Isolation and Structural Characterization of an Oligosaccharide Produced by Bacillus subtilis in a Maltose-Containing Medium

  • Shin, Kwang-Soon (Department of Food Science and Biotechnology, Kyonggi University)
  • Received : 2016.04.12
  • Accepted : 2016.05.30
  • Published : 2016.06.30


Among 116 bacterial strains isolated from Korean fermented foods, one strain (SS-76) was selected for producing new oligosaccharides in a basal medium containing maltose as the sole source of carbon. Upon morphological characterization using scanning electron microscopy, the cells of strain SS-76 appeared rod-shaped; subsequent 16S rRNA gene sequence analysis revealed that strain SS-76 was phylogenetically close to Bacillus subtilis. The main oligosaccharide fraction B extracted from the culture supernatant of B. subtilis SS-76 was purified by high performance liquid chromatography. Subsequent structural analysis revealed that this oligosaccharide consisted only of glucose, and methylation analysis indicated similar proportions of glucopyranosides in the 6-linkage, 4-linkage, and non-reducing terminal positions. Matrix-assisted laser-induced/ionization time-of-flight/mass spectrometry and electrospray ionization-based liquid chromatography-mass spectrometry/mass spectrometry analyses suggested that this oligosaccharide consisted of a trisaccharide unit with 1,6- and 1,4-glycosidic linkages. The anomeric signals in the $^1H$-nuclear magnetic resonance spectrum corresponded to ${\alpha}$-anomeric configurations, and the trisaccharide was finally identified as panose (${\alpha}$-D-glucopyranosyl-1,6-${\alpha}$-D-glucopyranosyl-1,4-D-glucose). These results suggest that B. subtilis SS-76 converts maltose into panose; strain SS-76 may thus find industrial application in the production of panose.


Supported by : Kyonggi University


  1. Mussatto SI, Mancilha IM. 2007. Non-digestible oligosaccharides: a review. Carbohydr Polym 68: 587-597.
  2. Kaneko T, Kohmoto T, Kikuchi H, Shiota M, Iino H, Mitsuoka T. 1994. Effects of isomaltooligosaccharides with different degrees of polymerization on human fecal bifidobacteria. Biosci Biotech Biochem 58: 2288-2290.
  3. Heincke K, Demuth B, Jordening HJ, Klaus B. 1999. Kinetics of the dextransucrase acceptor reaction with maltose-experimental results and modeling. Enzyme Microb Technol 24: 523-534.
  4. Schwengers DDCD, Zeuner BG. 1986. Suessungsmittel, verfahren zur herstellung und verwendung desselben. DE Patent 3446380 C1.
  5. Chung CH. 2006. Production of glucooligosaccharides and mannitol from Leuconostoc mesenteroides B-742 fermentation and its separation from byproducts. J Microbiol Biotechnol 16: 325-329.
  6. Robyt JF, Kimble BK, Walseth TF. 1974. The mechanism of dextransucrase action: direction of dextran biosynthesis the mechanism of dextransucrase action: direction of dextran biosynthesis. Arch Biochem Biophys 165: 634-640.
  7. Robyt JF, Eklund SH. 1983. Relative, quantitative effects of acceptors in the reaction of Leuconostoc mesenteroides B-512F dextransucrase. Carbohydr Res 121: 279-286.
  8. Fu DT, Slodki ME, Robyt JF. 1990. Specificity of acceptor binding to Leuconostoc mesenteroides B-512F dextransucrase: binding and acceptor-product structure of ${\alpha}$-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 by inversion of the hydroxyl and by replacement of the hydroxyl with hydrogen. Arch Biochem Biophys 276: 460-465.
  9. Chung CH, Day DF. 2002. Glucooligosaccharides from Leuconostoc mesenteroides B-742 (ATCC 13146): a potential prebiotic. J Ind Microbiol Biotechnol 29: 196-199.
  10. Goffin D, Delzenne N, Blecker C, Hanon E, Deroanne C, Paquot M. 2011. Will isomalto-oligosaccharides, a well-established functional food in Asia, break through the European and American market? The status of knowledge on these prebiotics. Crit Rev Food Sci Nutr 51: 394-409.
  11. Kothari D, Goyal A. 2015. Gentio-oligosaccharides from Leuconostoc mesenteroides NRRL B-1426 dextransucrase as prebiotics and as a supplement for functional foods with anticancer properties. Food Funct 6: 604-611.
  12. Seo DM, Kim SY, Eom HJ, Han NS. 2007. Synbiotic synthesis of oligosaccharides during milk fermentation by addition of leuconostoc starter and sugars. J Microbiol Biotechnol 17: 1758-1764.
  13. Seo YS, Shin KS. 2011. Optimal conditions and substrate specificity for trehalose production by resting cells of Arthrobacter crystallopoietes N-08. J Food Sci Nutr 16: 357-363.
  14. Bae BS, Shin KS, Lee H. 2009. Structural characterization of non-reducing oligosaccharide produced by Arthrobacter crystallopoietes N-08. Food Sci Biotechnol 18: 519-525.
  15. Kenzaka T, Ishidoshiro A, Tani K, Nasu M. 2009. Scanning electron microscope imaging of bacteria based on DNA sequence. Lett Appl Microbiol 49: 796-799.
  16. Perevedentseva E, Cheng CY, Chung PH, Tu JS, Hsieh YH, Cheng CL. 2007. The interaction of the protein lysozyme with bacteria E. coli observed using nanodiamond labelling. Nanotechnology 18: 315102.
  17. Kimura M. 1981. Estimation of evolutionary distances between homologous nucleotide sequence. Proc Natl Acad Sci USA 78: 454-458.
  18. Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406-425.
  19. Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791.
  20. DuBois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. 1956. Colorimetric method for determination of sugars and related substances. Anal Chem 28: 350-356.
  21. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  22. Jones TM, Albersheim P. 1972. A gas chromatographic method for the determination of aldose and uronic acid constitulents of plant cell wall polysaccharides. Plant Physiol 49: 926-936.
  23. Hakomori S. 1964. A rapid permethylation of glycolipid, and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide. J Biochem 55: 205-208.
  24. Waeghe TJ, Darvill AG, McNeil M, Albersheim P. 1983. Determination, by methylation analysis, of the glycosyl-linkage compositions of microgram quantities of complex carbohydrates. Carbohydr Res 123: 281-304.
  25. Pettolino FA, Walsh C, Fincher GB, Bacic A. 2012. Determining the polysaccharide composition of plant cell walls. Nat Protoc 7: 1590-1607.
  26. Sauer J, Sigurskjold BW, Christensen U, Frandsen TP, Mirgorodskaya E, Harrison M, Roepstorff P, Svensson B. 2000. Glucoamylase: structure/function relationships, and protein engineering. Biochim Biophys Acta 1543: 275-293.
  27. Kohmoto T, Fukui F, Takaku H, Machida Y, Arai M, Mitsuoka T. 1988. Effect of isomalto-oligosaccharides on human fecal flora. Bifidobact Microflora 7: 61-69.
  28. Miyake T, Yoshida M, Takeuchi K. 1985. Imparting low- or anti-cariogenic property to orally-usable products. US Patent 4518581 A.
  29. Higashimura Y, Emura K, Kuze N, Shirai J, Koda T. 2001. Fading inhibitors. CA Patent 2378464 A1.
  30. Machida Y, Fukui F, Komoto T. 1991. Use of oligosaccharides for promoting the proliferation of bifidobacteria. EP Patent 0242459 B1.
  31. Rabelo MC, Honorato TL, Goncalves LR, Pinto GA, Rodrigues S. 2006. Enzymatic synthesis of prebiotic oligosaccharides. Appl Biochem Biotechnol 133: 31-40.
  32. Chung CH, Day DF. 2004. Efficacy of Leuconostoc mesenteroides (ATCC 13146) isomaltooligosaccharides as a poultry prebiotic. Poult Sci 83: 1302-1306.
  33. Fernandes FAN, Rodrigues S. 2006. Optimization of panose production by enzymatic synthesis using neural networks. Process Biochem 41: 1090-1096.
  34. Hayashi S, Hinotani T, Takasaki Y, Imada K. 1994. The enzymatic reaction for the production of panose and isomaltose by glucosyltransferase from Aureobasidium. Lett Appl Microbiol 19: 247-248.

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