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Oligofructans are potential biological substances that due to their distinctive properties have a positive health-promoting effect. This research aimed to produce oligofructans using sugarcane juice fermentation with Bacillus subtilis TISTR 001 and to study the prebiotic properties in vitro. The results showed that the maximum total oligofructans in the form of free fructose was 2.57% (w/v) at the 84th hour of fermentation with 0.17 g/g reducing sugar and a production yield of 0.031 g/L/h and maximum levansucrase activity of 1.57 × 106