| 主权项 |
1. A bacterial composition which comprises from 104 to 1012 cfu/g of Pediococcus pentosaceus viable cells which have the ability to 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 106 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% CO2 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. 2.5×107 cfu of Pediococcus pentosaceus cells:106 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% CO2, 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 (IL1024 h−IL105 h)/IL105 h; wherein IL105 h and IL1024 h is the concentration of interleukin-10 in pg/ml at 5 and 24 hours, respectively. |
