Growth and metabolite production of Lactobacilli in minimal media supplemented with either glucose or scFOS as carbohydrate source. The growth kinetics of various Lactobacilli strains were studied for 24 hr in a specially designed minimal medium. The absorbance was recorded every hour in a multimode reader. Both strains could utilize scFOS for growth. Among the two strains, the growth of L. plantarum strain was higher than the L. fermentum. L. rhamnosus, on the other hand, exhibited restricted growth in presence of scFOS compared to glucose (Fig. 1A). The metabolite analysis also exhibited a similar profile. L .fermentum and L. plantarum could utilise scFOS and produce Lactic and Acetic acid, whereas L. rhamnosus did not show increase in metabolite production in presence of scFOS, while in presence of glucose a robust metabolite secretion was observed (Fig.1B) As witnessed in kinetic studies, L. fermentum had a slower growth rate; therefore it was obvious that the metabolite production will be reduced when compared to the other strains.
In vitro anti-inflammatory activity in presence of scFOS
The in vitro anti-inflammatory activity of Lactobacilli spp. was assessed by determining the effect of conditioned cell-free supernatant (CFS) of L. fermentum FS-10, L. plantarum Lpl-IC and L. rhamnosus GG on pro-inflammatory markers such as TNF-α, NO and anti-inflammatory IL-10. LPS-stimulated Raw and THP-1 macrophages were exposed to CFS and the effect on NO, TNF-α and IL-10 levels were analysed. The CFSs inhibited LPS-induced secretion of NO, TNF- α and increased IL-10 levels. (Fig. 4). Notably, the enhancement of IL-10 levels was evident in L. fermentum CFS in presence of scFOS compared to only bacteria. The in vitro findings indicate the potential of L. fermentum and L. plantarum to exert anti-inflammatory activity. It also indicates the possibility of combining the probiotics with scFOS for enhancing the efficacy. Further study was conducted in colitic mice model to understand the in vivo efficacy of the probiotics and scFOS, alone or in synbiotic combination.
Effect on Body weight and Disease Activity Index
Body weights were measured daily before vehicle and test item administration. After 7-days induction with 2.5% DSS, reduction in feed consumption, change in stool consistency and decrease in body weights of the mice indicated successful model establishment. Body weight loss was observed in all the groups from day 8 to day 14. At the end of the study significant body weight gain [p<0.05] was observed in all treatment groups when compared to DSS control. (Fig. 3A).
DAI was recorded during the entire experimental period. DAI was calculated from the percentage loss in body weight in combination with stool consistency and bloody stools. The DAI score increased significantly after DSS intake, in DSS control [2.5% DSS + Saline] and Vehicle groups, while it was markedly attenuated in the treatment groups. Treatment groups reduced the appearance of the colitis symptoms like appearance of hemoccult and diarrhoea which resulted in a significant reduction of DAI.
Measurement of TNF-α, IL-1β, IL-17, IL-6 and IL-10 in serum by ELISA
All the treatment groups showed significant reduction (p<0.05) in inflammatory cytokines- TNF-α, IL-1β, IL-17, and IL-6 levels in serum when compared to DSS control [2.5% DSS + Saline] and Vehicle control on day 15 and 21. After the treatment with lactobacillus alone and in combination with scFOS the levels of pro-inflammatory cytokines were close to that of the normal control group. The levels of pro-inflammatory cytokines (i.e., TNF-α, IL-1β, IL-17, IL-6) in serum were attenuated and anti-inflammatory cytokine IL-10 was upregulated in serum on both day 15 and 21 when compared to DSS control [2.5% DSS + Saline].
The synbiotic test samples shown significant decrease [p<0.05] in pro-inflammatory cytokines (i.e., TNF-α, IL-1β, IL-6) in serum when compared with respective probiotic samples whereas IL-17 was non-significant [p>0.05]. IL-10 levels of synbiotic (1 & 2) test samples shown significant increase [p<0.05] in serum when compared with respective probiotic samples on day 15 (Fig. 4).
Measurement mRNA expression of the cytokine in colon tissue by quantitative real time PCR(real-time PCR) analysis
The assessment of localized inflammation in the colon revealed that the mice fed with lactobacilli and scFOS, either alone or in combination displayed a significant down regulation of the pro-inflammatory markers TNF-α, IL-1β, IL-17, and IL-6 and upregulation of anti-inflammatory IL-10 when compared to DSS control [2.5% DSS + Saline]. As observed in systemic biomarker analysis, the synbiotic test samples showed significant decrease in relative mRNA levels [p<0.05] of TNF-α, IL-1β, IL-6 and IL-17 and increase in relative mRNA of IL-10 when compared (p˂0.05) with respective probiotic samples (Fig. 5).
Histopathology
At the end of the study, colon tissues were collected for HP from each animal, fixed in 10% Neutral Buffered Saline and subjected to histopathology. H&E staining: Hematoxylin and Eosin (H/E) staining of 4 micron-thick tissue sections were performed according to the standard protocols. The total histologic score was given as epithelium (E) + Infiltration (I).
Colon was assessed for pathology based on the following scores epithelium (E):
Microscopic examination of colon tissues from normal control group showed normal crypts in the mucosa, submucosa and muscularis mucosa. Colon tissues from disease control and vehicle control group showed mucosal erosion and loss of goblet cells, inflammation, oedema, infiltration of inflammatory cells in submucosa and muscularis mucosa when compared with normal control group animals. However, the incidence and severity of these lesions (recorded in G2 group) was reduced in treatment groups suggesting reversible nature of the change.