Probiotics are living microorganisms that confers beneficial effects when taken in adequate amount. Probiotic are the organisms which possess beneficial effect when ingested by the host. Lactobacilli are an extremely important group of probiotic bacteria. The aim of this study is to isolate lactobacillus strains from Vitis vinifera and Spinacia oleracea with de Man, Rogosa and Sharpe (MRS) broth to search for a new effective probiotic strains. Various strains of Lactobacilli identified and analysed for their probiotic properties including acid, salt tolerance and different fermentative test. The test sample showed significant activity against Escherichia coli (88%) Salmonella typhi (85%) and Staphylococcus aureus (84%). The test sample showed good (60%) phytotoxic activity against Lemna minor at 1000 µg/ml. To protect the isolated Lactobacillus strains encapsulation was done in the product’s environment to improve their survival. The results obtained from the different fractions of metabolites were selected for in vivo acute toxicity studies. The toxicological study revealed that the test samples are safe for animal use upto 800 mg/kg. The test sample showed dose dependent anti-nociceptive potentials, the significant effect was observed with 500 and 800 mg/kg b.w dose. The analgesic effect of diclofenac sodium (standard drug) was greater than test sample. The significant (P < 0.01) anti-nociceptive potentials was noticed at 800 mg/kg b.w. of the animal for normal saline (35.50 ± 0.84), chloroform (29.67 ± 1.22), ethyl acetate (30.67 ± 1.22) and (29.00 ± 0.93) chloroform fraction of L. plantarum secondary metabolites. The test sample reduced the motility of GIT in mice. The reduction in GIT motility is dose dependent and maximum effect was produced by 800 mg/kg dose. The test sample caused different degree of movement of charcoal meal in the intestine i.e. 24.17, 20.17 and 17.83cm at the tested doses of 200, 500 and 800 mg/kg, respectively. During isolation and characterization of chemical constituents (2-(3,4-dihydroxyphenyl)-3,5,7trihydroxy-4H-chromen-4-one),(1,2,3,4,5,6-Hexanehexol), (2-(3,4-dimetoxyphenyl)-5,7 dihydroxy-6-metoxy-4H chromen-4-one),(5-hydroxy-7-methoxy-2-(4-methoxyphenyl) 4H-chromen-4-one), (3S,3aS,5aR,9bS)-3,5a,9-trimethyl-3a,4,5,5a,7,8 hexahydronaphtho [1,2-b]furan 2,6(3H,9bH)-dione. Further investigations would help in the suitability of this isolate for human consumption and domestic animals feeds formulations as important probiotic.