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Sugarcane molasses is an agricultural by-product containing sucrose. In this study, the exopolysaccharide (M-EPS) produced by Leuconostoc citreum B-2 in molasses-based medium was characterized, optimized, and its application in set yogurt was investigated. The structure analysis, including gel permeation chromatography, Fourier transform infrared spectroscopy, and nuclear magnetic resonance, revealed that the M-EPS was a linear dextran composed of D-glucose units, which were linked by α-(1→6) glycosidic bonds with 19.3% α-(1→3) branches. The M-EPS showed a lower molecular weight than that produced from sucrose. The M-EPS was added into the set yogurt, and then the water holding capacity, pH, and microstructure of set yogurt were evaluated. Compared with the controls, the addition of M-EPS improved the water holding capacity and reduced the pH of set yogurt. Meanwhile, the structure of the three-dimensional network was also observed in the set yogurt containing M-EPS, indicating that M-EPS had a positive effect on the stability of set yogurt. The results provide a theoretical basis for the cost-effective utilization of sugarcane molasses. [ABSTRACT FROM AUTHOR]