Table 1.
Primers used during this study.
Fig 1.
Rats were divided to 6 groups (n = 6 per group), the groups received the probiotic isolates or PBS daily for 14 days then on day 15 of the experiment, the groups were induced for colitis by acetic acid followed by continues the administration of the probiotics to treatment groups and PBS to the ulcerative group for 4 days before be sacrificed. The control group received PBS only without any colitis induction. The treatment groups received the isolates orally before and after the colitis induction as follows; (A), WNYM01; (B), WNYM02; (C), WNYM03; (M), Mixture of WNYM (01, 02 and 03).
Table 2.
Screening the isolates for pH tolerance and the presence of folic genes (folk, folP) and riboflavin genes (ribA, ribB, ribG, ribH) within their genomic DNA by PCR using the specific primers as listed in Table 1.
Fig 2.
A: PCR products for detection of riboflavin and folic genes in the selected isolates using specific primers as listed in Table 1. Lane1, 1 Kb ladder; lane 2, 3, 4, PCR products for ribA gene from WNYM01, WNYM02, WNYM03; lane 5, 6, 7, PCR products for ribH gene from WNYM01, WNYM02, WNYM03; lane 8, 9, 10, PCR products for folK gene from WNYM01, WNYM02, WNYM03; lane 11, negative control; lane 12, 13, 14, PCR products for ribG gene from WNYM01, WNYM02, WNYM03. B: Continue PCR products for detection of riboflavin and folic genes in the selected isolates using specific primers as listed in Table 1. Lane1, negative control; lane 2, 3, 4, PCR products for ribB gene from WNYM01, WNYM02, WNYM03; lane 5, 6, 7, PCR products for folP gene from WNYM01, WNYM02, WNYM03; lane 9, 100bp ladder.
Fig 3.
A: PCR amplification of the 16S rRNA gene from the selected isolates using universal primer set as listed in Table 1. Lane1, 100bp ladder; lane 2, WNYM01, lane 3, WNYM02; lane 4, WNYM03; lane 5, negative control. B: Phylogenetic tree of the isolated strains for molecular identification based on their 16S rRNA gene. Blast search at NCBI data base indicate their 16S rRNA gene are 100% identical to that belong to Pediococcus acidilactici strains which their accession numbers are listed on the tree. Phylogenetic tree was performed using MEGA7 software.
Table 3.
Tolerance of isolated Pediococcus acidilactici strains to bile salts.
Table 4.
Susceptibility of isolated Pediococcus acidilactici strains to antibiotics.
Table 5.
Antimicrobial activity of the isolated Pediococcus acidilactici strains.
Table 6.
Detection of folic acid and riboflavin production by the isolated P. acidilactici strains was done using HPLC.
The culture supernatant from each strain was tested and1mg/ml of each folic acid and riboflavin was used as standard, the running volume was 10 μl, the retention time and the peak (area—height) listed.
Fig 4.
Gross photograph of rat’s colon segment showing the appearance differences among the groups in wall thickness, mucous membrane, muscle coat and serosa.
The rat groups received the probiotic isolates or PBS daily for 14 days then induction for colitis by acetic acid on day 15 of the experiment followed by continues the administration for 4 days before be sacrificed. The control group received PBS only without any colitis induction. The treatment groups received the isolates orally before and after the colitis induction as follows; (A), WNYM01; (B), WNYM02; (C), WNYM03; (M), Mixture of WNYM (01, 02 and 03).
Fig 5.
Rat’s colon segment showing the histo-morphological structures differences among the groups, including the mucosa (Lamina epithelialis, Lamina muscularis mucosa and lamina properia), the submucosa, the muscular coat (inner circular and outer longitudinal muscle fibers) and the serosa (arrows).
Scale bars 50 um, 100 um. The Rat groups received the probiotic isolates or PBS daily for 14 days then induction for colitis by acetic acid on day 15 of the experiment followed by continues the administration for 4 days before be sacrificed. The control group received PBS only without any colitis induction. The treatment groups received the isolates orally before and after the colitis induction as follows; (A), WNYM01; (B), WNYM02; (C), WNYM03; (M), Mixture of WNYM (01, 02 and 03).
Fig 6.
Levels of inflammatory markers in the cytoplasmic extracts from homogenate rat’s intestinal tissues.
(A) tumor necrosis factor-α (TNF-α) and tissue (B) interlukin-10 (IL-10). The rat experimental groups: Control, received PBS only; Ulcerative, received PBS and colitis induction; A, received WNYM01 + colitis induction; B, received WNYM02 + colitis induction; C, received WNYM03 + colitis induction. M, received Mixture of WNYM (01–03) + colitis induction. Three triplicate experiments were independently performed. Data are presented as the mean ± standard deviation.
Fig 7.
Levels of lipid peroxidation marker (TBARS) and oxidative stress biomarkers (GSH) in the cytoplasmic extracts from homogenate rat’s intestinal tissues.
(A), thiobarbituric acid reactive substances (TBARS) and (B), glutathione (GSH). The rat experimental groups: Control, received PBS only; Ulcerative, received PBS and colitis induction; A, received WNYM01 + colitis induction; B, received WNYM02 + colitis induction; C, received WNYM03 + colitis induction. M, received Mixture of WNYM (01–03) + colitis induction. Data are presented as mean ± S.D. (n = 3). Mean differences are significant (p < 0.05).