The short-chain FOSs with high purity were prepared using a two-step strategy: Aspergillus japonicus extracellular beta-fructofuranosidase-catalyzed synthesis of FOSs followed by cultivation with Pichia pastoris (P. pastoris). The higher FOSs content was obtained after 8 h under the catalysis of beta-fructofuranosidase at pH 5.5 and 55 degrees C. Successive P. pastoris cultivation exhausts almost all monosugars in 12 h at 30 degrees C, which increases the purity of FOSs, and also recovers beta-fructofuranosidase activity by ceasing the inhibition of glucose from catalysis of the enzyme, yielding more FOSs. Finally, the FOSs purity was increased from 56.55 to 84.45% (26.47% 1-kestose and 57.98% nystose).