2. NJ production culture

S. ficellus cultivation for NJ production was performed in a medium containing 13.6 g/L glucose, 40 g/L soluble starch, 8.0 g/L calcium carbonate, 20 g/L blackstrap molasses (EM Seikatsu, Nagoya, Japan), and 25 g/L additive reagent, adjusted to pH 7.2. For screening an effective additive reagent for NJ production, either BD Bacto™ Tryptic Soy Broth, BD Difco™ Marine Broth 2216, BD Difco™ Mueller Hinton Broth, Pharmamedia™ (Archer Daniels Midland Company, Chicago, IL, USA), polypeptone (Wako Pure Chemical Industries, Osaka, Japan), BD Difco™ Skim Milk, BD Bacto™ Tryptone, or BD Bacto™ Yeast Extract was used. The 20-times-diluted subculture was inoculated into fresh medium and cultured at 28°C and 170 rpm. In this study, we also evaluated the effect of removing soluble starch. For analyzing the time courses of cell growth, glucose, and NJ concentrations in S. ficellus culture in the medium supplemented by marine broth, we prepared an NJ production medium without soluble starch. For analyzing the NJ stability and bioactivity of S. ficellus culture, we also prepared NJ production medium without soluble starch.