A thermotolerant bacterium Paenibacillus thiaminolyticus with an ability to produce extracellular -mannanase was isolated from a soil sample. Bacterium produced 45U/mL -mannanase at 50 degrees C. The culture conditions for high-level production of -mannanase were optimized. Optimized MS medium [wheat bran 2% (w/v), ammonium sulfate 0.3% (w/v), yeast extract, and peptone (0.025% each) pH 6.5] was inoculated with 2% of 16H old culture. The culture was incubated at 50 degrees C for 48H resulting in 24-folds higher -mannanase production (1,100 +/- 50U/mL). Optimum pH and temperature for enzyme activity of the crude enzyme was 6.0 and 60 degrees C, respectively. The enzyme demonstrated 65% relative enzyme activity at 37 degrees C. The hydrolytic activity of the crude enzymatic preparation was assessed on various agro residues. Thin-layer chromatographic analysis showed that the enzyme activity to saccharify heteromannans resulted in production of a mixture of manno-oligosaccharides (MOS) and enzyme exhibited classic endo-activity. To evaluate the possible prebiotic potential of the MOS thus obtained, initial screening for their ability to support the growth of probiotics was carried out by the pure culture method. Bifidobacterium and Lactobacillus sp. responded positively to the addition of enzymatically derived oligosaccharides and their numbers increased significantly. (C) 2015 International Union of Biochemistry and Molecular Biology, Inc.