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In this work, we reported production of glucansucrases with molecular weights of about 300 kDa by strains of fructophilic lactic acid bacteria (FLAB) Lactobacillus pentosus AG8, Lactobacillus kunkeei AG9, AG10 and AG11 isolated from the gut of honey bee Apis mellifera. From sucrose, these enzymes synthesize average to high-molecular-weight dextran-type glucans ranging from 8.5 × 105 to >4 × 106 Da and containing 16 to 22% α-(1→3)-linked glucose units, with 5 to 8% branching in their structures. Enzyme preparations from the strains were applied in the synthesis of glucooligosaccharides (GOSs) with degrees of polymerization (DP) from 3 to 7 using maltose/sucrose ratios (M/S) from 8 to 0.12. The yields and composition of the obtained oligosaccharide products were strongly influenced by the ratios of the acceptor and donor of glucose units in the reactions. The highest yields of GOSs (>50 g/L) were achieved at M/S = 1, and the lowest content of α-(1→6) linkages (71%) combined with increased proportion of the products with DP > 3 were determined at M/S = 0.5. GOSs preparations from these two ratios were able to maintain the growth and acids production of three probiotic strains of lactobacilli, suggesting potential application as prebiotics.