||Fructooligosaccharides (FOS), an important prebiotics, can stimulate the proliferation of bifidobacteria in the intestine thus improve human health. The currently available FOS is primarily inulo-type FOS, which comprises 1-kestose, nystose and fructosyl nystose. Neo-FOS can be produced from sucrose through the catalytic action of 6G-fructofuranosidase (6G-FFase) from Xanthophyllomyces dendrorhous. This neo-FOS product consisted of neokestose, neonystose and small amount of 1-kestose. Neo-FOS has received increasingly interest due to its superior heat- and acid-resistance, and bifidogenetic activity compared to inulo-type FOS. All FOS can be analyzed by HPLC, using amino-type columns (75% acetonitrile as the mobile phase) or Hydrosphere C18 column (water as the mobile phase), which provided better resolution compared to amino-type column. Purification of neo-FOS can be performed by HPLC on a semipreparative ODS-AQ column.
A high-purity neo-FOS was produced by submerged culture of Xanthophyllomyces dendrorhous BCRC 22367. The fermentation medium consisted of 250 g/L of sucrose and small amount of nitrogen source. During the fermentation, sucrose was converted into neokestose, neonystose, 1-kestose, glucose and fructose through the catalytic action of intracellular 6G-FFase. Concurrently, glucose and fructose were consumed by the yeast cells. After 70 h of fermentation at 20oC, a neo-FOS product composed of 87% FOS on a dry weight basis was obtained.
Production of 6G-FFase was performed by submerged culture of Xanthophyllomyces dendrorhous BCRC 21346 using a 5-L fermentor. The culture medium consisted of 3% yeast extract and 6% sucrose. The fermentation was carried out at 20oC, 2 vvm, 300 rpm and the pH of the culture broth was controlled around 6.9. After 36 h of fermentation, total 6G-FFase of 37,000 U/L was obtained. Intracellular enzyme accounted for 95.6% of total enzyme acticity and other was extracellular enzyme. Because intracellular 6G-FFase located in the cell wall was very stable at 30oC and pH 6-7, and was most active at pH 6.5. Neo-FOS was produced by free-whole-cell biotrasformation at 30oC and pH 6.5, using 600 g/L of sucrose as the substrate. After 3 h of reaction, a neo-FOS mixture consisted of 374 g/L of FOS was obtained. Yeast cells were recovered and reused. In 10 cycles of repeated batch operations, more than 90% of FOS productivity as that obtained in the first batch reaction was always achieved.