(A) Colony morphology and SEM image of RSE02 probiotic strain. (B) Tn7 mediated RFP- gene transfer mechanism to fluorescence labelle RSE02 strain. (C) Phylogenomic tree based on whole genome sequences of the representative RSE02 strains in the TYGS tree inferred with FastME 2.1.6.1 17,24 from Genome BLAST Distance Phylogeny approach (GBDP); distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 93.7%. The tree was rooted at the midpoint20.

Probiotic potential of RSE02 bacterial endophyte. (A) Biochemical analysis of RSE02 bacteria. (B) Hydrophobicity and Auto-aggregation of strain RSE02 (dark grey bar: 0 h; grey bar: after 24 h). (C) pH tolerance (2 to 14) of strain RSE02.

Cholesterol utilization and Bile salt hydrolase activity test. (A) In-vitro cholesterol utilization by the RSE02 strain in different time (0, 6, 12, 18, 24 and 48 h) interval, where initial cholesterol concentration was 100 µg/mL in minimal medium. (B) Bile salt hydrolase (BSH) activity of the RSE02 strain was assessed by the qualitative direct plate assay.

Whole genome sequence analysis of the strain RSE02. (A) represents the circular genome map showing arrangement of CDs, RNAs etc. (B) represents abundance of major gene-subsystems (Where:1–25 represents N2 metabolism; respiration; stress response; protein metabolism; cell division and cell cycle; sulfur metabolism; phages, prophages, transposable elements, plasmid; secondary metabolism; iron acquisition and metabolism; membrane transport; phosphorus metabolism; RNA metabolism; carbohydrates; cell wall and capsule; DNA metabolism; regulation and cell signalling; fatty acids, lipids and isoprenoids; motility and chemotaxis; cofactor, vitamins, prosthetic groups, pigments; nucleosides and nucleotides; metabolism of aromatic compounds; virulence, disease and defense; potassium metabolism; amino acids and derivatives; dormancy and sporulation respectively. (C) represents presence of beneficial genes and gene clusters in respect of the probiotic potentials, and (D) shows representative biosynthesizing gene clusters of biotin, vitamin B12, folate and bacteriocin24.

(AI–IV) Cell adhesion assay of RFP-labelled RSE02 in Caco-2 cell line where, AI: Phase contrast, AII: DAPI staining, AIII: merge image of AI & AII and AIV: TRITC filter respectively under 40X magnification. (B) Kaplan-meir survival curve of zebrafish on exposure to RSE02 strain (PBS: Control, dose 1: 1 × 109 CFU/mL, dose 2: 1 × 1010 CFU/mL and dose 3: 2 × 1010 CFU/mL). (C) Cell cytotoxicity of Caco-2 cell line on exposure RSE02 bacterial cell free supernatant (control: PBS, 10, 20 and 30 μL csf: 1 × 109 CFU/mL bacteria. (DI & II) Transverse section of gut through the intestinal bulb segment showing RFP-labelled RSE02 (red) and nuclear DAPI staining (blue) in Control (PBS exposure) and RSE02-exposed adult zebrafish after 7 day treatment; GL, Gut lumen; Magnification 10x; Scale bar 100 μm. (EI–IV) Microscopic visualization RSE02 in zebrafish gut smear where, EI & II: gut smear of untreated fish under both TRITC & FITC filter and EIII & IV: gut smear of treated fish under both TRITC & FITC filter.

Morphological study of mice. (A–C) Morphology of mice control, HFD and HFD + RSE02, respectively. (D) Body weight of day 1, day 15 and day 30 of control, HFD and HFD + RSE02 mice, respectively. (E & F) Liver and brain weight of control, HFD and HFD + RSE02 mice, respectively. All data were taken from three random measurment. Mean values in each column do not differ significantly at p < 0.05. Comparison between control and treatment mean values was made by Dunnett’s multiple comparisons test. Where, “****” = p < 0.0001, “***” = p < 0.001, ““**” = p < 0.01, “*” = p < 0.1, “ns” = data is not significant. Data were subjected to analysis of variance (ANOVA ordinary one way) test.

Lipid profile and Liver Profile of Control, HFD and HFD + RSE02 mice. (A–E) Lipid profile (Total blood cholesterol, HDL, LDL, TG and VLDL) of Control, HFD and HFD + RSE02 mice respectively. (F–M) Liver profile (total blood CRP, bilirubin, alkaline phosphatase, SGPT, SGOT, globulin, albumin and protein) of Control, HFD and HFD + RSE02 mice, respectively. Mean (from three independent data point) values in each column do not differ significantly at p < 0.05. Comparison between control and treatment mean values was made by Dunnett’s multiple comparisons test. Where, “****” = p < 0.0001, “***” = p < 0.001, ““**” = p < 0.01, “*” = p < 0.1, “ns” = data is not significant. Data were subjected to analysis of variance (ANOVA ordinary one way) test.

Liver, Brain histology and Biochemical analysis of gut tissue of mice. (A–D) Biochemical analysis of total protein, MDA content, GSH content and catalase activity of gut tissue of mice, respectively. Mean values in each column do not differ significantly at p < 0.05. Comparison between control and treatment mean values was made by Dunnett’s multiple comparisons test. Where, “****” = p < 0.0001, “ns” = data is not significant. Data were subjected to analysis of variance (ANOVA one way) test by using the GraphPad Prism 8.0.1 version software. (E) Control liver having normal hepatic lobules. (F) is the liver section of HFD fed mice having lipid deposition (black circle) in intracellular space of hepatic cells. (G) is the liver section of probiotic fed mice with almost normal histoarchitecture having reduced number of lipid droplets (arrow heads) than the former. (H) Control brain depicting clear granular layer (G), molecular layer (M) and white matter (WM); (I) brain tissue section of HFD fed mice is having vacuolated (V) white matter while in case of HFD + RSE02 fed mice brain. (J) the reduced size and numbers of vacuoles in white matter were observed. (K-M) are images of white matter of control, HFD fed and HFD + RSE02 fed mice, respectively where L is having vacuolated white matter but the condition became better in M.