Aerobic fed-batch cultures were studied as a means of suppressing the production of lactate, which inhibits the growth of lactic acid bacteria (LAB). LAB produce lactate via lactate dehydrogenase (LDH), regenerating nicotinamide adenine dinucleotide (NAD(+)) consumed during glycolysis. Therefore, we focused on NADH oxidase (NOX), employing oxygen as an electron acceptor, as an alternative pathway to LDH for NAD(+) regeneration. To avoid glucose repression of NOX and NAD(+) consumption by glycolysis exceeding NAD(+) regeneration by NOX, glucose was fed gradually. When Lactococcus lactis MG 1363 was aerobically fed at a specific growth rate of 0.2 h(-1), the amount of lactate produced per amount of grown cell was reduced to 12% of that in anaerobic batch cultures. Metabolic flux analysis revealed that in addition to NAD(+) regeneration by NOX, ATP acquisition by production of acetate and NAD(+) regeneration by production of acetoin and 2,3-butanediol contributed to suppression of lactate production. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.