A loop of spores of the strain K10-0216 was inoculated into 100 ml of the seed medium, consisting of 2.4% starch (Wako Pure Chemical Industries, Osaka, Japan), 0.1% glucose (Wako), 0.3% peptone (Kyokuto Pharmaceutical Industrial, Tokyo, Japan), 0.3% meat extract (Kyokuto), 0.5% yeast extract (Oriental Yeast, Tokyo, Japan) and 0.4% CaCO3 (Wako) (adjusted to pH 7.0 before sterilization) in a 500 ml Erlenmeyer flask. The flask was incubated on a rotary shaker (210 r.p.m.) at 27 °C for 3 days. A 1 ml portion of the seed culture was transferred to 500 ml Erlenmeyer flasks (total 150) containing 100 ml of starch medium, consisting of 2% soluble starch (Wako), 0.5% glycerol (Wako), 1.0% defatted wheat germ (Nisshin Pharma, Tokyo, Japan), 0.3% meat extract, 0.3% dry yeast (JT Foods, Tokyo, Japan) and 0.3% CaCO3 (adjusted to pH 7.0 before sterilization) and fermentation was carried out on a rotary shaker (210 rpm) at 27 °C for eight days.

The cultured broth of K10-0216 strain (15 l) was centrifuged to separate the mycelium and supernatant. The supernatant was extracted three times with ethyl acetate (15 l). The organic layer was concentrated to dryness in vacuo to afford a crude material (6 g). The ethyl acetate extract was subjected to silica gel column chromatography FL100D (60 i.d. × 200 mm, Fuji Silysia, Tokyo, Japan), which was sequentially eluted with 1 l of a mixture of CHCl3-CH3OH (100:0, 100:1, 50:1 and 10:1) in that order. The eluate fraction (100:1) was concentrated to yield 550 mg and applied to an ODS column (40 i.d. × 150 mm; Senshu Scientific, Tokyo, Japan). After washing with 50% MeOH, the fractions containing 1 were eluted with 60% MeOH, followed by concentration in vacuo. The eluate fractions (278 mg) were purified by high performance liquid chromatography on an Inertsil ODS-4 column (14 i.d. × 250 mm, GL Sciences, Tokyo, Japan) with 40% MeOH at 9.3 ml min−1 and subsequently detected under UV light of 254 nm. The yield of 1 was 45.0 mg.

Log in or create a free account to read this content

Gain free access to this article, as well as selected content from this journal and more on nature.com

Access through your institution

or

Sign in or create an account
Continue with Google
Continue with ORCiD