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Exopolysaccharides (EPS) are well-known biopolymers secreted by several lactic acid bacteria with combination of various strains. The aim of this study is to increase EPS production by co-culturing Lactobacillus plantarum ATCC 8014 with Saccharomyces cerevisiae ICA-Y01 and study the changes in the functional characteristics of the EPS from both cultivations. In this study, the production and functional characteristics of EPS from co-cultivation culture of L. plantarum ATCC 8014 with S. cerevisiae ICA-Y01 were evaluated. The co-cultivation of L. plantarum ATCC 8014 with S. cerevisiae ICA-Y01was markedly increased EPS production up to 55.84% with 6.8 g/l yield after 20 hours cultivation. The pH of the co-cultivation culture was remained constantly at 5.2 until the end of cultivation. Furthermore, co-cultivation under pH 6 in the 16L bioreactor showed a higher growth rate of 0.214 h-1 and EPS production increased up to 104.44% when compared with single cultivation of L. plantarum ATCC 8014. This result clearly indicates the importance of growing the cells in the controlled pH condition when cultivated with S. cerevisiae ICA-Y01 to enhance EPS production. The functional characteristics of EPS secreted from both cultivation strategies were also evaluated. FT-IR spectroscopy confirmed EPS presence from both cultivations, indicating functional group of the polysaccharide with D-glucose units bound by α-(1→6). The EPS production from single cultivation showed a higher DPPH radical scavenging activity (88.21%) and IC50 (19.57%) as compared to EPS produced from co-cultivation with DPPH scavenging exhibited 32.45% with no IC50 value detected. Furthermore, solubility and water uptake of EPS from single cultivation are higher in comparison to co-cultivation. In conclusion, higher efficiency in the bioactivity of EPS from the single cultivation of L. plantarum ATCC 8014 was confirmed even though the EPS yield is low as compared to EPS synthesis through inter-kingdom cultivation.