Probiotic for infantile colic

Abstract

The invention provides a bacterial composition which comprises from 10 4 to 10 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.

1 . A freeze-dried bacterial composition which comprises from 10 4  to 10 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 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 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 7  cfu of  Pediococcus pentosaceus  cells: 10 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 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 24h −IL10 5h )/IL10 5h ; wherein IL10 5h  and IL10 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 2  incubator at the appropriate temperatures and % CO 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 4  to 10 12  cfu/g of cells of  Bifidobacterium longum  CECT 7894. 
     
     
         9 . The bacterial composition according to  claim 1 , wherein said composition comprises from 10 7  to 10 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 4  to 10 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 4  to 10 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 4  to 10 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 .