We report a method for suppression of lactate production by lactic acid bacteria (LAB) in culture. LAB produce lactate to regenerate NADD that is consumed during glycolysis. Glucose suppresses NADD regeneration pathways other than lactate dehydrogenase and non-glycolytic ATP production pathways. Therefore, the carbon source was changed to sucrose, and fed-batch culture was performed to limit the glycolytic flux and thus suppress lactate production. As a result, lactate productivity (i.e., the amount of lactate produced per amount of grown cell) in the sucrose/fed-batch culture was decreased compared to that in glucose/batch culture, in all five LAB strains examined. The productivity level decreased to 24% and 46% in Lactobacillus reuteri JCM 1112 and Lactococcus lactis JCM 7638, respectively. Metabolic flux analysis of Lactobacillus reuteri JCM 1112 revealed increased contributions of the mannitol production pathway to NADD regeneration and the arginine deiminase pathway to ATP production in the sucrose/fed-batch culture. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.