USES OF LIPOTEICHOIC ACID FROM BIFIDOBACTERIA

Abstract

The present invention relates to a lipoteichoic acid isolated from Bifidobacteria cultured in excess of glucose which has fat reduction activity, thus being useful for exploitation in the following application areas: food and beverages, animal feed, including pet food, nutritional supplements, infant nutrition, cosmetics (including nutricosmetics), medical foods and pharmaceutical and veterinary applications, among others.

Claims

1. A lipoteichoic acid (LTA) comprising a molar ratio Alanine/glucose of at least 0.5 and a molar ratio glycerol phosphate/glucose of at least 6.0.

2. LTA according to claim 1, wherein the molar ratio Alanine/glucose is at least 0.6, 0.7, 0.8, 0.9 or 1.0.

3. LTA according to claim 1 or 2, wherein the Alanines of the LTA are L-Alanine, D-Alanine or a combination of L- and D-Alanines.

4. LTA according to any one of claims 1 to 3, wherein the molar ratio glycerol phosphate/glucose is at least 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0 or 12.5.

5. LTA according to claim 4, wherein the molar ratio glycerol phosphate/glucose is 12.6.

6. LTA according to any one of claims 1 to 5, wherein the LTA is obtained from Bifidobacterium animalis, preferably from Bifidobacterium animalis subsp. lactis, more preferably, from the strain Bifidobacterium animalis subsp. lactis CECT 8145.

7. LTA according to any one of claims 1 to 5, wherein the LTA is obtained from Bifidobacterium longum, preferably, from the strain Bifidobacterium longum CECT 7347.

8. LTA according to any one of claims 1 to 7, wherein the LTA is heat-treated or lyophilized.

9. A composition comprising the LTA according to any one of claims 1 to 8.

10. Composition according to claim 9, further comprising a carrier or an excipient.

11. Composition according to claim 9 or 10, further comprising a bioactive compound.

12. Composition according to any one of claims 9 to 11, wherein the composition is a pharmaceutical composition or a nutritional composition.

13. A LTA according to any one of claims 1 to 8, or a composition according to any one of claims 9 to 12, for use as a medicament.

14. A LTA according to any one of claims 1 to 8, or a composition according to any one of claims 9 to 12, for use in the treatment and/or prevention of obesity, overweight or a related disease selected from the group consisting of diabetes, metabolic syndrome, hypertension, hyperglycaemia, inflammation, type-2 diabetes, cardiovascular disease, hypercholesterolemia, hormonal disorders and infertility.

15. A non-therapeutic use of a LTA according to any one of claims 1 to 8, or a composition according to any one of claims 9 to 12, for body fat reduction.

16. Use of a LTA according to any one of claims 1 to 8, or a composition according to any one of claims 9 to 12, for the elaboration of a food or feed product.

17. A process for obtaining a LTA according to any one of claims 1 to 8, comprising the following steps: (a) cultivating a Bifidobacterium in excess of sugars as carbon source, and (b) isolating the LTA from the cell wall of the bacterium.

18. Process according to claim 17, wherein the Bifidobacterium is Bifidobacterium animalis, preferably Bifidobacterium animalis subsp. lactis, more preferably, the strain Bifidobacterium animalis subsp. lactis CECT 8145.

19. Process according to claim 17, wherein the Bifidobacterium is Bifidobacterium longum, preferably, the strain Bifidobacterium longum CECT 7347.

20. Process according to any one of claims 17 to 19, wherein the sugars are selected from the list consisting of glucose, galactose, fructose, sucrose, lactose, maltose and trehalose.

21. A LTA from bifidobacteria cultured in excess of sugars as carbon source for use as a medicament, wherein the structure of the LTA is the following: ##STR00011## wherein GroP is glycerophosphate, X is Alanine (Ala) or Hydrogen (H), Gal is galactofuranan, each X is independently selected from hydrogen and Alanine (Ala), the number of molecules of Alanine is m or m-p, being m the number of repeating units of Gal and p the number of units of Gal in which X is hydrogen, Glc is glucan, m is the number of repeating units of Gal and is between 10 and 20, preferably, between 11 to 18, and n is the number of repeating units of Glc and is between 5 and 20, preferably, between 8 and 12.

22. A LTA from bifidobacteria cultured in excess of sugars as carbon source for use in the treatment and/or prevention of obesity, overweight or related diseases selected from the group consisting of diabetes, metabolic syndrome, hypertension, hyperglycaemia, inflammation, type-2 diabetes, cardiovascular disease, hypercholesterolemia, hormonal disorders and infertility, wherein the structure of the LTA is the following: ##STR00012## wherein GroP is glycerophosphate, X is Alanine (Ala) or Hydrogen (H), Gal is galactofuranan, each X is independently selected from hydrogen and Alanine (Ala), the number of molecules of Alanine is m or m-p, being m the number of repeating units of Gal and p the number of units of Gal in which X is hydrogen, Glc is glucan, m is the number of repeating units of Gal and is between 10 and 20, preferably, between 11 to 18, and n is the number of repeating units of Glc and is between 5 and 20, preferably, between 8 and 12.

23. A LTA from bifidobacteria cultured in excess of sugars as carbon source for use according to claim 21 or 22, wherein m is between 11 and 18, and n is between 8 and 12.

24. A LTA from bifidobacteria cultured in excess of sugars as carbon source for use according to any one of claims 21 to 23, wherein the molar ratio Alanine/glucose is at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0, and the molar ratio glycerol phosphate/glucose is at least 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0 or 12.5.

25. A LTA from bifidobacteria cultured in excess of sugars as carbon source for use according to claim 24, wherein the molar ratio glycerol phosphate/glucose is 12.6.

26. A LTA from bifidobacteria cultured in excess of sugars as carbon source for use according to any one of claims 21 to 25, wherein the Alanines are L-Alanine, D-Alanine or a combination of L- and D-Alanine.

27. A LTA from bifidobacteria cultured in excess of sugars as carbon source for use according to any one of claims 21 to 26, wherein the LTA is obtained from Bifidobacterium animalis, preferably from Bifidobacterium animalis subsp. lactis, more preferably from the strain Bifidobacterium animalis subsp. lactis CECT 8145.

28. A LTA from bifidobacteria cultured in excess of sugars as carbon source for use according to any one of claims 21 to 27, wherein the Bifidobacterium is Bifidobacterium longum, preferably, the strain Bifidobacterium longum CECT 7347.

29. A LTA from bifidobacteria cultured in sugars as carbon source for use according to any one of claims 21 to 28, wherein the LTA is heat-treated or lyophilized.

30. Non-therapeutic use of a LTA from bifidobacteria cultured in excess of sugars as carbon source for body fat reduction, wherein the structure of the LTA is the following: ##STR00013## wherein GroP is glycerophosphate, X is Alanine (Ala) or Hydrogen (H), Gal is galactofuranan, each X is independently selected from hydrogen and Alanine (Ala), the number of molecules of Alanine is m or m-p, being m the number of repeating units of Gal and p the number of units of Gal in which X is hydrogen, Glc is glucan, m is the number of repeating units of Gal and is between 10 and 20, preferably, between 11 to 18, and n is the number of repeating units of Glc and is between 5 and 20, preferably, between 8 and 12.

31. The non-therapeutic use according to claim 30, wherein m is between 11 and 18 and n is between 8 and 12.

32. The non-therapeutic use according to claim 30 or 31, wherein the molar ratio Alanine/glucose is at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0, and the molar ratio glycerol phosphate/glucose is at least 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0 or 12.5.

33. The non-therapeutic use according to claim 32, wherein the molar ratio glycerol phosphate/glucose is 12.6.

34. The non-therapeutic use according to any one of claims 30 to 33, wherein the Alanines are L-Alanine, D-Alanine or a combination of L- and D-Alanine.

35. The non-therapeutic use according to any one of claims 30 to 34, wherein the LTA is obtained from Bifidobacterium animalis, preferably from Bifidobacterium animalis subsp. lactis, more preferably from the strain Bifidobacterium animalis subsp. lactis CECT 8145.

36. The non-therapeutic use according to any one of claims 30 to 34, wherein the Bifidobacterium is Bifidobacterium longum, preferably, the strain Bifidobacterium longum CECT 7347.

37. The non-therapeutic use according to any of claims 30 to 36, wherein the LTA is heat-treated or lyophilized.

38. Use of a LTA from bifidobacteria cultured in excess of sugars as carbon source for the elaboration of a food or feed product, wherein the structure of the LTA is the following: ##STR00014## wherein GroP is glycerophosphate, X is Alanine (Ala) or Hydrogen (H), Gal is galactofuranan, each X is independently selected from hydrogen and Alanine (Ala), the number of molecules of Alanine is m or m-p, being m the number of repeating units of Gal and p the number of units of Gal in which X is hydrogen, Glc is glucan, m is the number of repeating units of Gal and is between 10 and 20, preferably, between 11 to 18, and n is the number of repeating units of Glc and is between 5 and 20, preferably, between 8 and 12.

39. Use according to claim 38, wherein m is between 11 and 18, and n is between 8 and 12.

40. Use according to claim 38 or 39, wherein the molar ratio Alanine/glucose is at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0, and the molar ratio glycerol phosphate/glucose is at least 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0 or 12.5.

41. Use according to claim 40, wherein the molar ratio glycerol phosphate/glucose is 12.6.

42. Use according to any one of claims 38 to 41, wherein the Alanines are L-Alanine, D-Alanine or a combination of L- and D-Alanine.

43. Use according to any one of claims 38 to 42, wherein the food or feed product is a nutritional supplement.

44. The use according to any of claims 38 to 43, wherein the LTA is obtained from Bifidobacterium animalis, preferably from Bifidobacterium animalis subsp. lactis, more preferably from the strain Bifidobacterium animalis subsp. lactis CECT 8145.

45. The use according to any of claims 38 to 43, wherein the Bifidobacterium is Bifidobacterium longum, preferably, the strain Bifidobacterium longum CECT 7347.

46. The use according to any one of claims 38 to 45, wherein the LTA is heat-treated or lyophilized.

47. The use according to any one of claims 21 to 29, the use according to any one of claims 30 to 37, or the use according to any one of claims 38 to 46, wherein the sugars are selected from the list consisting of glucose, galactose, fructose, sucrose, lactose, maltose and trehalose.

Description

DESCRIPTION OF THE DRAWINGS

[0206] FIG. 1. B. animalis subsp. lactis BPL1 functional activity relies on cell envelop component/s. A) Protease treatment of BPL1 cells. B) BPL1 cells treated with vancomycin and ampicillin at doses below their MIC. C) Glucose restriction (15 g/L, 10 g/L) conditions at the culture media (MRS-Cys). D) Cell growth curves of BPL1 strain obtained by quantification of OD at 600 nm in standard MRS-Cys culture medium (20 g/L glucose) or MRS-Cys at low glucose (10 g/L). Glucose levels were estimated at different times along the growth curve. Data are mean±sd. and were calculated from two biological independent experiments. E) Restriction of Fructose, saccharose, lactose, maltose, or galactose (10 g/L) at the culture media (MRS-Cys) F) Adherence to Caco-2 epithelial cells. BPL1 cultures at 5×10.sup.8 CFU/mL grown in glucose 10 g/L (restricted) or 20 g/L (standard) were used in the adhesion assays. Data are mean±sd of three independent experiments. For A), B), C), percentage of fluorescence in bacterial-fed nematodes (Wild-type-strain N2) is represented. Nile red was quantified at young adult stage. Orlistat (6 μg/mL) was used as positive control. Data are mean±sd and were calculated from two independent biological experiments. *P<0.05; **P<0.01; ***P<0.001, NS: not significant.

[0207] FIG. 2. Crude cell wall fraction from BPL1 exerts fat-reducing activity. Preparations of cell wall fraction of BPL1, of cells grown in standard conditions (MRS-Cys with 20 g/L of glucose) and low glucose (10 g/L). Percentage of fluorescence in cell wall fraction-fed nematodes (Wild-type-strain N2) is represented. Nile red was quantified at young adult stage. Orlistat (6 μg/mL) was used as positive control. Data are mean±sd. and were calculated from two independent biological experiments. **P<0.01; ***P<0.001, NS: not significant.

[0208] FIG. 3. Lipoteichoic acid (LTA) from BPL1 strain demonstrates fat reducing activity and preserves this capacity under different treatments. A) LTA fraction obtained from BPL1 in standard MRS-Cys (20 g/L) and from BPL1 cells grown in low glucose MRS-Cys (10 g/L). B) Heating or lyophilization of LTA fraction from BPL1 cells did not impact on its functionality. C) Fat-reducing effect of purified LTA at different doses. D) Representative images of Nile red staining of lipid content in live young adult C. elegans in a Wild-type N2 animal under fluorescence microscopy. Nematodes were fed with BPL1 cells, heat-treated BPL1 cells (HT-BPL1) or LTA. Scale bar 250 μm. Original image taken by the authors for this paper with a Nikon-SMZ18 Fluorescence Stereomicroscope. E) Quantification of triglyceride content (mM TG/mg protein) in C. elegans fed with purified LTA from BPL1. F) Purified LTA obtained from BPL1 in standard MRS-Cys (20 g/L) and from BPL1 cells grown in low glucose MRS-Cys (10 g/L). BPL1 cells grown in excess or restriction of glucose were included. For A), B), C), F) percentage of fluorescence in bacterial and LTA-fed nematodes (Wild-type-strain N2) is represented. Nile red was quantified at young adult stage. Orlistat (6 μg/mL) was used as positive control. Data are mean±sd. and were calculated from two biological independent experiments. **P<0.01; ***P<0.001; NS: not significant.

[0209] FIG. 4. Lipoteichoic acid (LTA) from BPL1 strain requires the Insulin-like signaling pathway (IGF-I) to exert its fat reducing effect, and has functional activity in hyperglycemic conditions. A) Feeding worms with LTA in mutant daf-2 and daf-16 strains; the same as with the live BPL1 cells and the heat-treated BPL1 cells. B) Fat content in C. elegans mutant for SKN-1 transcription factor treated with LTA from BPL1. C) Effect of treatments on a C. elegans hyperglycemic model. Nematodes of wild-type strain N2 grown in high glucose (100 mM). Metformin was used as positive control. For A), B), C) percentage of fluorescence in LTA-fed nematodes (Wild-type-strain N2, GR1307, daf-16 (mgDf50), CB1370, daf-2 (e1370), or LG333 Skn-1 (zu 135)). Nile red staining was quantified at young adult stage. Orlistat (6 μg/mL) was used as positive control. Data are mean±sd. and were calculated from two independent biological experiments. ***P<0.001, NS: not significant.

[0210] FIG. 5. LTA from BPL1 has fat-reducing activity and the mechanical disruption provides slightly more effective LTA. Cells, both alive (BPL1) and disrupted by PANDA homogenizer or Sonication, were included as positive controls. NGM negative control, Orlistat is a fat-reducing drug used as positive control in the assay.

[0211] FIG. 6. Fat reducing effect in C. elegans of LTAs fractions obtained from B. animalis subsp. lactis BPL1 (CECT 8145). Cells, both alive (BPL1) and heat-treated (HT-BPL1), were included as positive controls. NGM negative control, Orlistat is a fat-reducing drug used as positive control in the assay.

[0212] FIG. 7. Fat reducing effect in C. elegans of LTAs fractions obtained from B. animalis subsp—lactis BPL1 (CECT 8145) and other Bifidobacterium, Lactobacillus and Bacillus strains.

[0213] FIG. 8. Analysis of fat reducing effect in C. elegans of purified LTAs obtained from other Bifidobacterium strains, B. longum ES1 and B. animalis BPL1.

[0214] FIG. 9. NMR spectrum of BPL1-LTA.

[0215] FIG. 10. Functional evaluation of BPL1 cells obtained at different growth stages and under standard glucose medium (A) or glucose restrictions (B). ***Significant P value <0.001; **Significant P value <0.01; NS: Not significant.

[0216] FIG. 11. Purification and characterization of lipoteichoic acid (LTA). (A) Screening for phosphate content of hydrophobic interaction chromatography fractions after N-butanol extraction of BPL1 cell-wall material. (B) SDSPAGE and Alcian blue/silver staining.

[0217] FIG. 12. MALDI-TOF mass spectrum.

EXAMPLES

Example 1: Reduction of Fat Deposition by Lipoteichoic Acid (LTA) from Bifidobacterium animalis subsp. lactis CECT 8145 (BPL1)

I—Material and Methods

[0218] C. elegans Strains and Maintenance

[0219] Caenorhabditis elegans N2 wild-type strain (Bristol) and the mutant strains GR1307, daf-16 (mgDf50), CB1370, daf-2 (e1370), and LG333 Skn-1 (zu 135) were provided by Caenorhabditis Genetic Center (CGC), University of Minnesota (USA).

[0220] The nematode strains were routinely propagated on Nematode Growth Medium (NGM) plates with Escherichia coli strain OP50 as a food source at 20° C. Worms were synchronized by isolating eggs from gravid adults at 20° C., and eggs were hatched in NGM plates. In the experiments for fat quantification, the worms were fed with the different compounds from egg to adult stage.

[0221] To induce hyperglycemic conditions, nematodes were grown in NGM plates supplemented with 100 mM of glucose until reaching the young adult stage.

Bifidobacterium animalis Subsp. Lactis CECT 8145 (BPL1) Culture Conditions

[0222] B. animalis subsp. lactis CECT 8145 (BPL1) strain was originally isolated from feces of breastfed healthy babies as previously described (Martorell, P., et al. 2016, J Agric Food Chem 64: 3462-3472). The present study was conducted according to the Helsinki declaration and guidelines for the ethical conduct of medical research involving children. A written informed consent was obtained from the mother after receiving written information. All experimental protocols were approved by our institution committee (Biopolis Biosafety Committee) in accordance with relevant guidelines and regulations. For standard cultivation, bacteria were grown in MRS medium (Peptone from casein, tryptic digest 10 g/L; meat extract 10 g/L; yeast extract 10 g/L; D-glucose 20 g/L; K.sub.2HPO.sub.4 2 g/L; di-ammonium hydrogen citrate 2 g/L; sodium acetate 5 g/L; MgSO.sub.4 0.2 g/L; MnSO.sub.4 0.05 g/L; Tween 80 1 g/L) supplemented with cysteine (Sigma, 0.05% wt/vol)), MRS-Cys, for 18 hours at 37° C. in an anaerobiosis atmosphere generated by GasPak™ EZ Anaerobe Container System (BD).

[0223] Escherichia coli OP50 strain was cultured in LB broth (Bacto-tryptone 10 g/L; Bacto-yeast 5 g/L; NaCl 5 g/L) for 18 hours at 37° C. The E. coli OP50 culture was ready for use in seeding NGM plates.

Functional Evaluation of BPL1 Culture Supernatant

[0224] An overnight culture of BPL1 was obtained, and cells were pelleted by centrifugation at 3220×g for 10 minutes. Supernatant was collected in a new tube and pH was adjusted to 7. Finally, the BPL1 supernatant was filtered using a 0.22 pore size μm filter and tested in a C. elegans fat reduction assay. Three different doses (50, 100 and 200 μL/plate) were tested with Orlistat (6 μg/mL) as a positive control.

DNA Isolation from BPL1

[0225] DNA from BPL1 cells was isolated from overnight cultures using the High Pure PCR Template Preparation Kit (11796828001, Roche) according to manufacturer instructions. Once DNA was isolated and quantified using Nanodrop spectrophotometer (Thermofischer), three different doses (12.5 μg/plate, 25 μg/plate and 50 μg/plate) were tested on C. elegans fat reduction assay by adding directly DNA on the top of NGM agar plates, already seeded with E. coli OP50.

Influence of Glucose

[0226] To test the influence of glucose on the functional effect of BPL1 cells, glucose concentration was modified in the MRS-Cys medium. BPL1 was grown in MRS-Cys with different concentrations of D-glucose (20, 15 and 10 g/L), maintaining the other ingredients constant (see recipes above). After 18 hours at 37° C. in an anaerobic atmosphere generated by GasPak™ EZ Anaerobe Container System (BD), bacteria cultured under these glucose conditions were tested in the C. elegans fat reduction assay. Cells were harvested by centrifugation and washed three times with saline solution Consecutively, concentrated BPL1 treated cells (OD.sub.600=30) were tested on C. elegans fat reduction assay by adding 50 μL on the top of NGM agar plates with E. coli OP50.

[0227] To test the influence of glucose contained in the culture medium on the functional effect of LTA from BPL1, glucose concentration was modified in the MRS-Cys medium. BPL1 was grown in MRS-Cys with different concentrations of D-glucose (20, 15 and 10 g/L), maintaining the other ingredients constant (see recipes above). After 18 horas at 37° C. in an anaerobic atmosphere generated by GasPak™ EZ Anaerobe Container System (BD), cells were harvested by centrifugation and washed three times with saline solution and used for LTA isolation and purification.

Influence of Other Sugars Used as Carbon Source

[0228] The influence of other sugars used as carbon source in the culture of BPL1 strain was tested in modified MRS-Cys medium. D-glucose was replaced by 20 g/L and 10 g/L of fructose, saccharose, lactose, maltose or galactose, maintaining the other ingredients constant (see recipes above). After 18 hours at 37° C. in an anaerobic atmosphere generated by GasPak™ EZ Anaerobe Container System (BD), bacteria cultured under these glucose conditions were tested in the C. elegans fat reduction assay as detailed above.

Influence of Antibiotics

[0229] BPL1 was also cultured with MRS-Cys medium supplemented with ampicillin or vancomycin, using doses below MIC to Bifidobacterium genus. MRS-Cys medium was supplemented with 0.1 μg/mL and 0.25 μg/mL of each antibiotic and BPL1 cells were cultured for 18 hours at 37° C. anaerobically. After growth, cells were harvested by centrifugation and washed three times with saline solution. Consecutively, concentrated BPL1 treated cells (OD.sub.600=30) were tested on C. elegans fat reduction assay by adding 50 μL on the top of NGM agar plates with E. coli OP50.

Enzymatic Treatments

[0230] Proteinase K treatment of BPL1 cells was performed according Gopal et al. [Gopal, P. K., et al. 2001. Int J Food Microbiol 67, 207-216]. Overnight (18 hours, 37° C. anaerobically) cultures of BPL1 were treated with Proteinase K (P6556, Sigma). After growth, cells were harvested by centrifugation and washed three times with 50 mM phosphate buffer (pH 7). Stock solution of Proteinase K (1 mg/mL) was prepared in 50 mM phosphate buffer (pH 7). Then, washed cultures were incubated with Proteinase K for 30 minutes at 37° C. After incubation, cells were recovered by centrifugation and washed three times with M9 buffer (KH.sub.2PO.sub.4 3 g/L; Na.sub.2HPO.sub.4; 6 g/L; NaCl 5 g/L; 1 mL 1 M MgSO.sub.4) in order to stop the reaction. Treated cells were concentrated (OD.sub.600=30) and tested in C. elegans for fat reduction by adding 50 μL of cells on the surface of NGM plates with E. coli OP50.

Cellular Adhesion Assays in Caco-2 Cultures

Cell Culture and Preparation

[0231] Caco-2 cells were placed in Dulbecco's modified Eagle's minimal essential medium DMEM with L-glutamine supplemented with 10% (v:v) fetal bovine serum and 1% (v:v) nonessential amino acids solution. For adhesion assay, the Caco-2 cells were seeded at a concentration of 10.sup.5 cells/well in 24-well standard tissue culture plates. The cells were maintained for 2 weeks after the confluence, when they were considered to be fully differentiated M. Pinto, S. R.-L., et al. 1983. Biol. Chem, 323-330.

Adhesion Assay

[0232] Overnight cultures of BLP1 were centrifuged to remove culture medium. The bacterial pellet was washed with PBS buffer (NaCl 8 g/L; KCl 0.2 g/L; Na.sub.2HPO.sub.41.15 g/L; KH.sub.2PO.sub.4 0.2 g/L) and resuspended in cell culture medium and checked for optical density, to give about 1×10.sup.8, 5×10.sup.8 or 1×10.sup.9 cells/mL. Bacterial cells were then incubated with Caco-2 cells for 2 hours under standard conditions. Afterwards, the unattached BPL1 cells were removed by 3-fold washing with PBS. In order to enumerate the attached bacterial cells, monolayers in each well were recovered by pipetting. Mixtures of Caco-2 cells and attached probiotics were plated on MRS-Cys agar. To this end, serial decimal dilutions ranging from 104 to 107 CFU/mL were prepared. Material from each dilution was inoculated into Petri dishes by pouring, using MRS-Cys broth. Bacterial cultures were incubated under anaerobic conditions at 37° C. for 48 hours. The bacterial cell density from the stock culture used for the adhesion assay was also estimated. After incubation, the number of adhered bacteria was quantified. Based on the results, the number of adhering bacteria per 100 Caco-2 epithelial cells was calculated.

Fat Reduction Assays in C. elegans

[0233] Caenorhabditis elegans N2 wild-type strain (Bristol) and the mutant strains GR1307, daf-16 (mgDf50), CB1370, daf-2 (e1370), and LG333 Skn-1 (zu 135) were provided by Caenorhabditis Genetic Center (CGC), University of Minnesota (USA). The C. elegans fat content was measured using the Nile red (Sigma, St. Louis, Mo., USA) staining method, following the protocol previously described Martorell, P. et al. 2016 J Agric Food Chem 64, 3462-3472. The dye was added to the surface of NGM plates seeded with OP50 at a final concentration of 0.05 μg/mL. Positive control of the assay was NGM plates with E. coli OP50 with Orlistat (6 μg/mL).

[0234] Synchronized worms were incubated in these plates for three days until reaching young adult stage. After this period, nematodes were transferred to M9 buffer and the fluorescence was measured using an FP-6200 system (JASCO Analytical Instrument, Easton, Md., USA) with a λ.sub.ex=480 nm and λ.sub.em=571 nm. Two experiments were performed per condition to analyze a total of 120 nematodes per condition/treatment.

Triglyceride (TG) Quantification

[0235] Total TGs were quantified in nematodes fed with the isolated LTA from BPL1 cultured in excess of glucose using the Triglyceride Quantification Kit (Biovision, Mountain View, Calif., USA). Age-synchronized worms were cultured in NGM plates already seeded with E. coli OP50 or NGM plates supplemented with purified LTA (10 μg/mL, 1 μg/mL and 0.1 μg/mL). Worms at young adult stage were then collected and washed using M9 buffer. After worm settling, supernatant was removed, and 400 μL of the triglyceride assay buffer was added to worm pellet. Worms were sonicated with a digital sonifier (Branson Ultrasonics Corp., Danbury, Conn., USA) using 4 pulses of 30 s at 10% power. Protein content of each condition was measured using BCA Protein Assay Kit (Thermo Scientific, Rockford, Ill., USA). Samples were slowly heated twice at 90° C. for 5 min in a thermomixer (ThermoFisher) to solubilize all TG in the solution. After brief centrifugation, aliquots (50 μL/well) were used for the triglyceride assay following the manufacturer instructions. Four different biological replicates were included for each condition in four independent experiments.

Microscopy Analysis

[0236] A fluorescent stereomicroscope was used to visualize the Nile-red stained lipid droplets of nematodes under different treatments. Populations of worms incubated from egg to young adult stage in NGM plates with Nile red (0.05 μg/mL) with the same doses of BPL1, HT-BPL1 and LTA BPL1 tested in regular C. elegans fat reduction assay. Age-synchronized worms were transferred to a new agarose 1% (wt/v) plates and fluorescence was measured in a Fluorescence Stereomicroscope (Nikon-SMZ18), equipped with NIS-ELEMENT image software. A total of 30 worms were analyzed per condition. Orlistat (6 μg/mL) was used as positive control.

Cell Wall Fraction

[0237] BPL1 overnight cultures (100 mL) were boiled for 10 minutes and then centrifuged 14000×g for 8 minutes at 4° C. Pelleted cells were resuspended in 5% (wt/vol) sodium dodecyl sulfate (SDS) and boiled for 25 minutes. Then cells were recovered by centrifugation and resuspended and boiled again in 4% (wt/vol) SDS for 15 minutes. Insoluble material was washed five times with distilled water at 60° C. Afterwards, insoluble cell wall fraction was treated with 2 mg/mL of Pronase (10165921001, Roche) in order to remove covalently attached proteins for 1 hour at 60° C. Cell wall extract was recovered by centrifugation and washed once with distilled water and then insoluble pellet was resuspended in 400 μL of 48% (vol/vol) hydrofluoric acid (HF) (339261-100 ML, Sigma) and incubated for 24 hours at 2° C. Cell wall fraction was recovered by centrifugation and washed once with 50 mM Tris-HCl (pH 7) buffer and five times with cold water to eliminate the buffer. After the last wash, the insoluble cell wall containing fraction was normalized at 10 mg/mL using a standard curve of lyophilized Micrococcus lysodeikticus (M3770, Sigma) measuring absorption at 206 nm (Schaub, R. E. & Dillard, J. P. 2017. Bio Protoc 7, doi:10.21769/BioProtoc.2438). Finally, cell wall fraction was stored at −20° C. The cell wall fraction was evaluated in C. elegans fat reduction assay at a dose of 27.5 μg/mL.

LTA Purification and Analysis

[0238] The bacteria underwent butanol extraction and hydrophobic interaction chromatography as described previously (Kho, K., and Meredith, T. C. 2018, Journal of Bacteriology 200: e00017-00018). Briefly, bacterial BPL1 cells were recovered from overnight grown cultures, by centrifugation at 12,000×g 15 minutes and washed twice with sodium citrate 50 mM pH 4.7. The bacterial pellet was suspended in sodium citrate 50 mM pH 4.7 and disrupted in a PANDA PLUS 2000 homogenizer (GEA) at 1,000 bar. Insoluble cellular material pellet was collected by centrifugation at 12,000×g 90 minutes, suspended in sodium citrate 50 mM pH 4.7 and extracted for 45 minutes 37° C. with an equal volume of 1-butanol. The aqueous phase containing LTA was retrieved, freeze-dried, and dissolved in sodium citrate 50 mM pH 4.7 and loaded onto a hydrophobic interaction chromatography column (HiTrap Octyl FF).

[0239] LTA was purified with a linear 15%-65% 1-propanol gradient in sodium citrate 50 mM pH 4.7. Fractions containing LTA were determined by a phosphate assay as described elsewhere (Draing, C., et al. 2006, J Biol Chem 281: 455 33849-33859). Phosphate positive samples were pooled and dialyzed (FIG. 11A).

[0240] Purified LTA was analyzed by GC-Q-MS, after acid hydrolysis. Briefly, LTA was dissolved at 1 g/L in HCl 2M, and acid hydrolysis conducted at 100° C. for 2 hours. D-glucose, D-galactose, glycerol and glycerol-3-phosphate were determined as their trimethylsilyl derivatives (Fiehn, O., et al. 2000, Anal Chem 72: 3573-3580). GC was conducted in an Agilent 7820A gas chromatographer using a GC column DB5-MS, coupled to a 5977B mass detector, and identification by comparison with Agilent Fiehn GC/MS Metabolomics RTL Library.

Biochemical Characterization of LTAs.

[0241] LTA molecules were characterized by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry in the proteomics facility of SCSIE University of Valencia (this proteomics laboratory is a member of Proteored PRB3 and is supported by grant PT17/0019 of the PE I+D+I 2013-2016, funded by ISCIII and ERDF). Briefly, 1 μl of a sample and 1 μl of a matrix solution (10 mg/mL CHCA in 70% ACN, 0.1% TFA) were spotted onto the sample plate. After drying, the sample was analyzed with a mass spetrometer (5800 MALDI TOFTOF, ABSciex) in reflector mode, a mass range of 1,660-1,500 m/z, with lase intensity of 6,000.

[0242] LTA was further analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of LTA was shown in the SDS-PAGE gel imaged using the cationic dye Alcian blue coupled with modified silver staining for enhanced sensitivity (Kho and Meredith, 2018, cited ad supra). The structure of the LTA was assayed by 1H nuclear magnetic resonance (NMR) spectrometry. Briefly, 1H-NMR was performed on a Bruker AVANCE III 700 Ultrasield spectrometer (Bruekr BioSpin, Rheinstetten, Germany) operating at a 1H frequency of 700.13 MHz, and equipped with a 5 mm TCl (cryoprobe) with Z-gradient. The acquisition pulse sequence used were those from Bruker Topspin 3.6 with water presaturation and 2 s recycle time. Spectra was referenced using the TSP signal at 0 ppm (FIG. 9).

[0243] The purity of the LTA was determined by measuring its endotoxin content with the Limulus amebocyte lysate assay (Lonza Bioscience, Switzerland) since the assay is insensitive to LTA (Morath, S., et al. 2001, J Exp Med 193: 393-397). DNA and RNA contaminations were determined by measuring ultraviolet (UV) absorption at 260 nm and 280 nm (NanoDrop Spectrophotometer, Thermo Fisher Scientific, USA). Sugar component of PG backbone N-acetylmuramic acid was determined by liquid chromatography analysis carried out in an Alliance 2695 HPLC System (Waters Corporation, MA, USA)) coupled to a refractive index detector (model 2414, Waters Corporation MA, USA) and an ion-exchange column (Aminex HPX-87H, Bio-Rad, CA, USA). Aminoacids ornithine, lysine and serine were determined by GC-Q-MS, after acid hydrolysis of purified LTA (HCl 2M, 100° C., 2 hours) and trimethylsilyl derivatization (Fiehn, O. et al., 2000, cited ad supra). GC was conducted in a gas chromatographer (model 7820A, Agilent, CA, USA) using a GC column DB5-MS, coupled to a 5977B mass detector, and identification by comparison with Agilent Fiehn GC/MS Metabolomics RTL Library.

[0244] For enzymatic hydrolysis of peptidoglycan, sample of purified LTA was treated (0.04 mg/mL 37° C., 4 hours) with mutanolysin (SIGMA). After filtration (Amicon Ultra-0.5, Merck Millipore, Germany) soluble muropeptides were reduced by using sodium borohydride at a final concentration of 5 mg/mL. The reaction was stopped after 20 minutes by lowering the pH to 2-4 with phosphoric acid. Fragments were separated with HPLC and a reversed phase octadecyl silica (ODS) C18 column from Phenomenex (CA, USA) Elution was conducted at 30° C. as follows: linear gradient of 0 to 100% buffer B (50 mM sodium phosphate pH5.10 with 15% (v/v) methanol) over a period of 120 minutes after 10 minutes in buffer A (50 mM sodium phosphate pH4.33) with a flow rate of 0.5 mL per minute. Eluted compounds were detected by monitoring Abs 206 nm (Schaub and Dillard, 2017, cited ad supra).

Statistical Analysis

[0245] Results are given as the mean±standard deviation. Data on fat deposition in C. elegans were analyzed by One Way Anova test, using a Tukey's multiple comparison post-test. To compare effects among different C. elegans strains, Two-Way Anova test was used. Differences between groups in cell adhesion assays were analyzed by Student's t test. All the statistical analyses were performed with GraphPad Prism 4 software, setting the level of statistical significance at 5%.

II—Results

[0246] To identify the molecule(s) of the BPL1 probiotic strain responsible for the fat reducing functional activity, we used the nematode C. elegans as a simple and rapid model to evaluate the fat reduction produced by different cellular fractions. This nematode stores lipids in hypodermic and intestinal cells, easily detected by staining (Nile red). Many proteins involved in lipid synthesis, degradation and transport are conserved between C. elegans and mammals.

[0247] A priori the fat reducing activity might be present in bacterial cells and/or in the culture supernatant. Thus, we tested firstly the culture medium, i.e., age-synchronized nematodes of the wild-type strain N2 were reared and fed with the supernatant obtained after overnight culture of the probiotic BPL1 strain in MRS+Cys medium. No effect on fat reduction was observed in these nematodes when subjected to Nile red staining and subsequent fluorescence quantification in a spectrofluorometer (data not shown). Thus, we can discard that the activity was due to a secretable or released substance during the growth.

[0248] Taking into account that one of the main components of the cell is DNA and knowing that DNA from probiotic bacteria has been shown to exert some functional activities, we assayed in C. elegans, DNA isolated from BPL1. However, DNA did not display a fat reducing effect (data not shown).

[0249] To assess whether soluble or insoluble components of BPL1 cells were responsible of its fat reducing activity, we mechanically disrupted BPL1 cells obtained from an overnight culture and generated insoluble and soluble cellular fractions that were separated by centrifugation. Fluorescence assays revealed fat reduction activity was mainly present in the insoluble fraction (data not shown). This result suggests that the compound could more probably present in the cell envelope as part of the cell wall, membrane surface associated proteins or surface associated polysaccharides, among others.

[0250] To test if some proteins associated to cell surface of BPL1 could be responsible of the fat reducing effect, whole cells of BPL1 probiotic strain were treated with proteinase K (50 μg/mL), and then used as feed for nematodes. This treatment does not affect the functional activity of the cells, suggesting that the molecule responsible for the fat reducing effect is not a surface protein or at least it is not degradable by this protease (FIG. 1A).

[0251] Having gained evidence of the functional activity of BPL1 cell envelope, we designed different strategies to investigate the functionality of the strain when cultured in different media that could modify the composition of the cell envelope. Ampicillin interferes cell wall synthesis by inhibiting the transpeptidase. Vancomycin is a glycopeptide that inhibits cell wall synthesis by blocking the transglycosilation of N-acetylmuramic acid and N-acetylglucosamine. Thus, we cultured BPL1 in the presence of sub-lethal doses of ampicillin or vancomycin and cells were evaluated for functionality. In these conditions, cells can grow but it is expected to alter the properties of the cell envelope. Interestingly, cells obtained under these culture conditions did not exert fat-reducing effects in the nematodes (FIG. 1B). This result suggests that an alteration of the cell envelope affects dramatically the fat-reducing properties of the probiotic, reinforcing the hypothesis that the compound is a component of cell envelope.

[0252] A second strategy to alter the properties of cell envelope was to use glucose restriction conditions in the culture media since glucose is a precursor in the biosynthesis of many envelope components. Cells were overnight cultured in MRS+Cys medium and formulated with different concentrations of glucose. Remarkably, BPL1 cells cultured under glucose restriction lost their fat-reducing capacity in C. elegans (FIG. 1C). The functional activity of the cells clearly differed depending on glucose availability (FIG. 1D). Interestingly, BPL1 cells grown with 20 g/L of glucose (excess of glucose) exerted fat reducing activity when collected in all growth phases but BPL1 cells cultured with restriction of glucose (10 g/L), only exerted a fat reducing effect in the initial exponential phase, when glucose was mainly available (FIG. 10A y 10B). Such observation highlights that, in some cases, probiotic functional activity may require particular environmental conditions.

[0253] Furthermore, D-glucose was replaced in MRS-Cys medium by different sugars, as fructose, saccharose, lactose, maltose or galactose. Results indicate a loss of fat reducing phenotype of cells grown under sugar restriction in all cases (10 g/L), while being functional with high amount of sugar (20 g/L) (FIG. 1E). The result suggests that the composition of cell envelope is dramatically dependent of the presence of large amounts of sugars used as substrate.

[0254] Taking into account that cell adherence could be an important property to exert the fat-reducing activity we investigated if glucose restriction might alter the ability of BPL1 cells to adhere to epithelial cells of the intestinal tract. In order to evaluate this parameter, we used a conventional assay employed to characterize probiotic adhesion potential (Darfeuille-Michaud, A. et al. 1990. Infect Immun 58, 893-902). BPL1 grown in standard MRS-Cys medium (with 20 g/L glucose) exhibited significantly greater adhesion (P<0.01%) to Caco-2 cells compared to the strain grown with 10 g/L glucose (FIG. 1D). Therefore, these results reinforce again the idea that the composition of cell envelope from BPL1 is modulated by glucose and this modulation induces large differences in cellular adhesion capacity.

[0255] At this stage we investigated which component of cell envelope could be responsible of the observed properties. In this sense, we analyzed the BPL1 cell wall fraction containing peptidoglycan (PG).

[0256] To assess the potential activity of BPL1-derived PG on C. elegans fat reduction, we prepared a cell-wall fraction from BPL1 cultured in excess of glucose (MRS-Cys medium) and under restricted glucose concentration. Our results showed that fat-reducing activity was absent when cell wall fraction was prepared from BPL1 cells grown under glucose restriction, while being functional when cells were grown under glucose conditions (FIG. 2). This result points out that cell wall fraction or a component extracted together with this fraction is responsible of fat reduction.

[0257] To analyze differences within the cell wall fractions obtained with or without glucose restriction we determined the aminoacidic composition of cell wall fraction. However, enantiomeric analysis of amino acids revealed only minor differences between both fractions (data not shown) suggesting that most probably the peptide composition of PG is not responsible for such differences.

[0258] Conversely, when we treated cell wall fraction with a muramidase that should destroy its structure we did not observe any decrease in the fat-reducing activity (data not shown) suggesting that probably a component co-purified with the cell wall fraction, but not PG, could be responsible of the activity.

[0259] We then focused our interest on lipoteichoic acid (LTA) that sometimes is found as an impurity in cell wall fractions. LTA is an important cell envelope component that is anchored to cell membranes by lipidic moieties. LTA was obtained from the BPL1 cells grown with and without glucose restriction and assessed whether glucose restriction in the BPL1 culture medium affected LTA functionality. LTA fractions were obtained from BPL1 cultures in MRS-Cys with 10 g/L and 20 g/L of glucose and evaluated for their ability to reduce fat in C. elegans. LTA extracted from BPL1 cells grown in glucose medium was active, whereas LTA isolated from BPL1 grown in restricted glucose medium was non-functional (FIG. 3A and Table 1).

TABLE-US-00001 TABLE 1 Percentage of fat reduction in C. elegans provided by LTA obtained from BPL1 and BPL1 cultured with lower doses of glucose (10 g/L). HT-BPL1-Heat-B. lactis CECT8145. Conditions % Fat Reduction BPL1 (20 g/L glucose) 31.19 HT-BPL1 26.51 BPL1 LTA (20 g/L glucose) 25.60 BPL1 LTA (10 g/L glucose) 2.26

[0260] Furthermore, the fat reducing activity of LTA fraction was assessed after heat treatment and lyophilization, both preserving its activity (FIG. 3B). Finally, the fat-reducing effect of LTA was demonstrated after purification step (FIG. 3C). Higher doses of LTA were also tested (20 and 50 μg/mL), but similar reducing effect than 10 μg/mL was observed (data not shown). Lipid storage in nematodes fed with BPL1 cells, heat treated cells (HT-BPL1) and LTA from BPL1 is shown in FIG. 3D. As triglycerides (TGs) are the main constituents in lipid droplets stored in C. elegans, and lipid accumulation has been associated with increase in TG content in this nematode (Zhang, J., et al. 2011, J Mol Biol 411: 537-553), we further quantified the TGs levels in nematodes fed with the three LTA effective doses. Results indicated a significant reduction in total TG content in animals fed with the LTA (10 μg/mL; P<0.01; 1 and 0.1 μg/mL; P<0.05) (FIG. 3E). These results support the total fat reduction observed, and is are consistent with the fat-reducing effect of the probiotic strain BPL1 (and its heat-treated form).

[0261] Furthermore, LTA was purified from BPL1 cells cultured in MRS-Cys in glucose limiting conditions, vs standard MRS-Cys medium and evaluated for their ability to reduce fat in C. elegans. LTA extracted from BPL1 cells grown in standard glucose medium was active, whereas LTA isolated from BPL1 grown in restricted glucose medium was non-functional (FIG. 3F).

[0262] Having demonstrated the efficacy of LTA from the BPL1 strain in fat reduction, next we investigated the mechanisms underlying this functional effect. We have previously reported that the fat-reducing and antioxidant activities of BPL1 cells are dependent on the IIS pathway (Martorell, P. et al. 2016. J Agric Food Chem 64, 3462-3472). Due to the evolutionary conservation of the IIS pathway, study of compounds targeting this pathway in C. elegans is likely to shed light on its function in higher organisms and humans. To investigate the role of the IIS pathway in LTA-mediated fat reduction, DAF-2 (insulin receptor)/DAF-16, the key regulators in the IIS pathway were evaluated. The fluorescence assays showed that the DAF-16 mutation (daf-16 (mgDf50)) abolished the LTA-mediated fat-reducing effect (FIG. 4A). This was also the case for the BPL1 and HT-BPL1 cells. This result strongly suggests that fat reduction induced by BPL1 is DAF-16 dependent. DAF-2, is the human insulin receptor homolog upstream DAF-16 in the IIS pathway, and as in humans, mutations in the insulin receptor increase fat accumulation, showing an obese phenotype. Our results show that feeding DAF-2 mutant (daf-2 (e1370)) worms with LTA did not produce a fat-reducing phenotype, indicating that the LTA-mediated regulation of DAF-16 is dependent on DAF-2, and therefore, LTA effect requires the insulin/IGF-1-like signaling pathway (IIS) (FIG. 4A). Here, we observed a different response both with BPL1 and HT-BPL1 that is independent of SKN-1, as fat-reducing effects were still observed in a C. elegans mutant deficient in the ortholog of mammalian Nrf transcription factor (FIG. 4B). Similarly, LTA also reduce fat content in C. elegans mutant of SKN-1 transcription factor (FIG. 4B), suggesting that do not require SKN-1 for its function.

[0263] The insulin signaling pathway is also involved in glucose transport, playing a role in glucose homeostasis and insulin sensitivity. Therefore, it is a target pathway for the study of diabetes (or obesity-related diabetes) and the compounds targeting this pathway emerge as potential therapeutics. In C. elegans, glucose has been shown to shorten lifespan by up-regulating IGF-I pathway (IIS) activity or increasing reactive oxygen species (ROS) and because of that, it has been suggested that C. elegans is a good model system to evaluate glucose toxicity and to develop more efficient diabetes therapies.

[0264] Thus, we have used high glucose-fed nematodes to model diabetes and evaluate the efficacy of LTA. Taking into account that glucose is a known precursor of triglycerides (TGs) and TGs are the main components of lipid droplets in the nematode, we evaluated the effect of glucose (100 mM) on nematode fat content. Our results showed that fat content increased by 20%, in nematodes fed on high-glucose NGM, in agreement with other reports (FIG. 4C). Metformin (biguanide), a drug used in the management of type-2 diabetes, was included as positive control. Furthermore, we evaluated whether LTA could reduce fat content in a diabetic obese C. elegans model. Hyperglycemic nematodes fed with LTA showed a significant reduction in body fat content (FIG. 4C). This effect was also recorded in nematodes fed with BPL1 and HT-BPL1 cells (FIG. 4C). These results show the potential of BPL1 cells and its heat-treated form, HT-BPL1, as ingredients for therapeutic and/or preventive uses in diabetes-related obesity. Furthermore, and for the first time, the efficacy of LTA is shown in an in vivo hyperglycemic model, highlighting its relevance not only as a functional signaling molecule but also as a postbiotic with proven beneficial effects.

[0265] Overall, our findings reveal a previously unrecognized role for LTA as a new lipid modulator exhibiting fat-reducing properties in the pre-clinical model of C. elegans.

[0266] Our study is not only the first one to show a novel beneficial biological role of LTA from genus Bifidobacterium, but also the first one that demonstrates the involvement of LTA in fat-reducing activity using C. elegans as a host model. Our results illustrate the potential of LTA obtained from BPL1 probiotic strain as a new postbiotic, having therapeutic and/or preventive application in metabolic syndrome and diabetes-related disorders. Such applications include uses as an ingredient in human nutrition, including food and beverages, nutritional supplements and also medical foods.

Example 2: LTA from BPL1 has Fat Reducing Activity and the Different Extraction Methods and Treatments Applied Preserve this Capacity

I—Material and Methods

[0267] LTA was obtained from fresh cultures of BPL1 and heat-treated cultures of BPL1, following procedures here described. First, two different cell disruption methods, sonication and PANDA Homogenizer, were compared. Nematodes were cultured in the NGM plates (control conditions) and the NGM plates supplemented with the corresponding LTA from BPL1. Fat content was measured in the different nematode's populations by Nile Red staining method, a fluorescence dye which specifically binds to lipid droplets (Martorell P, et al., 2012, J Agric Food Chem. 60:11071-9).

II—Results

[0268] As can be seen from FIGS. 5 and 6, LTA from BPL1 cultured in excess of glucose has fat-reducing activity and the mechanical disruption provides slightly more effective LTA.

Example 3: LTA Extracted from Bifidobacteria, and LTAs Extracted from Lactobacilli and Bacilli Strains are Shown to Exhibit Fat-Reducing Capacity. Bifidobacterial LTAs Exhibit Higher Fat-Reducing Capacity

I—Material and Methods

Isolation of LTA

[0269] Bifidobacterium and Lactobacillus strains were cultured in MRS (10 g/L meat extract, 5 g/L yeast extract, 20 g/L D-glucose, 2 g/L K2HPO4, 2 g/L Di-ammonium hydrogen citrate, 5 g/L Sodium Acetate, 0.2 g/L MgSO4, 0.05 g/L MnSO4, 1 g/L Tween 80) supplemented with 0.05% cysteine). The commercial medium Brain heart infusion CM1135 B (Oxoid) was used to grow Bacillus strains. Cultures were overnight incubated under anaerobic conditions at 37° C. (except Bacillus strains, incubated in aerobic conditions). Cultures were adjusted to 1×10.sup.9 cells/mL and washed three times with saline solution. LTA extractions were adapted from Colagiorgi et al. 2015 (Colagiorgi A, et al., 2015. FEMS Microbiol Lett. 362: fnv141). Cells were harvested and mechanically disrupted in a PANDA Homogenizer. Afterwards, bacterial lysates were mixed with an equal volume of n-butanol and stirred from 30 minutes at room temperature. Phases were separated by centrifugation for 20 minutes at 13000×g at 4° C. The aqueous phase was recovered and subjected to freeze-drying or a further purification step.

[0270] For purification, lyophilized samples were suspended with chromatography start buffer (15% n-propanol in 0.1 M ammonium acetate, pH 4.7), and centrifuged at 10,000 rpm for 60 minutes and sterilized by membrane filtration (0.2 μm). The supernatant was subjected to hydrophobic interaction chromatography (HIC) on octyl-Sepharose column (GE Healthcare Life Sciences, UK). Elution was conducted by linear gradient from start buffer to elution buffer (60% n-propanol in 0.1 M ammonium acetate, pH 4.7). The obtained fractions were assessed by a molybdenum blue test to detect phosphate-containing fractions, which were pooled and lyophilized (Villéger R, et al. 2014. Antonie Van Leeuwenhoek. 106:693-706).

II—Results

[0271] LTA was obtained from B. animalis subsp. lactis BPL1 as described above and compared with other strains belonging to Bifidobacterium, Lactobacillus and Bacillus genera.

[0272] LTAs obtained were added to the nematode culture (NGM). Worms were fed in the different conditions and total fat content was measured by Nile Red staining, a fluorescence dye which specifically binds to lipid droplets, following the same protocol described in example 1.

[0273] Results indicated that, among Bifidobacterium strains, LTA from B. animalis subsp. lactis BPL1 was the most effective (30.79% of fat reduction), clearly showing the superior activity of BPL1 LTA versus other Bifidobacteria. Moreover, the LTA obtained from Bifidobacterium strains exhibited higher fat-reducing effect compared with the LTA from lactobacilli and bacilli strains (FIG. 7 and Table 2).

TABLE-US-00002 TABLE 2 Percentage of fat reduction in C. elegans provided by LTA obtained from BPL1 and other Bifidobacterium, Lactobacillus and Bacillus strains. % Fat Conditions Reduction LTA-Bifidobacterium animalis subsp. lactis (CECT8145 or BPL1) 30.79 LTA-Bifidobacterium animalis (BPL30) 21.05 LTA-Bifidobacterium longum (CECT 7347 or ES1) 26.71 LTA-Lactobacillus casei (BPL4) 13.75 LTA-Lactobacillus rhamnosus (LGG) 14.56 LTA-L. rhamnosus (BPL15) 6.51 LTA-L. rhamnosus (CNCM-i4036) 2.85 LTA-L. rhamnosus (CNCM-i4034) 3.55 LTA-Bacillus subtilis 168 16.65 LTA-B. subtilis CECT35 15.29 LTA-B..subtilis BPL83 9.79

[0274] LTAs from other bifidobacterial strains were isolated and purified following the previously described protocols. LTA from B. longum ES1 (CECT7347) was functionally evaluated for its fat-reducing capacity in C. elegans. Results showed that ES1-LTA significantly reduced nematode's fat content, but in a lesser extent than BPL1-LTA (FIG. 8).

Example 4: Fat-Reducing LTA (from BPL1) Shows Specific Structural Features when Cultured in Excess of Sugars as Carbon Source

[0275] I—Material and Methods

Isolation of the LTA from Bifidobacteria Culture in Excess of Glucose

[0276] The LTA from BPL1 was obtained as described in the above Examples.

Analysis of the LTA

[0277] The structure and composition of LTA obtained from BPL1 has been determined in accordance to the methods described in the literature and shown to contain D-glucose, D-galactose, glycerol, phosphorous and alanine.

[0278] Briefly, 1H nuclear magnetic resonance (NMR) experiments were performed on a Bruker AVANCE III 700 Ultrasield spectrometer (Bruekr BioSpin, Rheinstetten, Germany) operating at a 1H frequency of 700.13 MHz, and equipped with a 5 mm TCl (cryoprobe) with Z-gradient. The acquisition pulse sequence used were those from Bruker Topspin 3.6 with water presaturation and 2 s recycle time. Spectra were referenced using the TSP signal at 0 ppm.

[0279] D-glucose, D-galactose, glycerol, glycerol-P and total alanine were determined as their trimethylsilyl derivatives after acid hydrolysis of samples by gas chromatography performed with an Agilent 7820A gas chromatography system coupled to a 5977B mass detector using a DB5-MS column. The identification was carried out by comparison the retention time and spectral mass with those included in the Agilent Fiehn GC/MS Metabolomics RTL Library.

[0280] L- and D-isomers of alanine were determined liquid chromatography analysis carried out in an Alliance 2695 HPLC system, from Waters, coupled to a photodiode array detector (model 2996, Waters). The separation takes place in the chiral column, Chirex 3126 (D)-penicillamine, from Phenomenex, and the identification is by retention time and absorption spectrum, compared to D- and L-Alanine analytical standards

II—Results

[0281] The amount of the different chemical constituents of BPL1 LTA was determined by the techniques above described as is known to the skilled in the state of the art. Briefly, the amount of galactose, glycerol, alanine, glycerol-P, and phosphorous was determined in purified LTA samples, coming from BPL1 cultures grown in the presence of excess glucose (BPL1, 20 g/L glucose) and in limitation of glucose (BPL1, 10 g/L glucose).

[0282] As shown in Table 3, the relative content of glycerol, and glycerol-phosphate, vs glucose, and alanine, vs glucose, was found to be surprisingly higher in the LTA sample purified from BPL1 grown in excess of glucose, and exerting fat reduction in C. elegans.

TABLE-US-00003 TABLE 3 Compositional analysis of BPL1 LTA in different growth conditions. Source of Molar ratio to glucose % Fat LTA Galactose Glycerol Alanine Glycerol-P Phosphorous reduction B. animalis 0.8 2.2 1.0 12.6 26.5 26.9 BPL-1 (20 g/L glucose) B. animalis 0.6 0.7 0.2 1.1 2.6 2.0 BPL-1 (10 g/L glucose)

TABLE-US-00004 TABLE 4 Alanine isomer distribution % of total Alanine Source of LTA L-Alanine D-Alanine B. animalis BPL-1 (20 g/L glucose) 32.4 67.6 B. animalis BPL-1 (10 g/L glucose) 100 < LOD

[0283] Results are shown in FIG. 8.

[0284] To test this, we isolated and purified LTA from BPL1 strain overnight cultures grown in MRS-Cys .Isolation and purification of LTA was conducted by butanol extraction of the insoluble fraction of BPL1 cellular lysates, followed by hydrophobic exchange chromatography, as previously described to obtain high quality LTA (Morath, et al., 2001 (cited ad supra); Draing, et al. 2006 (cited ad supra); Gründling and Schneewind, 2007, Proceedings of the National Academy of Sciences 104: 8478-8483; Kho and Meredith, 2018 (cited ad supra)). A single elution peak was obtained by phosphate assay (FIG. 11A). The fractions forming the peak were pooled and used as purified LTA. The purity of LTA with respect to potential cross-contaminants, cell-wall material and nucleic acid was determined by specific analysis. Endotoxin contamination was excluded through an LAL assay and endotoxin content was <5 EU/mg in the lyophilized LTA. Nucleic acids contamination was determined by measuring UV absorption at 260 nm and 280 nm. DNA/RNA accounted for less than 1.5% wt/wt. Peptidoglycan contamination was excluded by analysis of derived muropeptides after mutanolysin treatment in which no peaks of peptidoglycan fragments were detected. In addition, N-acetylmuramic acid (MurNAc) or the aminoacids ornithine, lysine and serine, known to be present in the PG of BPL1 (previously determined by 1 D- and 2D-TLC of the total hydrolysate of the peptidoglycan of BPL1, data not shown), were not detected by gas or liquid chromatography of the total hydrolysate of purified LTA. To further characterize purified LTA, a sample was stained through Alcian blue/silver staining following SDS-PAGE (FIG. 11B), and analyzed using MALDI-TOF mass spectrometry and nuclear magnetic resonance (FIG. 12), revealing a compound with an estimated molecular mass distribution in the range of 8-10 kDa.