1:50 before the assay. The results are depicted in FIGS. 5A-B.

Example 5: Comparison of Growth of the L. fermentum Strains for Use According to the Present Invention in Different pH Environments

(26) MRS media (20 g/L dextrose, 10 g/L of pancreatic digest of casein, 10 g/L meat extract, 5 g/L yeast extract, 5 g/L sodium acetate, 2 g/L dipotassium hydrogen phosphate, 2 g/L ammonium citrate, 1 g/L Tween 80, 0.2 g/L magnesium sulfate heptahydrate and 0.05 g/L manganese sulfate heptahydrate) was adjusted to an initial pH of 6.8-8.8 with 1 M KOH. The adjusted media was inoculated with an overnight preculture of the L. fermentum strains (LB2-7=LF2-7) for use according to the present invention to OD 0.025. Cultures where incubated at 37° C. OD600 was determined spectrometrically after 1-6, 20 and 24 h. The results are summarized in FIG. 6 (the term “LB” as depicted in FIG. 6 refers to the same strains named “LF”).

Example 6: Fructose Depletion Assay for Selected L. fermentum Strains for Use According to the Present Invention

(27) The procedure of sugar utilization was carried out as described in Example 3 with LF4, LF6 and LF7. Growth of the cultures was stopped after 5, 7 and 9 hours with centrifugation at 3150 rcf at 4° C. The cell free supernatant was used for fructose assay. For analysis the fructose assay kit from BioAssaySys (Bioassay Systems LLC, USA) was used. All strains tested preferred fructose over glucose, as demonstrated in FIG. 7.