Levan-typed fructooligosaccharide (LFOS), a β-2,6 linked oligofructose, displays the potential application as a prebiotic and therapeutic dietary supplement. In the present study, LFOS was synthesized using levansucrase from Bacillus amyloliquefaciens KK9 (LsKK9). The wild-type LsKK9 was cloned and expressed in E. coli, and purified by cation exchanger chromatography. Additionally, Y237S variant of LsKK9 was constructed based on sequence alignment and structural analysis to enhance the LFOS production. High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis indicated that Y237S variant efficiently produced a higher amount of short-chain LFOS than wild type. Also, the concentration of enzyme and sucrose in the reactions was optimized. Finally, prebiotic activity assay demonstrated that LFOS produced by Y237S variant had higher prebiotic activity than that of the wild-type enzyme, making the variant enzyme attractive for food biotechnology.

With the aim to overcome the limitations of hydrogel chitosan beads (HGBs), various types of chitosan, core–shell chitosan beads (CSBs), and dried chitosan beads (DBs) were synthesized. Physical and chemical properties were compared with those of HGBs. CSBs were proved to be an effective support because they displayed higher stability and capacity over the HGBs, and thus, were selected for enzyme immobilization. Recombinant inulosucrase (INU) from Lactobacillus reuteri 121 was immobilized on CSBs using glutaraldehyde as a cross-linker. Immobilized biocatalysts (INU-CSBs) were then used for the synthesis of inulin-type fructooligosaccharide (IFOS). Biochemical characterization revealed that the optimum pH of both INU-CSBs and free enzyme was unaltered at 5.5 whereas the optimum temperature of INU-CSBs shifted from 50 °C to 60 °C. Moreover, pH stability and thermostability of INU-CSBs significantly improved. For batch synthesis of IFOS, INU-CSBs retained approximately 45% of their initial catalytic activity after being reused for 12 cycles. IFOS was also continuously synthesized in a fixed-bed bioreactor for a reaction duration of at least 30 h. The high efficiency of INU-CSBs makes them very attractive for industrial applications.

Alternan, an α-1,3- and α-1,6-linked glucan, is a polysaccharide that is produced by bacteria. Although the structure of alternan used in this study, an α-1,3- and α-1,6-linked glucan (hereafter referred to as alternan), has been comprehensively characterized, its function on cell biology, especially relative to cell growth and differentiation, has not been fully elucidated. In this study, we set forth to compare the effect of alternan versus chitosan on the biological properties of human mesenchymal stem cells (MSCs). The effect of chitosan on MSC differentiation has already been well characterized. The treated cells were determined for cell proliferation and differentiation capacity compared to untreated cells. The result showed that treatment by alternan or chitosan increased cell proliferation, as demonstrated by increased cell number and scratched regions that were fully restored in less time than it took to fully restore controls. Further investigation found that alternan and chitosan activates the toll-like receptor (TLR) pathway suggesting that these cells may be prone to differentiation. In agreement with this result, an increase in deposited calcium was observed in alternan- or chitosan-treated cells after osteogenic differentiation induction. However, adipogenic differentiation was significantly inhibited in the presence of chitosan, but no change was observed in alternan treatment. Taken together, these results demonstrate biological effects of alternan on human MSCs. Moreover, these novel roles of alternan may have important beneficial medical applications and may provide a basis from which stem cell therapies can be developed in the future.