To increase the hyaluronic acid (HA) yield from Streptococcus thermophilus YIT 2084, fermentation conditions (pH, temperature, agitation, aeration) were optimized in milk-based medium, and the effects of supplemental soybean peptides, which have different molecular weight distributions, were determined. HA production was enhanced to approximately 100 mg/l at pH 6.8 and 33-40 degrees C. Agitation speed and aeration rate slightly affected HA production. Soybean peptides including those of high molecular weight (approximately 27 to 130 kDa) further increased HA production to 208 mg/l under the optimal condition (pH 6.8, 35 degrees C, 100 rpm), which was 20-fold greater than non-optimal condition. HA production was no longer related to the specific growth rate. The HA produced under the optimal condition included a large amount of high-molecular-weight fraction of 100 to 2000 kDa, compared with under the basal condition without optimization. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.