PROBIOTIC FOR INFANTILE COLIC

Abstract

The invention provides a bacterial composition which comprises from 10.sup.4 to 10.sup.12 cfu/g of Pediococcus pentosaceus cells which have the ability to induce the production of interleukin-10 to reduce inflammation in the intestinal tract among other features. Thus, the bacterial composition is useful in the amelioration of excessive crying in infants. Particularly, Pediococcus pentosaceus cells are from the strain deposited as CECT 8330. The bacterial composition can be in the form of a food supplement, a medicament, an infant formula, an edible product and a food product. Particularly, the composition is in the form of an infant food supplement in the form of oily suspension.

Claims

1. A freeze-dried bacterial composition which comprises from 10.sup.4 to 10.sup.12 cfu/g of Pediococcus pentosaceus viable cells which induce the production of interleukin-10, wherein the production of interleukin-10 by THP-1 macrophages in the presence of Pediococcus pentosaceus cells as expressed as normalized increase is higher than the production of interleukin-10 by the negative control, which are THP-1 macrophages in the absence of Pediococcus pentosaceus cells, when the normalized increase is determined by the following steps: (a) differentiating THP-1 monocytes into macrophages by growing the THP-1 monocyte cell line obtained from the cell collection of the Public Health England, catalogue number 88081201, in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% Fetal Bovine Serum (FBS), and with phorbol 12-myristate 13-acetate (PMA) to a final concentration of 0.16 μM; (b) growing the THP-1 macrophages in RPMI 1640 medium with 10% FBS in 24-wells ELISA plates to a final concentration of 10.sup.6 macrophages/well; (c) incubating for 2.5 hours the THP-1 macrophages with lipopolysaccharide (LPS) at a final concentration of 10 ng/ml, and washing the THP-1 macrophages with Dulbecco's Phosphate Buffered Saline medium (D-PBS); (d) getting a culture of Pediococcus pentosaceus cells ready by having grown it overnight in Man, Rogosa and Sharpe medium (MRS) at 37° C. in a 5% CO.sub.2 atmosphere; (e) adding to each ELISA-well 500 μl of RPMI 1640 medium with 10% FBS and an appropriate amount of a dilution of Pediococcus pentosaceus cells to obtain a final ratio of 25:1, i.e. 2.5×10.sup.7 cfu of Pediococcus pentosaceus cells: 10.sup.6 THP-1 macrophages; (f) incubating the THP-1 macrophages with the Pediococcus pentosaceus cells for 2.5 hours at 37° C. or without the Pediococcus pentosaceus cells in the same conditions as negative control; (g) washing the THP-1 macrophages with D-PBS medium to remove the Pediococcus pentosaceus cells, subsequently adding to the THP-1 macrophages RPMI 1640 medium with 10% FBS supplemented with 50 μg/ml gentamicin, 10 μg/ml ampicillin and 12 μg/ml chloramphenicol, incubating at 37° C. at 5-7% CO.sub.2, and taking aliquots at 5 and 24 hours; (h) centrifuging the aliquots and assaying the supernatants for interleukin-10 quantification by flow cytometry; and (i) calculating the normalized increase of interleukin-10 concentration, with the formula (IL10.sub.24h−IL10.sub.5h)/IL10.sub.5h; wherein IL10.sub.5h and IL10.sub.24h is the concentration of interleukin-10 in μg/ml at 5 and 24 hours, respectively.

2. The bacterial composition according to claim 1, wherein the production of interleukin-10 by the THP-1 macrophages in the presence of Pediococcus pentosaceus cells as expressed as normalized increase is at least 2-fold higher than the production of interleukin-10 by the THP-1 macrophages in the absence of Pediococcus pentosaceus cells, when the normalized increase is determined by the steps (a)-(i) as defined in claim 1.

3. The bacterial composition according to claim 1, wherein the Pediococcus pentosaceus cells have the ability to antagonize Gram positive and Gram negative intestinal bacteria.

4. The bacterial composition according to claim 3, wherein the Gram positive bacteria comprises bacteria selected from the group consisting of Clostridium difficile and Enterococcus faecalis.

5. The bacterial composition according to claim 3, wherein the Gram negative bacteria comprises bacteria selected from the group consisting of Escherichia coli, Enterobacter aerogenes, Klebsiella oxytoca and Bacteroides vulgatus.

6. The bacterial composition according to claim 3, wherein the Pediococcus pentosaceus cells have the ability to antagonize Clostridium difficile, Enterococcus faecalis, Escherichia coli, Enterobacter aerogenes, Klebsiella oxytoca and Bacteroides vulgatus, wherein the ability to antagonize is determined by the following steps: (i) swabbing uniformly pathogen strains in plates containing Oxoid medium and growing to confluence in a CO.sub.2 incubator at the appropriate temperatures and % CO.sub.2 for the growth of each pathogen; (ii) placing two 6 mm diameter cylinder sections of a uniformly seeded confluent agar plate of the Pediococcus pentosaceus cells in contact with the pathogen seeded plate, confronting both (a) the grown side of one cylinder section against the pathogen seeded plate; and (b) the non-grown side of the other cylinder section against the pathogen seeded plate; and incubating overnight at 37° C.; (iii) measuring next day the inhibition zones by placing the agar plate over a flat rule; and (iv) calculating the growth inhibitory activity by subtracting the cylinder diameter (CD) from the inhibition zone diameter (IZD) measured in centimeters and dividing this difference by 2, following the formula GI=(IZD-CD)/2.

7. The bacterial composition according to claim 1, wherein Pediococcus pentosaceus is the Pediococcus pentosaceus deposited in the Spanish Type Culture Collection under the accession number CECT 8330.

8. The bacterial composition according to claim 1, which further comprises from 10.sup.4 to 10.sup.12 cfu/g of cells of Bifidobacterium longum CECT 7894.

9. The bacterial composition according to claim 1, wherein said composition comprises from 10.sup.7 to 10.sup.12 cfu/g of Pediococcus pentosaceus viable cells induce the production of interleukin-10.

10. The bacterial composition according to claim 1, wherein said composition is a food supplement or medicament.

11. The bacterial composition according to claim 1, wherein said freeze-dried composition is in the form of form of tablets, pills, capsules, lozenges, granules, powders, suspensions, sachets or syrups.

12. A bacterial composition which is in a form of an edible product comprising: (a) from 10.sup.4 to 10.sup.12 cfu/g of Pediococcus pentosaceus viable cells which induce the production of interleukin-10, wherein the production of interleukin-10 by THP-1 macrophages in the presence of Pediococcus pentosaceus cells as expressed as normalized increase is higher than the production of interleukin-10 by the negative control, which are THP-1 macrophages in the absence of Pediococcus pentosaceus cells, when the normalized increase is determined according to the procedure set forth in claim 1 and (b) a carrier selected from the group consisting of oat meal gruel, lactic acid fermented foods, resistant starch, dietary fibers, carbohydrates, proteins and glycosylated proteins.

13. The bacterial composition according to claim 12, wherein said edible product is in the form of an infant formula, food supplement or a food product.

14. A bacterial composition which is in the form of in the form of an oily suspension comprising from 10.sup.4 to 10.sup.12 cfu/g of Pediococcus pentosaceus viable cells which induce the production of interleukin-10, wherein the production of interleukin-10 by THP-1 macrophages in the presence of Pediococcus pentosaceus cells as expressed as normalized increase is higher than the production of interleukin-10 by the negative control, which are THP-1 macrophages in the absence of Pediococcus pentosaceus cells, when the normalized increase is determined according to the procedure set forth in claim 1.

15. A bacterial composition which is in the form of an infant formula comprising from 10.sup.4 to 10.sup.12 cfu/g of Pediococcus pentosaceus viable cells which induce the production of interleukin-10, wherein the production of interleukin-10 by THP-1 macrophages in the presence of Pediococcus pentosaceus cells as expressed as normalized increase is higher than the production of interleukin-10 by the negative control, which are THP-1 macrophages in the absence of Pediococcus pentosaceus cells, when the normalized increase is determined according to the procedure set forth in claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0115] FIG. 1. Pulsed-field gel electrophoresis patterns of Sma-I (left) and Not-I (right) restricted genomic DNA of, from left to right: Lactobacillus rhamnosus GG (LGG), Pediococcus pentosaceus CECT 8330 (8330), two strains of Pediococcus acidilactici as controls (1, 2) and molecular marker (M).

[0116] FIG. 2. Pulsed-field gel electrophoresis patterns of Xba-I (left) and Spe-I (right) restricted genomic DNA of, from left to right: Bifidobacterium longum CECT 7894 (7894), Bifidobacterium longum CECT 4551 (4551), and molecular marker (M).

[0117] FIG. 3. Reduction in average daily crying time and in duration of each episode. A) Reduction in average daily crying time (total minutes cried per day). B) Reduction in average duration of each episode (minutes per episode). Results expressed as means±standard error of the mean (SEM) for n=9 in placebo group and n=11 in probiotic formula group. PLA corresponds to placebo group. PRO corresponds to probiotic group.

EXAMPLES

[0118] The strain Lactobacillus reuteri ATCC 55730 is used as a control in some experiments.

Example 1. In Vitro Evaluation of the Ability to Induce IL-10 Production in an Intestinal Mucosa Model

[0119] The immunomodulatory capacity of the bacterial strains resulting from its interaction with the digestive tract's immune system (often referred to as gut-associated lymphoid tissue, GALT) was studied. More specifically, it was sought to test whether the bacterial strains have the ability to induce the production of anti-inflammatory IL-10 to reduce the inflammatory intestinal tract. The molecular basis for this is the interaction of probiotics cell surface receptors with TLR-2 and TLR-4 (Toll like receptor) that can be found on dendritic cells present in the Peyer's plates.

[0120] THP-1 Cell Line

[0121] The selected model was the cell line THP-1, which expresses TLR-2 and TLR-4. This model is sensitive to bacterial components like lipopolysaccharide -LPS- (as inducer of the inflammatory response), and is susceptible to modulate cytokine production when there are molecules in the medium suitable for the induction of the production of an anti-inflammatory cytokine pattern.

[0122] The term “THP-1 cell line” according to the art relates to a human monocytic cell line derived from an acute monocytic leukemia patient. It is used to test leukemia cell lines in immunocytochemical analysis of protein-protein interaction, and immunohistochemistry.

[0123] THP-1 cell line was obtained from the cell collection of the Public Health England (catalogue number 88081201). At the filing date of the present application the product catalogue for 88081201 from the provider Public Health England (www.hpacultures.org.uk) reads in relation to the THP-1 cells: “Human monocytic leukaemia. Derived from the peripheral blood of a 1 year old male with acute monocytic leukaemia”.

[0124] Mediums and LPS

[0125] THP-1 monocytes were grown in Roswell Park Memorial Institute (RPMI) 1640 medium+10% Fetal Bovine Serum (FBS). RPMI was a standard commercially available medium (RPMI 1640, ref. 61870-010 from Gibco). FBS was also from Gibco.

[0126] THP-1 monocytes were differentiated into macrophages by adding to the grow medium 5 mg of phorbol 12-myristate 13-acetate (PMA, ref. P8139 from SIGMA) to a final concentration of 0.16 μM and incubating for approximately 72 hours.

[0127] The bacterial strains were grown in MRS medium. It was a standard commercial available Man, Rogosa and Sharpe medium (MRS, Broth Oxoid ref. CM0359).

[0128] THP-1 macrophages were stimulated with LPS to induce an inflammatory response. Lipopolysaccharides (LPS), also known as lipoglycans, are large molecules consisting of a lipid and a polysaccharide joined by a covalent bond; they are found in the outer membrane of Gram-negative bacteria, act as endotoxins and elicit strong immune responses in animals. LPS used in this study was a standard commercial available lipopolysaccharide (ref. L4391 from Sigma).

[0129] Grow, Incubations and IL-10 Measurement

[0130] THP-1 macrophages were grown in RPMI 1640+10% FBS medium in 24-wells ELISA plates to a final concentration of 10.sup.6 macrophages/well. Final cell concentration was calculated by using Tripan blue dye and a Neubauer-counting chamber.

[0131] THP-1 macrophages were co-incubated with LPS (final concentration 10 ng/ml) for 2.5 hours. Then cells were washed with Dulbecco's Phosphate Buffered Saline medium (D-PBS, ref. 14190-094 from Gibco). Five hundred μl of RPMI 1640+10% FBS medium were added to each ELISA-well.

[0132] The bacterial strains were previously grown overnight in MRS medium at 37° C. in a 5% CO.sub.2 atmosphere. Bacterial strains appropriately diluted to obtain a final ratio of 25:1 (2.5×10.sup.7 cfu of bacteria: 10.sup.6 THP-1 macrophages) were added to each well. Concentration was calculated using a Neubauer-counting chamber.

[0133] THP-1 macrophages were then incubated for 2.5 hours at 37° C. with or without (negative control) bacterial strains. Subsequently, macrophages were washed twice with D-PBS medium to remove the bacterial strains. Then, RPMI 1640+10% FBS medium supplemented with gentamicin (50 μg/ml), ampicillin (10 μg/ml) and chloramphenicol (12 μg/ml) was added, incubated at 37° C. at 5-7% CO.sub.2, and aliquots were taken at 5 and 24 hours.

[0134] Aliquots were centrifuged and the supernatants assayed for IL-10 by flow cytometry by using the commercial kit Human IL-10 Flex Set (Bead B7 ref. number 558274 from BD Biosciencies) following manufacturer instructions.

[0135] Calculations

[0136] For interpretation of results, absolute values were not used. The most informative value is the evolution of cytokines, in this case IL-10 concentration, expressed as normalized increase taking the values at 5 and 24 h. This reflects what happens in gut and provides a standard value allowing a transversal comparison between experiments. The normalized increase is calculated following the formula, wherein IL10.sub.5h and IL10.sub.24h is the concentration of IL-10 in μg/ml at 5 or 24 hours, respectively:

(IL10.sub.24h−IL10.sub.5h)/IL10.sub.5h

[0137] Results

[0138] The higher the value, the higher the induction of IL-10. As shown in TABLE 1, LPS-induced THP-1 macrophages induced the production of IL-10 in the presence of bacterial strains, IL-10 induction being especially high in the presence of the strain CECT 8330. The induction caused by CECT 8330 is slightly higher than the caused by L. reuteri.

TABLE-US-00001 TABLE 1 Normalized increases of IL-10 in LPS-induced THP-1 macrophages. “Negative control” corresponds to THP-1 macrophages incubated without bacterial strains IL-10.sub.5 h in IL-10.sub.24 h in Normalized pg/ml pg/ml increase CECT 8330 30.83 140.18 3.54 CECT 7894 23.87 57.43 1.40 L. reuteri 30.31 122.17 3.03 Negative control 27.56 43.24 0.56

Example 2. Antagonism Capacity Against Intestinal Bacteria

[0139] The objective was to assess the ability of bacterial strains to antagonize undesirable members of species commonly abundant in infants with excessive crying.

[0140] The protocol used for detecting and evaluating these capabilities is known as Campbell protocol. This technique involves incubating the bacteria to be antagonized in Petri's plates with cylinder sections of uniformly seeded confluent agar plate of the probiotic strain. The halo of growth inhibition around the cylinder section is measured.

[0141] Medium

[0142] Pathogen strains were grown in Oxoid medium. It was a standard commercial available Oxoid medium (Oxoid CM0359).

[0143] Incubation and Measurement

[0144] Pathogen strains were swabbed uniformly in plates containing Oxoid medium and grown to confluence in a CO.sub.2 incubator at the appropriate temperatures and % CO.sub.2 for the growth of each pathogen. Then, two 6 mm diameter cylinder sections of a uniformly seeded confluent agar plate of the probiotic strains to be tested were placed in contact with the pathogen seeded plate, confronting the pathogen seeded plate with the grown side of one of the cylinder sections and with the non-grown side of the other cylinder section and incubating overnight at 37° C.

[0145] Calculations

[0146] Next day, inhibition zones were measured by placing the agar plate over a flat rule. Growth inhibitory activity (GI) was then calculated by subtracting the cylinder diameter (CD) from the inhibition zone diameter (IZD) measured in centimeters and dividing this difference by 2, following the formula GI=(IZD-CD)/2. The inhibiting capabilities of the strains of the invention were compared to that of the commercial strain L. reuteri. The final inhibitory activity was calculated as mean of the GI values for the two above-mentioned cylinder sections for each strain.

[0147] Results

TABLE-US-00002 TABLE 2 Growth inhibitory activity (GI) of probiotic strains. Pediococcus Bifidobacterium pentosaceus longum Lactobacillus Pathogen strain CECT 8330 CECT 7894 reuteri Gram negative bacteria Escherichia coli ATCC 10538 0.30 >0.6 n.i Enterobacter aerogenes ATCC 13048 0.08 >0.6 0.08 Klebsiella oxytoca KT 801 0.54 >0.6 0.13 Bacteroides vulgatus ATCC 8482 0.21 >0.6 n.i Gram positive bacteria Enterococcus faecalis ATCC 29212 0.35 >0.6 0.08 Clostridium difficile ATCC 9689 0.25 0.29 0.38 Results expressed in cm; “n.i.” denotes no inhibition

[0148] The strains displayed inhibitory activity against all the spectrum of pathogens studied. Therefore, the strains were effective inhibiting not only Gram positive but also Gram negative bacteria. This was not the case of L. reuteri which was inefficient inhibiting the growth of E. coli and B. vulgatus. This is of great interest as abnormal amounts of bacteria such as E. coli are commonly present in infants presenting excessive crying (De Weerth, C. et al. 2013 supra; Lehtonen, L. et al. “Intestinal Microflora in colicky and noncolicky infants: Bacterial Cultures and Gas-Liquid Chromatography”, Journal of pediatric Gastroenterology and Nutrition 1994, vol. 19, pp. 310-314). It is also noteworthy that, in general, CECT 8330 and specially CECT 7894, were more efficient inhibiting the growth of almost all the pathogen bacteria compared to L. reuteri. Moreover, it is also relevant that both strains of invention provide protection against Klebsiella and Clostridium, which are also abundant in the intestine of infants presenting excessive crying (De Weerth, C. et al. 2013 supra; Lehtonen, L. et al. 1994 supra).

Example 3. No Production of Gas

[0149] Heterofermentative bacteria produce CO.sub.2 and ethanol, as well as lactic acid, by glucose fermentation following the metabolic pathway:

1Glucose.fwdarw.1Lactic acid+1Ethanol/Acetic acid+2ATP+1CO.sub.2

[0150] The production of CO.sub.2 by the strains was determined. As it is shown in the formula, the production of CO.sub.2 is also informative of the production of ethanol. The production of CO.sub.2 was determined using the Durham Tubes technique, which is based on the incubation of the probiotic strain in heterofermentation broth in tubes containing smaller and inverted tubes inside, where the gas is accumulated when it is produced (Pilone, G. J., et al., “Characterization of wine lactic acid bacteria: single broth culture for tests of heterofermentation, mannitol from fructose, and ammonia from arginine” Am J Enol Vitic 1991, vol. 42, pp. 153-157).

[0151] The strains CECT 8330 and CECT 7894 did not produce gas. L. reuteri used as a control, did produce gas.

Example 4. Toxicity Assays

[0152] In contrast to bacteria from Bifodobacterium and Lactobacillus genus, Pediococcus pensotaceous is not commonly used as a probiotic for human consumption. Thus, although the probiotic strain CECT 8330 of the present invention belongs to a species which has QPS status additional toxicity assays were conducted to avoid any safety concern.

[0153] Given the high sensitivity of the babies due to their immature digestive tract, it was decided to develop a more appropriate model of acute toxicity using Wistar Han IGS Crl:WI neonatal rats (10 days after birth with a body weight range at the start of the experiment of 18-23 g), in order to ensure complete safety of the strains in infants.

[0154] Pregnant females were received at day 19th of gestation. After birth, litters were adjusted to 4 males and 4 females, mixing pups of all mothers in order to avoid maternal effects and achieve litters of equal size. Each lactating female were placed with 4 males and 4 females. Lactating females were fed with SAFE A03 diet and water ad libitum.

[0155] The experimental procedure comprised 4 groups: VEHICLE-translocation, VEHICLE-clinical signs, CECT 8330-translocation and CECT 8330-clinical signs.

[0156] Each group comprised a cage with a lactating female and a litter of 4 males and 4 females. CECT 8330 product was prepared daily at a final concentration of 0.5×10.sup.10 cfu/ml formulation. VEHICLE group received water instead of probiotic. All neonatal rats were administered with the VEHICLE or CECT 8330 treatments for 5 days (from day 0 to day 4 of the study) by oral gavage with an orogastric cannula at a fixed volume of 5 ml/kg (2.5×10.sup.10 cfu/kg in the case CECT 8330). The oral route was chosen for the study because it is the intended route of administration in humans.

[0157] Observations during the experiment were: morbidity/mortality; body weight; clinical signs (appearance of the pup including hydration and body condition; response to a stimulus; natural activity—ability to wriggle if put in supine- and skin color).

[0158] Animals were euthanized after two different periods of time: [0159] Groups of “translocation” were euthanized on day 4 of experiment (last day of the 5-day treatment) [0160] Groups of “clinical signs” were euthanized on day 11th of the study (one week after the last oral dosing).

[0161] Pups were euthanized by decapitation and a necropsy was carried out, including the examination of the intact animal and all its surface tissues, followed by an internal examination of the thoracic and abdominal cavities. In the animals belonging to the “translocation” group, immediately after euthanasia, the liver of the animals were collected and maintained at 2-4° C. until bacterial translocation analysis. Approximately 5 mg of each liver sample was homogenized in 1 ml 0.01% gelatin PBS. One hundred μl from this homogenate were plated either on McConkey plates or MRS plates. Colonies were counted after incubation at 37° C. for 48 h.

[0162] No spontaneous mortality or toxicity-related clinical signs were observed during the study. No differences on body weight between control (vehicle) and CECT 8330 were detected and the behavior of all animals was normal. Moreover, no differences were observed between control and CECT 8330 groups in the number of animals showing translocation of either lactic acid bacteria or enterobacteria in the liver.

Example 5. Isolation of Strains

[0163] Fresh stools were collected from 0-9 year-old children and dissolved in PBS buffer (pH 7.4), aliquoted and plated on MRS supplemented with various antibiotic combinations. Strains were cultured under microaerophilic conditions (5% CO.sub.2) at 37 or 30° C. Incubation time depended on the growth rate, but ran normally from 24 hours to 3 days. Gram staining was carried out in order to get a first identification. Once grown, isolated strains were stored by lyophilization in PBS 0.1× with 15% skim milk powder. The strains were grown on MRS agar supplemented with 10 μg/ml vancomycin. Microscopic examination revealed that Bifidobacterium longum CECT 7894 are Gram-positive bacilli, and Pediococcus pentosaceus CECT 8330 are Gram-positive coccus.

[0164] Genus and species identification was done by amplification of the 16S rRNA gene as previously described (Bosch, M. et al., Probiotic properties of Lactobacillus plantarum CECT 7315 and CECT 7316 isolated from faeces of healthy children. Lett App. Microbiol, 2012 vol. 54, pp. 240-6). SEQ ID NO: 1 corresponds to the 16S rRNA sequence of Pediococcus pentosaceus CECT 8330 and SEQ ID NO: 2 to the 16S rRNA sequence of Bifidobacterium longum CECT 7894.

[0165] Strain genotyping was performed by genomic digestion and pulsed-field gel electrophoresis (PFGE).

[0166] Pediococcus pentosaceus CECT 8330 was subjected to a previously described protocol (Rodas, A. M., et al., Polyphasic study of wine Lactobacillus strains: taxonomic implications. Int J Syst Evol Microbiol, 2005. 55(1): p. 197-207) with minor modifications. Since there were not commercial strains of Pediococcus pentosaceus available to use as controls, two commercial strains of Pediococcus acidilactici were included in the assay (1 and 2 in FIG. 1). Strains were grown on MRS agar plates and incubated at 37° C. 5% CO.sub.2 for 18 h. Cells were harvested and washed 3 times in 8 ml PET (10 mM Tris pH 7.6, 1 M NaCl) then centrifuged at 6000 rpm 10 min. Pellets were resuspended in 700 ml lysis buffer (6 mM Tris, 1 M NaCl, 0.1 M EDTA, 0.5% SLS, 0.2% deoxycholic acid; 1 mg/ml lysozyme; 40 U/ml mutanolysin; 20 mg/ml RNase). An equal volume of 1.6% low melting point agarose (FMC BioProducts, Rockland, Me., USA) was added to the resuspended cells and solidification was allowed at 4° C. for 1 h. Inserts were transferred to 2 ml lysis buffer II (0.5 M EDTA pH 9.2, 1% N-lauryl sarcosine and 1 mg/ml pronase) and incubated at 50° C. for 48 h. Then inserts were washed at room temperature with TE buffer (10 mM Tris, 1 mM EDTA pH 8.0). Total DNA digestion was performed separately by Sma-I and Not-1 restriction enzymes (Roche Diagnostics). Pulsed-field gel electrophoresis was carried out using CHEF DRIII apparatus (BioRad Laboratories). Inserts were loaded in a 1% agarose gel (SeaKem ME agarose, FMC BioProducts, ME, USA). TABLE 3 describes electrophoresis conditions for each enzyme. DNA molecular weight markers were Lambda ladder PFG Marker and Low Range PFG Marker (New England Biolabs). After electrophoresis, gels were stained with ethidium bromide and UV using GelDoc System (BioRad).

TABLE-US-00003 TABLE 3 Electrophoresis conditions Enzyme Block Initial Pulse (sec) Final pulse (sec) Time (hours) Not-I 1 2 25 18 Sma-I 1 0.5 5 16

[0167] Bifidobacterium longum CECT 7894 was characterized by PFGE using Xba I and Spe I as restriction enzymes as described by Briczinski, E. P. et al. “Technical note: a rapid pulsed-field gel electrophoresis method for analysis of bifidobacteria” J. Dairy Sci. 2006, vol. 89, pp 2424-2427. The resulting patterns were compared with those of B. longum CECT 4551.

[0168] The results are depicted in FIG. 1 and FIG. 2. Pulsed-field gel electrophoresis Not-I and Sma-I restriction patterns were different for the strain Pediococcus pentosaceus CECT 8330 and the commercial control strains belonging to Pediococcus acidilactici species (1 and 2). It was not possible to include Pediococcus pentosaceus strains as controls because they were not commercially available. PFGE allows distinguishing between strains of the same species, and thus can be used to uniquely identify a given bacterial strain within a bacterial species (Rodas, A. M., et al. 2005 supra).

Example 6. Preparation of an Oily Suspension

[0169] Four hundred ml of sunflower oil were introduced into a container provided with stirring means. Nine and a half g of colloidal silica were slowly added under stirring (150 rpm) to avoid the formation of lumps and agglomerations until complete homogenization. 13.3 g of Pediococcus pentosaceus CECT 8330 containing 5×10.sup.12 cfus were added to the container under slow stirring (50 rpm) until complete dispersion. Then, 42.75 g of Bifidobacterium longum CECT 7894 containing 5×10.sup.12 cfus were added to the container under slow stirring (50 rpm) until complete dispersion. The suspension was finally made up to 1000 ml with sunflower oil and stirred to homogenize the final suspension. The suspension was kept at room temperature.

Example 7. Clinical Study

[0170] Design of the Study

[0171] A pilot clinical trial was conducted to evaluate the efficacy and safety of the probiotic formula combining P. pentosaceus CECT 8330 and B. longum CECT 7894. The study was designed as a prospective double-blind, placebo-controlled, randomized clinical trial with two parallel arms which involved a total number of 8 participating centers from Catalonia (Spain). The study protocol was approved by the Ethical Committees from IDIAP Jordi Gol (Barcelona, Spain) and from Fundació Unió Catelana d'Hospitals (Barcelona, Spain) in compliance with the Helsinki Declaration.

[0172] Healthy term infants of both sexes meeting all of the following inclusion criteria were recruited: from 21 to 120 days old; minimum birth weight of 2.5 Kg; either breastfed or feed with infant formula (hydrolyzed or initiation formula); excessive crying and fussing according to the definition “intense, persistent and inconsolable crying, problematic for the normal family unit functioning, which implies at least 60 minutes per day in 3 or more episodes in 3 or more days observed during at least 1 week, previously ruling out an organic etiology, like intestinal intussusception or others”. Exclusion criteria were: Pre-term infants (born before 37 weeks); chronic illness; history of gastrointestinal disorders (not related to colic); immunosuppressed infants; previous or expected surgical intervention; having taken probiotics or antibiotics one week before the enrollment; infants whose parents or representatives were not able to appropriately follow the study requirements. Subjects were randomly assigned to either probiotic treatment group or placebo group. The treatment consisted in a composition as described in EXAMPLE 6. Placebo consisted in the same oily suspension without probiotic. Compositions were administered 30 minutes before feeding (5 drops/day) for 14 days. During the study, parents were asked to fill questionnaires recording the adhesion to the treatment, crying evolutions and adverse effects.

[0173] Data analysis was performed with IBM® SPSS Statistic v20 for Windows and results expressed as averages and standard errors. Average reduction in daily crying time during clinical trial was calculated as the difference between the average of the total number of minutes of crying per day during the last 3 days of the study (days 12, 13 and 14) and the average of total number of minutes per days during the first 3 days of study (days 1, 2 and 3). Average reduction in the duration of each episode was calculated as the difference between the average of the number of minutes lasting each episode during the last 3 days of the study (days 12, 13 and 14) and the average of minutes lasting each episode during the first 3 days of study (days 1, 2 and 3).

[0174] Results

[0175] At the beginning of the clinical trial it was confirmed that n=9 infants belonging to the placebo group and n=11 belonging to the probiotic formula group met the proposed definition of crying time and were therefore allowed to continue the study. The average crying time of this population at the beginning of the study ranged from 60 to 240 minutes. During the study, both placebo and probiotic formula were well tolerated and no adverse effects related to supplementation were observed. Moreover, as shown in FIG. 3, crying time was reduced in both placebo and probiotic group during the study. However, probiotic consumption caused a higher reduction in average crying time. A similar trend was observed for the duration of each episode.

[0176] The clinical effect observed supports the probiotic properties observed in vitro. These results are of relevant interest as this study presents some strength compared to other studies where probiotics have been used for treating colic.

[0177] For instance, the study included both breastfed and formula-fed infants, which is of relevant interest as current probiotic formulas have failed to display any improvement in formula-fed sub-populations. Moreover, participating infants were recruited based on clinical definition of infant colic more realistic according to daily clinical practice and the treatment period (14 days) was shorter than that of many other clinical trials (21-28 days).

BIBLIOGRAPHIC REFERENCES

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