Background: Levan is a promising safe, renewable, and environmentally friendly exocellular homopolysaccharides. It has recently gained increasing research interest because of its significant impact on biotechnology applications due to its biocompatibility and ability to biodegradability. Aim: The study aimed to determine some of the optimal cultural conditions for levan production by studying many parameters affecting production by local isolation Bacillus subtilis (B. subtilis Strain AE77). Methods: B. subtilis Strain AE77 It was obtained from the Department of Food Science/College of Agricultural Engineering Sciences as a local levan-producing isolate isolated from rotten sweets. The Mineral Salts Broth (MSB) medium was approved to study the optimized conditions for levan production. Then precipitation of levan from the fermentation medium. Then the identification of levan with High-Performance Liquid Chromatography (HPLC) technology by estimating the retention time of levan, compared with the standard model produced from the bacterial isolate Erwinia herbicola (E.herbicola). Besides, each of the standard solutions of sugars (fructose, glucose, and sucrose). Results: The study results proved that the polysaccharide produced from this isolate is pure and consists of fructose and is a homopolysaccharide with the HPLC technique. The best levan production was from B. subtilis Strain AE77 using a modified MSB medium of 20% sucrose in a 48 hours incubation period. An inoculum volume of 1x10(8) cell/ml at a concentration of 2% at an incubation temperature of 35 degrees C and pH = 7.0 and agitation speed of 150 rpm reached 4.80 g/100ml. Conclusion: HPLC was used to separate, identify and quantify both B.subtilis Strain AE77 and standard levan produced from the bacterial isolate E. herbicola, as well as standard fructose, glucose, and sucrose, respectively. The results showed that levan is a homopolysaccharide consisting of one type of monomer (the fructose sugar units). Conditions for levan production were determined by isolating B. subtilis Strain AE77 in a sucrose medium. The results showed that the optimal medium for production contained 20% sucrose without any nitrogen source, pH7.0, and inoculum volume 1x10(8) cell/ml at a concentration of 2% at a temperature of 35 degrees C agitation speed 150 rpm, and incubation period of 48 hours.
