Strain of <i>Bifidobacterium animalis </i>subsp. lactis CECT 8145 and use thereof for the treatment and/or prevention of excess weight and obesity and associated diseases

Abstract

The invention is applicable within the food and pharmaceutical industry. More specifically, it relates to a novel strain of the species Bifidobacterium animalis subsp. lactis CECT 8145, the cell components, metabolites and secreted molecules thereof, which, incorporated into food and/or pharmaceutical formulations, can be used in the treatment and/or prevention of excess weight and obesity and related diseases such as metabolic syndrome, hypertension, glycemia, inflammation, type 2 diabetes, cardiovascular diseases, hypercholesterolemia, hormonal alterations, infertility, etc.

Claims

1. A composition comprising: a strain belonging to the species Bifidobacterium animalis subsp. Lactis of accession number CECT8145; and a food formulation selected from the group consisting of fruit juice, vegetable juice, ice cream, infant formula, milk, yogurt, cheese, fermented milk, powder milk, cereals, bakery products, cereal-based products, nutritional supplements, soft drinks and dietary supplements.

2. The composition according to claim 1, wherein the strain is in the form of viable cells.

3. The composition according to claim 1, wherein the strain is in the form of nonviable cells.

4. The composition according to claim 1, wherein the strain is present in an amount of between 10.sup.5 CFU and 10.sup.12 CFU per gram or millilitre of the composition.

5. The composition according to claim 1, wherein the composition is a pharmaceutical composition.

6. The composition according to claim 1, comprising at least one other microorganism selected from the group consisting of Lactobacillus, Streptococcus, Bifidobacterium, Saccharomyces, Kluyveromyces, L. rhamnosus, L. delbrueckii subsp. bulgaricus, L. kefir, L. parakefir, L. brevis, L. casei, L. plantarum, L. fermentum, L. paracasei, L. acidophilus, L. paraplantarum, L. reuteri, S. thermophilus, B. longum, B. breve, B. bifidum, B. catenulatum, B. adolescentis, B. pseudocatenulatum, S. cerevisiae, S. boulardii, K. lactis, and K. marxianus.

7. A method for the treatment of overweight, obesity, or related diseases comprising administering to a subject in need thereof a therapeutically effective amount of the composition according to claim 1.

8. The method according to claim 7, wherein the related diseases are selected from the group consisting of metabolic syndrome, hypertension, glycemia, inflammation, type-2 diabetes, cardiovascular disease, hypercholesterolemia, hormonal disorders and infertility.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Screening of 23 strains of the genus Lactobacillus for body-fat reduction in C. elegans.

(2) FIG. 2: Screening of 15 strains of the genus Bifidobacterium for body-fat reduction in C. elegans.

(3) FIG. 3: Quantification of triglycerides in C. elegans wild-type N2 fed on strain CECT8145 (BIF-1) or given a control diet (nematode growth medium, hereafter NG medium).

(4) FIG. 4: Effect of a culture of strain CECT8145 (BIF-1) inactivated at 70 C. overnight on body-fat reduction in C. elegans.

(5) FIG. 5: Antioxidant activity of strain CECT8145 (BIF-1) estimated after subjecting C. elegans (wild type N2) to oxidative stress by applying hydrogen peroxide.

(6) FIG. 6: Reduction in body fat relative to C. elegans (wild type N2) and mutants.

(7) FIG. 7: Determination of body weight in obese Zucker rats treated with 10.sup.10 CFU/day (.square-solid.) of the strain CECT8145 (BIF-1) during the 17-week trial. A control group of obese Zcker rats (.circle-solid.) and a group of lean Zcker rats () were included in the trial.

(8) FIG. 8: Solid intake observed in obese Zucker rats treated with 10.sup.10 CFU/day (.square-solid.) of strain CECT8145 (BIF-1). A control group of obese Zcker rates (.circle-solid.) and a group of lean Zcker rats () were included in the trial.

(9) FIG. 9: Total cholesterol in obese Zucker rats treated with 10.sup.10 CFU/day of strain CECT (BIF-1) (gray bar), compared with control Zcker rats (black bar). A control group of lean Zcker rats (white bar) was included in the trial.

(10) FIG. 10: HDL cholesterol in obese Zucker rats treated with 10.sup.10 CFU/day of strain CECT8145 (BIF-1) (gray bar), compared with control Zcker rats (black bar). A control group of lean Zcker rats (white bar) was included in the trial.

(11) FIG. 11: Ratio total cholesterol:HDL cholesterol (Cardiovascular Risk Index) determined in obese Zucker rats treated with 10.sup.10 CFU/day of strain CECT8145 (BIF-1) (gray bar), compared with control Zcker rats (black bar). A control group of lean Zcker rats (white bar) was included in the trial.

(12) FIG. 12: Triglyceride concentration determined in obese Zucker rats treated with 10.sup.10 CFU/day of strain CECT8145 (BIF-1) (gray bar), compared with control Zcker rats (black bar). A control group of lean Zcker rats (white bar) was included in the trial.

(13) FIG. 13: Glucose concentration determined in obese Zucker rats treated with 10.sup.10 CFU/day of strain CECT8145 (BIF-1) (gray bar), compared with control Zcker rats (black bar). A control group of lean Zcker rats (white bar) was included in the trial.

(14) FIG. 14: Levels of TNF (marker of inflammation) in obese Zcker rats treated with 10.sup.10 CFU/day of strain CECT8145 (BIF-1) (gray bar), compared with control Zcker rats (black bar). A control group of lean Zcker rats (white bar) was included in the trial.

(15) FIG. 15: Adiponectin levels in obese Zucker rats treated with 10.sup.10 CFU/day of strain CECT8145 (BIF-1) (gray bar), compared with control Zcker rats (black bar). A control group of lean Zcker rats (white bar) was included in the trial.

(16) FIG. 16: Concentration of malondialdehyde (marker of oxidation) determined in obese Zucker rats treated with 10.sup.10 CFU/day of strain CECT8145 (BIF-1) (gray bar), compared to control Zcker rats (black bar). A control group of lean Zcker rats (white bar) was included in the trial.

(17) FIG. 17: Ghrelin levels (marker of appetite) determined in obese Zucker rats treated with 10.sup.10 CFU/day of strain CECT8145 (BIF-1) (gray bar), compared to control Zcker rats (black bar). A control group of lean Zcker rats (white bar) was included in the trial.

(18) FIG. 18: Resistance of strain BIF-1 to acidic pH levels.

(19) FIG. 19: Resistance of strain BIF-1 to bile salts.

(20) FIG. 20: Yogurt fermented with strain BIF-1 produces greater body-fat reduction in C. elegans (11.4%) than conventional commercial yogurt.

(21) FIG. 21: Fat-reducing effect of soymilk fermented with strain BIF-1 in C. elegans.

(22) FIG. 22: Fat-reducing effect of juice with strain BIF-1, live and inactivated cells, in C. elegans.

EXAMPLES

Example 1

(23) Screening Bacteria for Body-Fat Reduction in Caenorhabditis elegans.

(24) Twenty-three strains of the genus Lactobacillus and 15 strains of the genus Bifidobacterium was screened to analyze their effect on body-fat reduction after being ingested by the nematode Caenorhabditis elegans. Two commercial strains were included in the study, LGG (Lactobacillus rhamnosus) and Bb12 (B. animalis subsp. lactis).

(25) Caenorhabditis elegans accumulates fat in the form of droplets that can be visualized by staining with Nile red (fluorescent). The fluorescence emitted from said dye can be quantified by fluorimetry. Therefore, various microorganisms were assessed for their effect on body-fat accumulation and/or reduction in the nematode by analyzing the reduction in fluorescence in worms fed with different strains, compared to worms fed under control conditions (NG medium+Escherichia coli).

(26) The experiments consisted of feeding C. elegans with different microorganisms, for the period lasting from the egg to the young adult stage (3 days old). The standard feed was NG medium sown with the bacterium Escherichia coli.

(27) Fat droplets were stained by direct addition of Nile red dye to the plates of NG medium. Nematodes were incubated at 20 C. under the different feeding conditions throughout the test period. After the feeding period, samples of each condition were taken and the fluorescence emitted in each case was quantified. The control feeding condition (NG medium+Escherichia coli) was taken as reference to quantify and compare fluorescence under the experimental conditions.

(28) FIG. 1 shows the results obtained with Lactobacillus strains for body-fat reduction in C. elegans (expressed as a percentage of fluorescence reduction quantified in worms stained with Nile red dye.) The highest fat-reduction percentage corresponded to the LAC-1 strain (32.4% compared to control feeding conditions).

(29) FIG. 2 shows the screening of Bifidobacterium strains. The most effective strain for body-fat reduction was BIF-1 (40.5% compared to control feeding conditions).

(30) Based on the results obtained from the 38 strains tested, the strain Bifidobacterium BIF-1 was selected as the most effective against fat reduction. Accordingly we studied the functional and technological properties of this strain in greater depth.

Example 2

(31) Taxonomic Identification and Genomic Sequencing

(32) 2.1. Identification

(33) Strain BIF-1 was identified unambiguously at genus and species level by sequencing the ribosomal DNA (rDNA) 16S. The sequence was identified by comparing the BIF-1 strain sequence with the complete gene sequences deposited in public databases using the BLAST online (http://blast.ncbi.nlm.nih.gov/Blast.cgi), the highest homology (99%) was obtained with public sequences belonging to the species B. animalis subsp. lactis.

(34) 2.2. Genome Sequencing

(35) In order to characterize the genomic level and safety and functionality of strain BIF-1 we performed whole-genome sequencing of strain BIF-1 by pyrosequencing on a Life Science-Roche 454 platform. A total of 434,581 raw sequences were obtained. Further de novo sequence assembly organized sequences on five scaffolds, the largest being 1,923,368 nucleotides. The genome size of strain BIF-1 is estimated at 2.1 Mb. Genes encoding virulence factors were not detected nor were antibiotic resistance genes located in areas at risk of horizontal transfer.

Example 3

(36) Quantification of Triglyceride Reduction in BIF-1-Treated C. Elegans

(37) The effect of strain BIF-1 ingestion on triglyceride reduction was analyzed in C. elegans wild-type N2.

(38) Triglycerides were determined from synchronized young adult C. elegans populations. Nematodes from each condition were washed in PBS buffer and sonicated for lysate. Lysed samples were used to determine total triglycerides using a commercial kit based on fluorimetric determination. All samples were normalized for protein concentration.

(39) FIG. 3 shows triglyceride quantification for nematodes under control feeding conditions (NG medium) or fed on strain BIF-1. A reduction was observed in total triglycerides in the BIF-1-fed nematodes.

Example 4

(40) Body-Fat Reduction in C. elegans Treated with an Inactivated Culture of BIF-1

(41) The fat-reducing functional effect of inactivated BIF-1 cells was analyzed in C. elegans. Cells were inactivated by heat treatment at 70 C. for 18 hours.

(42) The tests consisted in feeding C. elegans with activated or inactivated BIF-1 from the egg to the adult stage (3 days). In control conditions, nematode were fed NG medium, containing Escherichia coli.

(43) Fat droplets were stained by direct addition of Nile red dye to the plates of NG medium. Nematodes were incubated at 20 C. under the various conditions during the test period. After the feeding period, samples were taken of each condition and the fluorescence emitted in each case was quantified. The control feeding condition (NG medium+Escherichia coli) was taken as a reference to quantify fluorescence of the other experimental conditions.

(44) The results (FIG. 4) show that cells of BIF-1 inactivated at 70 C. maintained their fat-reducing effect in the nematode, as the same percentage of fluorescence was observed as in live BIF-1 culture.

Example 5

(45) Antioxidant Activity of Strain BIF-1 in C. elegans

(46) We analyzed whether the ingestion of strain BIF-1 increased resistance to acute oxidative stress in C. elegans (wild-type N2).

(47) The tests were carried out following the Methodology described by Martorell et al. (2011). C. elegans wild-type N2 was used. Trials included a control (NG medium+E. coli strain OP50) and the BIF-1 strain. Trials were started with age-synchronized nematode populations, which were cultured in NG plates under the different feeding conditions. The plates were incubated at 20 C. for 7 days. After this period, oxidative stress was applied with H.sub.2O.sub.2 (2 mM), and nematode viability was determined after 5 hours of incubation. FIG. 5 shows the results obtained in nematode survival after applying hydrogen peroxide stress. Nematodes fed for 7 days with BIF-1 were much more resistant to oxidative stress, with increased survival as compared to the population under control-feeding conditions.

Example 6

(48) Transcriptomic Study in C. elegans with the Strain Bifidobacterium animalis subsp. lactis BIF-1

(49) We studied the effect of B. animalis subsp. lactis BIF-1 ingestion on the C. elegans transcriptome. Technology chips were used to study changes in gene expression, in metabolic pathways and biological processes in nematodes fed BIF-1 as compared to nematodes under control feeding conditions. The significance level P0.05 was used in the statistical analysis.

(50) 6.1. Differential Gene Expression in BIF-1-Treated Nematodes

(51) Nematodes fed strain BIF-1 showed a different gene-expression profile compared to nematodes under control feeding conditions. Thus, they presented 296 over-expressed genes and 26 under-expressed genes compared to control nematodes (Table 1).

(52) TABLE-US-00001 TABLE 1 Differential gene expression observed in C. elegans fed the BIF-1 strain. Number genes Number genes under- without differential Number over- expressed expression expressed genes BIF-1-treated 26 22303 296 vs Control

(53) Screening of the 296 genes over-expressed in BIF-treated nematodes revealed different functional groups. The aforementioned genes are related to proteolysis, reproduction, embryonic development, carbohydrate metabolism, molting cycle, body morphogenesis, locomotion, redox processes, protein metabolism, transport, glutathione metabolism, aromatic amino acid metabolism, response to gamma radiation, fatty acid metabolism and neuropeptide signalling pathways.

(54) The 26 under-expressed genes in BIF-1-treated C. elegans are mainly related to upregulation of growth.

(55) 6.2. Metabolic Pathways

(56) Concerning the metabolic pathways, it was determined that Nematodes fed BIF-1 exhibited 23 upregulated and 20 downregulated metabolic pathways compared to control nematodes (Table 2).

(57) Tables 3 and 4 list the upregulated or downregulated metabolic pathways after treatment with the BIF-1 bifidobacteria strain.

(58) TABLE-US-00002 TABLE 2 Number of metabolic pathways differentially expressed in C. elegans fed strain BIF-1. Number Number Number unaffected upregulated downregulated metabolic metabolic metabolic pathways pathways pathways BIF-1-treated 20 55 23 vs. control

(59) TABLE-US-00003 TABLE 3 List of upregulated metabolic pathways in C. elegans after BIF-1 treatment compared with the Control. ID: identification according to KEGG database. ID KEGG Metabolic pathways upregulated in BIF-treated vs. Control 00190 Oxidative phosphorylation 00480 Glutathione metabolism 00982 Drug metabolism - cytochrome P450 00980 Metabolism of xenobiotics by cytochrome P450 00983 Drugs metabolism - other enzymes 00670 Folate biosynthesis (vitamins and cofactors metabolism) 04142 Lysosome 00260 Glycine, serine and threonine metabolism 00330 Arginine and proline metabolism 00860 Porphyrin and chlorophyll metabolism 00270 Cysteine and methionine metabolism 01040 Unsaturated fatty acid biosynthesis 00040 Pentose and glucuronate interconversions 04146 Peroxisome 00590 Arachidonic acid metabolism 00053 Ascorbate and aldarate metabolism 00514 Other types of O-glycan biosynthesis 00910 Nitrogen metabolism 00250 Metabolism of alanine, aspartate and glutamate 00380 Tryptophan metabolism 00620 Pyruvate metabolism 00650 Butanoate metabolism 00410 Beta-alanine metabolism

(60) TABLE-US-00004 TABLE 4 List of downregulated metabolic pathways in BIF-1-treated C. elegans compared to the Control. ID: identification according to KEGG database. Metabolic pathways downregulated in BIF-treated vs. ID KGGE Control 04330 Notch signalling pathway 03440 Homologous recombination 04340 Hedgehog signalling pathway 03410 Damaged DNA repair (base excision repair) 04310 Wnt signalling pathway 03018 RNA degradation 04710 Circadian rhythm 04150 mTOR signalling pathway 03430 Damaged-DNA repair (mismatch repair) 03420 Nucleotide excision repair 03050 Proteasome 03013 RNA transport 04350 TGF-beta signalling pathway 03015 mRNA surveillance pathways 03040 Spliceosome 04120 Ubiquitin-mediated proteolysis 03030 DNA replication 04141 Protein processing in endoplasmic reticulum 04144 Endocytosis 04914 Progesterone-mediated oocyte maturation

(61) 6.3. Biological Processes

(62) In nematodes fed strain BIF-1, a total of 26 biological processes were over-expressed while 76 processes were under-expressed as compared to the Control (Table 5).

(63) TABLE-US-00005 TABLE 5 Number of biological processes differentially expressed in C. elegans fed strain BIF-1 compared to the Control. Under-expressed GO Over-expressed GO BIF-1-treated vs. Control 76 26

(64) Tables 6 and 7 list of the over-expressed and under-expressed processes in BIF-1-treated nematodes in detail.

(65) TABLE-US-00006 TABLE 6 List of the 26 biological processes over-expressed in BIF-1-treated C. elegans. GO: Gene Ontology (database). GO Name GO: 0030259 Lipid glycosylation GO: 0006937 Regulation of muscle contraction GO: 0042775 Mitochondrial ATP synthesis coupled to electron transport chain GO: 0009156 Ribonucleoside monophosphate biosynthetic processes GO: 0034220 Transmembrane ion transport GO: 0009072 Aromatic amino acid metabolism processes GO: 0030241 Skeletal muscle myosin thick filament assembly GO: 0009112 Nucleobases metabolism processes GO: 0015992 Proton transport GO: 0006508 Proteolysis GO: 0040018 Positive regulation of multicellular organism growth GO: 0034607 Behavior involved in mating GO: 0007218 Neuropeptide signalling pathway GO: 0046942 Carboxylic acid transport GO: 0072529 Catabolic processes of pyrimidine containing compounds GO: 0042398 Modified amino acid biosynthetic process GO: 0015833 Peptide transport GO: 0006754 ATP biosynthesis processes GO: 0009063 Cellular amino acid catabolic process GO: 0048521 Negative regulation of behaviour GO: 0055074 Calcium ion homeostasis GO: 0006637 Acyl-CoA metabolic processes GO: 0042338 Cuticle development involved in collagen and cuticulin-based cuticle molting cycle GO: 0006814 Sodium ion transport GO: 0036293 Response to decreased oxygen levels GO: 0009069 Serine family amino acid metabolic process

(66) TABLE-US-00007 TABLE 7 List of the 76 biological processes under-expressed in BIF-1-treated C. elegans compared with the Control. GO: Gene Ontology (database). GO Name GO: 0016477 Cell migration GO: 0008406 Gonad development GO: 0040027 Negative regulation of vulva development GO: 0042127 Regulation of cell proliferation GO: 0040020 Regulation of meiosis GO: 0006511 Ubiquitin-dependent protein catabolic process GO: 0045167 Asymmetric protein localization during cell fate GO: 0000070 Mitotic sister chromatid segregation GO: 0051729 Germinline cell cycle GO: 0007052 Mitotic spindle organization GO: 0007098 Centrosome cycle GO: 0070918 Production of small RNA involved in gene silencing GO: 0045144 Meiotic sister chromatid segregation GO: 0032465 Regulation of cytokinesis GO: 0000079 Regulation of cyclin-dependent protein serine/threonine kinase activity GO: 0009410 Response to xenobiotics GO: 0030261 Chromosome condensation GO: 0007606 Sensory perception of chemical stimulus GO: 0035046 Pronuclear migration GO: 0090387 Phagolysosome assembly involved in apoptotic cell clearance GO: 0045787 Positive regulation of cell cycle progression GO: 0006261 DNA replication GO: 0006898 Receptor-mediated endocytosis GO: 0001714 Cell fate GO: 0032320 Positive regulation of GTPase activity GO: 0000281 Cytokinesis after mitosis GO: 0090068 Positive regulation of cell cycle process GO: 0030703 Eggshell formation GO: 0018991 Oviposition GO: 0006997 Nucleus organization GO: 0000132 Mitotic spindle orientation GO: 0040022 Germline GO: 0006030 Chitin metabolism GO: 0032506 Cytokinesis GO: 0032880 Regulation of protein localization GO: 0040015 Negative regulation of multicellular organism growth GO: 0045944 Positive regulation of transcription GO: 0008630 DNA damage response GO: 0000122 Negative regulation of transcription GO: 0043066 Negative regulation of apoptosis GO: 0010638 Positive regulation of organelle organization GO: 0000398 Intron elimination/mRNA splicing via spliceosome GO: 0042464 Dosage compensation by hypoactivation of X chromosome GO: 0007127 Meiosis GO: 0042693 muscle cells fate commitment GO: 0032012 Regulation of ARF protein signal transduction GO: 0006310 DNA recombination GO: 0038032 G-protein coupled receptor signalling pathway GO: 0016331 Morphogenesis of embryonic epithelium GO: 0007219 Notch signalling pathway GO: 0008356 Asymmetric cell division GO: 0042026 Protein refolding GO: 0007040 Lysosome organization GO: 0045595 Regulation of cell differentiation GO: 0032446 Protein modification by small protein conjugation GO: 0034968 Histone methylation GO: 0008595 Specification of the anterior/posterior axis in embryo GO: 0001703 Gastrulation with mouth forming GO: 0042176 Regulation of protein catabolism GO: 0006606 Protein import into the neucleus GO: 0031114 Regulation of microtubule depolymerization GO: 0007411 Axon guidance GO: 0006200 ATP catabolism GO: 0016055 Wnt receptor signalling pathway GO: 0000212 Mitotic spindle organization GO: 0006911 Phagocytosis GO: 0046777 Protein autophosphorylation GO: 0035194 Post-transcriptional gene silencing by RNA GO: 0032269 Negative regulation of cellular protein metabolism GO: 0006289 Nucleotide excision repair GO: 0006661 Phosphatidyl inositol biosynthesis GO: 0048557 Embryonic gut morphogenesis GO: 0051295 Establishment of meiotic spindle localization GO: 0006906 Vesicle fusion GO: 0030071 Regulation of mitotic metaphase/anaphase transition GO: 0051053 Negative regulation of DNA metabolism

(67) In summary, the results of the transcriptomic study show that in the nematodes fed on strain BIF-1 there was an upregulation of the metabolic pathways and processes related to carbohydrate metabolism (oxidative phosphorylation, ATP synthesis, etc.) glutathione metabolism (decreased levels of oxidative stress), biosynthesis of cofactors and vitamins, lipid metabolism, nucleotide metabolism, glycosylation and membrane metabolism.

Example 7

(68) Metabolomic Study in C. elegans on Strain BIF-1

(69) We analyzed the changes in the metabolic profile of C. elegans after ingestion of strain BIF-1 compared with the profile of Control nematodes (fed NG medium+E. coli OP50).

(70) The trials involved feeding C. elegans with strain BIF-1 from the egg to the young adult stage (3-day-old). The control feeding condition was NG medium seeded with the bacteria Escherichia coli.

(71) After this time, nematodes were subjected to a metabolomic analysis, applying analytical techniques, LC-MS/MS (ESI+) (ESI) and GC-MS, and subsequent bioinformatic processing of the data.

(72) The results showed statistically significant changes, as listed below: Glutathione (GSH) metabolism and oxidative stress: In the study, the levels of -glutamyl-leucine and -glutamyl-methionine were higher in Nematodes fed BIF-1 compared with the Control, which would be consistent with a possible increase the -glutamyl-transferase (GGT) activity and thus, recycling of glutathione (GSH) in response to BIF-1. Furthermore, ophthalmate, a metabolite used for GSH synthesis, decreased significantly in the group fed BIF-1, which is consistent with a decrease in GSH biosynthesis. This is probably due to a lower demand for glutathione produced by a lower level of oxidative stress. This is supported by the observation of lower levels of GSSG (oxidized GSH) and cysteine-glutathione disulfide, biomarkers of oxidative stress in the group fed the BIF-1 strain. Carbohydrate metabolism: The group fed BIF-1 displayed changes in many of the metabolites involved in carbohydrate metabolism. Levels maltotetraose and maltopentaose exhibited high levels, whereas trehalose-6-phosphate and glucose levels were lower in the group fed BIF-1 compared to the Control. Other pathways affected were glycogen metabolism and the pentose phosphate pathway. Thus, 6-phosphogluconate showed a significant increase in the BIF-1 group. This fact together with the high levels of ribose and low levels of ribulosa-5-phosphate are consistent with a possible upregulation of the pentose phosphate pathway in the presence of BIF-1. Nucleotide Metabolism: Changes in nucleotide metabolism are a consequence of the changes observed in the activity of the pentose phosphate pathway. Nematodes fed BIF-1 showed higher levels of N-carbamoyl-aspartate and orotate, two intermediaries in pyrimidine synthesis. Similar changes were seen in purine metabolism. Thus, BIF-1-treated nematodes showed lower levels of allantoin (product of purine degradation). In addition, the group treated with BIF-1 had higher levels of purine nucleosides (adenosine and guanosine) bases (adenine and hypoxanthine) and nucleotides [adenosine 5-monophosphate (AMP) and guanosine 5-monofosfate (GMP)]. These results together with the observed increase in precursor amino acids (glutamate and glutamine), and the possible upregulation of the pentose phosphate pathway, supports a possible increase in purine biosynthesis, accompanied by a decrease in purine degradation. Metabolism of membrane and cholesterol: In nematodes fed BIF-1, we observed increased levels of choline and acetylcholine, which are involved not only in glycosylation processes, but also in membrane metabolism. Moreover, levels of 7-dihydrocholesterol, an intermediary in cholesterol biosynthesis, were increased in nematodes fed BIF-1, which is consistent with the effect of this probiotics on the modulation of cholesterol biosynthesis. Changes in cholesterol content in the membrane may affect the receptor environment, ion channels and other membrane proteins, and thereby alter their function. Furthermore, cholesterol metabolism affects lipid and hormone-related processes. Additional observations: In C. elegans BIF-1 increased levels of phosphopantetheine, 3-dephospho-coenzyme-a, and coenzyme A (CoA). Moreover BIF-1 led to an increase in flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), consistent with the upregulation of FAD biosynthesis. CoA and FAD are involved in the metabolism of carbohydrates, lipids and amino acids.

(73) In summary, feeding strain BIF-1 to C. elegans produces a series of metabolic changes related to the antioxidant metabolism, carbohydrate and nucleotide metabolism. Glutathione metabolism appears to be a target of the probiotic BIF-1 to reduce oxidative stress levels. Furthermore, the BIF-1 diet led to an upregulation of the pentose phosphate and glycosylation pathways. Additionally alterations were observed in the metabolism of glycogen, nucleotides, lipids and cofactors.

(74) These results are consistent with those observed in the transcriptomic study (Example 6).

Example 8

(75) Identification of Differentially Expressed Genes

(76) In order to explain the mechanism of action from the transcriptomics results described in Example 6, we undertook a trial to evaluate body-fat reduction in C. elegans fed strain BIF-1. In this experiment, we employed C. elegans wild-type N2 and different C. elegans mutants in the key genes highlighted by the transcriptomic study. A gene is essential to the mechanism of action of a certain ingredient when the functional effect observed in the C. elegans wild-type N2 wholly or partly disappears in the mutant of that gene. The results shown in Table 8 (attached), and FIG. 6 identify some of the target genes mutated in C. elegans, which are differentially expressed after ingestion of BIF-1 (transcriptomic study). These results explain the biological activities affected by ingestion of the strain of the present invention.

(77) TABLE-US-00008 TABLE 8 List of target mutated genes in C. elegans. TRIALS WITH BIF 1 (B. animalis subsp lactis CECT 8145) C. elegans OBESITY (name of mutated gene % reduction compared Biological processes appears in brackets) to Control Wild-type N2 29.21 B-oxidation fatty acids VC1785(Acox-1) 15.36 in peroxisome RB2015(Acs-5) 12.59 RB859(Daf-22) 19.03 Fatty acid desaturation BX153(Fat-7) 0.56 GR1307(Daf-16) 2.63 REDOX homeostasis VC175(Sod-4) 3.63 mechanisms RB1764(Trxr-2) 3.3 Oxidative RB2434(Asg-2) 5.39 phosphorylation Tryptophan metabolism GR1321(Tph-1) 18.19

(78) FIG. 6 quantitatively illustrates the percentage of body-fat reduction in C. elegans wild-type N2 and mutant strains with differential expression of the genes listed in Table 8.

Example 9

(79) Pre-Clinical Trial in a Murine Model

(80) A trial was undertaken in an obese Zucker rat model fed three different doses of the probiotic strain BIF-1 (10.sup.8, 10.sup.9 and 10.sup.10 CFU/day), and included two groups of lean Zucker rats as Control. The trial lasted 12 weeks, body weight was determined, and the solid and liquid intake during the test period was recorded. In addition, at the end of the trial biochemical data were determined: total cholesterol, HDL cholesterol, triglycerides, TNF factor (inflammation marker), malondialdehyde (marker of oxidative stress), adiponectin and ghrelin (markers of satiety).

(81) The results are shown in FIGS. 7 to 17.

(82) In summary, the results of pre-clinical study in the murine model showed a positive effect on weight reduction in obese Zucker rats fed BIF-1 at doses of 10.sup.10 CFU/day (reduction in weight gain of 6.42% for treatment vs. control group). In addition, animals fed BIF-1 had a lower solid intake. Moreover, the determination of biochemical parameters showed a decrease in total cholesterol, accompanied with an increase in HDL cholesterol in rats fed BIF-1, as well as a slight drop in triglycerides and glucose levels. Finally, BIF-1 treatment resulted in a reduction in levels of TNF factor, malondialdehyde and ghrelin, while there was an increase of adiponectin.

Example 10

(83) Safety Study

(84) The safety of strain BIF-1 was performed following FAO/WHO guidelines (FAO/WHO, 2002). Specifically, the production of unwanted metabolites was evaluated: lactic acid isomer production (Table 9), bile-salt deconjugation (Table 10) and biogenic amine production (Table 11), and the antibiotic resistance profile (Table 12).

(85) TABLE-US-00009 TABLE 9 Production of lactic acid isomers by strain BIF-1 Lactic acid (g/L of supernatant) STRAIN D-Lactic L-Lactic BIF-1 0.020 0.000 2.158 0.025

(86) TABLE-US-00010 TABLE 10 Bile-salt hydrolysis activity by strain BIF-1 (ND: not detected). BSH activity (U.I./mg of protein BSH activity in cell extract) (U.I./ml of supernatant) Sodium Sodium Sodium Sodium STRAIN glycocholate taurocholate glycocholate taurocholate BIF-1 0.597 0.028 0.127 0.004 ND 0.0 0.0

(87) TABLE-US-00011 TABLE 11 Biogenic amine production by strain BIF-1 (ND: not detected). Biogenic amines (g/ml of supernatant) STRAIN Putrescine Cadaverine Histamine Tyramine BIF-1 ND ND ND 0.38 0.14

(88) TABLE-US-00012 TABLE 12 Minimum inhibitory concentration of antibiotics obtained for strain BIF-1. Antibiotic CMI (g/mL) Gentamicin 64 Streptomycin 128 Erythromycin 0.5 Vancomycin 1 Ampicillin 2 Tetracycline 8 Kanamycin 128 Chloramphenicol 4 Clindamycin 0.25

Example 11

(89) Probiotic Properties of Strain BIF-1

(90) One of the main requirements for a strain to be considered probiotic is that it can survive gastrointestinal transit. Therefore, strain BIF-1 was tested for its resistance to digestive conditions. Accordingly, two tests were performed: one of resistance of low pH levels and the other of resistance to bile salts. In the first, the strain was put into contact with saline solution (0.09% NaCl) at decreasing pH levels for 15 minutes and the number of live cells (FIG. 18) was counted. In the second, strain BIF-1 was put into contact with saline solution with bile salts (Oxgall) in increasing amounts (FIG. 19) for 15 min. Results of these tests did not reveal significant differences in survival rates, except for incubation at pH 4, where a slight loss of viability was detected.

Example 12

(91) Functional Yogurt Fermented with Strain BIF-1 (Bifidobacterium animalis subps. lactis CECT 8145)

(92) First, the fermentative capacity of BIF-1 was analyzed in a milk matrix. To do so, a volume of commercial skim milk was inoculated with different doses of bacteria (10.sup.6, 10.sup.7 and 10.sup.8 CFU/mL) and incubated for 24 h at 37 C. The results showed a positive fermentation of the probiotic inoculated at 10.sup.7 and 10.sup.8 CFU/mL.

(93) Subsequently, functional yogurt was made by adding 10.sup.8 CFU/mL of BIF-1 and a mixture of commercial yogurt Bifidobacteria ferments on commercial skim milk and milk powder (0.6%). A control fermentation containing only commercial yogurt strains (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) was included in the study. Presence of strain BIF-1 was checked at the end of the fermentation by selective plate counting of Bifidobacterium.

(94) Finally, to analyze the effect of the yogurt obtained on reducing body fat, a functional study was performed in the pre-clinical model C. elegans. The results show that in C. elegans, the yogurt fermented with strain BIF-1 produced a reduction in body fat higher (11.4%) than the conventional commercial yogurt (FIG. 20).

(95) Also, the same degree of body-fat reduction was determined in C. elegans fed soymilk fermented with strain BIF-1 (FIG. 21).

Example 13

(96) Juice Supplemented with Strain BIF-1 (Bifidobacterium animalis subps. lactis CECT 8145).

(97) Commercial orange juice was supplemented with different doses (10.sup.6, 10.sup.7 and 10.sup.8 CFU/mL) of active and inactive cells of BIF-1 strain (Bifidobacterium animalis subps. lactis CECT 8145). In the latter (inactivated cells), the culture was inactivated by autoclave treatment at 121 C. for 30 min. For the functional analysis, the juice supplemented with strain BIF-1 at OD: 30 was added to the surface of the culture medium of C. elegans (NG medium). We studied the effect of juice containing thermally inactivated bacteria and live bacteria on body-fat reduction in C. elegans.

(98) The results (FIG. 22) show that nematodes fed the juice supplemented with 10.sup.7 CFU/mL of live cells of strain BIF-1 experienced a reduction in body fat of 10.3% over control conditions (NG medium). Furthermore, the reduction observed in nematodes fed juice with 10.sup.7 CFU/mL of inactivated cells of strain BIF-1 was very similar, showing a 7.2% reduction in fat compared to the Control.